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Annals of Pathology and Laboratory Medicine Nov-Dec. 2017; Vol. 4, Issue 6

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Contents

Original Article Analysis of double heterozygous haemoglobinopathies from a tertiary care center in A610-A614 North India Kaniyappan Nambiyar, Vijay Kumar, Sadhna Marwah, Abhay Shanker Nigam Role of Micronuclesus in Cervical Intraepithelial Lesions and Carcinoma Suganya Kuppovi Reddy, Surendra Kumar Verma, Sajini Elizabeth Jacob, Neelaiah Siddaraju, Dasari Papa, Suthanthira Kannan CD5 positive follicular lymphomas: A diagnostic dilemma in a resource restricted laboratory setting Sakthi Sankari Shanmuga, Arjunan Angaraju, Bhuvaneswari M Ganesan, Sindhuja Ramalingam, Rajeswari Thivya Dhanabalan Distribution Pattern of ER & PR Immunoexpression in Endometrial Biopsies of DUB and Infertile Patients from A Tertiary Care Centre Brijesh Thakur, Sanjay Kaushik, Sakshi Garg, Sanjeev Kishore Cytohistological Correlation of Palpable Breast Mass: A Study of 300 Cases Aditi Dharmesh Vasavada, Sheetal Kher Histomorphological Study Of Ovarian Tumors: At A Tertiary Care Centre Rashmi K Patil, Bhumika Jeevanraj Bhandari, Shreekant K Kittur, Rekha M Haravi, Aruna S, Meena N Jadhav Prognostic significance of prostate specific antigen in comparison with histological grade of prostatic adenocarcinoma: A Hospital based study Mohanrao Nandam, Vissa Shanthi, Bhavana Grandhi, Syama Sundara Rao Byna, Vijaya Lakshmi Muramreddy, Jyothi Conjeevaram ABO Blood groups and Malaria: Does it really matter? Chandrika Rao Relevance of Autopsy As a Diagnostic Tool in Present Times: A Study Kalpana Sharma, Aparna Rathi, Kusum Heda Heterometaplastic Bone Formation In Nephrolithiasis: Critical Review Of Pathology And Pathogenetic Mechanisms Nandkumar Vishwanath Dravid, Ashish V Rawandale, Arundhati S Gadre, Rajeshwari K, Kishor H Suryawanshi RBC Histogram as supplementary diagnostic tool with peripheral smear examination in evaluating anemias Byna Syam Sundar Rao, Vissa Santhi, Nandam Mohan Rao, Bhavana Grandhi, Vijaya Lakshmi Murra Reddy, Praveena Siresala Prognostic evaluation of vitamin D deficiency in breast cancer patients: a pilot study in India Bela Goyal, Sanjeev Garg A Comparative Study between Blood Donors and the General Population in Uttar Pradesh, India, to Analyse the Triggers for Donation Suparna Dubey, Seema Dua Evaluation of cervical smears by Conventional and Liquid Based Cytology Prakhar Srivastava, Rashmi Arora Spectrum of constitutive and inducible clindamycin resistance in Staphylococcus spp isolated from clinical samples and its relation with methicillin resistance. Monika Rajani, Malay Banerjee Impact of histopathological examination of appendix in context to clinical management of patients Mandakini M Patel, Rhuta J Shah Histopathological spectrum of Adult Nephrotic Syndrome over 16 years at a Tertiary care center in Mumbai with Clinicopathological ,Electron microscopy and Immunoflurescence Correlation of Renal biopsies Ganesh Ramdas Kshirsagar, Nitin Maheswar Gadgil, Sangeeta Ramulu Margam, Chetan Sudhakar Chaudhari, Prashant Vijay Kumavat, Sheela Jayawant Pagare Histopathological Analysis and Correlation of Ki67 and Progesterone Receptor Status with WHO Grading In Meningiomas Tamilselvi Veeramani, J Maheswari

III

A615-A620 A621-A625

A626-A631 A632-A637 A638-A645 A646-A650

A651-A655 A656-A661 A662-A667

A668-A672

A673-A677 A678-A685 A686-A691 A692-A698 A699-A704 A705-A713

A714-A720

Case Report

Diagnostic utility of Haematological scoring system (HSS) with clinicopathological and bacteriological evaluationin early diagnosis of neonatal sepsis Bhagyashree M Ahirrao, Nandkumar Dravid, Mahesh Ahirrao, Dhiraj Nikumbh, Arundhati Gadre, Shirish Gondane Role of Immunohistochemistry in trephine biopsies of Bone Marrow: A 5-year Retrospective study from a tertiary care hospital Ravi Teja J, Febe Renjitha Suman, Lenna Dennis Joseph, Jerusha Samuela Jacob, Rithika Rajendran, Jesu Magdalene S, Sai Shalini CN Role of renal biopsy in evaluation of morphological spectrum and pathogenesis of lupus nephritis Archana Chirag Buch, Rupali Bavikar, Shreya Rajesh Patel, Swapnil Karnik, Jehan Ansari Revisiting the Role of Chronic Kidney Disease and Its Association With Anemia in Diabetics and Non-Diabetics: A Cross Sectional Audit Of 450 Cases Lavanya Rajagopal, Sundaram Arunachalam, Shivashekar Ganapathy, Balaji Ramraj, Veena Raja Comparison of Papanicolaou and Acridine Orange stains in the diagnosis of Trichomonas vaginalis infection in vaginal discharge Neena Piyush Doshi Fine needle aspiration cytology of solitary thyroid nodule with histopathology correlation MS Susmitha, Veena S, Ramesh K Babu Rapid Cytodiagnosis By Different Staining Techniques In Comparison With Conventional Stains In Thyroid Cytology Veena Raja, Chinnaya Subramaniyam Babu Rajendra Prasad, Lavanya Rajagopal Diagnostic value of immunohistochemistry in soft tissue tumors Sridevi V, Susruthan Muralitharan, Thanka J Glioblastoma Multiforme: A clinico-pathological analysis Asha Shenoy, Shruti Shribhagwan Singhal Dermatological Manifestations In Human Immunodeficiency Virus Infected Patients: Histopathology And CD4 Cell Count Correlation Poonam Radadiya, Sheetal Kher Clinico Histopathological Correlation Study of Leprosy Bharat Ankush Ghodke, Arvind G Valand, Aradhana Bhubaneswar Deka, Sushma Nagsen Ramraje, Zeba Shamshad Ali Spectrum of Cervical Cytological Lesions in Premenopausal and Postmenopausal Women Vaishali Baburao Nagose, Nirvana Rasaily Halder, Shruthi Amit Deshpande, Shivanand Shriram Rathod, Varsha Ashok Jadhav Breast Adenomyoepithelioma with predominance of morules: a cytological dilemma Ganesh Ramdas Kshirsagar, Sheetal S. Yadav, Nitin Maheswar Gadgil, Chetan Sudhakar Chaudhari, Swati V. Patki, Prashant Vijay Kumavat Multiple Tuberous Xanthomas: A Diagnostic Dilemma on Cytology Richa Bhartiya, Pallavi Agrawal, Rajnish Kumar, Rajendra Prasad Dwivedi

A721-A726

Renal adysplasia in fetus: A rare autopsy case report Bhumika J Bhandari, Rashmi K Patil, Shreekant K Kittur

C172-C175

Urinary Bladder Cancer : Two Rare Cases Meenu Gupta, Gopinath Barui, Tushar Kanti Das

C176-C178

Letter to editor Rhino-Orbito-Cerebral Mucormycosis

Urshlla Kaul, Prabhakar Patro, Amruta Padhye, Reeta Dhar

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Website: www.pacificejournals.com www.apalm.org

E-mail: contactus@pacificejournals.com

A727-A731

A732-A741 A742-A748 A749-A754 A755-A760 A761-A767 A768-A773 A774-A781 A782-A787 A788-A794 A795-A800

C165-C168 C169-C172

L21-L22

Original Article DOI: 10.21276/APALM.1352

Analysis of Double Heterozygous Haemoglobinopathies from a Tertiary Care Center in North India Kaniyappan Nambiyar,1 Vijay Kumar2*, Sadhna Marwah2, Abhay Shanker Nigam2 1 Department of Histopathology, Post Graduate Institute of Medical Education and Research , Chandigarh, India Department of Pathology, Post Graduate Institute of Medical Education and Research & Dr RML Hospital, New Delhi, India

ABSTRACT Background: Haemoglobinopathies and thalassemias are inherited conditions, being diagnosed with increasing prevalence in India. The double heterozygosity for α and β chain variants leads to the formation of abnormal heterodimer hybrids, which can lead to diagnostic dilemmas. Hematological parameters of double heterozygous conditions have not been analysed much in the literature. Methods: This study is a retrospective analysis of haemoglobin High Performance Liquid Chromatography (HPLC) from January 2006 to August 2014. Hematological parameters of these patients were also analysed and correlated with respective haemoglobin HPLC findings. Family screening of cases was also done wherever possible. Result: Out of 6180 cases, 14 cases were found to be of double heterozygous with 10 cases of Hb E-β thalassemias, and 4 cases of Hb S-β thalassemias. In Hb E-β thalassemias, significant negative correlation was noted between haemoglobin and Red cell Distribution Width (RDW) and also between RDW and Red Blood Cell (RBC) count. In Hb S-β thalassemias, significant negative correlation was seen between Hb A2 level and RBC count. Conclusion: Although, haemoglobin chain disorders require combination of techniques, HPLC is a cost effective and powerful tool for characterization of these disorders. This study also highlights the importance of hematological parameters (Hb, RDW and RBC count) in elucidation of double heterozygous haemoglobinopathies from much commoner variants of haemoglobinopathies, particularly in under resourced areas. No similar studies correlating HPLC findings and RBC indices have been found in the literature. Keywords: HPLC, Double Heterozygous Haemoglobinopathies, Hb E-β Thalassemia, Hb S-β Thalassemia

Introducation

Haemoglobinopathies and thalassemias are inherited conditions of abnormal haemoglobin synthesis being diagnosed with increasing prevalence in India. They occur due to changes in the amino acid sequence of either globin chain or decrease in globin chain production.[1] Common haemoglobinopathies in India are thalassemia along with Hb S, Hb E, Hb D, and their combinations.[2] Cation exchange high performance liquid chromatography (CE-HPLC) is an excellent tool for accurate and reliable diagnosis of various haemoglobin (Hb) disorders. Haemoglobinopathies can occur either in heterozygous or homozygous states. The double heterozygosity for α and β chain variants leads to the formation of abnormal heterodimer hybrids.[1] This results in variable clinical and laboratory features leading to diagnostic dilemmas and delay in diagnosis. So, this study aimed to analyse the haematological and HPLC parameters in cases of double heterozygous haemoglobinopathy, and also to identify correlation between these parameters.

Materials and Methods

This study was a retrospective analysis of haemoglobin HPLC taken from the medical records of a tertiary care

hospital in North India from January 2006 to August 2014. A total of 6180 cases were included in the study. Complete haemogram with RBC indices was performed on automated cell counter for all the cases. HPLC was performed using VARIANTTM β-thalassemia short program and was used for identification of haemoglobinopathies in this study. Family screening of cases was also done wherever possible. Statistical analysis was performed using SPSS version 17 software. Pearson correlation was also done and a p value less than 0.05 was considered significant.

Results

Among 6180 patients only 249 (i.e. 4.02%) were found to have abnormal haemoglobin fractions. Among the patients having abnormal haemoglobin fractions on HPLC, 14 (0.22%) were found to have double heterozygous haemoglobinopathies. Hb E-β Thalassemia: Patient showing a peak at the HbA2/E position with increased HbA2 level (>15%) and increased Hb F level (5 - 87%) was labelled as a double heterozygous state of Hb E and β-thalassemia.[3] In the present study, 10 (0.16%) patients were diagnosed as Hb

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Reddy et al.

A-611 Hb S-β thalassemia: Patient showing a peak at the S-window with increased Hb A2 (3.5-5.5%) was labelled as a double heterozygous state of Hb-S and β-thalassemia. [4] In the present study, 4 (0.06%) patients were diagnosed as Hb S-β thalassemias. The mean value of Hb S and Hb A2 were found to be 62.8±14.8% and 4.7±0.6% respectively. The mean value of Hb F was found to be 16.3±5.8%. Complete haemogram revealed microcytic hypochromic anaemia in all the patients except for one case, with the mean haemoglobin of 8.3g/dl, ranging from 5.3 to 11 g/ dl. Mean value of HCT, RBC, MCV, MCH, MCHC, and RDW were 25.6%, 3.6 X1012/l, 69.4 fl, 23.3 pg, 33.5 g/dl and 25.6% respectively (Table 1). A negative correlation between Hb A2 and RBC count, as well as between Hb A2 and HCT (p value < 0.05) was noted.

E-β thalassemias. The mean value of Hb A2 and Hb F were 55.8±15.6 % and 27.2±17.4 % respectively. Complete haemogram revealed microcytic hypochromic anaemia in all the patients with the mean haemoglobin of 7.3g/dl, ranging from 3.1 to 9.5g/dl. Mean values of RBC count, hematocrit, Mean Corpuscular Volume (MCV), Mean Corpuscular haemoglobin (MCH), and Mean corpuscular Haemoglobin Concentration (MCHC) were 3.82 X1012/l, 22.8%, 60.3 fl, 19.5 pg and 32.3 g/dl respectively. RDW ranged from 21.1 to 40.5% with a mean of 32.1% (Table 1). Pearson correlation between RBC count and RDW showed a significant negative correlation (p value < 0.05). Pearson correlation between Hb level and RDW also showed a significant negative correlation (p value < 0.05).

Table 1: HPLC haemoglobin fractions and their haemogram values. S.no

Hb A2 (%)

Hb S (%)

Hb F (%)

Hb (g/dl)

RBC (X1012/l)

MCV (fl)

MCH (pg)

MCHC (g/dl)

RDW (%)

HCT (%)

Hb E-β thalassemia 1

81.1

9.7

9.3

4.52

60.4

20.7

34.3

21.1

27.3

74.3

8.5

4.69

56.8

18.2

28.2

26.7

69.5

15.7

6.5

4.41

47.5

14.8

31.3

20.9

59.3

20.1

7.8

4.31

60.4

18.2

30.2

31.4

53.5

37.2

3.1

1.71

64.3

21.2

39.3

52.7

17.3

6.3

2.86

68.6

40.5

19.6

49.3

45.2

9.5

4.68

63.1

20.4

32.4

24.5

29.5

48.2

44.3

7.3

3.85

58.9

19.1

32.4

35.5

22.5

40.5

58.5

6.8

3.55

58.9

19.2

32.6

30.9

20.9

11.3

3.68

21.7

33.4

35.8

23.9

Hb S-β thalassemia 11

5.4

5.3

2.19

68.6

26.7

38.9

22.9

5.1

69.6

19.1

3.04

64.4

19.9

30.8

19.6

4.3

62.2

23.2

4.90

22.5

31.2

22.7

35.3

4.1

42.4

11.9

10.9

4.53

72.6

24.2

33.3

32.8

Table 2: Comparison of prevalence with other studies. Diagnosis Hb E-β thalassemia

Hb S-β thalassemia

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Study

Prevalence (%)

Present

0.16

Mohanty D et al [7]

0.19

Baruah MK et al [8]

2.14

Present

0.06

Mohanty D et al [7]

0.02

Baruah MK et al [8]

0.6

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Analysis of Double Heterozygous Haemoglobinopathies

Table 3: Comparison of HPLC haemoglobin fraction with other study. Diagnosis Hb E-β thalassemia Hb S-β thalassemias

Study

Hb A2 (%)

Hb F (%)

Hb S (%)

Present

55.8±15.6

27.2±17.4

Baruah MK et al [8]

56.7±9.6

30.7±10.1

Present

4.7±0.6

16.3±5.8

62.8±14.8

Baruah MK et al [8]

6.3±0.9

18.9±8.7

69.3±9.2

Fig. 1: a) Double heterozygous state of Hb E and β-thalassemia b) Double heterozygous state of Hb-S and β-thalassemia.

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Reddy et al.

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Fig. 2: Family study of double heterozygous Hb E and β-thalassemia.

Discussion

The coinheritance of two different haemoglobinopathies is a rare occurrence. Although, they superficially resemble the non-coinherited variants, they show subtle variations in clinical, laboratory and management aspects. Among the laboratory investigations, HPLC is useful in presumptive diagnosis of various haemoglobinopathies. Although, DNA based techniques are needed for better delineation of these disorders, HPLC analysis in conjunction with clinical profile and different haematological parameters is pertinent for evaluation of double heterozygous haemoglobinopathies. The cumulative frequency of haemoglobinopathies in India has been found to be 4.2% in a previous study.[5] A comparable frequency of haemoglobin chain disorders (4.02%) was observed in our study.

and β-thalassemia. A study by Mohanty D et al [7] reported a prevalence of 0.19% which is similar to the present study (0.16%). Higher prevalence (2.14%) noted in the study by Baruah MK et al [8] is due to regional variation of haemoglobin disorders in India (Table 2). Mean values of HbA2 and Hb F in this study were similar to another study by Baruah MK et al [8] (Table 3). In addition, there was a negative correlation between RDW and Hb and also between RDW and RBC count (p value < 0.05). However, no studies correlating HPLC findings and RBC indices have been found in the literature. Family study of two cases in the same family has been depicted in figure 2. Similar family study for diagnosis of double heterozygous conditions has also been used in other studies.[1, 9]

Hb E-β Thalassemia: Patients having double heterozygous state of Hb E and β-thalassemia is characterized by marked clinical variability ranging from mild and asymptomatic anaemia to a life-threatening disorder requiring transfusions from infancy.[6] This is different from other double heterozygous states for structural β-chain variants

Hb S-β Thalassemia : Hb S-β thalassemia is a double heterozygous state of Hb S and β-thalassemia. The clinical course is similar to that of Hb SS, however anaemia is comparatively milder than in sickle cell anaemia [4]. A study by Mohanty D et al [7] reported a prevalence of 0.02% which compares favourable with our study (0.06%). Higher prevalence (0.6%) noted in the study by Baruah MK et al [8] is due to regional variation of haemoglobin

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Analysis of Double Heterozygous Haemoglobinopathies

disorders in India (Table 2). Previous study showed mean values of MCV for Hb S-β+ thalassemias and Hb S-β0 thalassemias to be 72 fl and 67.8 fl respectively [4] and a comparable mean value of MCV of 69.4 fl was noted in our study. Mean values of Hb S, HbA2 and Hb F in this study were similar to another study by Baruah MK et al [8] (Table 3). There was a negative correlation between Hb A2 and RBC count as well as between Hb A2 and HCT (p value < 0.05). No similar studies correlating these parameters have been found in the literature.

diagnostic evaluation and understanding of these disorders particularly in under resourced areas.

Conclusion

In conclusion, rarity of these disorders makes the diagnosis challenging. Haemoglobin chain disorders require a combination of techniques including HPLC, which is one of the most important diagnostic modality for elucidation of double heterozygous cases. Diagnostic difficulties are encountered by overlapping laboratory findings, prompting the search for newer parameters. Hematological parameters like haemoglobin level, RBC indices, and Hb A2 level and their correlation are important for characterization of double heterozygous state. This study highlights the significant negative correlation between RDW and Hb as well as RDW and RBC count in double heterozygous cases of Hb E-β thalassemia (p value <0.05), which helps particularly in elucidation of challenging cases. Role of family screening in a suspected case is also crucial. To the best of our knowledge, correlation of different hematological parameters for characterization of double heterozygous haemoglobinopathies has not been reported so far in the Indian literature. However, further studies including larger number of cases are required to validate the statistical correlation. This may unmask significant haematological parameters, which lead to better

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Mutreja D, Tyagi S, Tejwani N, Dass J. Double heterozygous hemoglobin Q India/hemoglobin D Punjab hemoglobinopathy: Report of two rare cases. Indian J Hum Genet. 2013;19(4):479-82.

Agarwal MB. The burden of haemoglobinopathies in India--time to wake up? The Journal of the Association of Physicians of India. 2005;53:1017-8.

Bain BJ. Other significant hemoglobinopathies. In: Bain BJ editor. Haemoglobinopathy diagnosis. 2nd edn. Massachusetts: Blackwell Publishing Ltd; 2006. 206-9.

Bain BJ. Sickle cell hemoglobin and its interactions with other variant haemoglobins and with thalassaemias. In: Bain BJ editor. Haemoglobinopathy diagnosis. 2nd edn. Massachusetts: Blackwell Publishing Ltd; 2006. 170-3.

Sarnaik SA. Thalassaemia and related haemoglobinopathies. Indian J Pediatr. 2005;72:319-24.

Olivieri NF, Pakbaz Z, Vichinsky E. Hb E/beta-thalassaemia: a common & clinically diverse disorder. Indian J Med Res. 2011;134:522-31.

Mohanty D, Colah RB, Gorakshakar AC, Patel RZ, Master DC, Mahanta J, et al. Prevalence of beta-thalassemia and other haemoglobinopathies in six cities in India: a multicentre study. J Community Genet. 2013;4(1):33-42.

Baruah MK, Saikia M, Baruah A. Pattern of hemoglobinopathies and thalassemias in upper Assam region of North Eastern India: high performance liquid chromatography studies in 9000 patients. Indian J Pathol Microbiol. 2014;57(2):236-43.

Rao S, Kar R, Gupta SK, Chopra A, Saxena R. Spectrum of haemoglobinopathies diagnosed by cation exchange-HPLC & modulating effects of nutritional deficiency anaemias from north India. Indian J Med Res. 2010;132:513-9.

*Corresponding author: Dr.Vijay Kumar, Room no: 315, Third floor, OPD building, Department of Pathology, Post Graduate Institute of Medical Education and Research & Dr RML Hospital, New Delhi, India– 110001 Phone: +91 9560204567 Email: vijaypgi1@gmail.com Date of Submission : 12.02.2017 Date of Acceptance : 09.06.2017 Financial or other Competing Interests: None. Date of Publication : 11.12.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Original Article DOI: 10.21276/APALM.1354

Role of Micronucleus in Cervical Intraepithelial Lesions and Carcinoma Suganya Kuppovi Reddy1*, Surendra Kumar Verma1, Sajini Elizabeth Jacob1, Neelaiah Siddaraju1, Dasari Papa1, Suthanthira Kannan2 1 ÂJIPMER, Puducherry, India Government Medical College, Kozhikode, India

ABSTRACT Background: Micronuclei (MN) are considered sensitive indicators of chromosomal damage. Studies have emphasized the utility of MNscoring in detection of increased risk of various cancers in humans. More recent studies have shown MN assay to be highly promising in cervical cancer screening. Aims: To evaluate the utility of micronuclei in distinguishing various squamous intraepithelial lesions and invasive squamous cell carcinoma of cervix. Methods: In this descriptive study, a total of 100 cases were studied, of which 50 were normal (control), 10 were high grade squamous intra epithelial lesions (HSIL) and 26 were invasive squamous cell carcinomas (SCC). Remaining 14 cases were equivocal entities comprising 3 ASCUS and 11 ASC-H cases. In each case, MN-count was done per 1000 epithelial cells and a consensus MN-score was taken after a consensus and careful review by 4 cytopathologists. Results: The mean MN-score showed a gradual increase from normal smear to invasive SCC cases. MN-score of HSIL and invasive SCC were significantly higher than the normal and ASC-H smears. Cases with premalignant/ malignant outcome had a significantly high MNscore as compared to cases with reactive outcome. Conclusion: MN-scoring is a simple, non invasive and cost effective test, which can be performed on an easily collected exfoliated cell sample. MN-assay in conjunction with conventional Pap test can be an effective tool for screening cervical cancer and identifying women at risk. Keywords: Micronuclei, MN-score, Cervical Cancer, Precancerous Lesions

Introduction

Micronucleus (MN) originates from chromosome fragments or whole chromosomes that are not included in the main daughter nuclei during nuclear division.[1] Its presence in a cell is a sensitive indicator of chromosomal damage. A direct association between increased MN frequency and cancer development has already been reported in head and neck,[2] colonic,[3] cervical,[4] and urothelial malignancies.[5] MN frequency is also increased in certain chromosome breakage syndromes such as Bloom syndrome and ataxia telangiectasia,as well as, in persons exposed to chemical carcinogens and ionizing radiation, indicating the increased risk of cancer.[6] Cervical cancer is one of the most common cancers in India and Papanicolaou (Pap) cervical cytology is an effective screening method for its early detection. Bethesda 2001 system for reporting cervical cytology has certain equivocal

entities like Atypical Squamous Cells of Undetermined Significance (ASCUS) and Atypical Squamous Cells cannot exclude HSIL (ASC-H).[7] Only a minor proportion of cases reported as ASCUS and ASC-H turn out to be cervical intraepithelial lesion (CIN) on histopathology.[8,9] HPV DNA testing is a useful adjunct in identifying those high risk cases, but it has cost limitations.[8] Therefore, there is a need for a simple, reliable, reproducible & cost effective method for identifying the true CIN cases, among those reported as ASCUS and ASC-H. Over the last decade, the wide applicability of the micronucleus test in peripheral blood lymphocytes and the simplicity of scoring has made it an attractive cytogenetic tool to assess occupational and environmental exposures to genotoxic agents. Micronucleus counting in cervical lesions, a simple non invasive procedure, helps to stratify pre-invasive lesions. Thus, this method in conjunction with present cytology-based test will be a helpful tool for triage, follow up or treatment of borderline or doubtful cases.[10]

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Aims and Objectives: To evaluate the utility of micronuclei in distinguishing various squamous intraepithelial lesions and invasive squamous cell carcinoma of cervix

Materials and Methods

stain deposits, bacterial colonies, cytoplasmic fragments, blood elements, sperms, apoptotic bodies, karyorrhetic material were carefully looked for and exempted from counting (Figure 3).

Our study is a descriptive study conducted in the Department of Pathology and Obstetrics & Gynaecology, JIPMER between January 2012 and March 2013. In this we compared the micronucleus scoring in the whole spectrum of cervical lesions.

Frequency of MN in various squamous intra-epithelial/ invasive lesions was compared among the various groups including the control group. Pre-biopsy cytologic interpretation and micronucleus frequency was compared with the histopathologic diagnosis.

A total of 100 cases interpreted as per the Bethesda 2001 system for reporting cervical cytopathology between January 2012 and June 2013 were examined. Among them 50 were normal smears without any intraepithelial abnormalities, 10 were premalignant lesions- high grade squamous intra epithelial lesions (HSIL) and 26 were frank invasive squamous cell carcinomas. Remaining 14 cases were equivocal entities including 3 cases of ASCUS and 11 cases of ASC-H.

Results

Smears with (i) scant cellularity, (ii) marked drying artifact, (iii) squamous cells obscured by intense inflammation and hemorrhage and (iv) poor staining were excluded from the study. These smears were subjected to routine Papanicolaou staining after fixation with 95% ethanol in the cytology laboratory. Final interpretation of all the cases was made after careful evaluation by four different cytopathologists. MN-frequency was studied with strict criteria in all the cases by counting 1000 squamous epithelial cells under oil immersion magnification (Figure 1 &2). Criteria for Micronuclei: The criteria for designating an extra-nuclear body as “micronucleus” were: [1,2,7] 1. Diameter 1/16 to 1/3 of the main nucleus 2 Staining intensity similar to, or slightly weaker than, that of the nucleus 3. Round-to-oval shape with a smooth perimeter i.e. the borders should be distinctly recognizable indicating the presence of a nuclear membrane 4. Color and texture same as that of the main nucleus 5. Close proximity, but no actual contact with the nucleus i.e. absence of overlap with or bridge to the nucleus 6. Plane of focus same as that of the main nucleus 7. Non refractile A consensus ‘micronucleus frequency’ was taken after the smears were evaluated by four cytopathologists independently. Cells with double or multiple MN were also given a score of one. Keratohyaline granules, candida,

The mean age of the patients in normal, ASCUS, ASC-H, HSIL, SCC of cervical lesions were 38.52±12.49, 45.67±15.63, 44.36±8.80, 40.90± 9.25, 56±9.89years respectively (Table 1). The mean age was more in patients with invasive SCC than in normal and precursor lesions. The mean MN-score in normal smears andvarious cervical lesions and its trend are shown in table-1 and figure-4; while, biopsy outcome of the patients with cervical lesions are shown in table-2.Biopsy was not available for control cases (normal smears) There was a gradual and stepwise increase in MNscore from normal to ASCUS to ASC-H to HSIL and to invasive SCC. Analysis of variance(ANOVA) was applied to analyse the difference in mean values of MN-scores among different groups. The increase in MN-score was more significant in invasive SCC group as compared to control and ASC-H groups, but the MN- score between the HSIL and invasive SCC group was not significant though it showed a gradual increase. We also correlated the MN-score with final biopsy outcome (Table-3). We noted a progressive increase in mean MN score from non-malignant to malignant cases. Two or more MN in a single cell wasa rare feature and was noted in Invasive SCC. Taking biopsy diagnosis as the gold standard, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of MN was calculated. MN were found to be 100% sensitive and 72% specific in detecting squamous intraepithelial lesions and SCC with a PPV and NPV of 95% and 100% respectively.

Discussion

Cervical cancer is one of the most common cancers in India[10]. The phenotypic changes of cervical cancer are always preceded by genetic damage caused by various carcinogens. Human papillomavirus (HPV) is a causal factor in the development of cervical cancer and its precursor lesion, cervical intraepithelial neoplasia (CIN).

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Table 1: Age-distribution and micronucleus frequency in cervical lesions. Group

No of Cases

Mean age ± Standard deviation (years)

Mean MN-Score ±Standard deviation

Normal

38.52±12.49

0.04± 0.19

ASCUS

45.67±15.63

1.67±1.53

ASC-H

44.36±8.80

5.27 ± 5.36

HSIL

40.90± 9.25

9.5 ±2.95

SCC

56.00±9.89

10.77 ± 3.96

P-Value

P<0.05

Table 2: Biopsy outcome of various cervical lesions. Biopsy outcome

Groups

No of cases

Normal

Chronic cervicitis

Metaplasia

Premalignant (CIN)

Malignant

ASCUS

ASC-H

HSIL

SCC

Table 3: Correlation of MN frequency with final biopsy outcome. Biopsy outcome

No of cases

MeanMN-Score±Standard deviation

Non malignant

0.71 ±1.25

Pre malignant

7.93 ±3.38

Malignant

11.10 ± 3.68

P-value P<0.001

Fig. 1: MN in the cervical smear of HSILcase.(Pap X1000). Inset shows HSIL cells.

Fig. 2.Cervical smear of an invasive SCC case showing MN. Inset shows a tadpole cell with aMN(Pap X1000).

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Fig. 3: Various mimics of MN. a. Multiple dot-likekerato-hyaline granules; b. A cell showing apoptotic bodies in a case of invasive SCC; c.Superimposed neutrophil and bacterial debris simulating micronucleus and d. Karyorrhexis in a case of invasive SCC (Pap x1000).

Though vaccination against HPV infection and periodical Papanicolaou cervical cytology screening are effective measures for preventing cervical cancer; there is a need for further improvement in the test in order to increase its sensitivity.[11] Since HPV infection induces cytogenetic instability in cervix cells, it can be evaluated by means of MN assay. Human bio-monitoring studies have shown that MN assay in exfoliated cells is a site-specific biomarker of exposure to genotoxic agents and cancer risk.[12]. Among the various non- invasive early detection methods, MN formed in vivo can be used as a suitable biomonitoring approach for detection of increased cancer risk in man, because >90% of all human cancers are of epithelial origin. In the present study, we noted a gradual increase in MNscoreaccording to the severity of phenotypic changes. Ourfindings are in concordance with those of previous

studies by Samanta et al[1], Guzman et al[13] and Leal Garza et al[14].We observed a significant difference in MNfrequency between HSIL/ invasive SCC groups and other groups. ASC-H with reactive outcome had a significantly lower MN-frequency as compared to those with CIN outcome. However, the MN-score between HSIL and invasive SCC was statistically not significant. Samanta et al[1] (2011) studied normal, inflammatory, ASCUS, ASC-H, LSIL and HSIL smears as separate groups and observed a statistically significant and gradually increasing MN score among these lesions. Leal Garza(14) found that MN-frequency in pap smear increases with increasing grade of cervical intraepithelial lesionsand there was a positive linear trend between the MN- frequency and increased cancer risk . Guzman(15) observed that MN-frequency of more than one was more

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Reddy et al. commonly found in LSIL (18%), HSIL (33%) and invasive carcinomas (16%) as compared to ASCUS (6%) and normal controls (3%). However there was no significant difference in the MN-frequencies of ASCUS cases when compared with normal smears. The wide variation in the MN-frequency among the individuals within the same group are attributed to various confounding factors like lifestyle, environmental exposure, micronutrient deficiency, genetic makeup, base line MNfrequencies and chromosomal damage.(1) The possible mechanisms(14) for progressive increase in MN in the cervical cancer patients are (1) the metabolic stress due to tumor growth, (2) the “clastogenic” product released by the tumor cells, (3) micronutrient deficiencies such as the folate and the vitamin B12 and (4) the presence of HPV. So it is evident that the cytomorphology together with MNfrequency helps in discriminating cellular atypia due to reactive change versus dysplastic change. The study of MN in Pap smears increases the sensitivity and specificity of cervical smears which could have an impact in diagnostics and secondary prevention of cervical cancer.(16) Hence, the frequency of MN can be used as an additional criterion for assessing the cervical cancer risk. Positive message from study: MN-scoring is a simple, non invasive and cost effective test, which can be performed on an easily collected cell sample. The processing and staining are less time consuming thanother test systems. The test can be performed in anyminimally equipped cytology laboratory. Though MN counting is time consuming and relatively tedious process with a need for diligent approach, it’sa worth while test in situations with genuine difficulty in distinguishing reactive versus ASC-H cases. Limitations of study: Relatively less number of samples and exclusion of inflammatory smears are a minor limitation of our study. As LSIL cases generally do not undergo biopsy follow up, they were not included in the study. Implication of this study: This simple test of detection of MN in conventional Papsmears can be used as a cost effective prognostic indicator during planning and validation of programs for cervical cancer screening, monitoring and prevention.

Conclusion

MN assay in conjunction with conventional Pap test can be utilized in screening cervical cancer and identifying women at risk. However, additional studies are needed to compare www.pacificejournals.com/apalm

A-619 spontaneous and induced MN in cervical epithelial tissues, to standardize protocols and validate the induction of MN in epithelial tissues as biomarker of cancer risk.

Refernces 1.

Samanta S, Dey P, Nijhawan R. Micronucleus in cervical intraepithelial lesions and carcinoma. Acta Cytol. 2011;55(1):42–7.

Delfino V, Casartelli G, Garzoglio B, Scala M, Mereu P, Bonatti S, et al. Micronuclei and p53 accumulation in preneoplastic and malignant lesions of the head and neck. Mutagenesis. 2002;17(1):73–7.

Karaman A, Binici DN, Kabalar ME, Calikuşu Z. Micronucleus analysis in patients with colorectal adenocarcinoma and colorectal polyps. World J Gastroenterol WJG. 2008 ;14(44): 6835–9.

Aires GMA, Meireles JRC, Oliveira PC, Oliveira JL, Araújo EL, Pires BC, et al. Micronuclei as biomarkers for evaluating the risk of malignant transformation in the uterine cervix. Genet Mol Res GMR. 2011;10(3):1558–64.

Arora SK, Dey P, Saikia UN. Micronucleus in atypical urothelial cells. Diagn Cytopathol. 2010;38(11):811–3.

Fenech M, Holland N, Chang WP, Zeiger E, Bonassi S. The HUman MicroNucleus Project--An international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans. Mutat Res. 1999;428(1-2):271–83.

Solomon D. The Bethesda system for reporting cervical cytology: definitions, criteria, and explanatory notes. New York: Springer; 2004.pp?

Manos MM, Kinney WK, Hurley LB, Sherman ME, ShiehNgai J, Kurman RJ, et al. Identifying women with cervical neoplasia: using human papillomavirus DNA testing for equivocal Papanicolaou results. JAMA J Am Med Assoc. 1999;281(17):1605–10.

Solomon D, Davey D, Kurman R, Moriarty A, O’Connor D, Prey M, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA J Am Med Assoc. 2002;287(16):2114–9.

10. Saxena U, Sauvaget C, Sankaranarayanan R. Evidencebased screening, early diagnosis and treatment strategy of cervical cancer for national policy in low- resource countries: example of India. Asian Pac J Cancer Prev APJCP. 2012;13(4):1699–703. 11. Leyden WA, Manos MM, Geiger AM, Weinmann S, Mouchawar J, Bischoff K, et al. Cervical cancer in women with comprehensive health care access: attributable factors in the screening process. J Natl Cancer Inst. 2005;97(9):675–83. 12. Fenech M, Kirsch-Volders M, Natarajan AT, Surralles J, Crott JW, Parry J, et al. Molecular mechanisms of

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micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis. 2011;26(1):125–32. 13. Guzmán P, Sotelo-Regil RC, Mohar A, Gonsebatt ME. Positive correlation between the frequency of micronucleated cells and dysplasia in Papanicolaou smears. Environ Mol Mutagen. 2003;41(5):339–43. 14. Leal-Garza CH, Cerda-Flores RM, Leal-Elizondo E, CortésGutiérrez EI. Micronuclei in cervical smears and peripheral

blood lymphocytes from women with and without cervical uterine cancer. Mutat Res. 2002;515(1-2):57–62. 15. Guzmán P, Sotelo-Regil RC, Mohar A, Gonsebatt ME. Positive correlation between the frequency of micronucleated cells and dysplasia in Papanicolaou smears. Environ Mol Mutagen. 2003;41(5):339–43. 16. Nersesyan AK. Possible role of the micronucleus assay in diagnostics and secondary prevention of cervix cancer: a minireview. T‪Sitologii‪a Genet. 2007;41(5):64–6.

*Corresponding author: Suganya Kuppovi Reddy, JIPMER, Puducherry, India Phone: +413 229 6562 Email: dr. suganya.k@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 12.02.2017 Date of Acceptance : 11.06.2017 Date of Publication :11.12.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Original Article DOI: 10.21276/APALM.1364

CD5 Positive Follicular Lymphomas- A Diagnostic Dilemma in a Resource Restricted Laboratory Setting Sakthi Sankari S1*, Arjunan A2, Bhuvaneswari M.G.2, Sindhuja R3, Rajeswari Thivya D4 Department of Pathology, PSG Institute of Medical Sciences and Research, Coimbatore, Tamilnadu, India 2 Department of Pathology, Coimbatore Medical college, Coimbatore, Tamilnadu, India 3 Department of Pathology, Thirunelveli Medical college, Tamilnadu, India 4 Department of Pathology, Karpaga Vinayaga Medical College, Kanchipuram, Tamilnadu, India

ABSTRACT Background: Follicular lymphoma is an indolent B Non Hodgkin lymphoma which has a characteristic follicular growth pattern and immunohistochemical expression. The neoplastic cells express CD20, CD10, and BCL2 and are negative for CD5, CD23 and Cyclin D1. CD5 is a useful marker in the differentiation of various B cell lymphomas. Small cell lymphoma and mantle cell lymphoma express CD5. Some of the follicular lymphomas express CD5 which results in diagnostic confusion Methods: 52 cases of lymphoma were diagnosed over a period of 2 years in a tertiary care centre in south India. Among these, 9 cases were diagnosed as follicular lymphoma based on the morphological and immunohistochemical study. A panel of antibodies was chosen based on the morphological diagnosis. The panel used for small cell lymphomas include CD20, CD3, CD5 CD23, Cyclin D1, CD10, BCL2 and Ki67. Result: Of the nine cases of follicular lymphoma, three were CD5 positive. Two showed bone marrow involvement. There was no significant morphological difference between CD5 positive and CD5 negative follicular lymphoma. Conclusion: The expression of CD5 by follicular lymphomas could be problematic when the follicular lymphoma as such lacks its characteristic follicular pattern andwhen there are no molecular studies available. Even though rare, follicular lymphoma is to be considered in the differential diagnosis of CD5 positive lymphomas. Keywords: Follicular Lymphoma, CD 5 Positive, Indolent Lymphomas, B Cell NHL, Immunohistochemistry

Introduction

Follicular lymphoma (FL) is the second most common Non Hodgkin lymphoma (NHL) in India. It is an indolent lymphoma derived from germinal centre derived B cells. FL has a characteristic morphology with effaced nodal architecture, follicular growth pattern recapitulating germinal centres. Genetically, the hallmark of FL is t(14;18)q(32;q21) which has been detectable in a significant number of cases accounting to about 80%. This genetic alteration is responsible for the deregulated expression of BCL2 which can be detected using immunohistochemistry. FL has characteristic immunophenotypic expression, positive for pan B cell markers, CD20, CD19, germinal centre associated antigens CD10, BCL6 and BCL2. FL do not express CD5, a T cell marker that has a key role in the differential diagnosis of small B cell Non Hodglkin lymphomas. [4-6] CD5 is a glycoprotein expressed on mature T cells and a small subset of B cells. CD5 positive cells in a lymph node are present in the follicular mantles. [7] Among the lymphomas, CD5 is considered a marker for small

lymphocytic (SLL) and mantle cell lymphoma (MCL). However a small subset of diffuse Large B cell lymphoma, Burkitt lymphoma and extranodal marginal zone lymphoma are found to have expressed CD5.[8,9] And also, cases of CD5 positive follicular lymphomas have been reported in small numbers in the studies performed even before the advent of molecular studies. [10-12] FL is basically a morphological diagnosis. Immunohistochemistry is used as an ancillary tool in the confirmation of diagnosis. CD5 expression however would result in diagnostic confusion when the morphology is not characteristic and flow cytometric and translocation studies cannot be done. In the present study, we describe morphologic and immunohistochemical features of 3 such cases of CD5 positive FL.

Materials and Methods

Fifty two cases of lymphoma were diagnosed over a period of 2 years in a tertiary care centre in south India. The lymphomas were reported based on the WHO classification of tumours (2008 classification). Among these, 9 cases were diagnosed as follicular lymphoma

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based on the morphological and immunohistochemical study. Histology was evaluated in formalin-fixed, paraffinembedded (FFPE) and H&E-stained sections. Grading of FLs was based on the number of centroblasts per highpower field. A panel of antibodies were chosen based on the morphological diagnosis. The panel used for small cell lymphomas include CD20, CD3, CD5 CD23, Cyclin D1, CD10, BCL2 and Ki67. For immunohistochemistry, 4Âľ sections were taken in specially coated slides. Slides were then deparaffinised and rehydrated in ethanol and xylene. Antigen retrieval was done by microwaving using two methods 1) by incubating sections with a Tris-EDTA solution, pH 9.0 for 20 minutes before staining with antibodies against CD10, CD23, BCL2, CD5, CyclinD1, CD3 (mouse, monoclonal, biogenex), and 2) by incubating sections with 0.01 M citrate buffer solution, pH 6.0, for 20 min before staining with antibodies against CD45, CD20 (mouse, monoclonal, biogenex). The slides were then incubated under humid conditions at room temperature for 90 minutes. The antibodies bound to antigens were detected by addition of secondary antibody conjugated with horse radish peroxidase polymer and diaminobenzidene substrate. The slides were counterstained with hematoxylin. Appropriate positive controls for the primary antibodies as per the manufacturerâ&#x20AC;&#x2122;s instruction manual (biogenex) were used. The immunohistochemically stained slides were analyzed for the presence/absence of reaction and cellular localization. The positive reaction in the neoplastic cells was assessed. This study was conducted with approval of the Institutional ethical committee, in accordance with the ICMR guidelines for human research.

Result

Of the nine cases of FL, 3 were CD5 positive. All the three patients were males with the age range of 37 to 53 presenting with lymphadenopathy. Histologically, all the three cases showed predominant follicular growth pattern, while one of them showed diffuse component accounting to less than 50 %.( fig 1 and 2). Histological grading was based on the number of centroblasts per high power field. On immunohistochemical study the cells were found to

express CD20, CD10 and BCL2. (Fig 3 and 4) BCL2 was expressed in the follicular centres. Approximately 70% of the cells were CD5 positive. CD3 immunostaining was included in the panel to assess the proportion of T cells in follicles. As the T cells in the follicular centre usually express BCL2, CD3 immunostaining was compared with BCL2 before interpreting BCL2 expression in order to avoid misinterpretation. The follicular centre also had CD3 positive T cells which also expressed CD5 (fig5). Cyclin D1 was negative in all the three cases ruling out mantle cell lymphoma. (Table 1) In case 2 there was weak expression of CD20 and CD5. Bone marrow trephine biopsies were subsequently received for these three cases. Two of the three cases showed bone marrow involvement, one was paratrabecular while the other was diffuse. There was no peripheral blood involvement. Extranodal involvement was not detected in any of the three cases. The other six cases which have not been described here showed typical histological and immunohistochemical features of lymphoma.

Discussion

Follicular lymphoma is a mature B cell lymphoma which accounts for 20% of NHL.It is usually a disease of adults having a slight female preponderance. FL predominantly involves the lymph nodes, involvement of bone marrow and other extranodal sites can happen in the setting of widespread disease. Primary Extranodal FL can also occur. [1] FL is a heterogenous neoplasm having a variable immunoarchitectural pattern. Typically the involved lymph nodes show complete effacement of nodal architecture by uniform sized follicles composed of centrocytes and centroblasts. These neoplastic follicles lack polarization and evidence of apoptotic activity (tingible body macrophages). The growth pattern can be follicular, follicular and diffuse and predominantly diffuse. CD23 immunostaining which highlights the follicular dendritic cells aids in assessing the extent of follicular component. [13, 14] The varied architectural patterns and variants lead to confusion with other small B cell lymphomas which include small lymphocytic lymphoma (SLL) and marginal

Table 1: Immunohistochemical feature of Follicular Lymphoma. Histology

CD20

CD10

CD23

CD5

BCL2

Follicular

+ in FDC

+ (70 % of cells)

Diffuse > 50%

+ (weak)

+ (70% of cells)

Follicular

+ in FDC

+ (50% of cells )

FDC- follicular dendritic cell.

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Fig. 1: Follicular growth pattern H& E, 10X magnification.

Fig. 2: showing follicular centres H&E, 40X magnification.

Fig. 3: Follicles expressing CD10 (IHC), 10X magnifiction.

Fig. 4: Follicles expressing BCL2 (IHC), 10X magnification.

Fig. 5: Neoplastic cells expressing magnification.

Fig. 6: Bone marrow trephine showing paratrabecular infiltrate of lymphoid cells.10X magnification.

CD5 (IHC) 10X

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zone lymphoma. The typical immunoprofile of FL includes expression of CD10, BCL6, BCL2 and CD20. The expression of these markers are stronger in the follicles when compared to the interfollicular cells. [15] CD10 is a germinal centre marker expressed both in reactive and neoplastic follicles. IT is also expressed in Burkitt and Diffuse Large B cell lymphoma. [15] BCL2 is an antiapoptotic protein which is negative in reactive germinal centres and overexpressed in FL, the expression of which is strong in grade 1 and 2 FL. It is a reliable marker for differentiating follicular lymphoma from follicular hyperplasia. T cells present within the follicular centres also express BCL2. [13] CD5 is involved in T- and B cell receptor signaling. CD5 is a valuable marker in the differentiation of non Hodgkin B cell lymphomas. However CD5 is a mature T cell marker, but is expressed in a subset of B cell lymphomas (SLL and MCL). CD5 along with CD23 helps in the differentiation of small lymphocytic and mantle cell lymphoma. It has also been found to be expressed in marginal zone and diffuse large B cell lymphoma. [1-6] CD5 is one of the negative markers for follicular lymphoma. But CD5 positive follicular lymphomas have been reported to be a rare phenomenon. In the present study, 3 out of 9 cases of FL were CD5 positive, which appears more frequent when compared to the previous published studies. They have been found to be associated with younger age, male predominance and grade 3 morphology. [15, 16] It has also been found to be expressed in follow up biopsies of FL which initially did not express. In the present study, the age ranged from 37 to 53. All the three cases were males. Morphologically, all the three cases had follicular growth pattern except for one which showed a predominant diffuse pattern. Two of the cases were grade 2, while the other was grade 3. CD5 positive FL has been reported to be associated with advanced disease and Extranodal involvement. One of the grade 2 and the grade 3 case showed bone marrow involvement. Bone marrow involvement was paratrabecular in grade 2 FL (figure 6) and diffuse in grade 3 case. None of them had any evidence of Extranodal disease at presentation. In the case 2 there was >50% diffuse component and immunohistochemically, there was weak expression of CD20. This weak expression of CD20 is characteristic of small lymphocytic lymphoma. This weak expression of CD20 along with CD5 positivity made SLL a strong possibility. However CD23 was negative in that case.

Hence it was diagnosed as follicular lymphoma. CD5 positive follicular lymphomas reported so far has been confirmed with translocation and flow cytometric studies. These investigations were not conducted in the present study. All the three cases showed morphological features of classical FL. No specific variants were found to comment on the association between CD5 positivity and any particular variant subtype. The other six cases of FL had wide age range (13 to 68), varied morphology, (4 of them grade 2 and the remaining two cases corresponding to grade 3). The final diagnosis obtained was based on morphological and immunohistochemical study. Flow cytometry and cytogenetics were not performed to identify the translocation t (14, 18) characteristic of FL. Follow up data was not available to understand the prognostic significance of CD5 expression in a follicular lymphoma.

Conclusion

CD5 positive FL can pose a diagnostic problem especially when the morphology is not characteristic including the presence of diffuse component and when genetic studies are not available. However the use of panel of markers can aid in the diagnosis. In summary CD5 positive Follicular lymphomas had the same morphological and immunophenotypic expression as that of CD5 negative FL. Eventhough the frequency is very low, we need to consider the possibility of follicular lymphoma in the evaluation of CD5 positive B NHL.

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Dong HY, Gorczyca W, Liu Z et al. B-cell lymphomas with coexpression of CD5 and CD10. Am J Clin Pathol. 2003; 119:218–230.

10. Sekiguchi Y, Imai H, Wakabayashi M et al. CD5- positive follicular lymphoma: a case report and literature review. Intern Med. 2011; 50:899–904. 11. Mayson E, Saverimuttu J, Cartwright K. CD5-positive follicular lymphoma: prognostic significance of this aberrant marker? Intern Med J. 2014; 44:417–422.

A-625 12. Takata K, Miyata-Takata T, Sato Y, Yoshino T. Pathology of Follicular Lymphoma. J Clin Exp Hematop. 2014; 54(1): 3-9. 13. Fouad-Younes S, Beck A, Lossos IS, et al. Immunoarchitectural Patterns in Follicular Lymphoma: Efficacy of HGAL and LMO2 in the Detection of the Interfollicular and Diffuse Components. Am J Surg Pathol. 2010; 34(9): 1266–76. 14. Rizzo K, Nassiri M. DiagnosticWorkup of Small B cell Lymphomas: A Laboratory Perspective. Hindawi Publishing Corporation Lymphoma. 2012; 15. Barry TS, Jaffe ES. CD5+ Follicular Lymphoma A Clinicopathologic Study of Three Cases. Am J Clin Pathol 2002; 118:589-598. 16. Yu Li, Shimin Hu, Zhuang Zuo, Ming Hong. CD5-positive follicular lymphoma: clinicopathologic correlations and outcome in 88 cases. Modern Pathology 2015;28, 787–798.

*Corresponding author: Sakthi Sankari S, Assistant Professor, Department of Pathology, PSG Institute of Medical Sciences and Research, Coimbatore, Tamilnadu, India, 641004, Phone: +91 0422 257 0170 Email: sakthissankari@gmail.com Date of Submission : 16.02.2017 Date of Acceptance : 10.06.2017 Financial or other Competing Interests: None. Date of Publication : 11.12.2017

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Original Article DOI: 10.21276/APALM.1374

Distribution Pattern of ER & PR Immunoexpression in Endometrial Biopsies of DUB and Infertile Patients from A Tertiary Care Centre Brijesh Thakur, Sanjay Kaushik*, Sakshi Garg, Sanjeev Kishore Shri Guru Ram Rai Institute of Medical & Health Sciences, Dehradun, India

ABSTRACT Objective: To study endometrial estrogen and progesterone immunoexpression in different phases of menstrual cycle, in cases of dysfunctional uterine bleeding and infertility. A comparative analysis was also done for calculating ER & PR expression, between two methods: quick score and percentage of immunopositive cells. Methods: Endometrial biopsies from 107 clinically diagnosed DUB cases and 23 infertile patients were included in the study. Tissue sections were analyzed for different phases of menstrual cycle and immunoexpression of ER & PR receptors was calculated in glandular epithelium and stromal cells, using percentage of positively stained cells and Quick score method. Endometrial sections of hysterectomy specimens of uterovaginal prolapse cases were used as control sections. Result: In the present analysis, secretory endometrium was the commonest finding histopathologically.Mean total ER & PR expression in cases of DUB was statistically higher in the proliferative phase using both methods(p<0.05).Mean total ER expression difference in both the phases of infertility cases was not statistically significant by the observation of percentage of positively stained cells.However, quick score revealed significant difference between both receptor expression in infertile patients. Conclusion: The percentage of positive cells for ER and PR expression plays a more determinant role in studying ER and PR expression in cases of DUB and infertility as compared to quick score. Keywords: Endometrium, DUB, Infertility, Quick score

Introduction

Infertility and dysfunctional uterine bleeding (DUB) together form the most common presenting complaint in obstetrics and gynaecology OPDs. Infertility is more of a social problem, whereas DUB is one of the most bothering complaints for any female, both mentally and physically. A common point, shared by these two conditions is that their prompt diagnosis and timely treatment can significantly raise the living standards of the patient. Dysfunctional uterine bleeding is defined as bleeding not associated with an organic cause in women of child bearing age.[1] Anovulatory cycles play an aetiological role in about 90% of cases of DUB. Infertility is termed when a couple fails to achieve pregnancy after 1 year of unprotected and regular intercourse.[2] Changing lifestyle of people has gradually increased the incidence of infertility. The uterine endometrium is exquisitely sensitive to estrogens and progesterone which play important role in sexual development and reproduction. The estrogen receptor (ER) and progesterone receptor (PR) expression and distribution pattern have been demonstrated to play

an important role in normal endometrial functioning. Immunohistochemistry has essential clinical implications since it supports the role of hormone receptors in the etiopathogenesis of DUB and also it could start a new field in the hormonal therapy.[3,4] The present study was aimed to analyze endometrial estrogen and progesterone immunoexpression in different phases of menstrual cycle, in cases of dysfunctional uterine bleeding and infertility. A comparison was also done for calculating ER & PR expression, between two methods: quick score and percentage of immunopositive cells.

Material and Methods

Total 150 clinically diagnosed cases of DUB and infertility over a period of two years were included in the present study. Out of them, 20 cases had fibroids as the cause of bleeding and were excluded from the study. Out of remaining 130 cases, 107 were DUB cases and 23 were infertility cases (20 cases - primary infertility and 3 cases - secondary infertility). All DUB cases presented with menorrhagia. Ten hysterectomy specimens of uterovaginal prolapse in the reproductive age group (21-

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50 yrs) were taken as controls. Control section was run with each set of 13 cases. Tissue samples were collected as endometrial biopsies (in premenstrual phase) and fixed in 10% formalin. Sections of 3-5 micron were made, stained with haematoxylin & eosin and were reported. For IHC, sections of 2-4 micron from the paraffin embedded blocks were taken on Poly-L lysine coated slides and subjected to ER & PR staining by indirect method of immunohistochemistry using rabbit monoclonal antibodies of Cell Marque; PR clone Y85 at dilution 1:100 and ER clone EP1 at dilution 1:150. The expression of ER & PR was assessed in the endometrial stroma and glands using semiquantitative method, Quick score of nuclear reaction. The percentage of positively stained cells and intensity of nuclear staining was recorded and final quick score was calculated by adding the obtained grading scores of percentage positive cells and the intensity.(Table 1) Subsequently the data was tabulated in mean values of to ER & PR expression and analysed. Statistical analysis was done using IBM SPSS software version 20. Student â&#x20AC;&#x2DC;tâ&#x20AC;&#x2122; test was used in the present study to find out the significance(p<0.05 taken as significant).

Results

In the present study, major proportion of subjects was young adults in age group of 21-40 years. Amongst DUB cases, maximum cases (50 cases, 46.73%), were seen in the age group 31-40 yrs and minimum cases (4 cases, 3.74%) were seen in the age group 21-30 yrs. 18 cases of infertility (78.26%) were seen in the 20-30 yrs of age group and rest of the 5 cases (13.04%) were in the age group of 31-40 yrs. According to histopathology, 60% of control cases were in proliferative phase and 40% were in secretory phase. In total 107 DUB cases, the maximum cases (36.4%) were in secretory phase, followed by irregular shedding endometrium (26.2%), simple hyperplasia without atypia (18.7%) and proliferative phase (18.7%). Out of total 23 infertility cases, the maximum cases (47.82%) were in secretory phase, followed by proliferative and

irregular shedding endometrium equally (21.73% each). Simple hyperplasia without atypia constituted 8.69% of infertile cases. In control cases, taking into account the percentage of positive cells, total mean ER & PR expression was significantly higher in the proliferative phase, as compared to secretory phase (for ER p=<0.0001, for PR p=<0.001). Mean total ER expression was 90.84% in proliferative phase while 39.13% in secretory phase. Mean total PR expression was 84.0% in proliferative phase while 63.13% in secretory phase in control sections. Mean total ER expression in cases of DUB was high in the proliferative phase (86.50%) while in the secretory phase was 59.68%. Mean total PR expression was also found high in the proliferative phase (79.70%) and 53.71% was in the secretory phase. These differences in ER & PR expression were found statistically significant in both phases of DUB cases. Mean total ER expression difference in both the phases of infertility cases (82.90% & 66.14%) was not statistically significant which denotes that the ER expression was consistently raised during the secretory phase. However, mean total PR expression was statistically significant in infertility cases in both phases (82.4% & 57.14%).(TABLE 2) Taking staining intensity into account, mean ER and PR staining intensity was higher in proliferative phase as compared to secretory phase but not, statistically significant in both phases of control and infertile cases. In DUB patients, ER and PR staining intensity results followed the same pattern as control cases except the fact that mean glandular PR expression intensity difference was statistically significant in between both phases.(Table 3) In control cases, distribution of quick score of mean total ER & PR expression was high in proliferative phase with statistically significant difference (p=<0.05) as compared to secretory phase. In DUB group, mean total ER and PR quick score was higher in proliferative phase being statistically very significant (p=<0.0001).In infertile patients, mean total ER expression score was significantly higher in the proliferative phase while on the other hand the mean total PR quick score was higher (p=<0.05) in the secretory phase.(Table 4)

Table 1: Grading of ER & PR expression according to positivity of cells and intensity of nuclear staining. Percentage of stained cells Grade 0

none stained

Grade 1 Grade 2 Grade 3 Grade 4 Grade 5

<1% of cells stained 110% of cells stained 1133% of cells stained 3466% of cells stained >66% of cells stained

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Intensity of nuclear staining Grade 0 Grade 1 Grade 2 Grade 3

negative weak reaction moderate reaction intense reaction

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Table 2: Distribution & comparison of ER & PR expression (% of positive cells) in Control, DUB & Infertility Cases. CONTROL DUB INFERTILITY PP SP PP SP PP SP Mean total ER expression 90.84 39.13 86.50 59.68 82.90 66.14 P Value <0.0001 <0.0001 >0.05 Mean total PR expression 84.00 63.13 79.70 53.71 82.40 57.14 P Value <0.001 <0.0001 <0.05 PP- PROLIFERATIVE PHASE SP- SECRETORY PHASE

Table 3: Distribution & comparison of ER & PR expression (Intensity of nuclear staining) in Control, DUB & Infertility Cases. CONTROL DUB INFERTILITY PP SP PP SP PP SP Mean total ER expression 1.67 1.00 1.92 1.56 1.60 1.00 P Value >0.05 >0.05 >0.05 Mean total PR expression 1.42 1.50 1.84 1.40 1.20 1.18 P Value >0.05 >0.05 >0.05 PP- PROLIFERATIVE PHASE SP- SECRETORY PHASE

Table 4: Distribution & comparison of ER & PR expression (QUICK SCORE) in Control, DUB & Infertility Cases. CONTROL DUB INFERTILITY PP SP PP SP PP SP Mean total ER expression 6.67 5.00 6.86 5.94 6.60 4.07 P Value <0.05 <0.0001 <0.05 Mean total PR expression 6.42 5.63 6.64 5.50 5.21 6.03 P Value <0.05 <0.001 <0.05 PP- PROLIFERATIVE PHASE SP- SECRETORY PHASE

Fig. 1: A. IHC Showing ER expression in endometrial glands and stroma in proliferative phase (100x). Inset showingER expression in endometrial glands in proliferative phase (400x).; 1.B. IHC Showing PR expression, in endometrial glands and stroma in proliferative phase(100x). Inset showingPR expression in endometrial glands(400x).; 1.C. IHC showing ER expression in endometrial glands and stroma in secretory phase (100x).; 1.D. IHC showing PR expression in endometrial glands and stroma in secretory phase (400x).

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Fig. 2: A. IHC showing ER expression in endometrial glands and stroma exhibiting simple hyperplasia without atypia (100x).; 2.B. IHC showing positive ER expression in myometrium (black arrow)and negative ER expression in myometrial vessel (red arrow) (100x).

Discussion

Despite the fact that the endometrium is a prime target organ for actions of estrogen and progesterone hormones and hormonal imbalance underlies many gynecological disorders, the clinical significance of endometrial ER and PR expression is underrated in literature. Functional disturbances with hormonal dysfunction may differ, but subtle morphological changes may not differ much. Study of endometrium for ER and PR expression may serve this purpose because the endometrial study has a distinct advantage over biochemical determination of hormones, because the actual effects of these hormones can be studied in the target tissue. In our study, out of the 107 cases of DUB, a major proportion of cases (50 cases, 46.73%) were from the age group 31-40 years. A comparable distribution of patients in different age groups has been reported by Gleeson et al, Chakraborty et al and Chakravarthy et al.[3,5,6] A few studies by Dhadhania et al and Gupta et al have however reported major number of patients from the age group of 41-50 yrs age group.[7,8] The literature reflects the sociocultural differences between Indian and western communities, as marriages occur earlier in India than western world. Maximum cases of infertility (18 cases, 78.26%) were in the age group of 21-30 years. Emokpae et al, also observed the mean age of their cases to be in the range of 21-30 yrs.[9]

pattern similar to the present study.[9] In our study we studied ER and PR expression in endometrial glands and stroma separately as well as a total expression. We found that the maximum ER and PR expression was noted in the glandular epithelium, except for PR expression in the secretory phase which was more in the stromal cells than glandular epithelial cells, a finding supported by Press et al.[12] Expression of these receptors was observed to be absent from vascular smooth muscle and endothelial cells in both endometrium and myometrium in the present study; also reported by Chakraborty et al, Press et al and Snijders et al.[5,12,13] It was also noted during our study that the method of endometrial sampling did not affect the demonstration of ER and PR expression, a finding corroborated by study of Garcia et al.[14] Antigen retrieval for ER is considered to be difficult as stated by Shet T et al,[15] but we did not find any significant discrepancy in the staining intensity for ER and PR. Although, staining for PR was more crisp and brighter than that for ER in most of our cases, which can be easily explained by the quantitative data from steroid-binding assays indicating that the content of PR in femtomoles per milligram cytosol protein is greater than ER. Press et al and Garcia et al also found that the PR staining was brighter and showed stronger intensity as compared to ER staining.[12,14]

Abdullah et al and Shanthala et al reported secretory phase as the most common pattern on histopathology in DUB cases as found in our study.[10, 11] Emokpae et al who also reported secretory endometrium as the most common

In this study, we examined the whole endometrial section for quantification of positively stained cells and results were expressed as percentage positivity, staining intensity and Quick score. Snijders et al and Lessey et al also used semiquantitative scoring methods in their studies of ER

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and PR expression, which accounted both intensity and percentage of stained cells.[13,16] However, we found that there was subjective and inter observer variation in assigning a score for intensity of staining and therefore did not correlate well with the percentage positivity. Also in some cases insufficient antigen retrieval resulted in weak staining. In the present study, the mean percentage positive ER and PR expression in control cases showed cyclical variation also reported by Press et al and Snijders et al.[12,13] Also in DUB cases, the trend of mean percentage positive ER & PR expression was high during the proliferative phase probably resulted from the increased local estradiol concentration. Similar findings were observed by Chakraborty et al, Shet T et al, and Gleeson et al, stating that the trend of ER and PR expression in DUB cases was consistent with the findings in normal control endometrium.[3,15,17] Mean staining intensity of ER & PR expression in DUB cases was found higher in the proliferative phase and low in the secretory phase. However, this pattern did not correspond statistically with the mean percentage positive cell pattern of the receptor’s expression in DUB cases, which may be due to the interobserver variation in assigning the staining score. In our present study on comparing the mean Quick scores of ER and PR expression in DUB cases, it was noted that the score was corresponding with the percentage positivity of ER and PR expression.The ER staining intensity did not match with the percentage positive expression of ER in both phases, which can be possibly due to the local increase of estradiol in DUB cases during the secretory phase. So a poor concordance among observers may be suggested in assigning score to intensity parameter except in very strongly positive or completely negative sections. In our study,quick score corresponded well with the percentage positive ER and PR expression in DUB cases, similarlyfound by Wells et al.[18] There was an increasing trend of ER and PR expression in proliferative phase and decreasing trend in secretory phase in infertile patients.The difference in ER expression in both the phases was however not statistically significant (p>0.05), which denotes that the ER expression was raised during the secretory phase. The mean percentage positivity of PR expression was slightly raised in the stroma in the secretary phase attributed to the increased PR expression in the stroma during the secretary phase. Margarit et al also found raised ER expression in infertile secretory endometrium; also observed a little different finding stating that the intensity of both ER and PR expression

intensity was increased in infertile ovulatory endometrium in secretory phase.[19] Our study suggests that mean Quick score for ER expression in glandular, stromal and in total was higher in proliferative phase in infertility cases while the mean total PR expression Quick score was higher in the secretory phase. However, Margarit et al and Papanikalaou et al in their study of ‘infertile endometrium’ observed increased expression of both receptors in the secretory phase.[19,20]

Conclusion

In conclusion, secretory phase is the appropriate phase for obtaining endometrial biopsy as overlap of ER and PR expression may appear in the proliferative phase. The percentage positive cells for ER and PR expression plays a determinant role in studying ER and PR expression in cases of DUB and infertility. The inter-observer variability while reporting mean staining intensity limits its utilisation. Since Quick score has staining intensity as one of its component we found that the percentage positivity of cells is a more reliable indicator during reporting. To the best of our knowledge we have not come across any study which has compared percentage positivity of cells, staining intensity and Quick score, all these parameters together, in both phases of menstrual cycle. However, a few studies have reported the singleton use of these parameters in cases of DUB and infertility.

References 1.

Rosai J. Female reproductive system. Uterus-corpus. In: Rosai and Ackerman’s Surgical Pathology. Vol 2. 9thed. New Delhi: Elsevier; 2004. p. 1569-635.

Dallenbach-Hellweg G. The Endometrium of Infertility. A Review. Pathol Res Pract 1984;178(6):527-37.

Chakraborty S, Khurana N, Sharma JB, Chaturvedi KU. Endometrial hormone receptors in women with dysfunctional uterine bleeding.Arch GynecolObstet 2005;272(1):17-22.

Mylonas I, Jeschke U, Shabani N, Kuhn C, Balle A, Kriegel S, et al. Immunohistochemical analysis of estrogen receptor alpha, estrogen receptor beta and progesterone receptor in normal human endometrium. ActaHistochem 2004;106(3):245-52.

Chakravarthy VK, Nag U, Rangarao D, Anusha AM. Estrogen and progesterone receptors in dysfunctional uterine bleeding. IOSR-JDMS 2013;4(3):73-6.

6. Gleeson N, Jordan M, Sheppard B, Bonnar J. Cyclical variation in endometrial oestrogen and progesterone receptors in women with normal menstruation and dysfunctional uterine bleeding. Eur J ObstetGynecolReprodBiol 1993;48(3):207-14. 7.

Dadhania B, Dhruva G, Agravat A, Pujara K. Histopathological study of endometrium in dysfunctional uterine bleeding. J Int Med Res 2013;2(1):20-4.

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8. Gupta A, Rathore AM, Manaktala U, Rudingwa P. Evaluation and histopathological correlation of abnormal uterine bleeding in perimenopausal women. IJBAR 2013;4(8):509-13.

15. Shet T, Agrawal A, Nadkarni M, Palkar M, Havaldar R, Paramar V, et al. Hormone receptors over the last 8 years in a cancer referral center in India: what was and what is?. Indian J PatholMicrobiol 2009;52(2):171-4.

Emokpae MA, Uadia PO, Mohammed AZ. Hormonal evaluation and endometrial biopsy in infertile women in Kano, Northern Nigeria: Acomparative study. Ann Afr Med 2005;4(3):99-103.

16. Lessey BA, Killiam AP, Metzger DA, Haney AF, Greene GL, McCarty KS. Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle. J ClinEndocrinolMetab 1988;67(2):334-40.

10. Abdullah LS, Bondagji NS. Histopathological pattern of endometrial sampling performed for abnormal uterine bleeding. Bahrain Med Bull 2011;33(4):1-6.

17. Gleeson N, Jordan M, Sheppard B, Bonnar J. Cyclical variation in endometrial oestrogen and progesterone receptors in women with normal menstruation and dysfunctional uterine bleeding. Eur J ObstetGynecolReprodBiol 1993;48(3):20714.

11. Shet T, Agrawal A, Nadkarni M, Palkar M, Havaldar R, Paramar V, et al. Hormone receptors over the last 8 years in a cancer referral center in India: what was and what is?. Indian J PatholMicrobiol 2009;52(2):171-4. 12. Press MF, Udove JA, Greene GL. Progesterone receptor distribution in the human endometrium. Analysis using monoclonal antibodies to the human progesterone receptor. Am J Pathol 1988;131(1):112-24. 13. Snijders MP, de Goeij AF, Debets-TeBaerts MJ, Rousch MJ, Koudstaal J, Bosman FT. Immunocytochemical analysis of oestrogen receptors and progesterone receptors in the human uterus throughout the menstrual cycle and after the menopause. J ReprodFertil 1992;94(2):363â&#x20AC;&#x201C;71. 14. Garcia E, Bouchard P, De Brux J, Berdah J, Frydman R, Schaison G, et al. Use of immunocytochemistry of progesterone and estrogen receptors for endometrial dating. J ClinEndocrinolMetab 1988;67(1):80-7.

18. Wells CA, Sloane JP, Coleman D, Munt C, Amendoeira I, Apostolikas N, et al. Consistency of staining and reporting of oestrogen receptor immunocytochemistry within the Europian Union: an inter-laboratory study. Virchows Arch 2004;445(2):119-28. 19. Margarit L, Taylor A, Roberts MH, Hopkins L, Davies C, Brenton AG, et al. MUC1 as a descriminator between endometrium from fertile and infertile patients with PCOS and endometriosis. J ClinEndocrinolMetab 2010;95(12):5320-9. 20. Papanikalaou EG, Bourgain C, Kolibianakis E, Tournaye H, Devroey P. Steroid receptor expression in late follicular phase endometrium in GnRH antagonist IVF cycles is already altered, indicating of early luteal phase transformation in the absence of secretory changes. Hum Reprod 2005;20(6):1541-7.

*Corresponding author: Dr Sanjay Kaushik, Shri Guru Ram Rai Institute of Medical & Health Sciences, Dehradun, India Phone: +91 0135 252 2110 Email: sanjaykaushik038@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 26.02.2017 Date of Acceptance : 21.08.2017 Date of Publication : 11.12.2017

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Original Article DOI: 10.21276/APALM.1385

Cytohistological Correlation of Palpable Breast Mass: A Study of 300 Cases Aditi Dharmesh Vasavada1*, Sheetal Kher2 Dept of Pathology, Gotri Medical College, Vadodara, India Dept of Pathology, GMERS Medical College Gotri, India

ABSTRACT Background: The present study is aimed at correlation of cytological and histological findings of palpable breast mass and evaluate efficacy of FNAC as a first line of investigation modality in breast lump. Material Method: Total 378 female patients with breast lump were subjected to fine needle aspiration cytology examination and reported by using five tire system for breast cytology. Out of 378 cases, in 300 cases cytological findings were correlated with histological diagnosis. Results: In present study, all benign, malignant and suspicious cases on cytology were well correlated with their histological diagnosis. The sensitivity, specificity and positive predictive value of FNAC for palpable breast lump are 97.7%, 98.8% and 97.7% respectively. Conclusion: FNAC of breast lump is an effective, rapid, cost effective, though simple diagnostic procedure with excellent patient compliance. It is giving high accuracy rates when practiced by and experienced hand; making it one of the most reliable modality for evaluation of palpable breast mass. Keywords: Breast Cytology, Five Tire System, Malignant Lesion.

Introduction

Palpable breast mass is the commonest presentation at surgical OPD. Breast malignancy is one of the commonest malignancy in women worldwide and its incidence increases with age.(1) It is very essential to evaluate palpable breast mass before any surgical intervention. Most of the countries are now adopting â&#x20AC;&#x153;triple testâ&#x20AC;? diagnostic approach for breast lesions i.e. clinical, radiological and pathological which include fine needle aspiration cytology as first line of investigation modality.(2) FNAC carried out by a well trained, experienced hand, has high accuracy rate observed in many series.(3,4) This makes it most reliable element of the triple test in cases where the three modalities are no concordant.(5,6) Moreover, FNAC is a cost effective, simple diagnostic procedure for palpable breast lumps,(7) which can be used on OPD basis without hospitalization of the patient. It is a minimally invasive procedure, less painful and having less chances of development of hematoma as compared to core needle biopsy. Patient compliance is best with this procedure even if it has to be repeated. However, fine needle aspiration cytology can be presumptive in some cases. It is not a substitute of core needle biopsy. Final diagnosis can be obtained by histological examination of biopsy tissue. So present study

is done to evaluate efficacy of FNAC in cases of palpable breast lesions, which is done by correlating cytological findings with histological diagnosis. Aim of study: 1) To analyse various cytological findings of aspirates from palpable breast masses presented to cytology department and categorised them according to Five tire reporting system of breast cytology. 2) To correlate cytological diagnosis with histological examinations of breast lesions. 3) To evaluate sensitivity, specificity and predictive value of fine needle aspiration cytology in diagnosis of breast lesion.

Materials and Methods

The present study is performed in a tertiary health care centre in central Gujarat, from December 2014 to December 2016. During this period total 1560 patients referred from various outdoor patient departments were registered in cytology section of pathology department. Out of total 1560 patient, 378 female patients with palpable breast lesions were included in present study. Male patients with breast lump are not included in this study. All the study participants were subjected to FNA examination after obtaining a written consent. The procedure of FNA was performed in supine position in a well lighted, properly ventilated room with maintaining privacy of the patients. The aspirations were taken using 23

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G needle attached to 10ml disposable syringe. The material obtained subsequently smeared on standard microscope glass slides, fixed with alcohol fixative and stained with H & E stain and modified Giemsa stain. A quick review of cellularity obtained in each aspiration was done on the spot immediately after staining the slides. The reporting of cytology slides was done using five tyre system for breast cytology. (table 1).

lesions are found. Total number of undiagnosed cases on FNAC was considered under C1 category which includes 32 cases in present study.

Out of these total 378 cases diagnosed cytologically, 300 cases subsequently subjected to histolopathological intervention. Cytological and histological diagnosis correlated in all the 300 cases. From the obtained data, statistical analysis was done.

Maximum number of malignant breast lesions was found between 5th and 6th decade of life. All the six cases above70 years of age were diagnosed malignant. Not a single benign case found in this age group. Maximum number of suspicious lesions (category C3,C4) were found between 35 to 50 years of age.

Result

Data recorded from all the 378 female patients with palpable breast mass, entered in Microsoft Excel sheet. The age of the female patients in included in the present study ranged from 12 to 73years. The age wise distribution of all the five categories (C1 to C5) is shown in a bar diagram, which shows more than 50 % patients with palpable breast mass falls in age range of 20 to 40years. This is the age range where maximum number of patients with palpable breast

The bar diagram also shows maximum numbers of benign breast lesions (C2 category) havebeen found in age range of 20 to 30years. Commonest benign breast lesion found in present study wasfibroadenoma.

Out of total 378 cases of breast FNAC, histological correlation was available in 300 cases (79.3%). Table 3 shows correlation between cytological and histological diagnosis of all the 300 cases in both malignant and benign categories. From the obtained data, sensitivity, specificity, positive predictive value, false negative rate and false positive rate for Breast cytology were calculated. (Table 4).

Table 1: Cytology categories according to five tire reporting system for breast cytology. Cytology categories

Explanation

Inadequate

Benign

Suspicious but probably benign

Suspicious but probably malignant

Malignant

Table 2: shows age wise distribution of various pathological categories. Cytology categories- according to Five Tire Reporting System Age in years

<20

21-30

31-40

41-50

51-60

61-70

>70

Table 3: Cyto-Histological correlation of 300 cases of Breast lesions. Cytological categories*

No. of patients Benign

Malignant

170

169

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Breast Cytology

Cytological categories*

No. of patients

Histology diagnosis

Benign

Malignant

Total

300

210

*C1 for inadequate; C2 for benign; C3 forsuspicious, probably benign; C4 for suspicious, probablymalignant; and C5 for malignant breast lesions

Table 4: Statistical analysis of data obtained in present study. Histological diagnosis

Cytological diagnosis

Total no. Of cases

Positive for malignancy

Negative for malignancy

TP= 86

FP= 2

FN=2

TN=178

180

268

Positive for malignancy(C5 +C4)* Negative for malignancy (C2+C3)# Total no. Of cases diagnosed

*C5- Malignant breast lesions; C4- suspicious probably malignant #C2-Benign breast lesions, C3-suspicious probably benign TP- True positive; FP- false positive; FN- false negative; TN- true negative Sensitivity of the test (FNAC)- [TP/TP+FN]x100= 97.7% Specificity of the test (FNAC)- [TN/ (TN+FP)] x 100= 98.8% Positive predictive value -[TP/ (TP+FP)] x 100= 97.7% False positive rate- [FP/FP+TP] x100= 2.27% False negative rate- [FN/FN+TN]x100= 1.11% Table 5: Comparison of statistical results of present study with other studies: Parameter

Results in present study

Chavda J (2013)

A.Daramola et al (2015)

N.Chauhan et al (2012)

Sensitivity

97.7%

95.2%

95.4%

96.6%

Specificity

98.8%

100%

88.9%

100%

Positive predictive value

97.7%

100%

99.6%

100%

False negative rate

1.11%

4.76%

0.8%

1.9%

False positive rate

2.27%

0.4%

Fig. 1: cytology smear of Malignant breast lesion, showing loosely cohestive clusters of ductal epithelial cells with moderate anisonucleosis, H & E stain, 400X.

Fig. 2: Cytology smear- Benign Fibroadenoma, Giemsa stain, 400X.

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Fig. 3: Cytology smear- Benign proliferative breast lesionwith apocrine changes, H & E stain, 100X.

Fig. 4: cytology smear- Inflammatory breast lesionshowing benign cohesive ductal epithelial clusters in inflammatory background, H& E stain, 100X.

Fig. 5: Histology of Malignant breast lesion- Invasive Ductal Carcinoma, H & E stain, 400x.

Fig. 6: Histology of Benign Phylloid Tumour, H & E stain, 100x.

Discussion

line pathological investigation in both screening and symptomatic populations. (5)

FNAC of palpable breast masses is considered as a quick, inexpensive, painless and safe procedure. Also, it gives reliable results as far as early detection of breast cancer is considered. This technique is very well accepted by the patient even if it had to be repeated. The only complication arises that is development of hematoma; which can be very well prevented by applying gentle pressure over the site of procedure for short duration.

In present study, we observed that maximum number of benign lesions were found in 2nd decade of life. Among these benign lesion, most common benign lesion was fibroadenoma. Similar findings have been observed by Ferguson et al. which shows commonest benign breast lesion is fibroadenoma occurring before age of 25years.(8)

Most countries have now adopted triple assessment approach (clinical, radiological, and pathological) for palpable breast masses, with FNAC as the first-

Assessing malignant breast lesions in present study, we observed that peak incidence of malignant breast lesion is in 5th to 6th decade of life. Similarly peak age of breast malignancies is 50.8 years in a study done by Murali and

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Breast Cytology

Cunden.(9). In present study, we obtained that most frequent malignant lesion was invasive ductal carcinoma of Not Otherwise Specified type (IDC-NOS). Such findings are comparable with a study done by Sigh et al, in which they found ductal carcinoma was most frequently diagnosed breast malignancy.(10) In present study, total 32 cases where FNAC smears were inadequate / unsatisfactory. That gave proportion of inadequate cases in present study was 10.5%. This rate is higher than that obtained in a study done by Daramola et al. (11) This propotion of undiagnosed cases on cytology can be reduced by immediate evaluation of cytology smear by pathologist using a rapid staining technique. This “onsite” evaluation makes FNAC even more cost effective modality and reducing chances of recalling patients for re-aspiration.(12) Technical skills of a cytopahtologist performing FNAC are having very much influence of diagnostic yield. Unsatisfactory cytological smears can be due to insufficient experience of a pathologist, poor technique in performance of FNAC or due to the nature of lesion itself. Provision of adequate sample by an experienced pathologist can prove FNAC as highly reliable diagnostic tool. (13) In present study, total two false negative cases were diagnosed. In one of that, we obtained heavily blood stained smears with mixed cytological features. Cytologically, this was diagnosed as a cystic lesion. On histological evaluation, it turned out to be an invasive ductal carcinoma of NOS type. Yeoh and Chan have also observed such pitfalls in cytodiagnosis. In their study, they got 6 false negative cases including 4 cases misdiagnosed as cystic lesions.(14) Such type of ‘missed diagnosis’ on cytology can be obtained due to either heavily blood stained smear hindering cytological features or the smear contains mixed cytological findings. Table 5 shows comparison of statistical data obtained in this study with the results obtained in those of other three study done by J Chavda, A. Daramola et al and N. Chauhan et al. (11,15,16). The table shows the results obtained in this study is quite comparable regarding sensitivity, specificity and positive predictive values of FNAC as a diagnostic modality in detective malignant breast pathologies.

Conclusion

Palpable breast mass is one of the common presentation at surgical OPD. Chances of a breast lump being malignant are definitely present. So the proper evaluation of a breast lump is very essential part of patient management. FNAC

of breast lump is now proved to be a rapid, reliable, cost effective diagnostic procedure with high degree patient compliance. Present study observed that the findings of breast FNACs are well correlated with histological diagnosis of respective breast lesions. This proves that cytodiagnosis by FNAC when in experienced hand are extremely useful in the evaluation of breast lumps. Moreover, a benign diagnosis on FNA allows a time period in which a surgery can be planned or delayed, while a positive diagnosis of carcinoma on cytology allows preoperative discussion/ counselling of the patient and further planning of the therapy and reduces morbidity.

Reference 1.

Globocan 2012 - Home [Internet]. [cited 2017 Jul 13]. Available from: http://globocan.iarc.fr/Default.aspx

Mitra S, Dey P. Fine-needle aspiration and core biopsy in the diagnosis of breast lesions: A comparison and review of the literature. Cytojournal. 2016;13:18.

Panjvani SI. Utility of Fine Needle Aspiration Cytology in the Evaluation of Breast Lesions. J Clin Diagnostic Res. 2013;2777–9.

Khan A, Jamali R, Jan M, Tasneem M. Correlation of Fine Needle Aspiration Cytology and Histopathology Diagnosis in the Evaluation of Breast Lumps. Int J Med Students. 2014;2(2):40–3.

Ahmed I, Nazir R, Chaudhary MY, Kundi S. Triple assessment of breast lump. J Coll Physicians Surg Pak. 2007 Sep;17(9):535–8.

Bukhari MH, Akhtar ZM. Comparison of accuracy of diagnostic modalities for evaluation of breast cancer with review of literature. Diagn Cytopathol. 2009 Jun 1;37(6):416–24.

Goyal P, Sehgal S, Ghosh S, et al., “Histopathological Correlation of Atypical (C3) and Suspicious (C4) Categories in Fine Needle Aspiration Cytology of the Breast,” International Journal of Breast Cancer, 2013, Article ID 965498, 5 pages, 2013. doi:10.1155/2013/965498.

Ferguson CM, Powell RW. Breast masses in young women. Arch Surg. 1989 Nov;124(11):1338–41.

Murali U, Cunden SM. Clinico-pathological Correlation of Breast lumps in Mauritian women. IOSR J Dent Med Sci Ver I. 2015;14(7):2279–861.

10. Singh P, Chaudhry M, Nauhria S, Rao D. Cytomorphological patterns of breast lesions diagnosed on fine-needle aspiration cytology in a tertiary care hospital. Int J Med Sci Public Heal Int J Med Sci Public Heal Online. 2015;4(5). 11. Daramola AO, Odubanjo MO, Obiajulu FJ, Ikeri NZ, Banjo AAF. Correlation between Fine-Needle Aspiration Cytology

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Vasavada et al. and Histology for Palpable Breast Masses in a Nigerian Tertiary Health Institution. Int J Breast Cancer. 2015;2015. 12. Nasuti JF, Gupta PK, Baloch ZW. Diagnostic value and costeffectiveness of on-site evaluation of fine-needle aspiration specimens: Review of 5,688 cases. Diagn Cytopathol. 2002 Jul;27(1):1–4. 13. Anderson L, Melamed MR. Koss’ apostrophe, Diagnostic Cytology and Its Histopathologic Bases. Pathology. 2006 Apr 1;38(2):193.

A-637 14. Yeoh GP, Chan KW. Fine needle aspiration of breast masses: an analysis of 1533 cases in private practice. Hong Kong Med J. 1998;4(3):283–8. 15. Srilakshmi HP, Chavda J. A Study Of Cyto-Histological Correlation Of Breast Lesions. 2013;4(September 2010):54–6. 16. Chauhan N, Pathak VP, Saini S, Singh Gaur D. Indian Medical Gazette Cytohistopathological Correlation in Palpable Breast Lesions. 2012;

*Corresponding author: Dr. Aditi Dharmesh Vasavada, Gotri Medical College, Vadodara, Gujarat 390021, India Phone: +91 0265 239 8008 Email: dharmeshvasavada@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 02.03.2017 Date of Acceptance : 06.08.2017 Date of Publication : 11.12.2017

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Original Article DOI: 10.21276/APALM.1412

Histomorphological Study of Ovarian Tumors at a Tertiary Care Centre Rashmi K Patil*, Bhumika Jeevanraj Bhandari, Shreekant K Kittur, Rekha M Haravi, Aruna S and Meena N Jadhav Department of Pathology, Belagavi Institute of Medical Sciences, Belagavi, Karnataka, India

ABSTRACT Background: Ovarian tumors account for about 30% of female genital tract tumors and is fourth leading cause of cancer deaths in females. This study was conducted to evaluate the frequency and distribution of histological types of ovarian tumors. Methods: This was a retrospective seven years observational study based on histomorphological evaluation of 151 ovarian tumours received in department of pathology. Statistical analysis was done and chi-square test was used to see the association. Results: Out of 151 cases, 149 were primary ovarian tumors and two were metastatic tumors to ovary. There were 124 benign tumors, one was borderline and 26 were malignant.Most common age group affected was 31 to 45 years. Benign tumors were common in 16 to 30 years age group, whereas malignant tumors in 46-60 years. For all age group, benign tumors were more common than malignant tumors. Surface epithelial tumors (72.2%) were the most common followed by germ cell tumors (19.9%) and then sex cord stromal tumors (6.6%). Serous cystadenoma (41.93%) was the most common benign tumor followed by mucinous cystadenoma (32.25%). Serous cystadenocarcinoma (38.46%) was the most common malignant tumor. Most common germ cell tumor was mature cystic teratoma (73.3%) and granulosa cell tumor (50%) was the most common sex cord stromal tumor. Conclusion: Diagnosis of neoplastic ovarian lesions requires correlation between clinical, gross and microscopy features as the morphologic diversity of ovarian tumors poses many challenges. In difficult cases, immunohistochemistry and molecular diagnosis may be often required. Keywords: Ovarian tumors, Surface epithelial, Serouscystadenoma, Endometroid

Introduction

Tumors of the ovary are amazingly diverse pathologic entities due to the three cell types that make up the normal ovary: the multipotential surface (coelomic) covering epithelium, the totipotential germ cells, and the multipotential sex cord/stromal cells. Each of these cell types gives rise to a variety of tumours.[1] In most of the population based cancer registries in India, ovarian cancer is the third leading site of cancer among women trailing behind cervix and breast cancer.[2,3] Ovarian tumors account for about 30% of female genital tract tumours and is the fourth leading cause of death among cancer deaths in females.[4,5] The age adjusted incidence rates of ovarian cancer vary between 5.4 and 8 per 100,000 populations in different parts of the country.[3] It is estimated that about 1 in every 70 women have a life time risk of developing ovarian cancer.[6] Ovarian tumorsare often difficult to detect until they are advanced in stage or size, as symptoms are vague and insidious and there is no definite screening programme for early detection. The incidence, clinical

appearance and the behaviour of the different types of ovarian tumors is extremely variable.[3,4]Knowledge of various histomorphological patterns of ovarian tumors is important for diagnosis as well as prognosis. Hence this study was conducted with the aim to evaluate the frequency and distribution of histological types of ovarian tumors at department of pathology of our tertiary care centre.

Materials and Methods

This was a retrospective seven years observational study based on histomorphological evaluation of 151 ovarian tumors received in department of pathology from January 2009 to December 2015. Institutional ethical committee approval was taken for the study.All histopathologically proven cases of ovarian tumors irrespective of the type of surgery done, were included in the study. Functional cysts and tumor-like condition of ovary were excluded. The clinical data was achieved from hospital records. The paraffin blocks and respective haematoxylin & eosin stained slides were retreived and studied. The world health organization (WHO) classification of ovarian tumours was used for classifying the tumors.

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Statistical analysis was done using statistical package for social sciences-22 (SPSS-22). Chi-square test was used to see the association. The p-value of less than 0.05 was considered statistically significant.

Results

Out of the total 151 cases studied, 149 were primary ovarian tumorsand two were metastatic tumors to the ovary. Among the 151 ovarian tumors, 124 were benign, one was borderline and 26 were malignant (Table 1).Age of the patient ranged from 12 years to 82 years. The most common age group affected was 31 to 45 years (Table 2). Benign tumors were most commonly seen in 16 to 30 years age group, whereas malignant tumors were more common in 46-60 years. Out of the total 109 surface epithelial tumors, 84.4% were benign, 0.91% were borderline and 14.67% were malignant. Benign surface epithelial tumors comprised 74.2% (92/124) of all benign tumors while their malignant counterpart formed 61.5% (16/26) of all malignant ovarian tumors. Serous cystadenoma (41.93%) was the commonest benign tumor followed by mucinous cystadenoma (32.25%)(Table 3). Among the malignant tumours, serous cystadenocarcinomas (38.46%) was the most common tumor (Table 4). Among the 30 germ cell neoplasms, 73.3%were benign and reported as mature cystic teratomas or dermoid cysts (Figure 1a, 1b). Most germ cell tumors, both benign and malignant, were seen in women younger than 30 years. Germ cell tumor was the most common malignant tumor upto 30 years of age. The youngest patient in the present study was a 12 year old girl with dysgerminoma (Figure 1c, 1d). There were ten cases of sex cord stromal tumors, all of which were benign. Most sex cord stromal tumors occurred in women above 30 years of age. Only two (1.3%) cases of metastasis to ovary was seen in the present study, both affecting patients above 30 years.

For all age group, benign tumors were more common than malignant tumors. Out of 52 patients below 30years, 46 (88.46%) had benign tumors and 6 (11.54%) had malignant tumors. Germ cell tumors was the most common malignant tumour below 30years, accounting for 83.33%. Out of 99 tumors affecting patients above 30years, 78 (78.78%) were benign, 1 (1.01%) was borderline and 20(20.20%) were malignant. Serous cystadenocarcinoma was the most common malignant tumor above 30 years accounting for 50%. The borderline surface epithelial tumour was of mucinous type. There were three cases of patient aged below 15 years. All three cases were of germ cell tumors. Surface epithelial tumors were more common in 31-45 years (37.61%), Germ cell tumors were more common in 16-30years (53.33%) and sex cord stromal tumors were more in 46-60 years (50%).

Discussion

Ovarian neoplasm has become increasingly important not only because of its large variety of histomorphological patterns but more because they have gradually increased the mortality rate in female genital cancers. A femaleâ&#x20AC;&#x2122;s risk at birth of having ovarian tumour sometime in her life is 6% to 7%, of having ovarian cancer is almost 1.5% and dying from ovarian cancer is 1%.[7] The poor survival is due to the fact that they do not clinically manifest early and approximately 60% to 70% of the neoplasm present as either stage III or stage IV.[8]Though certain investigations like peritoneal fluid cytology, estimation of serum lactate dehydrogenase, fibrin degradation products and immunological tests have been reported to be of some help in predicting the nature of the pathology, it is generally impossible to diagnose the nature of the ovarian tumor preoperatively just by clinical examination and exploration. Hence, one has to depend on the microscopic appearance of the tumor for further management of the ovarian tumors.[9]

Table 1: Frequency of different classes of benign and malignant ovarian tumors Classes of tumor

Benign (%)

Borderline

Malignant (%)

Total

Surface Epithelial tumors

92 (74.2%)

16(61.5%)

109

Germ cell tumors

22 (17.7%)

8(30.8%)

Sex cord stromal tumors

10 (8.1%)

2(7.7%)

124

151

Metastatic tumors Total

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Table 2: Frequency of different classes of ovarian tumors in different age group Age group in years

Surface epithelial tumor

Germ cell tumor

Sex cord stromal tumor

Metastatic tumor

Total

16-30

31-45

46-60

>60

Total

109

151

Upto 15

Table 3: Frequency of individual benign ovarian tumor in different age group Diagnosis

Upto 15 years

16-30 years

31-45 years

46-60 years

>60 years

Total

Serous cystadenoma

Mucinous cystadenoma

Benign Teratoma

Granulosa cell tumor

Fibroma

Fibrothecoma

Sex cord tumor with annular tubules

Total

124

Table 4: Frequency of individual malignant ovarian tumor in different age group Diagnosis

Upto 15years

16-30years

31-45years

46-60years

> 60years

Total

Serous cystadenocarcinoma

Mucinous cystadenocarcinoma

Endometroid tumor

Undifferentiated/poorly differentiated tumors

Immature teratoma

Teratoma with malignant transformation

Dysgerminoma

Yolk sac tumor

Mixed germ cell tumor

Metastatic tumors

Total

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Fig. 1: a) Cut section of mature cystic teratoma showing tuft of hair, b) Microphotograph of mature cystic teratoma (H and E, x100), c) Cut section of dysgerminoma, d) Microphotograph of dysgerminoma (H and E, x100).

Fig. 2: a) Cut section of sertoliform endometrid tumor showing solid and cystic areas, Microphotograph showing b) conventional endometrid carcinoma with sertoliform pattern (H and E, x200) c) EMA positivity (IHC, x400) d) CK positivity (IHC, x400).

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Fig. 3: a) Cut section showing cystic multiloculated (arrow) struma ovarii, b) Microphotograph showing struma ovarii (H and E, x100), Microphotograph showing thyroid follicular cells positive for thyroglobulin (c), negative for synaptophysin (d) and chromogranin (e) (IHC, x400).

Fig. 4: a) Cut section of granulosa cell tumor, b) Microphotograph showing granulosa cell tumor (H and E, x200), c) Gross photograph of SCTAT, d) Microphotograph of SCTAT (H and E, x200).

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In this study, 82.1% cases were benign, 0.7% were borderline and 17.2% were malignant. This was in concordance with study done by Modi et al, where 84.5% were benign, 2.1% were borderline and 13.4% were malignant.[9] In study done by Kuladeepa et al, 82.35% were benign , 3.68% were borderline and 13.97% were malignant.[4] However, in study done by Nishal et al (51%) and Ahmad et al (59.18%) , the incidence of benign tumours was less compared to the present study.[3,10]The incidence of borderline tumours was more in study done by Nishal et al (5%) and Mondal et al (7.3%) compared to our study. [3,11] Tejeswini et al andMalli et al didnâ&#x20AC;&#x2122;t find any borderline tumour in their study.[8,12] Tumors in the borderline category are characterised by epithelial proliferation greater than that of the benign tumor but an absence of destructive invasive stroma. In the present study, 98.68% tumors were primary and 1.32% tumors were metastatic to the ovary. Our findings corroborated well with study done by Tejeswini et al, wherein 98.92% were primary and 1.08% were secondary ovarian tumors.[8] Among the major histological classes, commonest type of ovarian neoplasm seen in the present study was surface epithelial tumors accounting for 72.2%, followed by germ cell tumors (19.9%) and sex cord stromal tumors (6.6%). Studies done by Modi et al and Yogambal et al had similar findings.[9,13] However Guppy et al documented higher incidence of surface epithelial tumors (90%) than in the present study.[14] Gupta et al documented lower incidence of surface epithelial tumors (48.8%).[15] In the present study, 84.4% of surface epithelial tumors were benign, 0.91% were borderline and 14.67% were malignant. This was similar to study done by Modi et al and Danish et al.[9,16] Serous cystadenoma was the most common surface epithelial tumour in our study as well as study done by Singh et al, Tejeswini et al and Modi et al.[6,8,9]Among the malignant surface epithelial tumors, endometroid tumor constituted 0.66% in the present study, similar to study done by Tejeswini et al and Pilli et al, constituting 0.72% and 0.7% respectively. [8,17] A case of sertoliform variant of endometroid tumor was diagnosed in a 55 year old lady who presented with torsion of ovary. On immunohistochemistry (IHC), the tumor cells showed positivity for epithelial membrane antigen(EMA) and cytokeratin(CK) and were negative for inhibin, thus confirming sertoliform endometroid tumor (Figure 2). www.pacificejournals.com/apalm

One case of struma ovarii was diagnosed in a 45 year female presenting with mass per abdomen and pain abdomen since 1 month. Due to suspected carcinoid foci along with struma ovarii, IHC was done. It was positive for thyroglobulin and negative for synaptophysin and chromogranin (Figure 3). In study done by Tejeswini et al, the incidence of strumaovarii was found to be 0.36%(1 case). In the present study, as well as study done by Tejeswini et al, one case of immature teratoma was reported.[8] We reported one case of mixed germ cell tumor having dysgerminoma and yolk sac component. Three cases were reported as teratoma with malignant transformation, two out of which was transformed into squamous cell carcinoma and one into adenosquamous carcinoma. The frequency of sex cord stromal tumors in our study was 6.6%. This value is comparable with study done by Modi et al (6.1%).[9] All the sex-cord stromal tumors in the present study were benign, 50% of which were granulosa cell tumour (Figure 4a, 4b). One rare case of sex cord tumor with annular tubules (SCTAT) was reported in a 26years nulliparous female who presented with amenorrhea and mass per abdomen since 3 months (Figure 4c, 4d). Undifferentiated/ poorly differentiated tumors were detected in 3 cases accounting for 11.53% of all malignant tumors. All three patients were aged more than 60 years. On conventional histomorphology, we could not further subtype these tumors. Due to financial constraints, IHC was not done, which was a limitation of our study. In study done by Danish et al, 19.2% of tumors were undifferentiated or poorly differentiated.[16] In the present study, we reported 2 cases of metastasis to the ovary. One case was of a 70 year female with papillary adenocarcinoma of fallopian tube and other was of a 40 year female with mucinous carcinoma of colon. In study done by Singh et al , Tejeswini et al and Gupta et al the incidence of metastatic tumors were 0.83%, 1.08% and 2% respectively.[6,8,15] Among the benign tumors, serous cystadenoma (41.93%) was the commonest followed by mucinous cystadenoma (32.25%) in the present study as well as study done by Modi et al.[9] Our study differs from Nishal et al and Mankar et al who documented mucinous cystadenoma as the most common benign tumor and from Jha et al and Ahmad et al who documented benign cystic teratoma as the most common benign tumor.[3,7,10,18] eISSN: 2349-6983; pISSN: 2394-6466

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Among the malignant tumors, serous cystadenocarcinoma (38.46%) outnumbered all others in our study. This observation is consistent with most other studies with few exceptions. In study done by Sharma et al, mucinous cystadenocarcinoma was the most common malignant tumour.[1] Swamy et al recorded granulosa cell tumours and Yasmin et al observed endometroid carcinoma as the most common ovarian malignancy.[19,20] In the present study, right ovary was more commonly involved than left ovary. Two cases of bilateral serous cystadenoma and 1 case of bilateral mucinous cystadenoma were seen in the present study. This differed from other studies which had higher incidence of bilateral tumours. Nishal et al reported 13% bilateral tumors, 1 out of which was benign and 1 borderline and rest were malignant.[3] The histopathological type of ovarian tumor correlates with the prognosis of the tumors. Role of histopathology is critical in recognizing the distinct patterns of ovarian tumors as they have different epidemiological and genetic risk factors, precursor lesion, patterns of spread, response to chemotherapy and prognosis.

Conclusion

It is concluded from this study that the benign ovarian tumors were more common for all age groups. Surface epithelial tumors were the most common class of tumors, similar to the western and local data from other medical institutions. Considering individual tumors, serous cystadenoma was the most common ovarian tumor overall as well as the most common benign tumor, whereas serous cystadenocarcinoma was the most common ovarian malignancy. Malignant ovarian tumors were more common above 40 years. Histopathological study is still the gold standard in diagnosing most of primary ovarian tumor. However, it may be supplemented by newer techniques like IHC, morphometric analysis, cytometric analysis of ploidy status to resolve the difficult, dilemmatic cases and to predict prognosis.

Abbreviations

WHO: World health organisation IHC: Immunohistochemistry EMA: Epithelial membrane antigen CK: Cytokeratin SCTAT: Sex cord tumor with annular tubules

References 1.

Sharma I, Sarma U, Dutta UC. Pathology of ovarian tumourA hospital based study. International journal of medical science and clinical invention 2014;1(6):284-6.

Basu P, De P, Mandal S, Ray K, Biswas J. Study of â&#x20AC;&#x2DC;patterns of careâ&#x20AC;&#x2122; of ovarian cancer patients in a specialized cancer institute in Kolkata, eastern India. Indian J Cancer 2009; 46:28-33.

Nishal AJ, Naik KS, Modi J. Analysis of spectrum of ovarian tumours: a study of 55 cases. Int J Res Med Sci 2015;3(10):2714-7.

Kuladeepa AVK, Muddegowda PH, Lingegowda JB, Doddikoppad MM, Basavaraja PK, Hiremath SS. Histomorphological study of 134 primary ovarian tumours. Adv Lab Med Int 2011;1(4):69-82.

Sen U, Sankaranarayanan R, Mandal S, Romana AV, Parkin DM, Siddique M. Cancer patterns in Eastern India. The first report of Kolkata Cancer Registy.Int J Cancer 2002;100(1):86-91.

6. Singh S, Saxena V, Khatri SL, Gupta S, Garewal J, Dubey K. Histopathological evaluation of ovarian tumors. Imperial journal of Interdisciplinary Research 2016;2(4):435-9. 7.

Jha R, Karki S. Histological pattern of ovarian tumors and their age distribution. Nepal Med Coll J 2008;10(2):81-5.

Tejeswini V, Reddy ES, Premalatha P , Vahini G. Study of morphological patterns of ovarian neoplasms. Journal of Dental and Medical Sciences 2013:10(6):11-6.

Modi D, Rathod GB, Delwadia KN, Goswami HM. Histopathological pattern of neoplastic ovarian lesions. International Archives of Integrated Medicine 2016;3(1):51-7.

10. Ahmad Z, Kayani N, Hasan SH, Muzaffar S, Gill MS. Histological pattern of ovarian neoplasms. J Pak Med Assoc 2000;50(12):416-9. 11. Mondal SK, Banyopadhyay R, Nag DR, Roychowdhury S, Mondal PK, Sinha SK. Histologic pattern, bilaterality and clinical evaluation of 957 ovarian neoplasms: A 10-year study in a tertiary hospital of eastern India. J Cancer Res Ther 2011;7:433-7. 12. Malli M, Vyas B, Gupta S, Desai H. A histological study of ovarian tumors in different age groups.Int J Med Sci Public Health 2014;3:338-41. 13. Yogambal M, Arunalatha P, Chandramouleeshwari K, Palaniappan V. Ovarian tumours- Incidence and distribution in a tertiary referral center in south India. Journal of Dental and Medical Sciences 2014:3(2):7480. 14. Guppy AE, Nathan PD, Rust n GJ. Epithelial ovarian cancer: A review of current management. Clinoncol 2005;17:399-411.

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15. Gupta N, Bisht D, Agarwal AK, Sharma VK. Retrospective and prospective study of ovarian tumours and tumour-like lesions.Indian J PatholMicrobiol 2007;50:525-7. 16. Danish F, Khanzada MS, Mirza T, Aziz S, Naz E, Khan MN. Histomorphological spectrum of ovarian tumors with immunohistochemical analysis of poorly or undifferentiated malignancies.Gomal J Med Sci 2012;10(2):209-15. 17. Pilli GS, Suneeta KP, Dhaded AV, Yenni VV. Ovarian tumours: a study of 282 cases: J Indian Med Assoc 2002;100:423-4.

18. MankarMankar DV, Jain GK. Histopathological profile of ovarian tumours: A twelve year institutional experience. Muller J Med Sci Res 2015;6:107-11. 19. Swamy GG, Satyanarayana N. Clinicopathological analysis of ovarian tumours- A study on five years samples. Nepal Med Coll J 2010;12(4):221-3. 20. Yasmin S, Yasmin A, Asif M. Clinicohistological Pattern of Ovarian Tumors in Peshawar Region. J Ayub Med CollAbbotabad 2008;20(4):11-3.

*Corresponding author: Dr. Rashmi K. Patil, Associate professor, Department of Pathology, Belagavi Institute of Medical Sciences, Dr.B.R.Ambedkar road, Belagavi â&#x20AC;&#x201C; 590001 Phone: +91 9986949387 Email: rashminimbal@rediffmail.com Date of Submission : 14.03.2017 Date of Acceptance : 19.07.2017 Financial or other Competing Interests: None. Date of Publication : 18.12.2017

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Original Article DOI: 10.21276/APALM.1428

Prognostic Significance of Prostate Specific Antigen in Comparison with Histological Grade of Prostatic Adenocarcinoma: A Hospital Based Study Mohan Rao. Nandam1*, Vissa Shanthi1, Bhavana Grandhi1, Syama Sundara Rao Byna1, Vijayalakshmi Muramreddy1 and Jyothi Conjeevaram2 Department of pathology, Narayana Medical College, Chinthareddypalem, Nellore, Andhra Pradesh (India) Department of Social And Preventive Medicine, Narayana Medical College, Chinthareddypalem, Nellore, Andhra Pradesh (India) 1

ABSTRACT Background: Prostate gland disease causes significant morbidity in elderly males. Our objective of the study was to evaluate the histopathological spectrum of prostatic lesions in correlation with prostate specific antigen and compare the prostate specific antigen with histological grade of prostatic adenocarcinoma. Methods: The present retrospective study was carried in the department of pathology, Narayana Medical College & Hospital, Nellore, and Andhra Pradesh, India from January 2015 to December 2015. 119 Prostatic biopsy specimens were analyzed histopathologically for diagnosis of types of prostatic lesion and correlated with serum PSA level. Prostatic adenocarcinomas were graded according to Gleason histological grading. Gleasonâ&#x20AC;&#x2122;s grading of tumors was correlated with serum PSA levels Result: Out of 119 patients, 95 cases had benign prostatic hyperplasia, 17 cases had prostatic adenocarcinoma, 7 cases had prostatitis. In our study, the maximum number of BPH cases (51.58%) showed PSA levels < 4ng/ml. Most of prostatic adenocarcinoma (82.35%) displayed PSA levels >10ng/ml. 5 cases (71.43%) of prostatitis showed PSA levels of 4 to 10ng/ml. Maximum cases of grade 2 and grade 3 prostatic adenocarcinoma had PSA range of 20-49.99 ng/ml. Conclusion: Benign prostatic hyperplasia is the most commonly diagnosed prostatic lesions. Investigation like serum PSA level detection can aid in the diagnosis, but accurate diagnosis of non-neoplastic and neoplastic lesions of prostate can be made by histopathological study of prostate biopsy.There is a positive relation was seen between higher levels of PSA and Gleason histopathological grade. Keywords: Prostate specific antigen, Gleason histopathological grade, Prostatic adenocarcinoma, prostatic lesions

Introduction

Recent reports around the globe suggest that prostate gland disease causes significant morbidity and mortality in elderly male. [1] In USA, it has been recorded as the important cause of cancer-related deaths in men after lung cancer. Among the total population, one out of six will be diagnosed with prostate cancer during lifetime. Annual checkup of Prostatic specific antigen levels in the men above 50 years of age was recommended by American cancer society.[2,3] For the diagnosis of prostatic cancer, PSA acts as an important tumor marker. [4] For every gram of malignant tissue, the PSA levels raised by 2-3ng/ dl which is 10 times more when compared to rise in PSA level for every gram of hyper plastic tissue.[5,6] Frequently encountered prostatic lesions are Benign Prostatic Hyperplasia, chronic prostatitis, granulomatous prostatitis and Adenocarcinoma.[1] Our main aim of this study was to evaluate the histopathological spectrum of prostatic lesions in correlation with PSA levels and prognostic significance

of PSA levels by comparing withhistopathological grade of prostatic adeno carcinoma.

Materials and Methods

The present retrospective study was carried in the department of pathology, Narayana Medical College & Hospital, Nellore, Andhra Pradesh, during the period of January 2015 to December 2105.Samples (serum and biopsy) from 119 patients, aged between 35 to 84 years, were examinedfor thelevel of serum PSA and histopathology of prostate biopsy was studied. Serum PSA was done on ACCESS-2 (BECKMAN COULTER) by chemiluminescence method. The range of PSA determination using this equipment is 0.1-150ng/ ml. Prostatic biopsy was collected from patients who underwent transurethral resection of the prostate for BPH, open Prostatectomy for prostatic lesion and transrectal ultrasound guided biopsy for suspicious malignancy.

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Prostatic biopsy specimens were kept in 10% neutral buffered formalin. Specimens were grossly examined for size/quantity and weight followed by processing, paraffin embedding and section cutting. 3-5 microns section were stained with Hematoxylin & Eosin and reported. Prostatic adenocarcinomas were graded according to Gleason’s histopathological grade. Statistical analysis: Data collected was entered in MS Excel and analyzed using SPSS-Version 22.0 Percentages and chi-square values were calculated. A P value of 0.05 was taken as significance.

Results

During the period of one year from January 2015 to December 2015, 119 prostatic biopsy specimens were received. The age ranges of patients were 31-90 years. The mean age of presentation was 64.46±10.70 years. Most common prostatic lesion is BPH [95 cases (79.83%)] with mean age 63.67±11.29 years. Second most common lesion is prostatic adenocarcinoma [17 cases (14.28%)] with mean age 68.88±6.49 years and least common is prostatitis [7 cases (5.88%)] with mean age 64.43±7.03 years (Figure1). In our study of 24 cases of prostatic lesions, 3 cases were prostatic adenocarcinomas, 19 cases BPH and 2 cases were prostatitis and occurred in the age group of 51-60 years. In the Age group of 61-70 years they were 45 cases of prostatic lesions, out of which 7 were prostatic adenocarcinomas, 34 were BPH and 4 cases were chronic prostatitis. The

age group of 71-80 years they were 35 cases of prostatic lesions, out of which 7 were prostatic adenocarcinomas, 27 were BPH and one case were Prostatitis. In our study 49 cases (51.58%) of BPH shows PSA levels <4 ng/ml, 41 cases (43.16%) of BPH shows PSA levels of 4-10ng/ml and 5 cases (5.26%) of BPH shows PSA levels of >10ng/ml. 14 cases (82.35%) of prostatic adenocarcinoma shows PSA levels of >10ng/ml, One case (5.88%) of prostatic adenocarcinoma shows PSA levels of <4 ng/ml and 2cases (11.76%) of prostatic adenocarcinoma shows PSA levels of 4-10 ng/ml . 5 cases (71.43% ) of prostatitis shows PSA levels of 4-10ng/ml and 2 cases of prostatitis shows PSA levels of <4 ng/ml. PSA levels are higher in prostatic adenocarcinoma when compared to BPH and prostatitis and this difference is statistically significant (p=0.000) (Table1). PSA levels in prostatic carcinoma were compared with Gleason’s histopathological grade of the tumors. Maximum number of prostatic adenocarcinoma were in grade 2 [12 cases (70.59%)]. Maximum cases of grade 2 and grade 3 prostatic adenocarcinomas had PSA range of 20-49.49 ng/ml. One case (5.88%) of grade 2 adenocarcinoma had PSA range of 0-3.98 ng/ml . One case (5.88%) of grade 1 and one case (5.88%) of grade 2 adenocarcinoma had PSA range of 4-9.99 ng/ml. 4 Cases (23.53%) of grade 2 prostatic adenocarcinoma had PSA range of 50- 99.99 ng/ ml. In our study there is a difference in PSA levels across the grades which is statistically significant (p=0.043) (Table 2 ).

Table 1: Histopathology diagnosis related with range of Prostate Specific antigen level: PSA

Adenocarcinoma

BPH

Prostatitis

0-4.0ng/ml

1(5.88%)

49(51.58%)

2(28.57%)

4-10.0ng/ml

2(11.76%)

41(43.16%)

5(71.43%)

10-20ng/ml

4(23.53%)

5(5.26%)

>20ng/ml

10(58.82%)

Total

17(100%)

95(100%)

7(100%)

P=0.000 significant PSA level are higher in prostate adeno carcinoma when compared to BPH & prostatitis and this difference is statistically significant (P=0.000).

Table 2: Correlation between serum PSA levels and Gleason grade of prostactic adeno carcinoma PSA Range(ng/ml)

Grade1

Grade 2

Grade3

0-3.99

1(5.88%)

4-9.99

1(5.88%)

10-19.99

3(17.65%)

1(5.88%)

20-49.99

3(17.65%)

50-99.99

4(23.53%)

Total (no=17)

1(5.88%)

12(70.59%)

4(23.53%)

There is a difference in PSA levels across the grades and this is statistically significant (P=0.043).

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Table 3: Comparison between different types of lesion with other studies S. No

Histopathological Diagnosis

Kshitij

BPH

85.8%

Prostatic adenocarcinoma

8.35%

Prostatitis

0.64%

Azmi .A

et al

Jevan

Haroun et al

[10]

[11]

Arunchitale

et al

Janardan

et al

[12]

Present study

et al

[13]

64.48%

83%

89%

93.9%

95(79.83%)

27.1%

17%

11%

6.06%

17(14.28%)

8.4%

7(5.88%)

Table 4: Benign and malignant prostatic lesion: Comparison between Prostate Specific Antigen level with other studies PSA range

BPH

Malignant prostatic lesion

(ng/ml)

Kshitij et al[10]

Ishtiaq Ali Khan et al[14]

Present study

Kshitij et al[10]

H.A Mwalyoma et al[9]

Sladana Zivkovic et al[15]

Present study

0-4.0

71.6%

51.58%

10.5%

2.50%

5.88%

4-10.0

22.6%

85%

43.16%

26.3%

5.3%

27.50%

11.76%

>10

15%

5.26%

63.15%

94.7%

70.0%

82.35%

Table 5: Comparison between our study and other studies in relation to PSA levels and Prostatic adenocarcinoma PSA levels (ng/ml)

SladanaZivkovic et al (2004) [15]

Shanthi.V etal(2012-2015) [16]

Our study(2015)

0-3.99

1(2.5%)

1(5.88%)

4-9.99

11(27.5%)

2(4.76%)

2(11.76%)

10-19.99

7(17.5%)

6(14.29%)

4(23.53%)

>20

21(52.25%)

34(80.95%)

10(58.82%)

Table: 6 Comparison between our study and Shanthi v et al[16]study in relation to serum PSA levels and Gleason grade of prostatic adeno carcinoma. PSA levels (ng/ml)

Grade 1

Grade 2

Grade 3

Shanthi V etal[16]

Our study

Shanthi V etal[16]

Our study

Shanthi V etal[16]

Our study

0-3.99

1(5.88%)

4-9.99

2(4.76%)

1(5.88%)

10-19.99

5(11.9%)

3(17.65%)

1(5.88%)

20-49.99

7(16.67%)

3(17.65%)

50-99.99

10(23.8%)

4(23.53%)

4(9.52%)

100-149.99

7(16.67%)

2(4.76%)

150-199.99

>200

5(11.9%)

Total

2(4.76%)

1(5.88%)

34(80.95%

12(70.59%)

6(14.29%)

4(23.53%)

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Fig. 1: Pie chart showing percentage of BPH, Prostatic Adenocarcinoma and Prostatitis

Discussion

The prostate is a pear - shaped glandular organ that weighs up to 20g in the normal adult male and it has been divided into anterior, middle, posterior and two lateral lobes by drawing divergent lines from the centrally located urethra. The prostate can be divided into four biological and anatomical zones, peripheral zone, central zone, transitional zone and periurethral gland regions. The transitional zone and periurethral regions are the exclusive sites of origin of nodular hyperplasia, whereas the peripheral zone is the one most susceptible to prostatitis and carcinoma. The glandular component of the organ is composed of acini and ducts, the latter subdivided into large (primary, major, excretory) and peripheral (secretory, minor). Both acini and ducts contain secretory cells, basal cells and scattered neuroendrocine cells. The secretory cells which are located in the luminal side of the gland, contribute a wide variety of products of the seminal fluid. They produce prostatic acid phosphate (PAP) and prostate specific antigen (PSA). PSA is glycoprotein that has been identified as a kallikrein like protease. In the present study most common lesion is BPHwith mean age 63.67Âą11.29 and BPH is more common between 51-80 years of age. Prostatic adenocarcinoma (Figure 2) is second most common lesion in our study. Prostatic adenocarcinoma mean age is 68.88Âą6.49 and more common between 61to 80 years of age in this study. In our study prostatic adenocarcinoma mean age figure are comparable with finding from other studies which report the mean age of 69 years by Thompson IM et al,[7]mean age of 65 years by Lyn et al,[8] and mean age of 68 years by H A Mwakyoma et al .[9] In the present study BPH cases are 79.83% which is nearby comparable with Kshitij et al,[10] Jeven et al,[12]Arunchitale et al[13] and Janardan et al studies. [13] In our study prostatic adenocarcinoma incidence was 14.28% which is nearby comparable with Jevan et al[12] and Arun Chitale et al studies.[13] (Table 3) .In our study www.pacificejournals.com/apalm

BPH cases are most commonly present between PSA level 0-4.0ng/ml (51.58%) Which is compared with study of Khitij et al,[10] and prostatic adenocarcinoma cases are more commonly present with PSA level >10.0ng/ml (82.35%) and it is nearly compared with Walyomaet al[9] Kshitij et al[10] and Sladana Zivkovic et al studies .[15] (Table 4). In the present study we evaluated the prognostic importance of serum PSA levels with grades of adenocarcinoma prostate. In our study maximum number of malignancies (58.82%) has serum PSA value of more than 20 ng/ml which coincided with study done by Sladana Zivkovic et al study (52.25%) [15] and lower than Shanthi V et al study (80.95%). [16] 2 cases (11.76%) of prostatic adenocarcinoma was detected with PSA values in the range of 4-9.99 ng/ml which is higher than the Shanthi V et al study (4.76%) [16] and lower than the Sladana Zivkovic et al study (27.5%). [15] 4 cases of (23.53%) prostatic adeno carcinoma was detected with PSA values in the range of 10-19.99 ng/ml which is higher than the SladanaZivkovic et al study (17.5%) [15]and Shanthi V et al study (14.29%).[16] (Table 5). In the present study, one case (5.88%) of grade 2 prostatic adenocarcinoma was noted with serum PSA range of 0-3.99 ng/ml. One case (5.88%) of grade 1 prostatic adenocarcinoma are noted with serum PSA range of 4-9.99 ng/ml which is slightly higher than Shanthi V et al study (4.76%).[16] One case (5.88%) of grade 2 prostatic adeno carcinoma . 3 cases(17.65%) of grade 2 prostatic adenocarcinoma were noted with serum PSA range of 10-19.99 ng/ml which is higher than the Shanthi V et al study (11.9%) .[16] One case (5.88% ) of grade 3 prostatic adeno carcinoma , 3 cases (17.65%) of grade 2 prostatic adeno carcinomas were noted with serum PSA range of 20-49.99 ng/ml which is slightly higher than Shanthi V et al study (16.67%).[16] 3 cases (17.65%) of grade 3 prostatic adeno carcinoma , 4 cases of (23.53%) grade 2 prostatic adeno carcinoma were eISSN: 2349-6983; pISSN: 2394-6466

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Prognostic Significance of PSA

noted with serum PSA range of 50-99.99 ng/ml which is similar to Shanthi V et al study (23.8%).[16] In our study histopathological grade 2 prostatic adeno carcinoma were restricted to PSA levels of 10ng/ml and above ,but grade 1 prostatic adeno carcinomas was restricted to PSA levels of < 10 ng/ml and grade 3 carcinomas were not having any correlation with specific PSA levels (Table 6). In studies done by Shanthi V et al (2012-2015)[16] and Lennox Anderson Jackson et al (2012),[15] histopathological grade 3 adeno carcinomas had a PSA range of 50-149.99ng/ ml and 76-190 ng/ml respectively . Our study coincided with the conclusion drawn from the studies of Shanthi V et al (2012-2015)[16] and Lennox Anderson Jackson et al (2012)[17] that histological higher grades of prostatic carcinomas are associated with higher PSA levels.

Conclusion

of Oncology. Advanced course. September, 2002: Education boo. 2000 ;101-4. 5.

Stamey TA, Yang N, hay AR, Mc Neal JE, Frina FS, Red wine E. Prostate specific antigen as a serum marker for adenocarcinoma of the prostate. N Engl J Med.1987:317:909-16.

WalshPC.Why make an early diagnosis of prostate cancer. JUrol 1990:147:853-4.

Thompson IM, Pauler DK, Goodmann PJ, et al. Prevalence of prostate cancer among men with a prostate specific antigen level=4.0ng/ml. Journal of Medicine .2004: 350(22):2239-2246.

Lyn NNK, Collins GN, Alex Ak et al. A comparative analysis of the role of prostate specific antigen parameters in clinical practice. The Prostate Journal 2000:2(4):205-210.

Mwaakyoma HA et al. Correlation of Gleason”s score and pretreatment prostate specific antigen in patients. Professional Med J Jun 2010; 17(2):235-240.

Benign prostatic hyperplasia was the most frequent lesion encountered followed by adenocarcinoma prostate . Prostatic adenocarcinomas were associated with raised prostate specific antigen more than 10ng/ml. In our study, there was a positive relation between higher levels of serum PSA and Gleason’s histopathological grade of prostatic adenocarcinoma. But poorly differentiated prostatic adenocarcinomas (higher grade) did not show correlation with serum PSA levels indicating that as the tumor becomes poorly differentiated, tumor cell production of PSA is reduced.

10. Jyotisapre KA, Agnihotri AS. et al: Utility of prostate specific antigen in different prostatic lesion: pathology and laboratory medicine: June 2011; 3(1):18-23.

Acknowledgement

13. Bhatt JV, Shah JM, Shah FR. Prostate –Assessment to management 2008.

The authors are thankful to the Miss. D. PADMAJA B. tech and all the faculty of department of pathology, Narayana Medical College and Hospital, Nellore.

References 1.

2. 3.

Anunobi CC, Akinde OR, Elesha SO, Daramola AO, Tijani KH, Ojewola RW. Prostate diseases in Lagos, Nigeria: a histologic study with PSA correlation. Nigerian postgrad Med J. 2011; 8(2); 98-104. Denis LJ. Diagnosing benign prostate hyperplasia versus prostate cancer. Br J Urol 1995:75(suppl1); 17-23. Akdas A. Turkan T, Turkeil, Cerviki, Biren T and Gurmen N. The diagnostic accuracy of digital rectal examination , transrectal ultrasonography, prostate specific antigen (PSA) density in prostate carcinoma. Br J Urol 1995:76:54-6. Radiae S. Prostate cancer molecular staging. State of-theArt in prostate and breast cancer treatment.European school

11. Haroun AA, Hadidy AS, Awwad ZM et al: Utility of free PSA serum level and its related parameters in the diagnosis of prostate cancer. Soudi journal of kidney diseases and transplantation. 2011:22(2);291-297. 12. Djavan B. Optimal predictors of prostate cancer on repeat prostate biopsy; a prospective study of 1051 men . Journal of urology (UNITED STATES) 2000; 163(4) ;1144-8.

14. Khan IA, Muhammadnasiret al: Carcinoma of prostate in clinically benign enlarged gland; J Ayub med coll Abottabad 2008; 20(2) ;90-92. 15. Zivkovic S et al: Correlation prostate specific antigen and histopathological difference of prostate carcinoma; arch oncol 2004; 12(3);148-51. 16. Shanthi V et al. prognostic importance of prostate specific antigen in assessing histological grade of prostatic adenocarcinoma. Int J Med Res Rev 2015; 3(3):268-272. 17. Jackson LA, McGrowder DA, Alexander-Lindo R. Prostate Specific antigen and Gleason Score in men with prostate cancer at a private Diagnostic radiology centre in Western Jamaica, Asian Pacific journal of cancer prevention.2012;13;1453-1456.

*Corresponding author: Dr. N. Mohan Rao, Associate Professor, Department of Pathology Narayana Medical College, Nellore Andhra Pradesh, India, Pincode:524003 Phone: +91 919849229201 Email: nmr2020kmc@gmail.com Date of Submission : 22.03.2017 Date of Acceptance : 20.07.2017 Financial or other Competing Interests: None. Date of Publication : 18.12.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Original Article DOI: 10.21276/APALM.1440

ABO Blood Groups and Malaria: Does it Really Matter? Chandrika Rao* Department of Pathology, K S Hegde Medical Academy Deralakatte, Mangalore, India

ABSTRACT Background: Malaria is most important parasitic disease affecting humans. The literature relating to malaria and the blood groups are sparse and have mixed response. Objectives: The study was undertaken to assess the distribution of ABO blood group and their relationship with malaria species and parasite load. Methods: In 200 malaria positive patients blood group analysis was done. Malaria parasite detection and load estimation was done using quantitative buffy coat (QBC) methodology using a fluorescent microscopy. ABO blood group was determined by agglutination test using antisera. Results: A total of 200 were included in the study of which 121 cases were positive for plasmodium vivax, 32 cases were positive for plasmodium falciparum and 47 patients had mixed infection. The results of blood groups showed, 63 malaria infected patients with A blood group, 50 with B blood group, 80 with O blood group and 7 with AB blood group. Maximum parasite load was noted in blood group O, followed by group A and B. The most common blood group infected with plasmodium vivax is blood group O, similarly with 3+ and 4+ parasite load is blood group O. Conclusion: This study suggests significantly higher proportion of O blood group malaria patients infected with plasmodium vivax with higher parasite load. Keywords: Malarial Parasites, ABO Blood Group, Malaria, Parasite Load

Introduction

Malaria is one of the most important parasitic disease affecting humans. Worldwide annually 8,81,000 deaths are related to malaria alone and in this India contributes a major share of incidence. According to WHO, in South East Asia region, 70% of malaria cases are from India. Karnataka constitutes 28 districts. Out of which Dakshina Kannada contributes 50% of the cases to the state malaria profile. Hence itâ&#x20AC;&#x2122;s a grave health problem that has tormented mankind for countless generations. [1] Despite high mortality and morbidity, certain individuals living in malaria endemic regions appear relatively protected compared to those who suffer frequent severe malaria attacks. Resistance to malaria is dependant on the development of an immune response by the host and to a variable extent on certain innate characteristics possessing protective value against infection. These factors include Sickle cell trait and disease, ABO blood type and levels of glucose- 6- dehydrogenase. It is thought that understanding nature of relationship between ABO blood groups and malaria should provide an invaluable scourge in the window and such studies in population of malaria endemic regions will be helpful in elucidating any such relationship.[2]

There is paucity of hospital based, comparative studies to investigate the relationship between blood group types and malarial infection in our population where malaria is endemic. The objectives of present study were, to assess the distribution of ABO blood groups in malaria patients and its relationship with malaria species and parasite load.

Materials and Methods

The hospital based retrospective review of electronic medical records of malaria patients who attended outpatient or admitted with malaria in a tertiary care teaching hospital in Dakshina Kannada district of Karnataka. Totally 200 malaria positive cases were included in the study. The malaria parasite detection and load estimation was done using QBC methodology using fluorescent microscope. The parasite species was confirmed on peripheral smear. The parasite load on QBC was reported as 1+ (<1 parasite/HPF), 2+ ( 1-10 parasites / HPF), 3+ ( 11-100 parasites /HPF) and 4+ ( >100 parasites /HPF).Commercial antisera was used for blood group determination ( tube method). Data analysis was done by Chi-Square test and p value of < 0.05 was considered as statistically significant.

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Result

respectively. Out of the cases, that had parasite load of 1+, 38.2% had blood group O, 34% and 25.5% cases had blood groups A and B respectively. Among cases with parasite load of 2+, 37.1had blood group A, 30%, 25.7% and 7.1% had blood groups O, B and AB respectively. Out of the cases that had parasite load of 3+, 51.6% had blood group O, and 23.3% each of blood group A and B. Parasite load of 4+ was seen in 43.4%, 30.4% and 26% had blood groups O, A and B respectively. There was no statistical significance between blood group frequency and parasite load (p = 0.321). (Table 2) (Figure3).

In our study malaria infection had male preponderance with 73.5% of the cases being males. Malaria affected all age groups and age ranged from 5- 82 years old. Out of 200 malaria positive patients 80 (40%) had blood group O followed by 63 (31.5%), 50 (25%) and 7(3.5%) patients having blood group A, B and AB respectively. (Table 1)(Figure 1).Among 200 patients, irrespective of blood group, Plasmodium vivax infection is seen in 121 (60.5%), P. falciparum in 32(16%) and rest 47 (23.5%) had mixed infection. Plasmodium vivax infected cases most commonly had blood group O (44.6%), with statistical significant relationship (p = 0.044) followed by blood group A (30.5%), B (22.5%). Out of total Plasmodium falciparum infected cases most common blood group was blood group A (34.3%). Among the cases having mixed infection, most cases had blood group O (38.2%) followed by 31.9% and 29.7% each of blood group A and B. (Table 1) (Figure2).

Discussion

Malaria has been known since antiquity. Much new information has emerged since relationship between ABO and malaria was first suggested >40 years ago. The observation by Miller et al that human erythrocytes lacking Duffy blood group antigens are refractory to invasion by Plasmodium vivax parasites indicates usefulness of studying the association of blood groups with malaria. In Indian scenario, the literature relating to malaria and the blood groups are sparse and have mixed results. [3]

In 35% of the cases, parasite load was 2+, followed by 30%, 22.5%, 11.5% with parasite load of 3+, 1+ and 4+ Table 1: Distribution of cases Blood group Sex

Total

147(73.5%)

53 (26.5%)

Vivax (V)

121(60.3%)

Falciparum (F)

32 (16%)

Mixed (M)

47(23.5%)

50 (25%)

80 (40%)

7 (3.5%)

200 (100%)

Type

63(31.5%) Table 2: Parasite load vs blood group type Blood group Parasite load in QBC

Total

16 (34%)

12 (25.5%)

18 (38.2%)

1(2.1%)

47 (23.5%)

26 (37.1%)

18 (25.7%)

21 (30%)

5 (7.1%)

70 (35%)

14 (23.3%)

31 (51.6%)

1 (1.6%)

60 (30%)

7 (30.4%)

6 (26%)

10 (43.4%)

23 (11.5%0

Table 3: Correlation of prevalence of malaria in different blood group Workers

A (%)

B (%)

O (%)

AB (%)

Singh et al (2015)

16.08

21.95

25.86

13.04

Gayathri et al (2013)

16.09

40.09

34.16

8.78

Deepa et al (2011)

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Workers

A (%)

B (%)

O (%)

AB (%)

Otajevvo et al (2013)

34.6

23.1

38.4

3.9

Sule Hussain et al (2014)

32.3

35.3

17.7

24.2

Tewodros et al (2011)

23.5

21.9

51.3

3.3

Present study

31.5

3.5

Fig. 1: Distribution of total cases according to blood groups

Fig. 2: Association between blood group and malaria species

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Fig. 3: Association of blood group and parasite load

In this study, high percentage of O blood group (40%) phenotype was observed among the study participants followed by A ( 31.5%), B (25%) and AB (3.5%). This agrees with some previous studies that also reported high prevalence of blood group O phenotype in tropical regions where malaria is rampant. [4] As regards to correlation there are some differences from other reports. High incidence of malaria in O blood group was found in the study of Singh et al, Tewodros et al, and Otejevvo et al. [2,4,5] Gayatri et al, Sule Hussain et al and Deepa et al however reported higher incidence in B blood group 40.97 %, 35.3%and 42% respectively.[3,6,7] (Table 3) According to the study conducted by our blood bank, most common blood group in our population is O, followed by B and A. Hence, highest number of malaria cases in blood group O, could be explained by the fact that blood group O is the most prevalent blood group in our population and South India. [8, 9] In this study, higher proportion of individuals with blood group A and B, were found to be infected with P. falciparum. This is also consistent with previous reports. [4, 10, 11] Several mechanisms relate to these associations including affinity for anopheles species, impairment of merozoite penetration of RBCs, as well as cytoadherence, endothelial activation and rosetting activity. [4]

Conclusion

This study lends further credence to earlier related studies that in any given population, the highest number of subjects

belongs to group O, while the least number belong to AB. The highest parasitemia rate was observed among group O. There was significant statistical relationship between blood group O being infected with P vivax (p=0.044) species. Since, blood group O is prevalent blood group in our population, this ostensibly implies that all ABO blood groups are equally at risk and therefore available malaria prophylactic and therapeutic strategies should be directed at individuals of all groups.

Acknowledgements

I acknowledge the support of Head of Department of Pathology and technical staff.

Reference 1.

Kumar S, BV R, Kumar A, Mukhta A, Suman D, Navya V. Malarial trend in Dakshina Kannada, Karnataka: An epidemiological assessment from 2004 to 2013.Indian J Health Sci 2015;8:91-4.

Otajevvo FD. Prevalence of malaria parasitemia and its association with ABO blood grouping among students of Igbinedion university Okada, Nigeria. Br J Med Res 2013;3:1164-77.

Deepa, Vanamala A, Karuna R, Cecil R. ABO blood group and malaria related outcome. J vector borne Dis 48, March 2011:7-11.

Tewodros Z, Abraham D, Berhanu E. Association of ABO blood group and Plasmodium falciparum malaria in Dore Bafeno area, Southern Euthopia. Asian Pac J Trop Biomed 2011;1;289-294.

Singh G, Urhekar AD, Singh R. A study on correlation of malaria infection with A,B,O, RH blood group system. J Parasitol Vector Biol 2015;7:67-73.

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Gayathri BN, Harendra KML, Gomathi N, Jeevan S, Reethesh PP. Relationship between ABO blood groups and malaria with clinical outcome in rural area of South India. Glob J Med. Public Health 2013;2:1-7.

Sule HA, Idachaba SO, Idoko T. Susceptibility of humans of the ABO blood groups to P falciparum infection among patients attending Ahmadu Bello University clinic (Sickbay), Samaru-Zaria, Kaduna state, Nigeria. Sch J App Med Sci 2014;2:1305-9.

10. Singh N, Shukla MM, Uniyal VP. ABO blood groups among malaria cases from district Mandla, Mandya Pradesh. Indian J Malariol 1995;32:59-63.

Sharanya H, Ruchi S, Shrijeet C, Lavnish O. Relationship between malaria and ABO blood group type. Int J Sci Res 2016;5:1041-44.

Chandrika R, Jayaprakash S. Frequency of ABO and Rhesus (D) blood groups in Dakshina Kannada district of Karnataka: A study from rural tertiary care teaching hospital in South India. NUJHS 2014;4:57-60.

11. Fry AF, Griffiths MJ, Auburn S, Diakite M, Forton JT, Green A, et al. Common variations in the ABO glycosyltransferase is associated with susceptibility to severe Plasmodium falciparum malaria. Hum Mol Genet 2008;17:567-76.

*Corresponding author: Dr Chandrika Rao, Assistant professor and Blood Bank Officer, Department of Pathology, K S Hegde Medical Academy Deralakatte, Mangalore, India Phone: +91 8242204490 Email: chandrika_valal@yahoo.com Date of Submission : 27.03.2017 Date of Acceptance : 07.08.2017 Date of Publication : 18.12.2017 Financial or other Competing Interests: None.

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Original Article DOI: 10.21276/APALM.1446

Relevance of Autopsy As a Diagnostic Tool in Present: Times A Study Kalpana Sharma*, Aparna Rathi, Kusum Heda Dept. of Pathology, Jawaharlal Nehru Medical College, Ajmer, Rajasthan, India

ABSTRACT Background: Autopsy rates have been declining world-wide; the ever-increasing availability of advanced diagnostic tests, complex legal procedure, and fear of medico-legal lawsuits appear to be some of the contributing factors. Clinical judgment errors however have shown no decline and continue to remain a source of concern. Mortality and morbidity data statistics become relevant in framing health policies only when validated by corresponding autopsy studies. The present study was conducted to ascertain the utility of autopsy as a modern diagnostic tool. Methods: A prospective study of hundred consecutive cases of medico-legal autopsies received in the Pathology Department was carried out for their demographic characteristics with a comparative analysis between the initial cause of death and final autopsy diagnosis. The utility of autopsy in discovering hitherto unknown incidental pathologies was also evaluated. Results: In 17.6 % of cases, the final cause of death was revealed only after autopsy. It proved especially beneficial in discovering latent cardiac illnesses, occult visceral bleed sites, and undiagnosed respiratory and infectious pathologies. Hitherto unknown incidental findings which were not directly responsible for death but nevertheless enhance our understanding of different pathologic processes were found in 16.5 % of cases. Conclusion: The study confirmed the utility of autopsy as an important diagnostic tool in revealing the true cause of death and contributing to our understanding of evolution of different disease processes. Simplifying the legal procedure will encourage more autopsies, stirring the clinicianâ&#x20AC;&#x2122;s interest and involvement in autopsy studies. Keywords: Autopsy; Cause of Death

Introduction

Autopsy studies are the most authentic means to confirm cause of mortality and evaluate ante mortem diagnosis made on the basis of clinical opinion and laboratory investigations. . It improves accuracy of medical auditing. [1] Each country has its own laws of land regarding the procedure that needs to be followed in conducting an autopsy. In India, the concept of medico-legal autopsy has been mentioned in the sections 174 and 176 of criminal procedure and is mostly done when there is a cause of doubt or suspicion regarding the cause of death. A postmortem examination can be conducted only after a written request has been made by the police, or by the order of the court. Availability of an ever increasing number of advanced, modern diagnostic tests with clinicians increasingly laying their faith in them to make a diagnosis, the complexity of the legal procedure involved in ordering an autopsy, compounded by the fear of autopsy revealing hitherto unknown findings/complications triggering law-suits are some of the reasons alienating clinicians from autopsy discussions. This declining trend in autopsy rates has been observed world-wide.[2] However, studies comparing

accuracy of the clinical diagnosis over different eras have shown no decline in the clinical judgment errors.[3, 4, 5, 6, 7, 8] The present study was conducted as an extension of the debate regarding relevance of autopsy in clinical practice and its role in unraveling hitherto unknown facts and gain insight into the morbidity patterns of various diseases. Aims & Objectives 1. To analyze the utility of autopsy in arriving at the cause of death, 2. To ascertain the extent to which autopsy provided information that was hitherto unknown. [Incidental findings]

Materials And Methods

Hundred consecutive cases that were received for medico legal autopsy histopathology at the Department of Pathology in a Medical College set-up were analyzed. In each case important information regarding age, sex, available clinical findings, suspected cause of death and postmortem findings were obtained from postmortem papers. Representative tissue specimens received from Brain, Lungs, Liver, Spleen, Kidneys and Whole Heart were studied. For whole heart dissection the inflow-outflow method was used. [9] All the tissue specimens were studied grossly and

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microscopically for different histomorphological changes. The formalin fixed,processed,paraffin tissue blocks were sectioned and stained with routine Hematoxylin and Eosin staining ; Special stains were used when required. Nine cases were found autolysed and unsuitable for any definite diagnosis. The findings on the autopsy table and the different histopathological features of the representative tissue specimens were analyzed to arrive at a final decision regarding cause of death; a comparative analysis between the initial basic cause of death which warranted autopsy and the final autopsy diagnosis was done according to Cordazzi et al. [3] The study was approved by the institutional review board.

Results

The majority of cases fell between the third and fourth decade, with males outnumbering females in a ratio of 2.4: 1. (Figures 1, 2) The cause of death as stated in the

postmortem papers which warranted the autopsy were varied and are represented incidence wise in % in the Figure-3. Death due to sudden, unknown cause followed by underlying chronic illnesses, suspected poisoning, cardiac causes and trauma were found to be the top five reasons. A comparison between the initial basic cause of death and the final autopsy diagnosis arrived revealed a consensus between ante mortem and postmortem opinion in 54.9 % with autopsy exclusively clinching the final cause of death in 17.6 %. It revealed important, hitherto unknown facts (incidental findings) in 16.5 %. Nine cases that were autolysed preventing a definite opinion were excluded. (Table-1) Many interesting findings which though not directly related to the cause of death but nevertheless have a significance of their own in studying the natural progression and morbidity patterns of various diseases were detected on tissue histopathology, some of them presented in a tabulated form in Table-2.

Table 1: Basic causes of death: correlation between initial diagnosis and autopsy findings: TYPE OF CORRELATION

NO. OF CASES

[%]

Correct diagnosis confirmed at autopsy

54.9

Autopsy added important data not suspected earlier

16.5

Diagnosis revealed only at autopsy

17.6

Autopsy diagnosis unclear

TOTAL NUMBER

91*

*Nine cases found to be autolysed completely were excluded.

Table 2: Some Incidental Findings On Autopsy. AGE(Years)/SEX

HISTORY/INDICATION FOR AUTOPSY

HISTOPATHOLOGY FINDING

38/M

Suspected poisoning

Emphysema

48/F

Suspected poisoning

Silicosis

40/M

Suspected poisoning

Chronic pyelonephritis

63/M

Suspected poisoning

Chronic glomerulonephritis

35/M

Cardiac cause

Asbestosis

55/F

Cardiac cause

Polycystic kidney disease

32/M

Trauma

Pulmonary tuberculosis

51/F

Trauma

Chronic pyelonephritis

19/F

Hanging

Multisystemic [lung, liver and heart] non-caseating epitheloid granulomas

6/F

Snake bite

Bronchopneumonia

45/M

Acute alcohol intoxication

Chronic pyelonephritis

55/F

Sudden death

Acute pneumonia

32/M

Sudden death

Hepatic cirrhosis

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Fig. 1

Fig. 2

Fig. 3

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Fig. 4: Showing variability in myocyte size, myocyte loss, interstitial fibrosis in a case of sudden death in a young male with a heavy, large, flabby heart [ 4a-H&E, 40x; 4b- Masson trichrome, 40x ].

Fig. 5: Myocardium showing dense, mononuclear interstitial infiltrate with myocyte necrosis (= Myocarditis) in a case of suspected infarction [H&E, 40x].

Fig. 6: Incidental findings of occupation induced interstitial lung diseases; 6a- Showing the coalescent collagenous nodules of silicosis [H&E,10x]; 6b- Characteristic ferruginous bodies (also in inset) with giant cell showing asteroid body in asbestosis [H&E,40x].

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Relevance of Autopsy As a Diagnostic Tool

Discussion

Analytical study comparing the initial basic cause of death and the final autopsy interpretation revealed a noteworthy agreement [54.9%] between pre and post mortem diagnosis which however was lower than the concordance rates observed by Cordazzi et al in two different periods [72.9% and 79.4% respectively ]. In 17.6 % cases, the final diagnosis regarding the cause of death was revealed only after autopsy, comparable to the 25 % found by them. [3] Previously unsuspected cardiac illnesses, the timely diagnosis of which could have averted / postponed sudden demise had they been suspected and treated ante mortem were discovered on autopsy. This was especially true in cases of sudden deaths of unknown cause in young adults. The spectrum of cardiac diseases detected were variable, from cardiomyopathy; cardiac hypertrophy [secondary to untreated essential hypertension]; myocarditis [misdiagnosed as myocardial infarction] to acute myocardial infarction [in a young male suspected of acute alcohol intoxication induced death] .Thus, autopsy proved beneficial in identifying silent cardiac killers. [Figures 4&5] In cases of sudden unexpected death, a detailed autopsy study helped identify the site / cause of internal bleed or hemorrhages which remained undetected ante mortem [they included cases of ileal perforation, hepatic laceration, extradural hematoma, hemothorax, and cirrhosis with variceal bleed]. Previously misdiagnosed / undiagnosed respiratory illnesses [comprising of cases of atypical pneumonia, chronic obstructive pulmonary disease (COPD), Tuberculosis] which were instrumental in causing death were also revealed on autopsy. The utility of autopsy as the sole diagnostic tool in ascertaining the final cause of death was underlined by a case of sudden death after a brief undiagnosed illness of two days, which revealed on histopathology, congested capillaries filled with parasitized red blood cells laden with malarial pigment in the visceral organs. Autopsy studies provide an insight into the different stages of disease progression, thus helping us know the natural evolution of untreated diseases validating their role as learning tools for research purposes.[10] Many interesting incidental findings were revealed in the present study too,which were unrelated to the primary cause of death [Some of which are tabulated in Table-2]. Roulson et al found 50 % autopsies revealing findings that were not suspected ante mortem. [1] Occupation induced interstitial lung diseases [Ex- Silicosis, Asbestosis] were found as an incidental finding in a

significant 3.2% in our study which is higher than similar studies on autopsy lung histopathology. [11] This could be because of the presence of many stone quarrying factories in the adjoining areas to which our medical college caters. These respiratory illnesses usually present with nonspecific symptoms and are usually misdiagnosed as COPDs, with autopsy studies revealing their true prevalence rate. [Figure 6] Morbidity and mortality statistics, thus acquire accuracy and significance when based on careful autopsies. [12] Autopsy helps reveal insidious onset diseases which have vague presentations. A case of death by hanging in a young female, revealed non-caseating epitheloid granulomas in multiple visceral organs[ lung, liver, heart], triggering suspicion of sarcoidosis which has significant incidence of neuropsychiatric manifestations like depression. [13] Autopsy studies by revealing the variable histopathological changes that occur in visceral organs in different disease processes facilitate improved understanding, making future diagnosis and correlation simpler. Of the 16 cases showing histological evidence of ischemic heart disease, corresponding lung histopathology revealed pulmonary edema and diffuse alveolar damage in 71.4%, similar to Soiero AM et al, [14] reiterating the fact that lungs are secondarily involved in all forms of terminal cardiac events. In 4 cases, lung histopathology revealed uncommon histopathological finding of cholesterol granulomas. Pulmonary hypertension has been implicated in their pathogenesis,but we did not find the characteristic lung pathology of plexiform arteriopathy though all had associated pulmonary edema and congestion and had died of cardio-respiratory failure,suggesting that apart from pulmonary hypertension certain other contributing factors like long standing severe illness, red blood cells /platelet lysis with release of membrane lipids might be contributing factors for development of these cholesterol granulomas. [15] Thus, autopsy exposes and helps a pathologist study rare histopathological entities, which otherwise are seldom encountered. It is, hence, an indispensable tool for its role in undergraduate and postgraduate medical education, the identification and characterization of new diseases and contribution to the understanding of disease pathogenesis. [16] In the present era of rising medical litigation cases with increasing allegations of death due to iatrogenic causes[3 cases in this study], autopsy studies prove beneficial in revealing the truth. None of the cases studied, revealed incidental neoplastic pathology in the present study; though they were detected to a variable degree in other similar studies .This could

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Sharma et al. be attributed to the variation in sample sizes, different demographic characteristics, and different study patterns. Visceral organs in nine cases showed varying degrees of autolytic changes preventing definite diagnosis, emphasizing the need to follow the tissue preservation protocols more stringently. This was specifically troubling in cases of death by drowning, where prolonged body submersion hastened autolysis. The clinicians’ indifference to autopsy, can be mitigated by certain strategies like making the legal procedure less complicated, swift communication of autopsy summaries, renewing their interest and contributing to upgraded medical understanding.

A-661 3.

Cordazzi AL, Morganti ALC, Montenegro MRG. Discrepancies between clinical diagnosis and autopsy findings. Braz J Med Biol Res 2003; 36: 385-391.

Sonderegger- Iseli K, Burger S, Muntwyler J, Salomon F. Diagnostic errors in three medical eras: a necropsy study. Lancet 2000; 355:2027-2031.

Baron JH .Clinical diagnosis and the function of necropsy.J R Soc Med 2000; 93:463-466.

Esteban A, Fernandez -Segoviano P.The autopsy as a tool to monitor diagnostic error. Intensive Care Med 1999; 25(4):343-344.

Kirch W, Schaffi C. Misdiagnosis at a university hospital in 4 medical eras.Medicine 1996; 75:29-40.

Goldman L, Sayson R, Robbin S, Cohn LH, Bettmann M, Weisberg M.The value of autopsy in three medical eras. N Engl J Med 1983; 308:1000-1005.

Ludwig J. Handbook of Autopsy Practice. 3 ed. Towata, New Jersey: Humana Press ; 2002.

Conclusion

Despite the rapid technological advancements, arriving at an accurate diagnosis shall always remain a challenge with autopsies continually revealing ante mortem diagnostic shortcomings. Autopsy studies serve in giving the final verdict regarding the cause of death especially those due to silent cardiac illnesses, occult visceral bleeds, undiagnosed infectious and respiratory illnesses. Often, hitherto unknown incidental findings with significant bearings are unveiled, paving way for better understanding of various disease processes. The present study reinforces the role of autopsy as a diagnostic tool that should be treasured and improvised.

Abbreviations & Symbols:

Chronic obstructive pulmonary disease (COPD)

Acknowledgements

Authors would like to thank Dr[Prof].Neena Kasliwal, Dr [Prof].Geeta Pachori,Department of Pathology, Jawaharlal Nehru Medical College[Ajmer] and Dr.Sushil Sharma, Reader, Department of Pharmacology,AFMC [Pune].

References 1.

Roulson J, Benbow EW, Hasleton PS. Discrepancies between clinical and autopsy diagnosis and the value of postmortem histology; a meta-analysis and review. Histopathology 2005; 47:551-559. Ermenc B .Comparison of the clinical and postmortem diagnoses of the causes of death. Forensic Sci Int 2000; 114:117-119.

10. Patel S, Rajalakshmi BR, Manjunath GV. Histopathologic findings in autopsies with interesting and incidental findings. Journal of Clinical and Diagnostic Research 2016; 10(11):EC08-EC12. 11. Mangal K, Dhakar P, Yadav A, Gupta K, Gandhi S. Magnitude of pulmonary diseases –Incidentally diagnosed on autopsy-at largest hospital and medical college of Rajasthan. International Journal of Current Research and Review 2016; 8(4):37-43. 12. Shojania KG, Burton EC, Mc Donald KM, Goldman L. The autopsy as an outcome and performance measure. Rockville (MD): Agency for Healthcare Research and Quality (US) 2002; 58:1998-2005. 13. Borson S, Randall Curtis J. Examining the link between sarcoidosis and depression. Am J Respir Crit Care Med 2001; 163 (2):306-308. 14. Soiero AM, Ruppert AD, Canzian M,Capelozzi VL, Serrano CV. Postmortem diagnosis of acute myocardial infarction in patients with acute respiratory failure - demographics, etiologic and pulmonary histologic analysis. Clinics 2012; 67(3):213-217. 15. Fischer EG, Marek JM, Morris A, Nashelsky MB .Cholesterol Granulomas of the Lung. Arch Pathol Lab Med 2000; 124:1813-1815. 16. Hill RB, Anderson RE. The autopsy - medical practice and public policy. Stoneham, MA: Butterworths Publishers;1988.

*Corresponding author: Dr. Kalpana Sharma, B-21,Arawali vihar , Near Lion’s bhawan , Vaishali nagar ,Ajmer , Rajasthan [India]-305004 Phone: +91 9414218849 Email: pkss75780507@gmail.com Date of Submission : 29.03.2017 Date of Acceptance : 06.08.2017 Financial or other Competing Interests: None. Date of Publication : 18.12.2017

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Original Article DOI: 10.21276/APALM.1466

Heterometaplastic Bone Formation in Nephrolithiasis: Critical Review of Pathology and Pathogenetic Mechanisms

Nandkumar V Dravid1*, Ashish V Rawandale2, Arundhati S Gadre1, Rajeshwari K1 and Kishor H Suryawanshi1 Department of Pathology, JMFâ&#x20AC;&#x2122;s ACPM Medical College, Dhule, Maharashtra, India Department of Urosurgery, Tejnaksh Institute of Urology, Dhule. Maharashtra, India

ABSTRACT Background: We critically analyze the incidence, presentation and histopathologic findings of heterometaplastic bone formation (HBF) in nephrolithiasis in the kidneys of patients undergoing percutaneous nephrolithitomy for stone disease. Methods: Percutaneous nephrolithitomy ( PCNL) was performed on 932 patients from August 2009 to October 2016 by a single surgeon. In 43 cases, heterometaplastic bone formation was seen to originate from urothelium and encompassing the renal calculi. Clinical workup, radiographic imaging, treatment modalities and histopathologic features in these patients were evaluated. Result: The patientsâ&#x20AC;&#x2122; age ranged from 14 years to 65 years (median age 33.7 years). The male to female ratio was 4.3: 1.Heterometaplastic bone formation (HBF) encompassing the stone was identified in 69.76% in right kidney, 25.58% in left kidney and 4.65% in both kidneys. Radiographic appearance of eccentric density surrounding hypodense area was observed in 32 of 43 cases (74.41%). Histopathological evaluation showed trabecular bone with surface osteoblastic activity and intra trabecular bone marrow, haemopoietic cells and adipose tissue encompassing birefringent crystal deposits in 22 cases (51.16%). Trabecular bone in intimate proximity of woven bone and haemopoietic cell islands partially encompassing birefringent crystal deposits was observed in 17 cases (39.53%). Woven bone with mineral deposits and fibro collagenous proliferation was seen in 4 cases (9.30%). Conclusion: Although reported infrequently, HBF in nephrolithiatic deposits has a high incidence in our patients. Pathogenetic mechanisms regarding transdifferentiating renal stem cells appears tenable in such a setup and is corroborated in our study. Keywords: Bone, Crystals, Heterometaplastic, Nephrolithiasis

Introduction

Heterometaplastic bone formation in stone pathology has been rarely described in the kidney. The first recorded bone formation in the pedicle attachment of a renal calculus to the kidney pelvis has been recorded in 2 cases of nephrectomy. [1] Stuart and Krikorian reported occurrence of true bone within a renal calculus. [2] Cifuentes Delatte et al have reported osseous and cartilaginous metaplasia in 1.17% of 1624 urinary stones. [3] With the advent of PCNL the reported incidence has shown a rise upto 3.4%. [4] The typical radiographic and histopathologic features have been found fairly commonly in our practice of renal stone surgeries. We present 43 cases of heterometaplastic bone formation predominantly encompassing renal stones requiring modified surgical interventions in 932 PCNLs performed in 2117 patients treated for renal stone disease at our institution. This series highlights the peculiarities of heterometaplastic bone formation and the implications of unexplored pathogenesis about epithelial-mesenchymal trans-differentiation of urothelial stem cells towards osteogenic lineage.

Materials and Methods

2117 patients of nephrolithiasis required therapeutic surgical intervention of various types. Nephrolithotomy (PCNL) was performed in 932 patients at the institute of urology between August 2009 and October 2016. All the patients presenting with renal stones underwent preoperative clinical assessment, excretory urography, plain X ray (KUB) and CT scan. PCNL was performed under general anaesthesia with endotracheal tube. Retrograde pyelography was performed prior to PCNL access. Access was acquired under fluoroscopic guide using 17Fr or 26Fr nephroscope. Stones were fragmented with pneumatic lithoclast. After clearance and debulking, the pelvicalyceal system was inspected with a nephroscope. Whenever abnormal appearing hard pelvic tissue, bleeding stone, adherent stone was visualized, biopsy was taken and sent for histopathological examination. Bony tissue when identified, the undecalcified tissue was processed for paraffin embedded section and stained by haematoxylin and eosin, trichrome stain and visualized by light and polarizing microscopy.

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Decalcification was avoided for proper visualization and evaluation of heterometaplastic bone. 43cases revealing heterometaplastic bone with crystalloid deposits have been documented. The clinical, radiographic and operative records were reviewed for patientâ&#x20AC;&#x2122;s age, sex, location of stones and the operative findings. These findings and histopathological details were evaluated.

cells and adipose tissue completely encompassing birefringent crystal deposits in 22 cases (51.16%). (Figure 1A, 1B, 1C and 1D). Pattern II showed trabecular bone in intimate proximity of woven bone and haemopoietic cell islands partially encompassing the birefringent crystal deposits was observed in 17 cases (39.53%) (Figure 2A, 2B and 2C)

Result

Heterometaplastic bone formation and crystal deposits were seen in 43out of 932 consecutive PCNL procedures (4.61%). These HBF with renal stones included one child (2.32%) and 42 adults (97.68%) with a mean age of 33.7 years(age range 14 to 65 years). The male to female ratio was 4.3:1.The duration of symptoms in these 43 patients ranged from 3 months to 42 months. Preoperative urine analysis showed Calcium Oxalate (CaOx) crystals in 27 out of 43 cases. Bacteriological culture identified urinary tract infection in 8 patients preoperatively. HBF was identified in 69.76% in right kidneys, 25.59%.in left kidneys and 4.65% in both kidneys. Preoperative nephroscopic appearance was that of a hard looking stone adherent to the pelvic mucosa. During pulverization it was found that the stone had an admixture of bony hard tissue. During removal, the white hard tissue was in continuation with the mucosal lining of the papilla/renal pelvis. The removal was accompanied with operative area bleeding which was duly controlled. Of the 43 cases, the HBF encompassing the stone was located near the renal papillae in the pelvicalyx in 40 cases and 3 near the right PUJ .These stones were grey to dark brown in colour, 4mm to 25mm in size and weighing from340mg to 2100mg.

Pattern III showed woven bone with mineral deposits and fibro collagenous proliferation was seen in 4 cases(9.30%) (Figure 3A and 3B). In 39 specimens from the first and second group, bony areas were clearly seen in continuation with the urothelium. (Figure 1B). Chronic inflammatory changes, urothelial proliferation and subepithelial spindle cell proliferation was evident in 28 cases (71.79%).

Discussion

Metaplastic changes along urothelium denote a deranged epithelial response to injurious stimuli. These changes have been variously named as ectopic renal ossification, [3] bone formation in ureter, [5] ossification in the kidney stone, [1] pyelic osseous formation, [6] bone metaplasia, [7,8] extra osseous metaplasia [9] and extra osseous bone formation in renal pelvis . [4] The terminology of Heterometaplastic bone formation used in the present study denotes formation of tissue foreign to the part where it is formed. [10] These responses initially were hypothesized as Randallâ&#x20AC;&#x2122;s plaque. [11] The sites of interstitial crystal deposition were shown to be near the tip of papillae. He conjectured CaOx stone formation at these sites which was confirmed by others [12-16] and recognized this discovery adequately to be an important step in our understanding of pathogenesis of HBF.

The X ray findings were available in 43 cases where typical findings were seen in 32 cases (74.41%), where plain films revealed radio-opaque eccentric halo with radiodensity showing connection with the urothelium. 11 cases (25.58%) revealed only fluffy radio-opaque densities. Stone analysis in the 43 cases showed predominantly CaOx in 35(81.39%) with additional urates in 14(32.55%), mixed struvite stones in 8(18.60%) cases. In all the surgical resections, the tissue was hard and brittle in consistency. . Histopathologic evaluation showed 3 patterns-

Over the last three decades, various hypotheses and observations have been put forward as 1) hypothesis of physico-chemical imbalance; 2) fixed particle theory; 3) role of defective renal tubular cells; & 4) discovery of crystal growth and aggregation inhibitors including macromolecules such as osteopontin, nephrocalcin, bikunin and BMP-2. [17-26]

Pattern I showed trabecular bone with surface osteoblastic activity and intra trabecular bone marrow, haemopoietic

Gambaro et al have been unable to combine these hypotheses with the hypothesis that Randallâ&#x20AC;&#x2122;s plaque

Table 1: Showing Histopathological patterns in heterometaplastic bone formation of 43 cases. Serial No.

Histopathological patterns

No.of cases

Percentage

Continuation with urothelium

Pattern I

51.16%

Present

Pattern II

39.53%

Present

Pattern III

9.30%

Absent

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Heterometaplastic Bone Formation In Nephrolithiasis:Critical Review Of Pathology And Pathogenetic Mechanisms

Fig. 1: Pattern I-Trabecular bone (arrow) completely encompassing birefringent crystal deposits , Fig. 1A (H & E X 400), Fig. 1B (Polarising microscopy X 400), bony areas (arrow) were clearly seen in continuation with the urothelium. Fig. 1C (H & E X 400) birefringent crystal deposits with trabecular bone (arrow) Fig. 1D (Polarising microscopy X 400).

Fig. 2: Pattern II-Trabecular bone (arrow) partially encompassing birefringent crystal deposits Fig. 2A (H & E X 400), trabecular bone in intimate proximity of woven bone and haemopoietic cell islands (arrow) Fig. 2B (H & E X 400), trabecular bone (arrow) partially encompassing birefringent crystal deposits Fig. 2C (Polarising microscopy X 400).

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Fig. 3: Pattern III-Woven bone (arrow) with significant mineral deposits and fibro collagenous proliferation (arrow) Fig. 3A (H & E X 400), Fig. 3B (Polarising microscopy X 400).

allows CaOx stones to form and grow in the renal pelvis. [17] In vitro models, oxalates have been shown to trigger inflammatory, oxidative, chemotactic and fibrogenic loops. [18-20] Gambaro et al have hypothesized regarding condition which may trigger the trans differentiation of tubular cells the origin of which is mesodermal despite their epithelial appearance. [21] Thus the epithelial cells may be induced to undergo epithelial-mesenchymal differentiation under paraphysiological oxalate concentrations similar to idiopathic CaOx stone formers ( ICSF). The renal interstitial myofibroblasts like the liver Ito cells are thought to be pericyte like cells. [22, 23] Notably such pericytes have the ability to undergo osteoblastic differentiation and mineralization. [24-25] Cultured artery smooth muscle cells, similar to multipotent interstitial cells in the kidney are also induced to become osteogenic by inflammatory stimuli, reactive oxygen species and hypoxia. [26]

Taking into consideration the peculiar physiological condition of the papillae, of low oxygen tension; and a sub-ischaemic environment, the pericyte like stem cells are sensitive even to mild toxic insults, or to high CaOx or phosphate concentrations and their propensity towards osteogenesis should explain such high occurrence of HBF in renal stones in our cases.

Given its particular conditions of low oxygen tension, the papilla is a niche for stem cells, which have been shown to differentiate into myofibroblasts and cells expressing neuronal markers and to spontaneously form cellular spheres. These renal stem cells can migrate to other parts of the kidney and to the medullary tubular epithelia in particular. [27-28] Since stem cells recovered from other tissues can differentiate along the bone lineage, the third cell population potentially capable of mineralizing in the kidney is that of papillary stem cells.

Huggins has confirmed the sequential steps of ectopic osteogenesis. [29] The appearance of small cysts to calcification, then to organized osseous tissue was confirmed in experiments on urothelial tissue. It was hypothesized that the primary osseous metaplasia has served as a focus for superimposed stone formation. This view was supported by Fernandez Conde et al ,who have suggested deposition of woven bone which later on was remodeled to form lamellar bone. [8] This ossification nidus perforating the urothelium comes in contact with urine. The direct and continuous action of urine induces bone formation. In our series, the HBF was found in renal papilla and in the renal pelvis. The complete encompassment of the stone by heterometaplastic bone suggests the involvement of multi-potent papillary stem cells. These stem cells convert to osteoblastic cells due to humoral inductions. Bone encompassing the kidney stones has been studied using undecalcified biopsied material. This approach has been found to conserve cellular and extracellular details and has been utilized in the present study. [8]

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In a single case of extraosseous bone formation by Stuart et al and Plata, the entire stone was described as osseous tissue. [2, 9] In the present study we have seen the stone encompassed by heterometaplastic osseous tissue, crystals forming the nidus confirmed by polarizing microscopy. In the present study, sections from all stones revealed firm adhesions with urothelial mucosa by fibrous tissue or bone. [7] The characteristic radiological density of stone and an eccentric halo surrounding the area of low radiologic density as identified by LubnaSamad et al and Garcia-Cuerpo et al is observed in32 (74.41%) cases in the present study. Garcia-Cuerpo et al. [4, 7] The higher incidence of HBF has been thought to be due to the increasing usage of PCNL technique for stone disease. [4] This corroborates with our findings from the high endemic zone in India. [30] However, the peculiar findings of HBF in the present study need to be further evaluated for the hypothetical role played by the third type of renal stem cells. [17]

Conclusion

Although rarely reported in the literature, metaplastic bone formation has been found in relation to renal pelvis and proximal to PUJ. The frequency of heterometaplastic bone formation encompassing crystal deposits in our patients is unique and significantly higher. The pathogenetic mechanisms; calcium oxalate crystallization, Randall’s plaques and stem cells of renal pelvis and papilla differentiating along bone lineage have been hypothesized. In depth analysis has opened up the unexplored avenue of epithelial-mesenchymal trans-differentiation of urothelial stem cells in HBF in renal stone disease. We corroborate these findings in our study.

Reference

Garcia-Cuerpo E, Lovaco F, Berenguer A, Garcia-Gonzales R. Bone metaplasia in the urinary tract- A new radiological sign. Jour Urol.1988;139:104.

Fernandez-Conde M, Serrano S, Alcover J, Aaron JE. Bone metaplasia of the urothelial mucosa- An unusual biological phenomenon causing kidney stones. Bone.1996;18:289.

Plata AL, Faerber GJ, Koo HP, Putzi M. Extra osseous metaplasia of the renal pelvis in a child. Journal of Urology.1999;161:1295.

10. Dorland WAN. Dorland’s Illustrated Medical Dictionary. Editor W A N Dorland Edition 31, Publisher Saunders, 2007. 11. Randall A. The origin and growth if renal calculi. Ann Surg.1937;105:1009-1027 12. Cifuentes Delatte L, Minon- Cifuentes J, Medina JA.New studies on papillary calculi Journal of Urology.1987;137:1024-29. 13. Stoller ML, Low RK, Shami GS et al. High resolution radiography of cadaveric kidneys: Unravelling the mystery of Randall’s plaque formation. Journal of Urology.1996;156:1263-1266. 14. Gusek W, Bodew, Matouschek E et al .Concentrically layered micro concrements in the renal medulla of nephrolithiasis patients. A contribution to the renal stone pathogenesis. Urologe A.1982;21:137-141(German). 15. Evan AP, Lingeman JE,Coe FL et al. Randall’s plaque of patients with nephrolithiasis begins in the basement membranes of thin loops of Henle. J Clin Invest. 2003;111:607-616. 16. Evan AP, Coe FL, Lingeman JE et al. Mechanism of formation of human calcium oxalate renal stones on Randall’s plaque. Anat Rec.2007;290:1315-1323 17. Gambaro G, Antonia F, Cataldo A et al. Pathogenesis of nephrolithiasis: Recent insight from cell biology and renal pathology. Mini review. Clinical cases in mineral and bone metabolism.2008;5(2):107-109.

Phemister DB. Ossification in kidney stones attached to the renal pelvis.AnnSurg.1923;78: 239.

18. Umekawa T, Chegini N, Khan SR. Oxalate ions and calcium oxalate crystals stimulate MCP-1 expression by renal epithelial cells. Kidney Int.2002; 61:105-112

Stuart G, Krikorian KS. The occurrence of true bone within a renal calculus. The Journal of Pathology and Bacteriology.1932;35:373-378

19. Jonassen JA, Cao LC ,Honeyman T, Scheid CR. Mechanisms mediating oxalate-induced alterations in renal cell functions. Crit Rev Eukaryot Gene Expr.2003;13:55-72.

Cifuentes DL, Minon JL, Santos M and Traba ML. Ectopic renal ossification as a nucleus of urinary stones. Journal of Urology.1976;116:398.

Lubna S, Mohammed A, Zafar Z. Extra osseous bone formation in the renal pelvis. Journal of Urology. 2007;178(5):2124-2127.

20. Bhandari A, Koul S, Sekhon A, Pramanik SK et al. Effects of oxalates on HK-2 cells, A line of proximal tubular epithelial cells from normal human kidney. Journal of Urology. 2002;168:253-259.

Klinger ME. Bone formation in ureter- a case report. J Urol.1956;75:793.

Schulman CC, Wieser M. Pyelic osseous formation. ActaUrol Belg.1971;39:322.

21. Gambaro G, D’Angelo A, Fabris A et al. Crystals, Randall’s plaques and renal stones: Do bone and atherosclerosis teach us something? Journal of Nephrology.2004;17:774-777. 22. Iwano M, Plieth D, Danoff TM et al. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest. 2002;110:341-350.

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Dravid et al. 23. Rockey DC. The cell and molecular biology of hepatic fibrogenesis. Clinical and therapeutic implications. Clin Liver Dis.2000;4:319-355. 24. BostrĂśm K, Watson K, Horn S et al. Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest.1993;91:1800-1809. 25. Doherty MJ et al. Vascular pericytes express Osteogenic potential in vitro and in vivo. Journal bone miner research.1998;13:828-838. 26. Proudfoot D, Davis JD, Skepper JN et al. Acetylated lowdensity lipoproteins stimulates human vascular smooth muscle cell calcification by promoting osteoblastic

A-667 differentiation and inhibiting phagocytosis. Circulation. 2002;106:3044-3050. 27. Oliver JA, Maarouf O, Cheema FH et al. The renal papilla is a niche for adult kidney stem cells. J Clin Invest.2004;114:795-804. 28. Angalani F, Forino M, Del Prete D et al. In search of adult renal stem cells. J Cell Mol Med.2004;8:474-487. 29. Huggins CB. The formation of bone under the influence of epithelium of the urinary tract. Arch Surg;.1933;27:203. 30. Raguraman G, Singh SK. Epidemiology of stone disease in northern India- Urolithiasis; Basic science and clinical practice. Ed: Springer:2012:39-46.

*Corresponding author: Dr. Nandkumar V Dravid, Professor and Head, Department of Pathology, JMFâ&#x20AC;&#x2122;s ACPM Medical College, Dhule. Maharashtra, India-424001 Phone: +91 9422289277 Email: nandudravid25@gmail.com Date of Submission : 11.04.2017 Date of Acceptance : 28.08.2017 Financial or other Competing Interests: None. Date of Publication : 18.12.2017

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Original Article DOI: 10.21276/APALM.1468

RBC Histogram as Supplementary Diagnostic Tool with Peripheral Smear Examination in Evaluating Anaemia Byna Syam Sundara Rao*, Vissa Shanthi, Nandam Mohan Rao, Bhavana Grandhi, Vijayalakshmi Muramreddy, Sirasala Praveena Dept of pathology, Narayana medical college, Nellore (India)

ABSTRACT Background: Red blood cell(RBC) histogram provide an idea about morphological changes of red blood cells in hematological disorders. Peripheral smear examination findings are usually correlated with complete blood cell counts by automated analyzer. To known the utility and advantage of red cell histogram and correlation of microscopic examination of peripheral smear with automated histogram pattern. Methods: Blood sample was collected from 220 anemia patients in ethylene diamine tetra acetic acid (EDTA) tubes for peripheral smear examination and ran in Beckman coulter LH 780 automated hematology analyzer for obtaining histogram, complete blood count includes hemoglobin, total leucocyte count, Platelet count, red blood cell indices and red cell distribution width. This study was undertaken for a period of one month of November 2016 in department of pathology, central laboratory, narayana medical college & hospital, Nellore. Results: This study of histograms of 220 different types of anemia consisted predominantly females 154 (70%) , and males 64(30%). Maximum cases of anemia were noted in 30-40 years of age range. Microcytic hypo chromic anemia was the most common (63.63%) followed by normocytic normochromic anemia (19.4%), macrocytic hypochromic anemia (2.2%), dimorphic anemia (12.72%) and pancytopenia (1.8%). Left shifted curve and broad base mostly seen in microcytic anemia, right shift curve seen in macrocytic anemia and bimodal peak mostly seen in dimorphic anemia. Conclusion: Histogram can be used as an important screening test for hematology and can become a new parameter in the diagnosis of anemia though peripheral examination remains the definitive diagnostic test for evaluation. Keywords: Histogram, RBC, Anemia, Peripheral Smear

Introduction

Evaluation of anemia for diagnosis and management of various red blood cell (RBC)disorders through red blood cell histogram is a vital part. Complete blood count parameters like red cell distribution width (RDW) and mean corpuscular volume (MCV) are useful along with histogram for interpretation of abnormal red blood cell morphology.[1,2] The Normal Red cell distribution curve is a bell shape and curve peak should be within the normal MCV of 80.0-100.0fl. The narrow red cell distribution curve indicates homogenous population of cells and wider redcell distribution curve indicates a heterogenous population of red cell. In megaloblastic anemia where the red blood cells are larger, the histogram curve will shift to right and the curve will move to left if the cells are smaller than normal like in microcytic anemia. After treatment of underlying cause in anemia, the curve should shift toward the normal range . In dimorphic anemia the histogram curve may show multiple peaks due to two distinct red cell populations.After treatment of the cause of an anemia curve should move toward the normal range.Even when

MCV is normal, RDW is a good indicator of anisocytosis. [3] Higher RDW represents dual population of cells like small cells, some normal size cells, and immature red cells during degenerative response to anemia which are larger than normal. Histogram can be useful for monitoring the reliability of results ofanalyzer, potential causes of results and arriving at the probable diagnosis.[3]

Materials and methods

The data of 220 samples has been taken from the period of November-2016 to December-2016, central laboratory, Narayana General Hospital & Medical College, Nellore Andhrapradesh, India. In this Retrospective study, The Red blood cell histograms of all anemic patients visiting the central diagnostic laboratory of Narayana general hospital were analyzed. The Red blood cell histogram correlated with peripheral smear interpretation when the haemoglobin is low. The spectrum of variations in Red blood cell histograms tabulated with types of anemias based on peripheral smear interpretation.

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Complete blood count including haemoglobin , total leucocytes count (TLC), differential leucocytes count(DLC), platelet count and blood indices were obtained by Beckman coulter LH 780 automated analyser along with histogram. To maintain validity of LH 780 Automatic analysis machine quality control was done. Peripheral smear examination was also done. Normally volume of RBCs from 34fl to 150fl represents a good cell distribution ideally. If the graph starts before 34 fl and touches the baseline before 150fl are considered to be a left shift indicates microerythrocytosis. If the graph starts after 34fl and touches the base line after 150fl are considered to be a right shift indicates macro erythrocytosis. If graph starting at 34fl and ending between 225-250 fl are considered to be a broad base. If RBCs Populations has two morphologies then the graph will have two peaks representing to their respective morphology and called as dimorphic red cells. If the RBCs population will have single lineage of cells seen, the graph will be constraint and will look like short peak. Data was analysed using SPSS 22 and P-value was calculated using chi-square & degree of freedom. p value of 0.05 or less was considered statistically significant. Inclusion Criteria: All cases of anemia with low haemoglobin. For men anemia is defined as haemoglobin level less than 13.5 gm/100ml and in women as haemoglobin of less than12.0 gm/100ml. Based on peripheral smear interpretation ,different types of anemias like normocytic normochromic, microcytic hypochromic, macrocytic, dimorphic and pancytopenia were included in our study. Exclusion Criteria: Patient having leukocytosis, leukemaiod reaction, leukaemia, parasites and platelet disorders

Results

In our study of histograms of various types of anemia total 220 cases were studied. This study includes predominantly females 154 (70%) and males 64 (30%). Maximum number of anemia cases were noted in 30-40 years of age

range (Table-1).All cases had anemia with hemoglobin less than 12 gm/dl. All these cases consists of Normocytic normochromic anemia, Microcytic hypochromic anemia, dimorphic anemia and pancytopenia were diagnosed by peripheral smear. In our study 43 (19.54%) cases are normocytic normochromic anemia, 140(63.63%) cases are of microcytic hypochromic anemia, 5 (2.2%) cases are of macrocytic anemia and 28(12.72%) cases are of dimorphic anemia. Pancytopenia was seen in 4 (1.8%) cases (Table-2). Our study of 220 cases showed normal curve in 39(17.7%) cases, left shift in 64(29%), right shift in 12(5.45%), Broad base in 83 (37.72), short peak in 6(2.7%) cases and bimodal in 6(7.27%) cases (Table-3). In our study in normocytic normochromic anemia, cases shows MCV, Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC) and RDW within normal limits with few cases showing mild increase in RDW. In Microcytic hypochromic anemia MCV, MCH, are less than normal range with normal MCHC and increased RDW due to ansiopoikilocytosis. In Macrocytic anemia increase in MCV, MCH, RDW with normal MCHC due to variation in size & shape of the RBCs.In Dimorphic anemia, MCV, MCH, MCHC were in the normal limits andRDW was increased due to high degree of ansiopoikilocytosis. In pancytopenia only change noticed in red cell indices are increased RDW with normal MCV, MCH, and MCHC.In our study out of 43(19.54%) cases of normocytic normochromic anemia, 26(11.8%) showed normal curve and 17(7.72) % showed broad base curve. Out of 140(63.23%) cases of microcytic hypochromic anemia 7(3.18 % ) were normal, 60 (27.27%) showed left shift curve, 60 (27.27%) showed broad base curve, 7 (3.18%) showed bimodal curve histogram. and 6 ( 2.72% )showed short peak . Out of total 28 (12.7% ) cases of dimorphic anemia ,9(4)% showed normal curve, 4(1.81%) showed left curve, right curve 9(4% ), broad base curve 4 (1.81%) and 2 (0.9% ) showed bimodal curve. All 5 (2.2%) cases of macrocytic anemia showed right shift curve. All pancytopenia cases were broad base curve (Table-4). P value of <0.001 and chi square value 74.28 were considered statistically significant (Table-4).

Table 1: Age & sex wise distribution of anemia. Age

10-20

21-30

31-40

41-50

51-60

Total

100

220

4.54

22.72

45.45

18.18

9.09

100

M(Male)

F(Female)

156

M:F

2:3

1:2.3

1:3

1:1.6

1:3

1:2.4

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Table 2: Distribution of cases per types of anemia. Types of anemia

Percentage

Normocytic

43 cases (19.54%)

Microcytic

140 cases (63.63%)

Macrocytic

5 cases (2.2%)

Dimorphic

28 cases (12.72%)

Pancytopenia

4 cases (1.8%)

Table 3: Types of histogram abnormality in the study. Types of histogram

Percentage

Normal curve

39 (17.7%)

Left shift

64 (29.0%)

Right shift

12 (5.45%)

Broad base

83 (37.72%)

Bimodal

16 (7.27%)

Short peak

6 (2.7%)

Table 4: RBC histogram variations in different anemia. Normal curve

Left shift

Right shift

Broad base

Bimodal

Short peak

Normocytic

26 (11.81%)

17 (7.72%)

Microcytic

7 (3.18%)

60 (27.27%)

7 (3.18%)

6 (2.72)

Macrocytic

5 (2.27%)

Dimorphic

6 (2.72%)

4 (1.81%)

7 (3.18%)

2 (0.90%)

9 (4.0%)

4 (6.2%)

Pancytopenia P value - < 0.001

Chi square value - 74.28

Table 5: Comparitive study of anemias based on peripheral smear. Anemias

Sandhya et al Et al

JitendraChavda et al

Out study

Normocytic anemia

17%

17.4%

19.4%

Microcytic

61%

65%

63.63%

Macro etal

3.6%

2.2%

Dimorphic

15%

14%

12.7%

Pancytopenia

1.8%

Table 6: Comparative study of histogram curves in different studies. Histogram

Sandhya et al

Jitendra Chavda et al

Our study

Normocytic

15%

19%

17.7%

Left shift

30%

27%

29.0%

Right shift

07%

5.45%

Broad base

40%

38%

37.72%

Bimodal

7.27%

Short peak

2.7%

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Discussion

The RBC histogram is a graphic representation of particle size distribution in automated cell analyser. This is a standard part of complete blood picture. It provides clues in management and diagnosis of various red cell disorders and give information regarding to RBC parameters like RDW, MCH and MCV. [1, 2] Normal curve is symmetrical bell shaped or Gaussian distribution. Normal curve falls within normal MCV range 80-100fl.The RBC histogram in the cell counter displays the cell ranges for RBC are between 24fl and 360fl. The instrument counts only those cells with volume sizes between 36fl to 360fl as red cells .Those cells counted in the range 24fl to 36 fl are rejected and not include in the RBC count. Normally below 36fl size space is clear, but histogram begin above base line indicates presence of small particles like microspherocytes, malaria parasite, platelet clumps, normoblast, elliptocytes, bacteria, etc.[3,4] RBC histogram follows well known coulter principle of counting and sizing red cells providing the basis for generating the histogram. This method relies on the change in conductance as each cell passes through an aperture. The change in conductance results in an electrical pulse, the amplitude of which is proportional to the cell volume. The y axis represents the number of cells per channel, with each cell being stored in the representing its size ,so that after data is further processed by the computer, and the RBC Curve is smoothed by a moving average technique and displayed on a data management system. WBCs presented in RBC channel and counted with red blood cells. RBC Histogram is affected if WBCs count is more than 50000 cells. The presence of right sided shoulder usually corresponds to reticulocytosis and a trailer of erythrocyte population on the far right of the histogram correlates to red cell agglutination.

a higher deviation and hence higher RDW. In our study out of 43(19.54%) cases of normocytic normochromic anemia, 26(11.8% ) showed normal curve and 17(7.72)% showed broad base curve. These finding were correlated with study carried by Chavda J. [6] In the microcytic hypochromic anemia, MCHC may be normal but MCV and MCH are decreased. RBC population with low MCV will be shifted toward left. A broad base curve because of high RDW represents anisocytosis. RDW is also increased when there is increase number of smaller cells like in iron deficiency anemia. In present study MCV and MCH were less than normal in microcytic anemia with normal MCHC and increased RDW due to ansiopoikilocytosis as noted in peripheral smear study. Out of 140 (63.23%) cases of microcytic hypochromic anemia 7(3.18 % ) were normal, 60 (27.27% ) were left shift curve, 60 (27.27%) showed broad base curve, 7 (3.18%) showed bimodal curve histogram. and 6 ( 2.72% ) showed short peak. .These finding were correlated with study carried out by sandhya [5] and Chavda J.[6] The short peak well correlated with low haemoglobin and red cell count.

In our study out of 220 anemiacases, 43 (19.54%) of the cases are Normocytic normochromic anemia, 140(63.63%) of the cases are Microcytic hypochromic anemia, 5 (2.2%) Cases with macrocytic anemia and 28 (12.72%) cases are dimorphic anemia. Pancytopenia seen in 4 (1.8%) of the cases .Our findings regarding distribution of anemia cases were correlated with sandhya [5] and Chavda J[6] (Table-5). Our study out of 220 cases showed normal curve39 (17.7%), left shift 64(29%), right shift 12 (5.45%) Broad base 83 (37.72%), short peak 6 (2.7%) and bimodal 6(7.27%). Our findings regarding to RBC histogram were correlated with other studies like sandhya [5] and Chavda J[6] (Table-6). In normocytic normochromic anemia, the red cell indices like MCV, MCH and MCHC were within the normal limits with some cases showing mild increase indices. The population of the cells would be variable in size. Like some microcytic cells with predominance of normal size cells that results in

In dimorphic anemia the histogram may have 2 or more red cell populations, whereas in dual populations, the histogram has 2 distinct red cell populations. In dimorphic blood picture there may be dual population of microcytic & normocytic or normocytic & macrocytic red cells or admixture of small, normal and large cells of different sizes or admixture of patient and donor red cells. In our study in dimorphic anemia, a MCV, MCH and MCHC were normal and increased RDW due to marked ansiopoikilocytosis. The dimorphic RBC showing bimodal curve along with some cases showing left and right shifting of curve. The reason for dimorphic population may be nutrional anemia, recent blood transfusion or response therapy to nutrional anemia. [7]Out of total 28 (12.7%) cases of dimorphic anemia 9(4)% showed normal curve, 4(1.81%) showed left curve 04% showed right curve 9(4% ), broad base curve 4(1.81%) and 2 (0.9% )showed bimodal curve. These finding were correlated with study carried out by sandhya [5] and Chavda J. [6] In macrocytic anemia right shift with broad based curve means low Hb and macrocytic blood picture. As Causes of macrocytosis vary from benign to malignant, complete approach is essential to determine the etiology.[8, 9] Macrocytosis may occur at any age, but it is more prevalent in old age. [10,11,12] In our study macrocytic anemia due to variation in size and shape of the RBCâ&#x20AC;&#x2122;s, increased MCV, RDW, MCH were noted with normal MCHC. All 5 (2.2%) cases of macrocytic anemia showed right shift curve. This finding was correlated with Sandhya. [5] Right shift curve correlated well with increased MCV and MCH. All pancytopenia cases were broad base curve and correlated with Sandhya. [5]

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Histogram plays a vital role as working tool in early stage of morphological analysis when combined with concept of normal curve and the knowledge of particular complete blood count parameters like red cell distribution width and red cell indices. Presumption of presence of fragments, microcytichypochromic, macrocytic or dimorphic red cells, and different combinations of cells is possible by observations of outlines of histograms. In addition MCV and MCV provide an idea about distribution of red cell histogram.

Fossat C, David M, Harle JR, Sainty D, Horschowski N, Verdot JJ, Mongin M et al. New parameters in erythrocyte counting value of histograms. Archpathol Lab Med. 1987;111(12):1150-54

Gulati GL, Hyun BH; The Automated CBC. A current perspective. HematoloncolClin North Am.1994;8(4):593-603

Bessman JD. Red bllod cell fragmentation: Improved detection and identification of causes.Am j clin pathol.1988:90(3):268-73

Sandhya I, Muhasin T.P. Study of RBC Histogram in various anemias. Journal of Evolution of Medical and Dental sciences 2014; 3( 74),15521-34

Chavda J, Goswami P, Goswami A. RBC histogram as diagnostic tool in anemias. IOSR Journal of Dental and Medical Sciences .2015;14(10), 19-22

Benie T. Constantino SH. The Red Cell Histogram and The Dimorphic Red Cell Population .LAB MEDICINE.2011;42(5):300-8

Brigden ML. A Systemic approach to macrocytosis: sorting out the causes. Postgrad Med. 1995;97(5):171-84.

Kaferle J, Strzoda CE. Evaluation of macrocytosis. Am Fam Physician. 2009; 79(3): 203-8.

Conclusion

Along with peripheral smear examination,diagnosis ofRBC disorders is supplemented by histogram and they provide guidance regarding RBCS morphology, blood indices and Hb value. While interpreting microscopic examination of peripherial smear, good idea can be obtained from reviewing histograms.The speed and reliability of analyzers allow time to evaluate abnormal blood smears and correlate with histograms with confidence and efficiency Peripherial smear interpretation was significantly correlated with histogram curve in 220 anemia cases (p value <0.001, chi square value 74.28). Histogram Changes correlated well with peripheral smear findings in majority of the cases. It is used for screening but not considered diagnostic for any pathological condition.

References 1.

Bessman JD, Gilmer PR Jr, GardnerFH. Improved classification of anemias by MCV and RDW.Am J clin pathol.1983: 80(3):322-6

10. Argento V, Roylance J, Skudlarska B, et al. Anemia prevalence in a home visit geriatric populations. J Am Med Dir Assoc, 2008;9(6):422-6 11. Younis M, Daugher GA, Dulanto JV, Njeim M, Kuriakose P. Unexplained macrocytosis. South Med J.2013;106(2):121-5 12. McNamee T, Hyland T, Harrington J, Cadogan S, Honari B, Perera K, et al. Haematinic deficiency and macrocytosis in middle-aged and older adults. PLoS One. 2013; 8 (11): e77743

*Corresponding author: Dr. Byna Syam Sundara Rao, Narayana medical college, Associate Professor, Dept of Pathology, Nellore, Andhra Pradesh 524003 (India). Phone: +91 09493517944 Email: syam.byna@gmail.com Date of Submission : 11.04.2017 Date of Acceptance : 01.09.2017 Financial or other Competing Interests: None. Date of Publication : 19.12.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Original Article DOI: 10.21276/APALM.1483

Prognostic Evaluation of Vitamin D Deficiency in Breast Cancer Patients: A Pilot Study in India Bela Goyal1 and Sanjeev Garg2* Department of Biochemistry, Govt. Medical College and Hospital, Sector-32, Chandigarh, India 2 Department of Pathology, Grecian superspeciality Cancer hospital, Mohali, India

ABSTRACT Background: Vitamin D deficiency has been associated with poor prognosis in breast cancer. A new molecular classification of breast cancer is being increasingly used that is based on expression of estrogen receptors, progesterone receptors and Her2/neu amplification and is found to correlate well with prognosis and outcome of disease. The present study was designed to determine serum vitamin D deficiency in these molecular subtypes of breast cancer. Methods: Estimation ofserum vitamin D levels by chemiluminiscence was done in a cohort of 99 breast cancer patients and 25 age matched healthy controls. Breast cancer patients were further grouped into luminal A, luminal B, Her2/neu enriched and triple negative subtypes based on immunohistochemistry results. Non-parametric tests were performed to compare vitamin D levels in different groups. Results: 54.6%, 60%, 66.6% and 84.2% of patients with luminal A, luminal B, Her2/neu enriched and triple negative subtypes respectively were found to be vitamin D deficient (<20ng/mL) as compared to only 20% in healthy controls. More aggressive Her2/neu enriched and triple negative subtype patients had significantly lower serum vitamin D levels than the luminal subtypes of breast cancer. Conclusion: Vitamin D deficiency is more prevalent and significantly lower levels are found in more aggressive subtypes of breast cancer. Vitamin D supplementation must be considered in these forms of breast cancer to improve outcome. Keywords: Vitamin D Deficiency, Breast Cancer, Her2neu Enriched Breast Cancer, Triple Negative Breast Cancer

Introduction

In addition to maintaining calcium and phosphate homeostasis, vitamin D has been proposed to have antiproliferative effects on various cancer subtypes. It is believed to retard tumor cell growth and proliferation, promote apoptosis, prevent tumor cell stimulation by growth factors and regulate inflammation, thus affecting tumor microenvironment[1].Low levels of vitamin D have been shown to be associated with increased mortality and poor prognosis in breast cancer in various studies in different parts of the world.[2,3,4] Breast cancer is the most common cancer among Indian urban females.[5] It has classically been classified based on morphologyor histological grade into various types taking into account tumor cellâ&#x20AC;&#x2122;s mitotic index, degree of differentiation and nuclear pleomorphism.[6] However, within morphological and histological subtypes, variation was observed in terms of prognosis and response to therapy. With the advent of microarray based gene expression profiling, various classification systems emerged. Recently, a microarray based molecular classification proposed by Perou and colleagues[7] and validated by

others has become the most acceptable classification to determine prognosis. It classifies the breast cancer into luminal A, luminal B, Her2neu enriched and basal type/ triple negative breast cancer (TNBC) based on the tumor cells expression of estrogen receptor(ER), progesterone receptor (PR) or Her2/neu amplification. The status of ER, PR and Her2neu expression is routinely determined by immunohistochemistry and fluorescence in situ hybridization(FISH).[8] Based on a case study in California, that has shown deficient levels of vitamin D in breast cancer patients subtypes according to the above mentioned classification,[9] the present study was planned to determine vitamin D levels in the breast cancer patients as per the molecular classification, to compare the serum vitamin D levels among the various groups of breast cancer patients and also with age matched healthy controls in India.

Materials and Methods

A consecutive case series study was conducted at a tertiary care hospital in North India in the year 2012-2014. Blood samples of 99 newly diagnosed breast cancer patients

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Vitamin D Deficiency in Breast Cancer

presenting to the hospital were collected in vacutainers. Blood was allowed to clot for 20-30 min and centrifuged at 3000Xg for 20 min. to separate the serum. Serum total Vitamin D estimation was performed by fully automated chemiluminescence based autoanalyser, Advia centaur XP (Siemens, USA). The performance of the instrument and quality controls were satisfactory before analyzing the samples. The cohort of breast cancer patients was divided into 4 groups based on the immunohistochemical analysis on breast cancer cells. Luminal A group showed positivity for Estrogen Receptors (ER) and/or Progesterone receptors (PR) and were negative for Her2/neu overexpression. Luminal B group showed positivity with ER, PR as well as Her2/neu overexpression. Her2/neu enriched group showed Her2/neu overexpression but negative for ER and PR. Triple negative breast cancer (TNBC) group showed negative status with ER,PR as well as Her2/neu. Total 25 voluntary healthy controls were also enrolled in the study and their serum vitamin D levels were measured. Based on the serum vitamin D levels, these groups were subdivided into vitamin D deficient (vitamin D ≤20ng/ mL), vitamin D insufficient (vitamin D between 20-29 ng/ mL), and vitamin D sufficient (vitamin D ≥30ng/mL).[10] Statistical analysis was performed using SPSS v.16 (SPSS, USA). Non-parametric Mann-Whitney U test was carried out to compare the various groups of breast cancer patients with healthy controls. Unpaired student t- test was performed to match the age of breast cancer patients and healthy controls. Non-parametric kruskal- wallis test followed by Mann-Whitney U test was done to compare these groups with each other. P value <0.05 was considered statistically significant.

Results

The mean age of patients with breast cancer was found to be 55.8 with standard deviation of 10.09. Table 1 shows the sample size, mean, standard deviation and 95% confidence interval of serum vitamin D levels of all the breast cancer patients groups and healthy controls. The mean serum vitamin D levels in all the groups of breast cancer patients was found to be significantly lower than the healthy controls with p value < 0.05 as shown in table 2 and figure 1. Furthermore, serum vitamin D levels in Her2/ neu enriched group and Triple negative breast cancer group were significantly lower (p<0.05) than vitamin D levels in the sera of Luminal A and B type of breast cancer patients. On subdividing the groups based on the levels of vitamin D, it was observed that luminal A type breast cancer patients had 54.6% (n=35) patients with deficient vitamin D, 32.8% (n=21) had insufficient serum vitamin D levels and only 12.5% (n=8) were having sufficient vitamin D levels. Luminal B type breast cancer patients group had 60% (n=6), 30%(n=3) and 10%(n=1) patients showing deficient, insufficient and sufficient levels of serum vitamin D levels respectively. However, in the Her2/neu enriched and TNBC type breast cancer patients, none of the patients showed sufficient serum vitamin D levels. Rather 66.6% (n=4) of Her2/neu enriched and 84.2% (n=16) of TNBC patients were deficient in vitamin D levels. This is in contrast to the healthy controls group where only 20% (n=5) were deficient in vitamin D levels, whereas, 48%(n=12) and 32% (n=8) of the total subjects showed insufficient and sufficient levels of serum vitamin D levels as also depicted in figure 2. This clearly shows that vitamin D deficiency is more prevalent in breast cancer patients as compared to age matched healthy controls with Her2/neu enriched and TNBC patients showing even lower values of serum vitamin D than in luminal types of breast cancer.

Table1: Mean, SD, 95% Confidence interval of serum Vitamin D levels for Breast cancer patients groups and Healthy controls. Groups Luminal A Luminal B Her2neu enriched Triple negative Healthy Controls

Sample size 64 10 6 19 25

Mean±SD of Vitamin D (ng/mL) 19.2±8.57 18.9±6.52 15.7±5.98 13.31±5.19 25.95±8.09

95% Confidence interval 17.05-21.34 14.26-23.59 9.44-21.99 10.8-15.81 22.61-29.29

Table2: Mann whitney U test comparing Breast cancer patients groups with healthy controls. Groups Luminal A Luminal B Her2neu enhanced Triple negative

Mann-Whitney U 471.0 60.5 22.5 42.0

Wilcoxon W 2.55 115.5 43.5 232.0

Z value -3.0 -2.35 -2.62 -4.63

*indicates statistically significant at p<0.05

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P value 0.003* 0.018* 0.009* 0.000*

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Fig. 1: Box plot showing Mean±SEM of Vitamin D levels (ng/ml) in Breast cancer patients groups and healthy controls. Solid bold line represents mean serum vitamin D in different groups.

Fig. 2: Clustered Bar diagram showing number of subjects with Deficient Vitamin D levels(≤20ng/mL), Insufficient Vitamin D levels(20-29ng/mL) and Sufficient Vitamin D levels(≥30ng/mL) in Breast cancer patients groups and healthy controls.

Discussion

With a mean of 17.8 ng/mL of serum vitamin D in the breast cancer patients as compared to the mean of 25.9 ng/ mL in healthy controls, the present study shows agreement

with previous studies in Saudi Arabia[11] and in other parts of the world.[2,3,4] The present study also shows that levels of vitamin D in triple negative breast cancer with mean of 13.3ng/mL is lowest among all the subtypes. This finding conforms to the earlier studies where vitamin D levels in TNBC have been found to be lowest.[9] Furthermore, there are studies that show that TNBC has the worst prognosis than its other counterparts[12]. Her2neu overexpression is long known to have contribution to aggressive behavior of breast cancer with poor outcomes[13]. Her2neu enriched subtype of breast cancer has been also shown to have unfavourable prognosis as compared to the luminal types. [14] Pertaining to the anti-proliferative and protective effect of vitamin D on cancer cells, its deficiency implies higher proliferation in cancer cells and this could explain lower

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Despite the availability of a large number of studies on the role of vitamin D in breast cancer across the globe, the studies in India are limited. With the advent of new classification based on the receptor status in breast cancer patients, the current study was planned to look for the vitamin D deficiency in various subtypes of breast cancer. In a country like India where skin pigmentation, milk intolerance along with lifestyle and avoidance of sun exposure already predispose to low vitamin D levels, the deficiency of vitamin D in breast cancer patients can result in poorer prognosis.

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levels of vitamin D in the Her2/neu enriched and TNBC subtypes that are more aggressive and are associated with poor outcomes. Contrary to our study, a study by Kim HJ in korea has shown relation of vitamin D deficiency with poor prognosis of luminal types but no relation was shown with her2/neu enriched or TNBC subtype. They have hypothesized it to be due to effect of vitamin D on suppressing estrogen mediated proliferation of breast cancers that are ER positive i.e. luminal type but they have not explained any mechanism for ER negative cancers.[15]However, In a recent study, it was shown that by upregulating the vitamin d receptors in murine model of TNBC, antiproliferative effect is enhanced[16] and thus explaining the effect of vitamin D in TNBC subtype breast cancer. Further studies explaining the exact molecular mechanism underlying such observations needs to be carried out. Moreover, studies with larger sample size involving larger geographical distribution have to be carried out to validate our results. Studies are also available that show that vitamin D supplementation in breast cancer patients improved their prognosis.[17] One such study showed that intake of high dose weekly vitamin D supplementation can be helpful not only in breast cancer but also in reducing treatment related arthralgias and fractures.[18] In the current study, a lower mean level of vitamin D was observed in both the breast cancer patients as well as healthy controls than the previous studies in western population. The plausible explanation for this could be socio-cultural taboos and vegetarian diet combined with lack of awareness regarding vitamin D supplements in Indian population as explained elsewhere.[10]Their study also showed that mean levels of vitamin D in Punjab, Haryana are around 25.3ng/mL which is close to the mean vitamin D levels in healthy controls (25.95ng/mL) in our study. The drawback of the current study includes the lower sample size, single geographical area and determination of a single parameter to evaluate prognosis in breast cancer patients. Therefore further studies considering these aspects must be carried out. However, to the best of our knowledge, this is the first study in India, evaluating role of vitamin D deficiency in breast cancer taking into account the molecular subtypes of breast cancer. To conclude, Vitamin D deficiency is associated more with breast cancer as compared to age matched healthy controls. The Triple negative and her2/neu enriched subtypes that

carry poorer prognosis shows higher prevalence and significantly lower mean values of serum vitamin D than the luminal types.

Acknowledgement

There is no funding or conflict of interest involved.

References 1.

Moukayed M, Grant WB. Molecular link between vitamin D and cancer prevention. Nutrition. 2013;5(10):3993–4021. doi:10.3390 /nu5103993.

Tretli S, Schwartz GG, Torjesen PA, Robsahm TE. Serum levels of 25- hydroxyvitamin D and survival in Norwegian patients with cancer of breast, colon, lung, and lymphoma: a population-based study. Cancer Causes Control ;2012: 363–370.

Peppone LJ, Rickles AS, Janelsins MC, Insalaco MR, Skinner KA. The association between breast cancer prognostic indicators and serum 25-OH vitamin D levels. Ann. Surg. Oncol.2012;19:2590–2599.

Yao S, Ambrosone CB. Associations between vitamin D deficiency and risk of aggressive breast cancer in African–American women. J. Steroid Biochem. Mol. Biol. 2013;136:337–341

Consolidated Report of Population Based Cancer Registries 2001–2004. National Centre for Disease Informatics and Research National Cancer Registry Programme (Indian Council of Medical Research); 2006.

Rakha EA, Reis-Filho JS, Baehner F, et al. Breast cancer prognostic classification in the molecular era: the role of histological grade. Breast Cancer Res 2010; 12: 207.

Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature 2000; 406: 747–52.

Cummings MC, Chamber R, Simpson PT, Lakhani SR. Molecular classification of breast cancer. Is it time to pack up our microscopes? Pathology. 2011;43(1):1–8.

Rainville C, Khan Y, Tisman G. Triple negative breast cancer patients presenting with low serum vitamin D levels: a case series. Cases J. 2009;2:8390.

10. Ritu G, Gupta A. Vitamin D Deficiency in India: Prevalence, Causalities and Interventions. Nutrients 2014; 6:729-775. 11. Yousef FM, Jacobs ET, Kang PT, Hakim IA, Going S, Yousef JMet al. Vitamin D status and breast cancer in Saudi Arabian women: case–control study. Am. J. Clin. Nutr. 2013;98:105–110. 12. Rakha EA, Ellis IO. Triple-negative/basal-like breast cancer: review. Pathology .2009; 41:40–47. 13. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire RL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235:177–182. 14. Lukong KE. Understanding breast cancer – The long and winding road. BBA Clinical.2017;7: 64 –77.

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Goyal et al. 15. Kim HJ, Lee YM, Ko BS, Lee JW, Yu JH, Son BHet al. Vitamin D Deficiency is Correlated with Poor Outcomes in Patients with Luminal-type Breast Cancer Surg. Oncol.2011; 18 :1830–1836. 16. LaPorta E, Welsh J. Modeling vitamin D actions in triple negative/basal-like breast cancer. J. Steroid Biochem. Mol. Biol. 2014;144:65–73.

A-677 17. Gissel T, Rejnmark L, Mosekilde L, Vestergaard P. Intake of vitamin D and risk of breast cancer a meta-analysis. J. Steroid Biochem. Mol. Biol. 2008;111:195–199. 18. Peppone LJ, Huston AJ, Reid ME, Rosier RN, Zakharia Y, Trump DL, et al. The effect of various vitamin D supplementation regimens in breast cancer patients. Breast Cancer Res Treat. 2011;127:171–7.

*Corresponding author: Dr Sanjeev Garg, H.No.111,sector 46A, Chandigarh Phone: +91 9878517283 Email: lifeline.sanjeev@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 17.04.2017 Date of Acceptance : 20.07.2017 Date of Publication : 19.12.2017

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Original Article DOI: 10.21276/APALM.1487

A Comparative Study between Blood Donors and The General Population in Uttar Pradesh, India, to Analyse the Triggers for Donation Suparna Dubey1* and Seema Dua2 Department of Pathology, School of Medical Sciences and Research, Sharda University, G. Noida, U.P., India Department of Transfusion Medicine, Super-speciality Pediatric Hospital and Postgraduate Teaching Institute, Noida, U.P., India 1

ABSTRACT Background: Despite its large population, access to safe blood is disproportionately low in India. To recruit more members from the general population into the pool of voluntary donors, it is essential to understand the differences in their knowledge, attitudes and practices; and utilise the existing resources to eliminate them. Methods: A quasi-experimental study was conducted on 180 donors and an equal number of potential donors randomly selected from the community, using a structured questionnaire containing questions on attitudes and practices. Knowledge was assessed by interview by a single observer. Results: Donors were found to have a highly significant difference (p<0.00001) from the general population in age, gender distribution, marital status and socioeconomic status and significant difference in educational level (p<0.05). Knowledge about blood donation practices was significantly higher in donors (p=0.0002). Replacement donors constituted 82.2% of the donor category, while voluntary donors dominated (52.3%) in the community. Commonest reasons for non-donation in both groups included apathy. Misconceptions regarding pain, weakness, and increased chances of infection were prevalent. Donors were less likely to perceive blood donation as risky (p<0.00001). There was a highly significant difference in the attitude towards incentives (p<0.00001), the donor group supporting and the community denouncing them. Replacement credits were the most popular incentive. In the donor group, a markedly higher (p=0.0003) proportion had a history of previous donations (55% vs.36.1%). Most of them had a higher number (p<0.00001) and greater frequency of donation. Donors generally reported a better donation experience (p=0.00003) and less complications than the general population (p=0.0002). Conclusions: There is a need to recruit women and young donors from the community, and promote donor retention. Blood donation drives play important role in creating awareness, educating the masses and dispelling myths and misconceptions. Keywords: Blood Donation, Blood Donors, Voluntary Donation

Introduction

Human blood is considered the â&#x20AC;&#x153;liquid of lifeâ&#x20AC;? as it cannot be synthesised or substituted by any artificial means. The need for an adequate supply of safe blood has increased worldwide due to advancements in surgery and medical care. Simultaneously, increasingly stringent donor selection criteria to ensure decrease in transfusiontransmitted infections (TTI) have been enforced, which has led to an imbalance in the supply and demand of blood in many countries.[1] A lower prevalence of TTIs has been reported among voluntary donors, with the lowest rates being found among regular donors.[2,3] Therefore, the World Health Organisation (WHO) in 1975, laid down the goal of obtaining all blood supplies from voluntary, non-remunerated blood donors by 2020 and directed all countries to frame their national policies accordingly. [4] Despite this, only 35% of the 192

Member States have a national blood policy, relevant legislation and one specific organization responsible for the national blood programme.[5] According to the WHO, there is a great disparity in the access to safe blood. Only 45% of the global blood supply is collected in developing countries, which are home to more than 80% of the worldâ&#x20AC;&#x2122;s population. The average number of blood donations is 11 times higher in high income countries than in low-income countries. Moreover, 92% of donations in developed countries are from voluntary unpaid donors as compared to about 67% in developing and transitional countries.[6] In India, blood transfusion services are affected by the multiplicity of controls, with licensing being under Drug Controller General of India, policy under National and State Blood transfusion Councils and implementation with the states.[3,7] A mere 2760 blood banks cater to a population of 1.33 billion.[8,9] The annual requirement of blood in the

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country is estimated at 12 million units of blood, but only 10.9 million units were collected in the year 2015-16, 79% of which was through non-remunerated donation.[10] The disparity is even more conspicuousin the state of Uttar Pradesh (UP), where this study was conducted. Despite having the highest population, it has only 240 licensed blood banks, with a total annual collection of approximately 0.9 million units, ranking third nationally on both scores. [7,8,10] Voluntary donation shows a dismal figure of 44%, with only 6 out of 37 states ranking below it.[10]

except that on knowledge. Thereafter each subject was interviewed about blood donation and transfusion by the same principal investigator, to avoid observer bias, and graded on a three-part Likert scale.

To overcome this deficit, it is essential to understand the knowledge, attitudes and practices (KAP) of the existing donors and compare them with the community at large. The available resources can then be tailored to minimise the differences observed, thereby recruiting the general population to the pool of voluntary donors. In a country like India, where myths and superstitions pose a challenge to delivery of healthcare, KAP studies also help to identify the factors and myths that enable or prevent blood donation.

On comparing the demographic characteristics of the 180 donors with those of the general population, the differences were numerous and marked [Table 1]. Despite the apparent similarity in the mean age (29.45±7.50 vs. 28.09±10.43 years) and age range (19-65 vs.18-60), the difference in age was highly significant (p<0.00001). It is noteworthy that the majority of donors (97.2%) were below 45 years of age, whereas the distribution was more uniform in the case of the control group. Gender distribution, marital status and socioeconomic status were seen to have a highly significant (p<0.00001) and educational level a significant (p<0.05) association with donation behaviour.

Material and Methods

This was a quasi-experimental study conducted by the Department of Pathology in a tertiary care centre in Greater Noida in Uttar Pradesh, India from March to September 2016 after obtaining permission from the Institutional Ethics Committee. Considering the blood donation rate of 12.7% in previous studies, taking a confidence level of 95% and a margin of error of 5%, the calculated sample size is 167.[11] The study was conducted on two groups after taking informed written consent - 180 donors who donated blood in the institutional Blood Bank, and an equal number of potential donors randomly selected from the community, 90 from among the relatives accompanying patients and 90 from community visits. A structured questionnaire was designed by the authors, keeping in mind the various parameters enumerated in the “Methodological Guidelines for Socio-cultural Studies on Issues Related to Blood Donation”.[12] It had three sections containing multiple-choice questions regarding donor demographics, source and level of knowledge on the topic, and attitudes, including factors which motivate or hinder blood donation, perceptions about risks involved and role of incentives. There was an additional section for assessment of donation practices, to be attempted only by participants who had previously donated blood, containing questions regarding the reasons for donation, number and frequency of previous donations, level of satisfaction with services at donation locations and details of complications, if any.

Statistics-The data collected was analysed using SPSS (version 17) software. Descriptive statistics, Chi-square test and, where applicable, Fisher’s exact test, were used in this study to analyse the data.

Results

Two aspects of the knowledge about blood donation were studied - the level and the source of this information. Donors were significantly better informed than the general population (p=0.000214) [Figure 1]. Friends played an important role, providing donation-related information to 35% of donors and 25% of the general public, but the main source of knowledge was the family in donors (53.9%), and blood donation camps in the general community (37.9%) [Figure 2]. The attitudinal assessment also revealed many differences [Table 2]. In this study, replacement donors, who formed the majority (82.2%) in the donor group, were outnumbered by voluntary donors (52.3%) in the community. Detailed questioning revealed that 2.2% of donors were actually commercial donors.

The questionnaire was administered individually to the participants and they were directed to complete all segments

Among those who had not donated earlier, indifference or apathy, indicated by the statement “I was not asked to donate”, was the commonest reason, both among the donor group and the general population. Lack of time was another important demotivating factor amongst donors (33.3%) while fear contributed to 19.1% non-donations in the community. This included fear related to the procedure, like fear of injections in 2 (1.7%) cases and fear bred by misconceptions like weakness in 9 (7.8%), pain in 6 (5.2%), and increased chances of infection in 5 (4.3%) cases. 7.8% subjects in the general community had been rejected, the prime cause of deferral being anaemia in 7 (6.1%) cases, followed by ongoing medication in 2 (1.7%) cases, the records of which were not available.

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Perception of risk associated with blood donation was studied in all the subjects in both the groups and the difference found to be highly significant (p<0.00001). The degree of risk perceived also varied greatly, with one case (0.6%) each in the donor group labelling it as medium and low risk, and none grading it as high-risk. On the other hand, of the 44 (24.4%) members of the general population who feel that it is a â&#x20AC;&#x2DC;riskyâ&#x20AC;&#x2122; procedure, it was graded as high, medium and low risk by 3 (1.7%), 38 (21.1%) and 3 (1.7%) persons respectively. However, both groups perceived similar risks- anaemia by both donors (1.7%) and 38 (21.1%) in the community, and infection by 6 (3.3%) members of the public.

leave from work by 10 (5.6%), free haematinics by 5 (2.8%) and certificates by 2 (1.1%) members. A marked difference in blood donation practices was noted when previous donation behaviour in the donor group and the community was compared [Table 3]. The majority (55%) of donors had donated earlier (p=0.00032). A large number of them had donated twice (39.4%), thrice (30.3%) or more times (28.3%) prior to this donation. The highest number of previous donations was 21. The fact that the last donation had occurred within the last six months in 79.8% of the cases indicates a higher frequency of donation. On the other hand, the donors in the community were generally casual donors, with the majority having donated only once (64.6%), the highest number of donations being 6, and 83.1% having donated more than six months back.

Attitudes towards the role of incentives, if any to, to promote blood donation, also varied considerably (p<0.00001). However, the incentives suggested by both groups were similar, with the majority- 160 (88.9%) donors and 27 (15%) community members proposing free replacement of blood for family or friends. Free blood test was proposed by 3 (1.7%) donors and 10 (5.6%) community members. Other suggestions from the general public included paid

A statistically significant difference was observed in the donation experience (p=0.000034) and the post-donation complications reported by both groups (p=0.000169). In the general population, 6 (3.3%) donors complained of weakness and 3 (1.7%) complained of pain at the phlebotomy site, both of which were self-resolving.

Table 1: Demographic profile of the study population. Age (years)

Gender Marital status Education

Donors (%)

General population (%)

p value

18-25

58 (32.2)

106 (58.9)

p<0.00001

26-35

95 (52.8)

39 (21.7)

36-45

22 (12.2)

12 (6.7)

46-55

4 (2.2)

21 (11.7)

>55

1 (0.6)

2 (1.1)

Male

178 (98.9)

140 (77.8)

Female

2 (1.1)

40 (22.2)

Married

99 (55.0)

156 (86.7)

Unmarried

81 (45.0)

24 (13.3)

Below 12th std.

108 (60.0)

130 (72.2)

Graduate

58 (32.2)

43 (23.9)

Postgraduate

13 (7.2)

4 (2.2)

Professional

1 (0.6)

3 (1.7)

Family Income

< 10

9 (5.0)

65 (36.1)

(x10Âł per month)

10-50

47 (26.1)

68 (37.8)

50-100

115 (63.9)

27 (15.0)

>100

9 (5.0)

20 (11.1)

p<0.00001 p<0.00001 p=0.018346

p<0.00001

Table 2: Attitudes towards blood donation. Reasons for donation

Reasons for non-donation

Donors (%)

General population (%)

Voluntarily

28 (15.6)

34 (52.3)

For relative/ friend

148 (82.2)

31 (47.7)

For monetary gain

4 (2.2)

0 (0.0)

I was rejected as donor

0 (0.0)

9 (7.8)

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Dubey et al.

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General population (%)

I was not asked to donate

54 (66.7)

66 (57.4)

I do not have time

27 (33.3)

18 (15.7)

I am afraid

0 (0.0)

22 (19.1)

Others

0 (0.0)

Yes

2 (1.1)

44 (24.4)

178 (98.9)

136 (75.6)

Yes

163 (90.6)

54 (30.0)

17 (9.4)

126 (70.0)

Risks due to blood donation Should incentives be given? Table 3: Practices of blood donation.

Donors (%) Previous donation Number of donations

Last donation

General population (%)

Yes

99 (55.0)

65 (36.1)

81 (45.0)

115 (63.9)

2 (1.1)

42 (64.6)

39 (39.4)

11 (16.9)

30 (30.3)

8 (12.3)

â&#x2030;Ľ4

28 (28.3)

4 (6.2)

< 6 months back

79 (79.8)

11 (16.9)

6 months-1 year

20 (20.2)

31(47.7)

>1 year back

0 (0.0)

23 (35.4)

Bad

0 (0.0)

Satisfactory

10 (10.1)

24 (36.9)

Good

89 (89.9)

41 (63.1)

Donation experience

Complications

Yes

0 (0.0)

9 (5.0)

99 (55.0)

56 (31.1)

Fig. 1: Level of knowledge about blood donation.

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Differences in Blood Donors and General Population

Fig. 2; Source of Knowledge about Blood Donation.

Discussion

India is among the first in the South-East Asia region (SEAR) to attempt a reorganisation of its blood transfusion system.[13] In its landmark judgement of 1996, the Supreme Court of India banned professional blood donation, introduced mandatory licensing of blood banks, and directed the government, through National AIDS Control Organization (NACO), to establish the National and State Blood Transfusion Councils (NBTC/SBTC), to develop policies and programmes for improvements in blood banks. In 2002, NACO adopted the WHO Guidelines on the Clinical Use of Blood. In 2003, the Government of India framed and adopted the National Blood Policy (NBP). In 2013, India was among the 51 countries of WHO participating in the ‘Rome declaration’ on ‘achieving self-sufficiency in safe blood and blood products based voluntary non-remunerated donation.’ Despite this, there is limited access to safe blood in rural areas in states like UP, Uttarakhand, Jharkhand, Bihar and Chhattisgarh.[14] Hence this study was planned in a non-profit tertiary-care centre in a semi-urban region in UP, which mainly caters to the adjacent rural population. This study demonstrated that young, unmarried males with a higher educational and socioeconomic level tend to be donors. India has a high proportion of youth. However the majority (52.8%) of the donors were of the age group 26-35 years, similar to a study in north-east India,[12] but in contrast to studies in urban north India[14,15] and south

India, where subjects below 25 years formed the largest proportion (61.3%) of voluntary donors.[16] This shows that the large pool of potential donors below 25 years of age (58.9%) has not been tapped in the rural areas of north and north-east India. Males have shown greater donation behaviour in almost all studies in developing countries like India[12, 14-17] and SEAR,[13] Nigeria,[18] Israel,[19] Iran [20] and Greece [21] which is not the case in western countries.[5] In our study, 98.9% of the donors were males, similar to but higher than the values of 93%,[16] 90%, [14] 84.2%, [12] and 76%,[15] in other Indian studies. This may be attributed to multiple factorshigh rates of illiteracy, which promote myths, sociocultural taboos, which restrict mobility, and malnutrition, anaemia and multiple pregnancies, which cause deferral in women, especially in rural areas. In our study 55% of the donors were married but unmarried men were more likely to donate (p<0.00001), which was similar to other studies. [15,17,22] Owing to a high school dropout rate in India, the majority of subjects in both categories had education below the twelfth standard. However, donors were likely to have a higher educational level (p<0.05), with 40% of them having attended college. Similarly they were likely to enjoy a higher socio-economic status (p<0.00001). This association of donation behaviour with education [12,14,16,17,22] and income [12] has been welldocumented, but it is noteworthy that both these factors are dependent variables, and one may well lead to the other.

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Dubey et al. Knowledge about Blood Donation: The fact that donors have a significantly higher (p<0.05) level of knowledge about blood donation services is supported by numerous other studies [14,15, 20,23] and refuted by some.[2] However, our study demonstrated that the knowledge level in a majority of subjects in both groups was just satisfactory. Only 41.1% of donors and 26.7% of the general population had a high level. There is a need to act upon this lacuna by education programmes. The peer group was useful in dissemination of information, and possibly motivation, to all, but the majority of donors were motivated by their family, and the general public by blood donation camps organised in the region. The important role of television and media, highlighted in other studies,[15] have a lesser impact in rural areas. However this study shows that blood donation drives and propaganda by blood banks may contribute significantly in educating the masses, thereby recruiting them into the donor pool.[24] Attitudes Towards Blood Donation: Most developing and transitional countries depend heavily on replacement donation, [13,14,18,25-27] and India is no exception. In our study, 82.2% of the donors were replacement donors, similar to 93.3% in another north Indian study.[14] It is worth mentioning here that the donor group in this study comprised of only those donors who visited the blood bank in the hospital, possibly driven by a need for replacement credit; proportion of voluntary donations is much higher in blood donation camps but no such camps were organised during this period. However, voluntary donors comprised 52.3% of donors in the community, similar to 47.2% in a study from south India.[16] This indicates that members of the community are motivated for voluntary donation by blood donation drives, but not sufficiently to visit blood banks of their own accord to donate blood. Other studies have also cited the role of drives or reminders by blood bank personnel in motivating voluntary donation.[21,24] The presence of 2.2% commercial donors also reveals that though donation for money is illegal,[3] such donors often find entry under the disguise of replacement donors. [12,13]

A-683 in many studies [14,15,25,28] and refrained 33.3% of the present donors in this study from donating earlier. This shows that donations can be sought by arranging blood donation camps at convenient locations. Fear, much of it caused by misconceptions regarding pain and syncope during the procedure on one hand, and myths about weakness, loss of immunity, transmission of HIV and infertility on the other, are prevalent, both in India [13-16] and abroad. [20, 22,23,26-28] They contributed to 19.1% detentions in the community, but strangely, no subject reported fear of infertility, which has been reported in all erstwhile Indian studies.[13-15] Hopefully, this myth has been busted, at least in this region. Furthermore, steps to eradicate malnutrition and the resultant anaemia, which has been the major cause for deferral, will help not only in nation-building, but in recruiting more female donors. This study showed that donors were less likely to view blood donation as a risky procedure (p<0.00001), and if they did, they were likely to grade the degree of risk lower than the general population. This has been documented in previous studies.[16] Much of this perception of ‘risk’ is due to the myths and misconceptions mentioned earlier. These issues need to be addressed and irrational fears allayed. Our study also unveiled a highly significant difference in the attitude towards incentives for blood donation (p<0.00001), the donor group promoting and the community denouncing them. While interpreting this data, we must however, bear in mind the fact, that the donor group consisted chiefly (82.2%) of replacement donors, who understandably asked for free blood for family or friends as an incentive (in 88.9% cases). On the other hand, the donors in the community were mainly (52.3%) voluntary donors, and therefore shunned the idea of incentives (in 70% cases). Some studies supporting incentives can be found in literature.[12] However, like all other studies, our study also showed replacement credits to be the most popular incentive. [12,14,15,21,25] Other suggestions, which have been proposed in other studies too, include free tests, [15,21] paid leave,[12,21] free haematinics and certificates.[12,14]

The commonest reason for not donating blood earlier, both among the donor group (66.7%) and the general population (57.45%), was an apathetic rejoinder- ‘I was not asked to donate blood.” A similar attitude of indifference has been noted in many Indian[14,15] and global studies as the chief deterrent.[18,21] This points towards a lack of awareness of the shortage of blood and its life-saving properties. Convenience-related issues like lack of time, distance of the blood bank, transport issues, have been cited as causes

Practices of Blood Donation: A far more important issue than donor recruitment is donor retention. Only 5-10% donors in the SEA Region are repeat donors.[13,16] In our study, most of the donors in the donor group were seen to be regular donors, with a history of past donations (p<0.05) and boasting of a significantly greater number (p<0.00001) and higher frequency of blood donations. The donors in the community were generally casual donors-64.6% had donated once, 16.9% twice and 6.2% thrice, which is comparable to the figures of 68%, 13.3% and 6.7%registered in the general population in a similar

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Differences in Blood Donors and General Population

study in urban north India.[14] It is essential to prevent the dropout of these casual donors and convert the oncedonors to regular voluntary donors, to be able to attain the goal of 100% voluntary non-remunerated blood donation laid down by the WHO. In this study, donors had a significantly better donation experience than the general population (p=0.000034), similar to other studies.[15] This is actually a reflection of the fact that a positive donation experience increases the probability of a repeat donation, [29] Thus, it is important to ensure that the donation process is hassle-free, the donation area has a pleasant atmosphere and the staff is courteous. Only 3.3% donors from the community complained of minor complications (p<0.05). [16] Follow-up in the community is important to emphasize the transient and self-resolving nature of these complaints. This study suffers from the limitation of sampling bias. Larger studies may further help evaluate the differences in the blood donating mindset between the donors and the general population.

Conclusion

The majority of donors in India are replacement donors. Young, unmarried males with a higher educational and socioeconomic level and having a greater knowledge of blood donation practices tend to donate blood. There is a need to recruit women and potential donors below the age of 25 years into the pool of voluntary donors. This is possible by blood donation drives in the community, which will reach out to the masses, create an awareness about the importance of blood donation, serve as reminders, educate them, and dispel the prevalent myths and misconceptions. It is important to ensure convenience of blood donation services and a hassle-free procedure for retention of the recruited donors as regular voluntary donors.

References 1.

Riley W, Schwei M, McCullough J. The United States’ potential blood donor pool: estimating the prevalence of donor-exclusion factors on the pool of potential donors. Transfusion. 2007;47:1180-8.

World Health Organization. Universal Access to Safe Blood Transfusion. Geneva, Switzerland:WHO; 2008. Available from: http://www.who.int/bloodsafety/publications/ UniversalAccesstoSafeBT.pdf. Accessed on 10 April 2017.

Nair SC, Mammen JJ. Repeat voluntary non-remunerated blood donor is the best quality indicator for blood safety. Indian J Med Res. 2015;141:749-52.

WHA28.72 Utilization and supply of human blood and blood products. Available from: www.who.int/bloodsafety/ en/WHA28.72.pdf Accessed on 10 April 2017.

Utilisation and supply of human blood and blood products. Available from: http://www.who.int/bloodsafety/en/ WHA28.72.pdf. Accessed on 10 April 2017.

Blood safety. Available from: http://www. who.int/bloodsafety/StrategicPlan20082015AccessSafeBloodTransfusion.pdf. Accessed on 10 April 2017.

7. Blood-transfusion-services. http://www.naco.gov.in/ NACO/Blood_Safety/Blood_Safety_More/ Accessed on 10 April 2017. 8.

Blood Banks in India. Available from: http://www.cdsco. nic.in/writereaddata/BLOOD BANKS INDIA feb 2015.pdf Accessed on 10 April 2017.

Total Population- Both Sexes. World Population Prospects, the 2015 Revision. https://esa.un.org/unpd/wpp/ Accessed on April 10, 2017.

10. Blood Transfusion Services (BTs) data for FY 2015-16. Available from: http://www.naco.gov.in/NACO/Divisions/ Blood_Safety/ Accessed on April 10, 2017. 11. Policy on research for health. Available from: http://www1. paho.org/hq/dmdocuments/2009/F4942/ Accessed on April 10, 2017. 12. Shenga N, Pal R, Sengupta S. Behavior disparities towards blood donation in Sikkim, India. Asian J Transfus Sci. 2008 Jul; 2: 56–60. 13. Bharucha ZS. Donor management in South-East Asia region (SEAR). DevBiol. 2005;120:145-53. 14. Sharma R, Madan N, Venkatesh S, Ichhpujani RL, Lal S. Factors influencing blood donations and the rational use of blood. J Commun Dis. 2010;42:185-90. 15. Dubey A, Sonker A, Chaurasia R, Chaudhary R. Knowledge, attitude and beliefs of people in North India regarding blood donation. Blood Transfus. 2014;12Suppl 1:s21-7. 16. Uma S, Arun R, Arumugam P. The Knowledge, Attitude and Practice Towards Blood Donation Among Voluntary Blood Donors in Chennai, India. J Clin Diagn Res. 2013; 7: 1043–1046. 17. Raghuwanshi B, Pehlajani NK, Sinha MK. Voluntary Blood Donation among Students - A Cross-Sectional Study on Knowledge and Practice vs. Attitude. J Clin Diagn Res. 2016;10:EC18-EC22. 18. Sekoni AO, Balogun MR, Odukoya OO, Inem V, Onigbogi OO. Blood donation practices and willingness to donate among residents of an urban slum in Lagos Nigeria. Niger Postgrad Med J. 2014;21:21-7. 19. Weinberg I, Zarka S, Levy Y, Shinar E. Why would young people donate blood? A survey-based questionnaire study. Vox Sang. 2009;96:128-32. 20. Shahshahani HJ, Yavari MT, Attar M. Knowledge, attitude and practice study about blood donation in the urban population of Yazd, Iran, 2004. Transfus Med. 2006;16:403–9.

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Dubey et al. 21. Marantidou O, Loukopoulou L, Zervou E, Martinis G, Egglezou A, Fountouli P,et al. Factors that motivate and hinder blood donation in Greece. Transfus Med. 2007;17:443-50. 22. Vรกsquez M, Ibarra P, Maldonado M. Blood donation: knowledge and attitudes of a university population in Chile. Rev Panam Salud Publica. 2007;22:323-8. 23. Wiwanitkit V. Knowledge about blood donation among a sample of Thai university students. Vox Sang. 2002;83:97-9. 24. Glynn SA, Kleinman SH, Schreiber GB, Zuck T, Combs SM, Bethel J,et al. Motivations to donate blood: demographic comparisons. Transfusion. 2002;42:216-25. 25. Sampath S, Ramsaran V, Parasram S, Mohammed S, Latchman S, Khunja R,et al. Attitudes towards blood

A-685 donation in Trinidad and Tobago. Transfus Med. 2007;17:83-7. 26. Alam M, MasalmehBel D. Knowledge, attitudes and practices regarding blood donation among the Saudi population. Saudi Med J. 2004;25:318-21. 27. Salaudeen AG, Odeh E. Knowledge and behavior towards voluntary blood donation among students of a tertiary institution in Nigeria. Niger J Clin Pract. 2011;14(3):303-7. 28. Mathew SM, King MR, Glynn SA, Dietz SK, Caswell SL, Schreiber GB. Opinions about donating blood among those who never gave and those who stopped: a focus group assessment. Transfusion. 2007;47:729-35. 29. Schlumpf KS, Glynn SA, Schreiber GB, Wright DJ, Randolph Steele W, Tu Y,et al. Factors influencing donor return. Transfusion. 2008;48:264-72.

*Corresponding author: Dr. Suparna Dubey, H-20, Kailash Colony, N. Delhi-110048, India. Phone: +91 9311310507 Email: drsprn@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 19.04.2017 Date of Acceptance : 21.08.2017 Date of Publication : 19.12.2017

eISSN: 2349-6983; pISSN: 2394-6466

Original Article DOI: 10.21276/APALM.1488

Evaluation of Cervical Smears by Conventional and Liquid Based Cytology Prakhar Srivastava* and Rashmi Arora Vardhman Mahavir Medical College and Safdarjung Hospital, West Ansari Nagar, New Delhi, India

ABSTRACT Background: Cervical cancer is the second most common cancer. Screening for cervical cancer is done by Pap [Papanicolaou] test by Conventional Pap Smear [CPS] or by newer technique Liquid Based Cytology [LBC]. The aim of the study was to learn and compare cervical lesions on both the techniques. Methods: This was a prospective study conducted over a 2 months period. Cervical smears were taken from 625 patients. On 25/625 patients both CPS and LBC were done. CPS and LBC were done on 300/625 patients each. The smears were processed and stained with Papanicolaou stain. Smears were studied for adequacy, cell types, inflammatory/ haemorrhagic background, organisms and reactive changes. The cervical lesions were categorised according to the Bethesda System of classification. The two techniques were compared based on certain defined parameters. Results: The techniques of CPS and LBC were learnt and cervical cells were appreciated on smears. Various epithelial abnormalities could be identified. Inflammation, bacterial vaginosis, candidiasis and reactive changes were noted. Out of 25 cases taken for comparison, 24 [96%] and 25 [100%] cases were satisfactory on CPS and LBC respectively.CPS showed more cases of severe inflammation and hemorrhagic background as compared to LBC. Conclusions: We conclude that both CPS and LBC are effective screening modalities for cervical lesions. Despite better performance of LBC in many aspects, it was felt that CPS is an equally effective tool in our settings due to the cost constraints. Keywords: Cytodiagnosis, Pap Test, Cervical Cancer, Mass Screening

Introduction

Cancer of the cervix is the most common cancer cause of death in the developing countries. [1] Every year in India, 122,844 women are diagnosed with cervical cancer and 67,477 die from the disease. [2] There are several methods to screen cervical cancer including Pap [Papanicolaou] test by Conventional Pap Smear [CPS] or by Liquid Based Cytology [LBC]. The screening coverage in Asian countries is low and varies from 50 per cent in Singapore to 2.6-5 per cent in India. [3,4,5] Though data from 20 populations based cancer registries in India indicate a steady decline in cervical cancer incidence rates over the last two decades, it still occupies number two position and the risk of disease is still high. [6] The clinical performance of CPS is not without limitations. A broad range of sensitivity [30%â&#x20AC;&#x201C;87%] has been reported for the detection of high-grade lesions by the conventional Pap test. [7] The conventional Pap test was also found to have a false-negative rate of about 14% to 33%, approximately two-thirds of which is due to limitations of sampling or slide preparation. [8] To overcome these limitations, Liquid Based Cytology was introduced. It is a new technique

in India. The shift from CPS to LBC is because LBC provides better sample quality, reproducibility, sensitivity, and specificity, as well as the ability to perform molecular testing. [9] The Bethesda system [TBS] is a system used for reporting Pap smear results.[10] Latest guidelines followed are of 2014, which are an update of the 2001 Bethesda System terminology, refinements of morphologic criteria, and incorporation of revisions and additional new information into a third edition of the Bethesda atlas for cervical cytology. [11] The following study was carried out to get a greater insight into the procedure and the analysis of cervical smears by both CPS and LBC and their applications in a developing country like India. The aims of the study were: 1. To learn the techniques of LBC and CPS., 2. To identify the cells in LBC and CPS., 3. To categorise the cervical lesions according to the Bethesda system.

Material and Methods

The study was conducted in the Department of Pathology and the Department of Obstetrics & Gynaecology of

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

Srivastava et al. Vardhman Mahavir Medical College & Safdarjung Hospital, New Delhi. The study was a prospective study conducted for 2 months in which smears from 625 patients were studied. Study Population: Patients visiting the OPD of the Department of Obstetrics & Gynaecology, Safdarjung Hospital. Inclusion Criteria: 1. Routine gynaecological examination of females above 25 years of age coming to the OPD. & 2. Patients with complaints of discharge or bleeding per vagina. Exclusion Criteria: 1. Smears obscured by blood or mucus. & 2. Very thick smears.

Methods

After obtaining the permission and clearance from the Institute Ethics Committee of our institution, 625 patients who attended the gynaecology out-patient department during a period of 2 months from June 2016 to July 2016 were randomly selected. On 300 patients, CPS was performed. On another 300 patients, LBC was performed. On the remaining 25 patients, both CPS and LBC were performed for comparison. The chief complaints of these patients were bleeding per vaginum, white discharge, irregular menstrual cycles, lower-abdominal pain etc. CPS was made using an Ayerâ&#x20AC;&#x2122;s spatula and an endocervical brush, which was immediately fixed using the cyto preservative. LBC sample was collected using the detachable brush provided by the manufacturer. The plastic brush was rinsed in vial of LBC medium containing cytorich preservative and was kept for minimum 40 minutes. LBC Smears were prepared using BD Prep Stain TM Slide Processor [SurePath Technique] [12] according to the directions in the operatorsâ&#x20AC;&#x2122; manual provided by the manufacturer. CPS were stained by Pap staining method. [13] The slides were studied for the cell morphology. The cervical lesions were categorised according to the Bethesda System of classification. [11] Only satisfactory smears were included for statistical analysis.

Results

A-687 All the smears were studied in detail for the following parameters: 1. 2. 3. 4. 5. 6. 7.

Adequacy Cell types Inflammatory background Haemorrhagic background Organisms Impression Reactive changes

The superficial cells were seen as large polygonal cells, with eosinophilic, transparent cytoplasm and small dark nuclei. Intermediate squamous cells were similar in size to superficial cells or somewhat smaller. They had a vesicular nucleus with usually a basophilic cytoplasm [Fig. 2]. Parabasal cells occurred singly and were usually round or oval in shape, with smooth cytoplasmic borders and bland and homogenous nuclei [Fig. 3]. Basal cells had scant basophilic cytoplasm and large nuclei with chromatin granules and tiny round nucleoli. Endocervical cells were seen as columnar cells, arranged in palisades or in honeycomb pattern . CPS: Out of 300 cases of CPS, 29 [9.7%] were unsatisfactory. For the 29 unsatisfactory cases, the main causes were scant cellularity of squamous cells in 15 [51.7%] cases and haemorrhage in 10 [34.5%] cases. The diagnosis was made as Negative for intraepithelial lesion or malignancy [NILM] in 265 [97.8%] of cases. Atypical glandular cells of unknown significance [AGUS] were reported in 2 [0.7%] cases. Atypical squamous cells of unknown significance [ASCUS] were reported in 2 [0.7%] cases. High-grade squamous intra epithelial lesion [HSIL] was reported in 2 [0.7%] cases which showed nuclear changes like increased N/C ratio and irregular coarsely clumped chromatin. The background was seen for the presence of inflammation or haemorrhage. The background was clear in 38 [14%] cases. The severity of inflammation was graded as 1+, 2+ and 3+ based on the visual impression of the extent of neutrophils present. Inflammation was 1+ in 126 [46.5%] cases, 2+ in 98 [36.1%] cases and 3+ in 29 [10.8%] cases. Haemorrhage was seen exclusive of inflammation in 20 [7.4%] cases.

CPS covered the entire slide while LBC smears had a monolayer of cells spread over 13 mm diameter only [Fig. 1AC]. LBC smear showed lesser debris, cell clumps and obscuring elements microscopically. The background was cleaner in LBC as compared to CPS [Fig. 1B, 2D]. Immediate liquid fixation in LBC prevented artefacts such as air-drying.

The background was analysed for the type of organisms present. Mixed flora was found in 6 [2.2%] cases, fungal Candida hyphae were reported in 2 [0.7%] cases and shift in flora was reported in 34 [12.5%] cases. The reactive changes such as atrophy and therapy associated changes were appreciated in 8 [2.9%] cases.

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Conventional PAP vs LBC.

LBC: Out of 300 cases of LBC, 14 [4.7%] were unsatisfactory, the main cause being scant cellularity of squamous cells in 11 [78.6%] out of 14 unsatisfactory cases. The diagnosis was made as Negative for intraepithelial lesion or malignancy [NILM] in 282 [98.6%] of cases. Atypical squamous cells of unknown significance [ASCUS] were reported in 2 [0.7%] cases. Low-grade squamous intra-epithelial lesion [LSIL] [Fig. 4] was reported in 1 [0.3%] case which showed the presence of koilocytes. Squamous cell carcinoma [Fig. 5] was reported in 1 [0.3%] case which showed abnormal cells, necrosis and mitosis. The background was clear in 39 [13.6%] cases. The background showed inflammation as 1+ in 110 [38.4%] cases, 2+ in 109 [38.1%] cases and 3+ in 38 [13.3%] cases. Haemorrhagic background was seen in 4 [1.4%] cases. Table 1: Comparison between CPS and LBC [N=25]. S. No. CRITERIA CATEGORISATIONS Satisfactory 1. Adequacy Unsatisfactory NILM Impression 2. Bacterial vaginosis 1+ Inflammatory 3. 2+ background 3+ 4. Haemorrhage 5. EC cells

Mixed flora was seen in 4 [1.4%] cases, Shift in flora was reported in 31 [10.8%] cases and budding yeast and Candida hyphae were seen in 1 [0.3%] case. The reactive changes such as atrophy and therapy associated changes were appreciated in 9 [3%] cases. Out of 25 cases taken for comparison, 24 [96%] cases of CPS were satisfactory while 25 [100%] of LBC cases were satisfactory. Other details are given in Table: 1.

Discussion

The importance of Pap smears in the screening of cervical cancer was reported in the studies by Manjit et al. [14] and Mulazim et al. [15] Out of 9.7% unsatisfactory cases of CPS, the main causes were scant cellularity of squamous cells in 15 [51.7%] cases and haemorrhage in 10 [34.5%] cases. CPS [N=25] 24 [96%] 1 [4%] 24 [100%] 1 [4%] 11 [46%] 6 [25%] 7 [29%] 1 [4%] 17 [71%]

LBC [N=25] 25 [100%] 0 25 [100%] 0 19 [76%] 5 [20%] 1 [4%] 0 21 [84%]

Fig. 1: Comparison between CPS and LBC. [A] CPS Slide. [B] A negative CPS [pap, x400]. [C] LBC Slide. [D] A negative LBC Smear [pap, x400].

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Fig. 2: An LBC smear showing intermediate squamous cells [Pap, x400].

Fig. 3: An LBC smear showing parabasal cells [Pap, x400].

Fig. 4: An LBC smear showing LSIL [Pap, x400].

Fig. 5: An LBC smear showing squamous cell carcinoma of the cervix [Pap, x400].

The Jyotsna et al. [16] study also noted haemorrhage, dense inflammation, drying artefacts and scant cellularity in the unsatisfactory cases of CPS. Similarly, the Gupta N et al. [17] study reported that the main cause of unsatisfactory rate in CPS was low cellularity followed by low cellularity with excess blood. The interpretation was made as NILM, AGUS, ASCUS and HSIL. No case of LSIL or carcinoma was reported probably because of the smaller sample size and limited duration of the study.

[18]

study also found increased cases of haemorrhage and inflammatory samples in CPS. Out of 4.7% unsatisfactory cases of LBC, the main cause was scant cellularity of squamous cells in 78.6% of unsatisfactory cases. The studies done by Siebers et al. [19] and Gupta N et al. [17] found scant cellularity as the sole cause for unsatisfactory LBC, similar to our study.

In CPS, inflammation was 1+ in 46.5% cases and 3+ in 10.8% cases. Haemorrhage was seen in 7.4% cases. Since the smears are directly prepared from the brush, and stained and visualised as such, the background is inflammatory and hemorrhagic in so many cases. The Singh VB et al.

In LBC, the background was clear in 39 [13.6%] cases. 1+ inflammation was found in 110 [38.4%] cases and 3+ in 38 [13.3%] cases. Haemorrhagic background was seen in 1.4% cases. The background was clearer and showed lesser inflammation due to the removal of the obscuring materials like blood and mucus during the processing of the sample. Despite this, inflammation was usually not

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missed out in LBC because as reported by other authors, neutrophils are clearly visualised on LBC although their number is reduced. [16, 20]

features were appreciated. Different cervical lesions were seen and their characters were analysed and diagnosed according to the Bethesda classification.

Mixed flora, shift in flora, budding yeast cells and Candida hyphae were reported were reported with both CPS and LBC. While the test may detect infections, this is not its primary purpose. [11] The reactive changes such as atrophy and therapy associated changes were also appreciated.

Some advantages of LBC that were identified are higher satisfactory rate, better spread of cells, cleaner background, smaller screening area and easy to review over CPS. Despite these advantages, it is felt that CPS remains as an equally effective tool for cervical screening in developing countries like India due to the significantly higher costs of LBC.

Out of 25 cases taken for comparison, 24 [96%] cases of CPS were satisfactory while 25 [100%] of LBC cases were satisfactory. Most of the other studies reported higher satisfactory rates with LBC as compared to CPS. [16, 18, 19, 21, 22, 23] The diagnosis was NILM in 24 [100%] of the satisfactory CPS cases and in 25 [100%] of the satisfactory LBC cases. No difference in interpretation of Pap lesion was reported in our comparative study. However, in studies done by Abulafia et al. [24], Obwegeser et al. [25], Vincenzo et al. [26], Davey et al. [27], an interpretation of ASCUS was more frequent with CPS, while no significant difference was reported in LSIL/HSIL detection. Our study group was small and so probably larger study group is required for a definite opinion. Bacterial vaginosis was reported in 1 [4%] case of LBC while none of the CPS smear showed any shift in flora. Similarly, in the Sherwani et al. [28] study, where only infectious organisms were taken into account, CPS detected organisms in 3.1% smears while LBC detected them in 8.7% cases. CPS showed more cases of severe inflammation whereas inflammation was appreciated as mild in most of the LBC smears. In CPS, the background showed inflammation as 1+ in 11 [45.8%] cases, 2+ in 6 [25%] cases and 3+ in 7 [29%] cases. In LBC, the background was 1+ in 19 [76%] cases, 2+ in 5 [20%] cases and 3+ in 1 [4%] case. Similarly, the study by Jyotsna et al. [16], reported 3+ inflammation more in cases of CPS [42%] than in LBC [20%] while 1+ was more in LBC [52%] than in CPS [31%]. Haemorrhagic background and RBCs were encountered more frequently in CPS [1 [4%]] whereas none of the LBC smear showed haemorrhage. More number of endocervical cells was reported in LBC, which is in accordance to the studies by Bergerone et al. [29] and Sharma J et al. [16] However, the study by Strander et al. [30] found that most LBC smears lacked endocervical cells as compared to CPS.

Conclusion

Both the techniques of CPS and LBC were performed and learnt in detail on 625 patients. Different cell types were identified and their staining and cytomorphological

Acknowledgement

Dr Indrani Dhawan, Head of the Department, Department of Pathology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi

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6. Three year report of population based cancer registries 2006-2008. New Delhi: ICMR; 2010. National Cancer Registry Programme. 7.

American College of Obstetricians and Gynecologists. ACOG Practice Bulletin No. 99: management of abnormal cervical cytology and histology. Obstet Gynecol 2008; 112:1419–1444.

Hartmann K, Hall SA, Nanda K, et al. Systematic Evidence Review Number 25: Screening for Cervical Cancer. Rockville, MD: US Department of Health and Human Services; [Accessed June 10, 2016].

Gibb RK, Martens MG. The Impact of Liquid-Based Cytology in Decreasing the Incidence of Cervical Cancer. Reviews in Obstetrics and Gynecology 2011;4[Suppl 1]:S2-S11.

10. The 1988 Bethesda System for reporting cervical/vaginal cytological diagnoses. National Cancer Institute Workshop. JAMA. 1989 Aug 18;262[7]:931-4. 11. Wilbur DC, Nayar R. Bethesda 2014: improving on a paradigm shift. Cytopathology 2015 Dec;26[6]:339-42. 12. BD PrepStain Slide Processor Operator’s Manual; Becton, Dickinson and Company; 2007. 13. Koss LG, Melamed MR. Koss Diagnostic Cytology and its Histopathologic Bases. Fifth ed: Lippincott Williams and Wilkins Publications; 2005.

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Srivastava et al. 14. Bal MS, Goyal R, Suri AK, Mohi MK. Detection of abnormal cervical cytology in Papanicolaou smears. Journal of Cytology / Indian Academy of Cytologists 2012;29[1]:45-47. 15. Bukhari MH, Saba K, Qamar S, Majeed MM, Niazi S, Naeem S. Clinicopathological importance of Papanicolaou smears for the diagnosis of premalignant and malignant lesions of the cervix. Journal of Cytology / Indian Academy of Cytologists 2012;29[1]:20-25. 16. Sharma J, Toi PCh, Siddaraju N, Sundareshan M, Habeebullah S. A comparative analysis of conventional and SurePath liquid-based cervicovaginal cytology: A study of 140 cases. J Cytol 2016;33[2]:80-4. 17. Gupta N, Bhar VS, Rajwanshi A, Suri V. Unsatisfactory rate in liquid-based cervical samples as compared to conventional smears: A study from tertiary care hospital. Cytojournal 2016 Jun 10;13:14. 18. Singh VB, Gupta N, Nijhawan R, Srinivasan R, Suri V, Rajwanshi A. Liquid-based cytology versus conventional cytology for evaluation of cervical Pap smears: experience from the first 1000 split samples. Indian J Pathol Microbiol 2015 Jan-Mar;58[1]:17-21. 19. Siebers AG, Klinkhamer PJ, Vedder JE, Arbyn M, Bulten J. Causes and relevance of unsatisfactory and satisfactory but limited smears of liquid-based compared with conventional cervical cytology. Arch Pathol Lab Med 2012 Jan;136[1]:76-83. 20. Roghaei MA, Afshar Moghaddam N, Pooladkhan SH, Roghaie SH. Adequacy criteria and cytomorphological changes in Liquid-Prep TM versus conventional cervical cytology. SEJM 2010;11:173–82. 21. Sigurdsson K. Is a liquid-based cytology more sensitive than a conventional Pap smear? Cytopathology 2013 Aug;24[4]:254-63. 22. Ronco G, Confortini M, Maccallini V, Naldoni C, Segnan N, Sideri M, Zappa M,Zorzi M, Calvia M, Giorgi Rossi

A-691 P. [Health technology assessment report. Use of liquidbased cytology for cervical cancer precursors screening]. Epidemiol Prev 2012 Sep-Oct;36[5 Suppl 2]:e1-e33. 23. Budak MŞ, Senturk MB, Kaya C, Akgol S, Bademkiran MH, Tahaoğlu AE, Yildirim A, Büyükbayram H. A comparative study of conventional and liquid-based cervical cytology. Ginekol Pol 2016;87[3]:190-3. 24. Abulafia O, Pezzullo JC, Sherer DM. Performance of ThinPrep liquid-based cervical cytology in comparison with conventionally prepared papanicolaou smears: A quantitative survey. Gynecol Oncol 2003:137–44. 25. Obwegeser JH, Brack S. Does liquid-based technology really improve detection of cervical neoplasia? A prospective, randomized trial comparing the ThinPrep Pap Test with the conventional Pap Test, including follow-up of HSIL cases. Acta Cytol 2001 Sep-Oct;45[5]:709-14. 26. Ilter E, Midi A, Haliloglu B, Celik A, Yener A, Ulu I, et al. Comparison of conventional and liquid based cytology: Do the diagnosis benefits outweigh the financial aspect? Turk J Med SCI. 2012;42:1200–6. 27. Davey E, Barratt A, Irwig L, Chan SF, Macaskill P, Mannes P, et al. Effect of study design and quality on unsatisfactory rates, cytology classifications, and accuracy in liquidbased versus conventional cervical cytology: A systemic review. Lancet 2006;367:122–32. 28. Sherwani RK, Khan T, Akhtar K, Zeba A, Siddiqui FA, Rahman K, et al. A comparative study of conventional pap smears and liquid based cytology. J Cytol 2007;24:167–72. 29. Bergeron C, Fagnani F. Performance of a new, liquid-based cervical screening technique in the clinical setting of a large French laboratory. Acta Cytol 2003;47:753–61. 30. Strander B, Andersson-Ellström A, Milsom I, Rådberg T, Ryd W. Liquid-based cytology versus conventional Papanicolaou smear in an organized screening program: A prospective randomized study.Cancer 2007;111:285–91.

*Corresponding author: Prakhar Srivastava, D-44, Dhanwantri Block, New Boys Hostel, Safdarjung Hospital, West Kidwai Nagar, New Delhi, India- 110023 Phone: +91 07042875202 Email: sriprakhar1996@gmail.com Date of Submission : 19.04.2017 Date of Acceptance : 14.08.2017 Financial or other Competing Interests: None. Date of Publication : 19.12.2017

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Original Article DOI: 10.21276/APALM.1489

Spectrum of Constitutive and Inducible Clindamycin Resistance in Staphylococcus Spp Isolated from Clinical Samples and Its Relation with Methicillin Resistance Monika Rajani1* and Malay Banerjee2 Department of Microbiology, Career Institute Of Medical Sciences & Hospital, Lucknow 2 Professor & Head, Career Institute Of Medical Sciences & Hospital, Lucknow

ABSTRACT Backgound: Increasing Staphylococcal infections with changing patterns of antimicrobial resistance has led to renewed interest in macrolidelincosamide-streptogramin group B (MLSB) antibiotic. Misuse of MLSB antibiotics has increased resistance of Staphylococcus spp. to these drugs. Failure to detect inducible clindamycin resistance leads to therapeutic failure. Determine the prevalence of constitutive and inducible clindamycin resistance in clinical isolates of Staphylococcus spp. Ascertain the relationship between methicillin-resistance in Staphylococcus spp with constitutive and inducible clindamycin resistance. Study Aimed: Study aimed to determine the prevalence of constitutive and inducible clindamycin resistance in clinical isolates of Staphylococcus spp. and ascertain the relationship between methicillin-resistance in Staphylococcus spp with constitutive andinducible clindamycin resistance. Methods: Prospective study was carried over a period of six months with 722 clinical specimens. A total of 184 Staphylococcal isolates were identified. Staphylococcal isolates were speciated as S aureus, S epidermidis and S saprophyticus. Antibiotic susceptibility testing was done by Kirby-Bauerâ&#x20AC;&#x2122;s disc diffusion method using cefoxitin, erythromycin, and clindamycin. A disc approximation test was performed for detection of inducible clindamycin resistance for all strains resistant to erythromycin but sensitive to clindamycin. Results: Out of 184 staphylococcal isolates, 128 (69.5%) S aureus and 56 (30.4%) Coagulase negative Staphylococcus spp were identified. 114 (61.9%) of the isolates were susceptible to both clindamycin and erythromycin. Overall 70 (38.04%) isolates were resistant to erythromycin. Out of these,38 (54.2%%) of strains depicted cMLSB phenotype being resistant to both erythromycin and clindamycin.20 (28.5%) isolates showed inducible clindamycin resistance while 12(17.1%) isolates indicated MS phenotype. Percentage of both constitutive and inducible clindamycin resistance was found to be higher in methicillin resistant staphylococcal isolates than methicillin sensitive isolates. Conclusions: We recommend that the clinical microbiology laboratories should test the isolates for inducible clindamycin resistance by D test, for all isolates that appear erythromycin resistant and clindamycin susceptible in vitro.

Keywords: Clindamycin Resistance, Constitutive Resistance, Macrolide-Lincosamide-Streptogramin, Methicillin-Resistance, D-Test

Introduction

Staphylococcus aureus and coagulase negative staphylococcus spp are virulent pathogens that are currently significant etiology for a variety of infectious diseases.[1] The antimicrobial resistance problem in methicillin resistant Staphylococcus aureus(MRSA) and methicillin resistant coagulase negative staphylococcus spp (MRCONS) isolates leads to high morbidity and mortality. [2] Increasing frequency of such infections and changing patterns in antimicrobial resistance have led to renewed interest in the use of macrolide-lincosamide-streptogramin group B (MLSB) antibiotics to treat such infections.[3] Clindamycin is the preferred antibiotic for the treatment of methicillin resistant strains due to its excellent pharmacokinetic properties with optimum tissue penetration and high concentration in abscess.[4]MLSB antibiotics inhibit bacterial protein synthesis by binding to the 23S rRNA of the 50S ribosomal subunitÂ .[5] However, the

misuse of MLSB antibiotics has led to increased resistance of Staphylococcusspp. to these drugs due to a variety of resistance mechanisms. Ribosomal target site modification is the most common mechanism of resistance to MLSB in staphylococci. MLSB resistance can be either constitutive (cMLSB) or inducible (iMLSB). [6] In vitro, isolates with constitutive expression are resistant to erythromycin and clindamycin due to presence of erm (erythromycin ribosome methylase) gene. In constitutive resistance, rRNA methylases are always produced unlike in inducible resistance where they are produced only in presence of an inducer.[7]Isolates with inducible resistance are resistant to erythromycin but appear falsely susceptible to clindamycin in vitro.[8] Inducible clindamycin resistance is not recognized by using standard susceptibility test methods.[9] Failure to identify iMLSB may lead to clinical failure of clindamycin therapy.[9] Apart from this, macrolide resistance can also be mediated by efflux mechanisms expressed by msr gene (MS phenotype), where only

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Rajani et al. macrolides are resistant in vitro while clindamycin remains susceptible.[10] The Clinical Laboratory Standards Institute (CLSI) has recommended the erythromycin –clindamycin disc approximation test (D-zone test) to detect the inducible clindamycin resistance.[11]The aim of this study was to determine the prevalence of constitutive and inducible clindamycin resistance in clinical isolates of Staphylococcus spp in our region. There is scarcity of data regarding this prevalence from our geographical region. This will help avoid and clinical failure and retain the usefulness of clindamycin. We also tried to ascertain the relationship between methicillin-resistance in Staphylococcus spp with constitutive and inducible clindamycin resistance.

Methodology

This prospective observational study was conducted in the department of microbiology over a period of six months from January 2016 to June 2016 on the clinical samples from inpatient and outpatient departments. A total of 184 Staphylococcal isolates from 722 clinical specimens like pus, urine, blood, sterile body fluids, sputum, peripheral and central line catheter tips, wound swabs, respiratory aspirates were included in the study. Isolates were identified as Staphylococcus spp and further speciated as S aureus, S epidermidis and S saprophyticus by standard biochemical techniques like Gram staining, catalase test, slide and tube coagulase test, growth on Mannitol Salt agar and novobiocin (5µg) disc susceptibility testing.[12]Antibiotic susceptibility testing was done on Mueller Hinton Agar (Himedia Laboratories Pvt. Ltd, India) by Kirby-Bauer’s disc diffusion method using cefoxitin (30µg), erythromycin (15µg), and clindamycin(2µg).The zones were interpreted as per CLSI 2015 guidelines.[11] Mec A mediated Oxacillin resistance was determined among the isolates using 30ug cefoxitin disc inoculated on Mueller Hinton Agar (Himedia Laboratories Pvt. Ltd, India) and incubating the plates at 33-350C in ambient air for 16-18 hrs. The zones were interpreted as follows: sensitive (S) ≥ 22 mm, and resistant (R) ≤ 21 mm to cefoxitin, for Staphylococcus aureus; S ≥ 25 mm and R ≤ 24 mm to cefoxitin, for coagulase-negative Staphylococcus spp.[11]The antibiotic discs were procured from Hi-media Laboratories Pvt. Ltd, India. Quality control (QC) was achieved with S. aureusATCC 25923 (American Type Culture Collection, Manassas, VA, USA).All isolates those were resistant to both erythromycin (zone size ≤13 mm) as well as clindamycin (zone size ≤14 mm) were labeled under cMLSB phenotype. [8]

A-693 placed 15 mm away from the edge of a 15µg erythromycin disc. The culture plates were incubated aerobically overnight at 37°C and were examined under transmitted light the following day. The different phenotypes were appreciated as follows: 1. MS (Macrolide Streptogramin) phenotype: Such isolates were termed as D test negative. 2. Inducible MLSB (iMLSB) phenotype: Such isolates were termed as D test positive. [Figure 1] Statistical analysis was done with relevant tests. This study was approved by institutional ethics committee.

Results

Out of total 722 different clinical samples received, 184 staphylococcal isolates were identified. The distribution of positive samples according to the source of sample was found to be significantly unequal (p<0.001). The maximum positive samples were seen in swabs (62.1%) followed by venous tips (53.9%). [Table 1] Out of the total positive isolates, 128 (69.5%) S aureus and 56(30.4%) coagulase negative Staphylococcus spp were identified. Among 128 S aureus isolates, 84 (65.6%) were phenotypically identified as MSSA while 44(34.3%) as MRSA. Among 56 Coagulase negative staphylococcus spp, 48 (85.7%) were phenotypically identified as S epidermidis while 8(14.2%) as S saprophyticus. Among 48 S epidermidis strains, 38 (79.1%) were phenotypically identified as MSSE, while 10 (20.8%) as MRSE. Among 8 S saprophyticus isolates, all 4 isolates were methicillin sensitive.The distribution of MSSA according to the source of sample was found to be significantly unequal (p<0.001) and same case were for MRSA, MSSE, MRSE and MSSS. [Table 1]

A disc approximation test was performed for detection of inducible clindamycin resistance for all strains that were resistant to erythromycin but sensitive to clindamycin as per CLSI 2015 guidelines. [11] A 2 µg clindamycin disc was

114 (61.9%) of the isolates were susceptible to both clindamycin and erythromycin. Overall 70(38.04%) isolates were resistant to erythromycin.No significant relationship was found between the type of isolate and Erythromycin resistance (p=0.516). Out of the total erythromycin resistant strains,38 (54.2%) of strains depicted cMLSB phenotype.20 (28.5%) isolates showed inducible clindamycin resistance while 12 (17.1%) isolates indicated MS phenotype.Statistically, the proportion of phenotype among the erythromycin resistant isolates was highly significant (p<0.001).Percentage of both constitutive and inducible clindamycin resistance was found to be higher in methicillin resistant staphylococcal isolates than methicillin sensitive isolates. However statistically no significant relationship was found between the individual isolates and type of erythromycin resistance (p=0.136). [Figure 2, 3, Table 2]

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Clindamycin Resistance in Staphylococcus Spp

Table 1: Sample source and distribution of isolates of Staphylococcus spp. Total No. of Total samples Source of sample MSSA MRSA samples positive Urine 257 16 8 0 Blood 164 44 20 16 Pus 137 60 28 16 Swabs 58 36 16 10 Sputum 39 4 2 2 Body fluids 41 10 6 0 Venous tips 26 14 4 0 Total 722 184 84 44 chi sq chi sq chi sq = 131, chi sq = 45.2, Significance = 45.0, = 80.3, p<0.001 p<0.001 p<0.001 p<0.001

MSSE

MRSE

MSSS

MRSS

0 8 12 6 0 2 10 38 chi sq = 25.7, p<0.001

0 0 4 4 0 2 0 10 chi sq = 14.6, p=0.023

8 0 0 0 0 0 0 8

0 0 0 0 0 0 0 0

MRSA = methicillin resistant S.aureus, MSSA = methicillin sensitive S.aureus, MRSE = methicillin resistant S epidermidis, MSSE = methicillin sensitive S epedermidis, MSSS= methicillin sensitive S saprophyticus, MRSS= methicillin resistant S saprophyticus

Table 2: MLSB phenotypes of Staphylococcal isolates and their relationship with methicillin resistance Isolates Overall % of isolates Erythromycin resistance (%) cMLSBType iMLSBType MSSA (84) 45.7 32(38%) 14(43.7%) 10(31.2%) MRSA (44) 23.9 16(36.3%) 10(62.5%) 6(37.5%) MSSE (38) 20.7 12(31.5%) 6(50%) 2(16.6%) MRSE (10) 5.4 6(60%) 4(66.6%) 2(33.3%) MSSS (8) 4.3 4(50%) 4(100%) 0(0) MRSS (0) 0.0 0 0 0 Total(n=184) 70 (38.04%) 38(54.2%) 20(28.5%) chi sq = 3.26, chi sq = 12.4, Significance p=0.516 p=0.136 Test of phenotype proportion : chi sq = 15.20, p<0.001

Fig. 1: The D- test showing positive result.

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MS Type 8(25%) 0(0) 4(33.3%) 0(0) 0(0) 0 12(17.1%)

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Fig. 2: Distribution of MLSB resistance phenotypes in Staphylococcal isolates.

Fig. 3: Association Between type of isolate and type of methicillin resistance.

Discussion

In addition, methicillin sensitive strains were more common both among S aureus as well as Coagulase negative staphylococcus spp.In our study 34.3% of S aureus isolates were methicillin resistant while 20.8% of Coagulase negative staphylococcus spp isolates were methicillin resistant. Various Indian studies concluded that MRSA as one of the common cause of hospital-acquired infections and different hospitals have reported a prevalence of about 30%- 80% methicillin resistance.[13,14,15]Newer antibiotics

like vancomycin, linezolid, and pristinomycin have been advocated in the management of such infections, but recent resistance trends are alarming.[16,17]It raises real concerns over how long these uniform susceptibilities will hold good. In such a scenario, clindamycin becomes a good cost effective choice. Good oral absorption makes this drug an important option in outpatient therapy and as a follow-up after intravenous therapy.[18]In recent times, clindamycin has become the drug of choice for StaphylococcalÂ skin and soft tissue infections as it directly inhibits the Staphylococcal toxin production. Clindamycin attains high concentrations in abscesses and requires no renal dose adjustments .It can be used as an alternative in penicillin-allergic patients. [18] It is effective against both methicillin resistant and the methicillin sensitive Staphylococcal isolates.[5]

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Out of the 184 Staphylococcal isolates obtained from various sites, 69.5% were identified as S aureus and 30.4% as Coagulase negative Staphylococcus spp. S aureus was the most common isolate in our study.

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In our study 38.02% of isolates were resistant to erythromycin. Our findings were supported by studies of Prabhu et al and Deotale V et al where the prevalence of erythromycin resistance was 28.4% and 32.4% respectively.[8,19]The prevalence of constitutive clindamycin resistance was found to be 54.2% in our study which was higher than what is reported in other studies.[19,20,21]Inducible clindamycin resistance was observed in 28.5.% of the isolates in our study which was in concordance with the resistance rates of 28.2% reported by Prabhu et al.[8]However lower (10.5%) and higher rates(50.6%,49%) of inducible clindamycin resistance have been reported by other authors.[8,10,22] Our observations suggest that skipping the D test will lead to nearly one third of the erythromycin resistant isolates misidentified as clindamycin sensitive resulting in therapeutic failure. Misreporting of Staphylococcal isolates susceptible to clindamycin without checking for inducible resistance may result in institution of inappropriate clindamycin therapy. Not performing D test would underestimate clindamycin resistance. On the other hand, negative result for inducible clindamycin resistance confirms clindamycin susceptibility.

B phenotypes were more in MRSA (74%) as compared to MSSA (45%).[29] This indicates that the prevalence of resistance phenotypes vary among methicillin sensitive and methicillin resistant staphylococcal isolates among different geographical regions. Induced clindamycin resistant Staphylococcal spptend to be multidrug resistant because of the overlapping binding sites of macrolides, lincosamides, and streptogramins B in 23S rRNA. This accounts for cross-resistance amongst the three classes of drugs.[30] Such a situation limits treatment options and increases the likelihood of inadequate antimicrobial therapy.

The constitutive clindamycin resistance was found to be higher in MRSA and Methicillin resistant S epidermidis (MRSE) as compared to MSSA and Methicillin sensitive S epidermidis (MSSE) (62.5% and 66.6% vs 43.7% and 50%). This is in concordance with findings by Azap et al (64% vs 4.6%), Yilmaz et al (44.2% vs 4.5%), Gupta et al (46% vs1 0%) ,and Farooq S et al (49.52% vs 8.27%) who reported a higher prevalence of c MLSB phenotype in MRSA as compared to MSSA.[21,23,24,25]Constitutive mutants can be selected in vitro in the presence of clindamycin or any other non-inducer macrolide as they are widespread among methicillin-resistant strains.[26]

Appropriate therapy decisions demands vigilance on the resistance trends. The pattern of macrolide resistance in Staphylococcus spp varies in different regions. This makes prescription patterns for clindamycin very variable. There is no substantial data regarding clindamycin prescription from India.[5,8]It is kept as a reserve drug and is usually advocated in severe in-patient MRSA infections depending upon the antimicrobial susceptibility testing. The patients attending our hospital are generally financially burdened. Thus by prescribing clindamycin, use of reserve antibiotics can be spared. However, expression of inducible resistance to clindamycin could limit its effectiveness. Inducible resistance is often missed by the clinical microbiologists in routine testing when erythromycin and clindamycin discs are placed in nonadjacent positions. So, clinical microbiology laboratories should correctly report inducible clindamycin resistance in Staphylococcus spp, and D-test can be used as a simple, cost effective and reliable method to delineate inducible and constitutive clindamycin resistance in routine clinical laboratories.

Similarly, the inducible clindamycin resistance was higher in MRSA and MRSE as compared to MSSA and MSSE (38% and 33.3% vs 31.2%% and 16.6%) .This was in concordance with few other studies. [24, 27, 20] In studies by Yilmaz et al , Braun et al and Gadepalli et al inducible resistance to clindamycin in MRSA isolates was found to be 24.4% ,30% and 30% respectively.[24,27,20] However lower prevalence of inducible resistance to clindamycin in MRSA has also been reported in literature. [23,28] High frequency (63%) of iMLS B resistance is reported among S. aureus isolates with an erythromycin-resistant and clindamycin-susceptible phenotype. Inducible MLS

We observed a high prevalence of MS phenotypes (25% and 33.3%) in our study among MSSA and MSSE isolates respectively. This could be attributed to the empirical and inappropriate use of macrolides. Macrolides are frequently used over the counter and usage for non bacterial infections leads to selection of resistant mutants. Hence it is emphasized that macrolide antibiotics should always be used with caution during empirical therapy. Also, due to the shared site of activity, MLS group of drugs can be antagonistic to each other and lincosamides should not be administered concurrently with erythromycin.

Conclusions

We recommend that the clinical microbiology laboratories should test inducible clindamycin resistance by D test for all isolates that appear erythromycin resistant and clindamycin susceptible in vitro to avoid therapeutic

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failure. This will also prevent an underestimated clindamycin resistance rate in different geographic regions. Macrolides should be used with caution as an empirical therapy. Clindamycin should not be administered concurrently with erythromycin as these drugs can be antagonistic.The prevalence of both constitutive as well as inducible clindamycin resistance is higher in methicillin resistant strains which limit the treatment options and demands prompt vigilance of resistance trends. Prevalence of macrolide resistant phenotypes should be evaluated in CA MRSA and HA-MRSA strains in our community especially in the background of high constitutive resistance when selection of resistant mutants can be fast. Our study gives a spectrum of clindamycin resistance among clinical isolates of S. aureus from this region of the country which will help clinicians choose an appropriate therapy.

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Sasirekha B, Usha MS, Amruta JA, Ankit S, Brinda N, DivyaR.Incidence of constitutive and inducible clindamycin resistance among hospital-associated Staphylococcus aureus. 3 Biotech. 2014;4(1):85-89.

Vivek JS, Rajesh GN, Mukesh S, Manpreet K, Misra RN, Matnani GB et al. The prevalence of inducible clindamycin resistance among community-and hospital-associated Staphylococcus aureus isolates in a tertiary care hospital in India. Biomedical Res 2011; 22:465-469.

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Leclercq R. Mechanisms of Resistance to Macrolides and Lincosamides: Nature of the Resistance Elements and Their Clinical Implications .Clin Infect Dis.2002: 482-492.

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Vandana KE, Singh J, Chiranjay M, Bairy I. Inducible Clindamycin Resistance in Staphylococcus aureus: Reason for Treatment Failure. J Glob Infect Dis. 2009;1(1): 76–77.

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10. Goyal R, Singh N P, Manchanda V, Mathur M. Detection of clindamycin susceptibility in macrolide resistant phenotypes of Staphylococcus aureus. Indian J Med Microbiol 2004;22:251-4. 11. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial Susceptibility testing, 17th informational supplement (M100-517). 2015, Wayne Pa: Clinical and Laboratory Standards Institute. 12. Baird D. Staphylococcus:Cluster forming Gram positive cocci. In: Mackie & McCartney, Practical Medical Microbiology. 14th edition. pp245-261. 13. Blomquist PH. Methicilli resistant staphylococcus aureus infections of the eye and orbit (an american ophthalmological society thesis). Trans Am Ophthalmol Soc. Dec; 104: 322–345. 14. Pramodhini S., Thenmozhivalli PR, Selvi R., Dillirani V., Vasumathi A., Agatha D. comparison of various phenotypic methods and mec A based PCR for the detection of MRSA. Journal of Clinical and Diagnostic Research , 2011; 5:1359-1362. 15. Brumfitt W, Hamilton-Miller J. Methicillin-resistant Staphylococcus aureus. N. Engl. J. Med. 1989; 320:1188-1196. 16. Tarai B, Das P, Kumar D. Recurrent Challenges for Clinicians: Emergence of Methicillin-Resistant Staphylococcus aureus, Vancomycin Resistance, and Current Treatment Options. J Lab Physicians. 2013 Jul-Dec; 5(2): 71–78. 17. Kaur DC, Chate SS. Study of Antibiotic Resistance Pattern in Methicillin Resistant Staphylococcus Aureus with Special Reference to Newer Antibiotics. J Glob Infect Dis. 2015 Apr-Jun; 7(2): 78–84. 18. Rayner C, Munckhof WJ. Antibiotics currently used in the treatment of infections caused by Staphylococcus aureus. . Intern Med J. 2005;35Suppl 2:S3-16. 19. Deotale V, Mendiratta DK, Raut U, Narang P. Inducible clindamycin resistance in Staphylococcus aureus isolated from clinical samples. Ind J Med Microbiol. 2010;28:124-6. 20. Gadepalli R, Dhawan B, Mohantay S, Kapil A, Das BK, Chaudhary R. Inducible clindamycin resistance in clinical isolates of Staphylococcus aureus. Indian J Med Res. 2006;123:571–73. 21. Gupta V, Datta P, Rani H, Chander J. Inducible clindamycin resistance in Staphylococcus aureus: A study from north india. J Postgrad Med. 2009;55:176–79. 22. Ajantha GS, Kulkarni RD, Shetty J, Shubhada C, Jain P. Phenotypic detection of inducible clindamycin resistance among Staphylococcus aureus isolates by using the lower limit of recommended inter-disk distance. Indian J PatholMicrobiol 2008;51:376-8. 23. Azap OK, Arslan H, Timurkaynak F, Yapar G, Oruc E, Gagir U. Incidence of inducible clindamycin resistance in erythromycin-resistant isolates of Staphylococcus aureus. J ClinMicrobiol. 2005;43:1716–24.

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24. Yilmaz G, Aydin K, Iskender S, Calyan R, Koksal I. Detection and prevalence of inducible clindamycin resitance in staphylococci. J med Microbiol. 2007;56:342–45. 25. Farooq S, Saleem M. Prevalence of constitutive and inducible clindamycin resistance among clinical isolates of staph aureus in Kashmir valley: a hospital based study. J. Evolution Med. Dent. Sci. 2016; 5(17):828-831. 26. Ciraj A M, Vinod P, Sreejith G, Rajani K. Inducible clindamycin resistance among clinical isolates of staphylococci. Indian J PatholMicrobiol 2009;52:49-51. 27. Braun L, Craft D, Williams R, Tuamokumo F, Ottolini M. Increasing clindamycin resistance among methicillin resistant Staphylococcus aureus in 57 northeast United

States military treatment facilities. Pediatr Infect Dis J 2005;24:622-6. 28. Fokas, S. Fokas, M. Tsironi, M. Kalkani, M. Dionysopouloy. Prevalence of inducible clindamycin resistance in macrolideresistant Staphylococcus spp.ClinMicrobiol Infect, 11 (2005), pp. 337–340 29. Ajantha GS, Kulkarni RD, Shetty J, Shubhada C, Jain P. Phenotypic detection of inducible clindamycin resistance amongst Staphylococcus aureus isolates by using lower limit of recommended inter-disk distance. Indian J PatholMicrobiol. 2008;51:376–8. 30. Parasa LS, Tumati SR. Prevalence of induced clindamycin resistance in methicillin resistant Staphylococcus aureus from hospital population of coastal Andharapradesh, Southindia. Archives of clinical microbiology. 2011;2( 1).

*Corresponding author: Dr. Monika Rajani, 19/789 Indira Nagar, Lucknow (India) 226016. Phone: +91 07755091159 Email: drmrajani@rediffmail.com

Financial or other Competing Interests: None.

Date of Submission : 21.04.2017 Date of Acceptance : 29.08.2017 Date of Publication : 19.12.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Original Article DOI: 10.21276/APALM.1496

Impact of Histopathological Examination of Appendix in Context to Clinical Management of Patients Mandakini M Patel and Rhuta J Shah* Dept. of Pathology, Govt. Medical College, Surat, India

ABSTRACT Background: Acute appendicitis is one of the common conditions requiring emergency surgery. In routine practice , appendix is sent for histolopathological examination only when the operative findings are inconclusive. In view of this trend in clinical practice this study was carried out to assess the value of routine histopathological examination of surgically resected appendices, to review spectrum of histopathological diagnosis of appendectomy done at our institute over a 22 months period, some of which would further have effect on patient management and prognosis. Methods: A retrospective study was done including appendicectomy specimens received at histopathology section of Department of Pathology at Tertiary care centre in South Gujarat during January 2015 till October 2016 . Result: Total 400 cases were reviewed. Out of these, clinically suspected appendicitis was found in 365 (91.3 %) cases including spectrum of appendicitis (acute, subacute, chronic, ulcerative, suppurative, necrotizing, gangrenous, acute with periappendicits, acute on chronic). Unusual unexpected findings were found in 20 (5 %) cases[tuberculosis, amoebiasis, faecolith, congestion with sickle RBCs, mucocele and neoplastic lesions including carcinoid, adenocarcinoma, mucinous cystadenoma (low grade appendiceal mucinous neoplasm)]. Conclusion: Though majority of cases had the usual features, 20 of these 400 specimen (5 %) had an impact on patient management or outcome. They were not suspected on macroscopic examination at the time of surgery and would have been missed had the specimens not been examined microscopically. Intraoperative diagnosis of surgeon is therefore unreliable in detecting abnormalities of appendix. This study supports the sending of all appendicectomy specimens for routine histopathological examination. Keywords: Appendicitis, Unusual Findings, Value

Introduction

Acute appendicitis is one of the common conditions requiring emergency surgery.[1] The practice of sending all appendix specimens for routine histopathological examination depends on the concerned clinician and is variable. Matthyssens et al are against this policy and suggest that appendices should be sent for examination only if there is an obvious macroscopic abnormality at surgery [2,3]. They justify their opinion by the rarity of aberrant findings, together with the significant costs of specimen processing. However, a number of other papers have found such aberrant incidental findings to be more common, and suggest that failure to histopathologically examine all appendices would lead to many significant pathologies being missed and cause an impact on patient management [2,4-6]. There is no authoritative data or previous studies which deal with this issue in India. Hence, a query still remains whether this policy should be adhered to in poor resource countries like India also. Keeping in mind the above facts, we undertook this study to shed some light

on this topic by evaluating the current scenario concerning this issue in India. Histopathological examination still remains the gold standard method for the confirmation of the appendicitis. Not only the pathologic diagnosis of acute inflammation, at times unusual findings such as incidental tumours noted in the appendix highlights the importance of the pathologic analyses of every single resected appendix. This study aims to determine the various histologic diagnoses of all surgically removed appendices and to find out the age and sex related incidence of appendicitis, rate of negative appendicectomies and unusual findings which would have effect on patient management and care[1]

Aims & Objectives

(1) To study spectrum of histopathological lesions in appendicectomy specimens. (2) To analyze the proportion of various lesions, age and sex distribution in resected specimens of appendix. (3) To find out proportion of unusual findings in appendicectomy specimen , some of which would have effect on patient management and prognosis.

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Materials and Methods

A retrospective study was done including appendicectomy specimens received at histopathology section of Department of Pathology at Tertiary Care Center in South Gujarat during 22 months period from January 2015 till October 2016. Total 400 appendicectomy specimens were included in study. Inclusion Criteria: All emergency appendectomies and interval appendectomies performed on clinically suspected appendicitis were included. Exclusion criteria: Incidental appendectomies which were performed during other abdominal or pelvic surgeries were excluded . Relevant clinical data, intraoperative findings and gross findings were noted . Appendicectomy specimens are prepared according to a hospital-defined protocol, involving immediate fixing in formalin prior to transport to the pathology laboratory. Specimens are sectioned at the tip, body and base, processed, slides stained with hematoxylin-eosin and were examined by a consultant or senior pathologist. Data were analysed by Ms Excel and SPSS software. Negative appendectomy was defined as one which is performed for a clinical diagnosis of acute appendicitis but in which the appendix is found to be normal on histopathological examination. The analysis focused on the confirmation of acute appendicitis, incidental unexpected incidental findings other than inflammation, whether these abnormalities were suspected on gross examination at the time of surgery, and the effect on patient management and prognosis.[7]

Result

Total of 400 specimens of appendix were received in the histopathology department during the period of 22 months from January 2015 to October 2016. There were 284 (71%) males and 116 (29%) females among 400 cases of appendicitis with the male: female ratio of 2.4 : 1. Overall, a greater number of appendectomies (71%) were performed in males than in females (29%).

The peak age incidence of appendicitis was found in the age group of 21 to 30 years. More than 80% cases of appendicitis occurred below the age of 40 years. The youngest patient was two years old and the oldest was seventy-five years of age. Many patients presented with multiple and overlapping clinical symptoms. The most common symptom was pain in abdomen seen in 395(98.5 %) followed by fever 225(56.2%) and vomiting 118(29.6%). Patients presenting with intestinal obstruction were 3(0.75%) patients and perforation were 9(2.25 %). As shown in table 1, out of total 400 cases reviewed ,clinically suspected appendicitis was proven histologically in 365 (91.3 %) cases including spectrum of appendicitis (acute, subacute, chronic, ulcerative, suppurative, necrotizing, gangrenous, acute with periappendicits, acute on chronic). Unusual unexpected findings were found in 20 (5 %) cases ( tuberculosis, amoebiasis [figure 1], faecolith, congestion with sickle RBCs, mucocele and neoplastic lesions including carcinoid, adenocarcinoma, mucinous cystadenoma(low grade appendiceal mucinous neoplasm)[figure 2]). As shown in table 2, total 20(5%) cases out of 400 were found to have aberrant/ unusual findings. These include 4 cases of Tuberculosis,3 cases showed trophozoite forms of Amoebiasis. Faecolith was found in 3 cases, retention mucocele in 3 cases. One appendix showed congestion with sickle RBCs. Total 6 (1.5 %) out of 400 turned out to be neoplastic which include 4 cases of Mucinous cystadenoma (low grade appendiceal mucinous neoplasm), 1 case of Carcinoid tumour and 1 case of Adenocarcinoma. Negative appendectomy was defined as one which is performed for a clinical diagnosis of acute appendicitis but in which the appendix is found to be normal on histopathological examination.11 cases out of 400 in our study accounted for negative appendicectomy. This implies that appendicitis was not cause of acute abdomen

Table 1: Histologic findings of appendicectomy specimen. Diagnosis

No of cases

% of cases

Acute appendicitis

142

35.5 %

Acute Ulcerative appendicitis

131

32.7 %

Subacute appendicitis

9.5 %

Acute necrotizing appendicitis

Acute appendicitis with periappendicitis

2.5 %

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Diagnosis

No of cases

% of cases

Acute suppurative appendicitis

2.2 %

Acute on chronic appendicitis

1.7 %

Gangrenous appendicitis

1.2 %

Chronic appendicitis

1.7 %

NO Appendix

NAD

2.7 %

UNUSUAL FINDINGS

Table 2: Unusual findings in appendicular specimen. Unusual findings

No of cases

% of cases

Tuberculosis

Amoebiasis

0.75 %

Congestion with sickle RBC

0.25 %

Faecolith

0.75 %

Retention Mucocele

0.75 %

NEOPLASTIC

1.5 %

Carcinoid tumour

Adenocarcinoma

Mucinous Cystadenoma (Low grade appendiceal mucinous neoplasm)

Table 3: Comparison of histopathological findings. Histopathological findings Inflammatory lesions UNUSUAL FINDINGS

Present study

Divya R et al (2016, Aligarh, India)

Hanish Chavda(2015, Bagalkot, Karnataka, India)

Alun Jones(2007, England)

365 (91.3 %)

300 (92.3 %)

94.6 %

77 %

20 ( 5 %)

8 (2.5 %)

4.6 %

3.75 %

Fig. 1: Trophozoites of Entamoeaba histolytica in appendiceal lumen (H&E,40x)

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Fig. 2: Mucinous cystadenoma (H&E , 10x).

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Discussion

The current study is a two year retrospective study and presents the data on histopathological analyses of 400 appendectomy specimens received in the Department of Pathology at Tertiary Care centre in South Gujarat. The histopathological examination of the appendix serves two purposes. First it allows the diagnosis of acute appendicitis to be confirmed; Second, histopathological examination may disclose additional pathologies that may not be evident intraoperatively which may impact patient management.[8] In our study, age incidence of appendicitis was higher in 20-30 yrs age group. Similar results were seen in study done on 325 cases at Jawaharlal Nehru Medical College by Divya Rabindranath[2] in Aligarh, India. Our result are concordant with study done on 930 cases by Shrestha R et al[1] in bharatpur, Nepal and by Mohsin-ul-Rasool[8] on 440 cases in Srinagar, India. In our study , maximum number of patients presenting with complaints of suspected Acute appendicitis were males(71 %). Similar findings were observed in study done by Divya Rabindranath[2] in Aligarh, India(58.7 %), Mohsin-ul-Rasool [8]on 440 cases in Srinagar, India(68.2 %) and by Hanish Chavda[9] in Bagalkot, Karnataka(60.95 %) . In our study most common presenting complaint was abdominal pain.This finding was concordant with study done by Mohsin-ul-Rasool[8] (Srinagar, India) and Edino et al, Nigeria[10]. Negative appendectomy was defined as one which is performed for a clinical diagnosis of acute appendicitis but in which the appendix is found to be normal on histopathological examination. In our study , rate of negative appendicectomy was 2.7 %. In study done by Mohsin-ul-Rasool[8] (2014, Srinagar, India) reported rate for negative appendicectomy was 5.7 % and 10.8 % in study done by Shretha R et al[1] (2012, Bhartapur, Nepal). Negative appendicectomy implies that appendicitis was not cause of acute abdomen and other investigations need to be performed if symptoms persist . In our study , clinically suspected appendicitis appendicitis were histologically correlated in 365 (91.3 %) cases as shown in table 3.This findings were concordant with those of Divya R et.al(92.3 %) [2], Hanish Chavda (94.6 %) [9] and Mohsin-ul-Rasool(77 %) [8](Table 3)These include spectrum of inflammatory lesions of appendix , of which acute appendicitis(35.5 %) and acute ulcerative appendicitis(32.7 %), constitute majority of cases. Other in spectrum, constitute minority of proportion including acute suppurative, necrotizing, with periappendicitis, gangrenous and chronic appendicitis.

As shown in table 3, in our study aberrant/unusual findings were found in 20 (5 %) of cases. Divya R et.al[2] found such unusual histological features in 2.5 % of cases. In study done by Hanish Chavda [9], unexpected histological features were found in 4.6 % and 3.75 % in Alun Jones [7]. Results are concordant Another important incidental diagnosis in our study was granulomatous appendicitis which was reported as Tuberculosis due to confirmation on Ziehl-Neelsen staining. Incidence of this rare condition has been reported as 0.14% to 0.3% in Western countries and as 1.3% to 2.3% in underdeveloped countries [7,11,]. It can be caused by various infectious and noninfectious factors. Systemic conditions, such as Crohnâ&#x20AC;&#x2122;s disease and sarcoidosis, may be associated with granulomatous inflammation of the appendix. However, infectious causes like Mycobacterium tuberculosis, Yersinia spp, blastomycosis, Schistosoma spp, Actinomyces spp, Campylobacter spp, and Histoplasma capsulatum form a much more important cause in our country [7, 12]. Since Tuberculosis is endemic in our country, our case was also suspected to suffer from intestinal tuberculosis. Patient was subsequently investigated for the same and our suspicions were found to be correct(positive Ziehl-Neelsen staining). The reported incidence of appendicular Tuberculosis varies from 0.1% to 3.0% among all appendectomies performed. An accurate diagnosis is usually established only after histopathological examination of a specimen. Some studies report that no further treatment after appendectomy is necessary for primary appendicular disease. In contrast, Jones et al described a case of appendicular Tuberculosis in their study who subsequently underwent right hemicolectomy for treatment. Hence, no consensus has been reached yet about the treatment of appendicular Tuberculosis [7,11]. Acute appendicitis may be the mode of presentation of appendix neoplasms particularly adenocarcinoma [4] as was also seen in our study. One case that was suspected to be acute appendicitis was finally revealed to have adenocarcinoma on histopathological examination. patient with adenocarcinoma should undergo subsequent right hemicolectomy Carcinoids are the most common tumors of appendix and are typically small, firm, circumscribed yellow brown lesions. An appendiceal carcinoid tumor is found in 0.3%2.27% of patients undergoing an appendectomy [14].in our study, we had one case of carcinoid. It has been suggested that carcinoid tumors may present as appendicitis because of luminal obstruction or elevated levels of 5

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hydroxy tryptamine, histamine and kinin as these are all potent mediators of inflammation [13]. Characteristics of appendiceal carcinoids predicting aggressive behavior include tumor size, histological subtype, and mesoappendiceal involvement. Carcinoid tumors are smaller than 1 cm in 70%-95% of cases [14], including ours. Such small tumors are easily missed on gross examination intraoperatively. The calculated risk of metastasis from tumors 1 cm or smaller are reported to be nearly zero and therefore may be managed with a simple appendectomy. An increase in metastasis risk of up to 85% occurs with a tumor double the size or larger. An appendiceal carcinoid tumor larger than 2 cm should be managed with a formal right hemicolectomy [14, 15 ] . Since our particular case was about 1 cm in size, no further management was needed.. Our study also included three cases of mucocele. A mucocele of the appendix denotes an obstructive dilatation of the appendiceal lumen due to abnormal accumulation of mucus, which may be caused either by a retention cyst, endometriosis, mucosal hyperplasia, cystadenoma, or a cystadenocarcinoma. The incidence of mucocele has been reported to range from 0.2% to 0.3% of all appendectomy specimens. We have seen 3 cases of retention mucocele in our study Mucoceles are often asymptomatic and discovered only as incidental findings at appendicectomy, or during laparotomy for another indication or at histological examination of an operative specimen. However, they may also be diagnosed clinically from features of acute appendicitis. Confirmative diagnosis of mucocele and its cause is possible only after histopathology. Appendectomy is the treatment of choice for mucinous cystadenoma, whereas a cystadenocarcinoma requires a right hemicolectomy. Because of the high association of mucinous cystadenoma with colon and ovarian malignancy, follow-up Computed tomography, Ultrasonography, and colonoscopy examinations must be performed during the postoperative period [14]. Hence, our study demonstrated that histopathological examination led to the incidental diagnosis of many important lesions that would have been otherwise missed by the surgeon. These diagnoses led to significant effect on patient management. These included conditions like Low grade Appendiceal Mucinous neoplasm (Mucinous cystadenoma) and mucinous cystadeno carcinoma which have a high risk of association with other neoplasms. Hence, their diagnosis is imperative for adequate patient management. Also, few conditions can be first diagnosed in appendix only, like granulomatous appendicitis (due to tuberculosis or crohn`s disease), amoebiasis, cancers www.pacificejournals.com/apalm

like carcinoid and adenocarcinoma .Thus, such incidental detection can lead to early treatment of these conditions. It becomes obvious from the above discussion that it is highly beneficial to send all appendectomy specimens for histopathological examination. When we weigh the cost of the procedure against the possible benefits, it becomes clear that the benefit far outweighs the cost in this situation. Early diagnosis and treatment of a lesion would prevent the added costs the patient would have to bear if the diagnosis was late and the disease had spread to other organs.

Conclusion

Despite of advances in technology and imaging modalities there is dilemma in the clinical diagnosis of acute appendicitis. Histopathological examination still remains the gold standard method for the confirmation of the appendicitis. The histopathological examination of the appendix serves two purposes. First, it allows the diagnosis of acute appendicitis to be confirmed, especially where this is not evident intra-operatively. Second, histopathological examination may disclose additional pathologies that may not be evident on gross examination intra-operatively but may affect subsequent clinical management of the patient.

Implication of Research

Abberant/unusual findings discovered at Histopathological analysis were not suspected on macroscopic examination at the time of surgery and had an impact on patient management and outcome. These would have been missed had the specimens not been examined microscopically. The intraoperative diagnosis of the surgeon is therefore unreliable in detecting abnormalities of the appendix. This study supports the sending of all appendicectomy specimen for routine histopathological examination and meticulous examination of all of those.

Acknowledgements

We acknowledge support of our departmental head and technical assistance of staff concerned with histopathology processing unit.

Reference 1.

Shrestha R, Ranabhat SR, Tiwari M: Histopathologic analysis of appendectomy specimens. Journal of Pathology of Nepal. 2012:2;215 -219

Rabindranath D, Khan AA, Ansari H, Senthil P. Unusual incidental findings of routine histopathological examination of appendectomy specimens- a 2-year retrospective analysis with review of the literature. Int J of Allied Med Sci and Clin Res 2016; 4(1):90-98.

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Matthyssens LE, Ziol M, Barrat C, Champault GG: Routine Surgical Pathology in General Surgery. Br J Surg 2006; 93:362-368

A 5 Year Study. Scholars Journal of Applied Medical Sciences. 2014; 2(1B):176-180

Chan W, Fu KH: Value of Routine Histopathological examination of appendices in Hong Kong. J Clin Pathol 1987; 40:429-433.

Duzgun AP, Moran M, Uzun S, Ozmen M, Ozer VM, Seckin S, Coskun F: Unusual findings in appendicectomy specimens: Evaluation of 2458 cases and review of the literature. Indian J Surg 2004; 66(4):221-226.

Nemeth L, Reen DJ, O’Briain D, McDermott M, Pui P: Evidence of an Inflammatory Pathologic Condition in “Normal” Appendices following Emergency Appendectomy. Arch Path Lab Med 2001; 125(6):759-764. Jones AE, Phillips AW, Jarvis JR, Sargen K. The value of routine histopathological examination of appendicectomy specimens, Bio Medical Central surgery 2007;7:17 Sharma S, Mahajan D, Mohsin-ul-Rasool, Bashir S, Hafiz A, Wajahat M. Histopathology of Appendicectomy Specimen:

Chawda HK, Miskin AT, Dombale VD. Spectrum of histopathological lesion in surgically removed appendix, Journal of Drug Discovery and Therapeutics Available. 2015:3;53-56

10. Edino ST, Mohammed AZ, Ochicha O, Anumah M. Appendicitis in Kano, Nigeria: A 5 year review of pattern, morbidity and mortality, Ann Afr Med 2004;3:38-41.. 11. Gaffar AB. Granulomatous diseases and granulomas of the appendix. Int J Surg Pathol2010; 18: 14-2 12. Tucker ON, Healy V, Jeffers M, Keane FB. Granulomatous appendicitis. Surgeon2003;1(5):286–289 13. Cortina R, McCormick J, Kolm P, Perry RR. Management and prognosis of adenocarcinoma of the appendix. Dis Colon Rectum 1995; 38: 848-52 14. Sieren LM, Collins JN, Weireter LJ, Britt RC, Reed SF, Novosel TJ, Britt LD. The incidence of benign and malignant neoplasia presenting as acute appendicitis. Am Surg 2010; 76: 808-811

*Corresponding author: Dr. Rhuta J Shah, Navkar, Opp. Deep Kiran Appt. Bhagyoday Soc. Chala, Vapi-396191, dist-Valsad, Gujrat(India) Phone: +91 09428159081 Email: dr.rhutashah@gmail.com Date of Submission : 23.04.2017 Date of Acceptance : 09.09.2017 Financial or other Competing Interests: None. Date of Publication : 19.12.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Original Article DOI: 10.21276/APALM.1326

Histopathological spectrum of Adult Nephrotic Syndrome over 16 years at a Tertiary Care Center in Mumbai with Clinicopathological, Electron Microscopy and Immunoflurescence Correlation of Renal Biopsies Ganesh Ramdas Kshirsagar*, Nitin Maheswar Gadgil, Sangeeta Ramulu Margam, Chetan Sudhakar Chaudhari, Prashant Vijay Kumavat and Sheela Jayawant Pagare Department Of Pathology, Lokmanya Tilak Municipal Medical College and General Hospital, Sion , Mumbai (Maharashtra), India.

ABSTRACT Background: The pattern of diseases causing adult nephrotic syndrome varies globally as well as in India. The aim of our study was to analyze the spectrum of patients with biopsy proven nephrotic syndrome in adults over 15 years, in respect with incidence, age distribution and correlate the clinicopathological features, electron microscopy and immunofluorescence. Methods: We have evaluated and analyzed retrospectively 263 renal biopsies of adult nephrotic syndrome over a consecutive period of 16 years (January 2000 to December 2015) in our tertiary care Hospital. Result: In our study of 235 (89.35%) adequate renal biopsy cases overall male predominance was seen (M: F ratio 1.7:1) with maximum males noted in diabetic nephropathy (M: F ratio 4:1) while SLE was seen exclusively in female (M: F ratio 0:6). Minimal change disease (26.38%), followed by MPGN (16.17%) and FSGS (15.74%) were the common histopathological lesions. In 15-45 years age majority of 78.72% cases were observed with prominently histomorphological pattern as MCD( 25.10%),followed by FSGS ( 13.61%) & MPGN (13.19%). In 45-85 years age , 21.28% cases majority were of membranous glomerulonephritis (5.10%) and diabetic nephropathy (4.25%). Primary glomerular diseases accounted for 78.3% cases commonest was MCD (26.38%) and secondary glomerular diseases in 21.7% of cases, most common being amyloidosis (7.23%) Light microscopy, immunopathology findings correlated with electron microscopy findings in 79 cases (91.86%) out of 86 cases. Sample error was main reason of non correlation of EM & LM diagnosis, especially in FSGS. Conclusion: This data analysis is essential to study the prevalence of biopsy proven renal diseases and its variation and distribution as per age .Which can improve the understanding of utility of renal biopsy for future research of renal parenchymal diseases in adults. Keywords: Nephrotic Syndrome, Renal Biopsy, Minimal Change Disease, Glomerulonephritis, Electron Microscopy.

Introduction

Richard Bright (1827) correlated for the first time the frequent occurrence of renal lesion in patient with dropsy and albuminous urine,[1] which was called Brightâ&#x20AC;&#x2122;s disease. Nephrotic syndrome is characterized as heavy proteinuria, hypoalbuminemia, hypercholesterolemia (serum cholesterol >200 mg), edema and hypertension. Quantitative estimation of 24 hour urinary proteins is usually > 3.5 gms /1.73 m2 of body surface area or > 50 mg/kg/day. Proteinuria less than this range, but associated with serum albumin < 3.0 g/dl was also classified as nephrotic range. [2] Most common causes and conditions associated with nephrotic syndrome were minimal change disease(MCD), focal segmental glomerulosclerosis (FSGS), membranous glomerulonephritis (MGN), membranoproliferative glomerulonephritis (MPGN), diabetic nephropathy (DN),

amyloid nephropathy, Systemic lupus erythematosis(SLE) in advanced stage & IgA nephritis. MCD, also known as nil disease is more common in children also seen in adult and only 60% cases respond well to steroids and more likely to relapse. Diabetes is common cause of renal failure and nephrotic syndrome in adults. Clinical features of nephrotic syndrome were periorbital and lower limb edema, ascitis, pleural effusion, hypoalbuminemia, tiredness, breathlessness, fluid overload, acute renal failure, pulmonary thromboembolism & dyslipidemia. Complications of nephrotic syndrome were hypercoagubility, atherosclerosis, renal vein thrombosis and high susceptibility for infections. Renal biopsy is useful for identifying the specific diagnosis, assessing the level of disease activity, and for allowing specific decisions about treatment to be made.[3] Definitive diagnosis of nephrotic syndrome can be done on histopathological

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examination EM & IF is essential for correct diagnosis. Glomerular diseases in tropical countries is vastly different in epidemiology, etiology and natural history from those seen in temperate countries; and their prevalence also varies according to socio-economic conditions, race, age and indications for renal biopsy.[4] Over the last few years, studies have shown a changing pattern of these diseases. The present study was conducted to know the histopathological spectrum of nephrotic syndrome in adults, in relation to the incidence and distribution in various adult age group, gender with clinicopathological, electron microscopy and immunoflurescence correlation at our institute during a 16 years period to ascertain any changes in the spectrum if any of these diseases.

Materials and Methods

All adults between 15 years and 85 years of age with nephrotic syndrome undergone renal biopsy over the 16 years period from, January 2000 to December 2015 were included in this retrospective study. Blood samples were checked for hemoglobin, platelet count, ESR, serum creatinine, blood urea, lipid profile and 24 hour urine protein for all patients. The percutaneous biopsy was done under continuous monitoring (real time USG procedure). At the time of biopsy whenever possible two cores were obtained for electron microscopic examination and immunofluorescence using antibodies for IgG, IgA, IgM and C3. None of the patients had a previous biopsy and as per the protocol of the treating adult nephrologists none underwent a repeat biopsy. Biopsies were processed and stained with routine hematoxyline and eosin stain, special stains like periodic acid schiff (PAS) and silver impregnation were done whenever required. On light microscopy the lesions were classified as adequate if five complete glomeruli were seen. However the changes, if diagnostic and pathognomic of the lesion were seen even in one glomerulus then, the biopsy was termed adequate in spite of not fulfilling the above criterion. Cases were classified on histomorphology into various groups like MCD, FSGS, MGN, MPGN, diabetic nephropathy, amyloid nephropathy, IgA nephropathy, SLE etc. Electron microscopy (EM) and Immuonofluorescence (IF) findings were correlated with histopathology. All data was entered on Microsoft excel sheet and analyzed by using descriptive statistic.

Result

Total 634 biopsies were received in our institute over the study period of 16 years from January 2000 to December 2015. Out of which 263 (41.48 %) biopsies performed were from more than 15 years adult patients with nephrotic syndrome. On light microscopy, total 235 (89.35 %) renal

biopsies were adequate. The ratio of male (148 patients) to female (87 patients) was 1.7:1, highest male predominance was noted in diabetic nephropathy which was 4:1. While nephrotic syndrome associated SLE was noted exclusively in female in our study. Clinically, all the patients had edema, 26% were hypertensive while 36 % had oliguria. Microscopic hematuria was observed in 23 patients (9.78%). Average 24 hr urine protein excretion was 5.8 g, serum creatinine was 2.15 mg/dl, while one patient was positive for hepatitis B surface antigen.

Spectrum of Glomerular Lesions

Primary glomerular diseases accounted for 184(78.3%) cases, the commonest histomorphological pattern was MCD 62 (26.38%) cases followed by MPGN(38 cases) & FSGS(37 cases) and secondary glomerular diseases for 51(21.7%) of cases most common in amyloidosis 17(7.23%) cases followed by diabetic nephropathy in 15 cases( 6.38% ). Nine cases of nephrotic syndrome were in end stage renal disease at the time of diagnosis[Table 1]. In age group of 15-25 years minimal change disease followed by focal segmental glomerulosclerosis and membranoproliferative glomerulonephritis were common. Diabetic nephropathy and membranous glomerulonephritis were predominantly seen in late adulthood [Table 1]. Electron microscopy was available in 86 cases (36.6%) out of 235 cases. Ten (9%) biopsies were inconclusive and 15 (13.5%) biopsies were inadequate. High rate of positive correlation with light microscopic findings was observed with MCD, MPGN, MGN, Amyloidosis, and SLE. In FSGS, diabetic nephropathy, IgA nephropathy correlation was not observed in 33% cases [Table 2]. Light microscopy findings correlated with electron microscopy findings in 79 cases (91.86%) out of 86 cases where electron microscopy was available. However, discrepancy was seen in 11 cases (12.79%) out of 86 cases. Final diagnosis was given based on light microscopy, immunofluorescence, electron microscopy and serological findings. Poor outcome of diabetic nephropathy, focal segmental glomerulosclerosis and amyloidosis was noted while outcome of minimal change disease was good in our study.

Discussion

This work reports on a 16 years of retrospective analysis of 634 cases of adult renal biopsies in a single tertiary care referral institute in Mumbai. Out of which 235 cases (89.35%) were adequate biopsies. Age more than 15 years is an indication for biopsy; hence all cases were subjected to biopsy. Of all the renal biopsies done over this period, nephrotic syndrome was the most frequent indication for renal biopsy accounting for 263 cases (41.48%) in adultâ&#x20AC;&#x2122;s age group [Table 1]. Sabir S et al [5] from their study from a

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Kshirsagar et al. tertiary care naval hospital in Karachi, Pakistan found that the most common indication of renal biopsy was nephrotic syndrome (43.3%) and Primary glomerulonephritides (76.6%) were predominant overall lesions. N Balkrishnan et al, [6] observed nephrotic syndrome in 65.40% of cases of various clinical syndromes. This is similar to that reported in various studies around the world, including India and Pakistan. [ 6, 7] Age and gender wise distribution: As age advances from 15 years to 85 years incidence of nephrotic syndrome decreased sequentially in recent study [Table 2]. In our study overall male predominance was seen with male to female ratio of 1.7: 1. Highest male predominance (4:1) was seen in diabetic nephropathy followed by Amyloidosis (2.4:1) & in membranous glomerulonephritis (2.1:1). While systemic lupus nephritis was seen exclusively in female (0:6, 100%) in our study [Table 3].This reflects the increased prevalence of SLE in females. All recently published studies worldwide showed a similar pattern. [ 6, 7] A.R.Reshi et al, [7] also observed male predominance and N Balkrishnan et al, observed marked female predominance in SLE similar to our study. In our study, In 15-45 years of age group 185 out 235 (78.72%) cases were seen.MCD was the most common glomerular legion in patients less than 45 years of age while MGN was most common in patients greater than 45 years [Figure 2]. N Balkrishnan et al, observed that majority minimal change disease, membranoproliferative glomerulonephritis in age group 15-34 years similar to our study. Histopathological distribution of glomerular lesions: Primary glomerular diseases was the most prominent renal disease in our study accounted for 78.3% cases of nephrotic syndrome as well as in all other studies,[ 8 , 9, 10 ,11] while amyloidosis was the most common secondary cause. The underlying etiology of nephrotic syndrome is variable all over the world. In our study, the most commonest cause was minimal change disease (62 cases, 26.38%), followed by membranoproliferative glomerulonephritis (38 cases, 16.17%) and focal segmental glomerulonephritis (37 cases, 15.74%) were the commonest histological type [ Table 2]. Reshi A.R. et al , Agarwal S.K.et al, Dash S. C. et al ,Chang Jae Hyun et al [12] observed that minimal change disease was 33.52%, 37% & 15.5% respectively was the most common histological type of nephrotic syndrome in their studies similar to our study.

A-707 and C3 negative. Electron microscopy was available in 31 cases, which revealed flattening and fusion of the visceral epithelial cells (Figure 1B). In membranoproliferative glomerulonephritis, increase in mesangial cellularity and matrix on hematoxylin and eosin staining (Figure 2A). Silver staining showed splitting of glomerular capillary basement membrane typically described as tram tracking (Figure 2B). Immunofluorescence was available in 15 cases, out of which 10 cases showed linear C3 deposits along capillary wall. Five cases showed IgG deposits in the mesangium, out of which 3 were weakly positive. Eight cases had IgM deposits in mesangium and 3 showed IgA deposits) (Figure 2C). Electron microscopy was available in 11 cases and revealed thickened capillary due to interposed mesangial cells, mesangial matrix and electron dense subendothelial deposits (Figure 2D). Histology of FSGS, revealed 15 cases with peripheral sclerosis (Figure 3B), 7 cases with collapsing glomerulopathy (Figure 3A), 8 cases with perihilar sclerosis (Figure 3C) and 5 cases with tip lesion. Immunofluorescence was available in 13 cases. IgM was positive in mesangium in 8 cases with 5 cases showed C3 positivity, 4 showed IgG positivity. Electron microscopy was available in 10 cases. Positive correlation was seen in 6 cases. Two showed focal areas of sclerosis (Figure 3D). Wrinkling of glomerular basement membrane was seen in 4 cases. One case showed duplication wrinkling and showed focal areas of sclerosis. Membranous glomerulonephritis, histology revealed on H and E thickening of basement membrane , confirmed on periodic acid Schiff (PAS) staining (Figure 4A). Silver stain showed spikes along the capillary basement membrane (Figure 4B). Immunofluorescence was available in 12 cases. Seven cases showed granular deposits in IgG and 6 cases showed C3 positivity while 2 cases showed IgM positivity (Figure 4C). Electron microscopy was available in 9 cases. Showed subepithelial deposits in contact with and indenting the visceral epithelial layer (Figure 4D).

Histopathology of minimal change disease revealed unremarkable glomeruli on hematoxylin and eosin, PAS and silver staining (Figure 1A). Immunofluorescence was available in 12 cases, out of which 7 cases showed positivity for IgM. Ten cases showed IgG negative, 12 cases were IgA

Diabetic nephropathy microscopy revealed increase in solid spaces of tuft and Kimmelstiel-wilson nodules (Figure 5A) with few glomerular lobular sclerosis confirmed on periodic acid Schiff (PAS) stain. Immunofluorescence was available in 6 cases. Four cases showed IgG diffusely deposited along the basement membrane, 3 cases showed IgM positivity and 3 cases showed IgA positivity. Deposition of protein seen in linear pattern, narrowing and thickening of renal vasculature. Electron microscopy was available in 5 cases. Four cases showed positive correlation with light microscopy and showed showing increase in mesangial cellularity with thicker capillary basement membrane. One case was diagnosed as membranoproliferative glomerulonephritis on electron

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microscopy. Lupus nephritis (SLE) histology revealed showed diffuse lobular accentuation. WHO class IV showing glomerular capillary wall thickened (wire loop) (Figure 5B) and increase in mesangial matrix. PAS stain showed splitting of capillary wall and silver stain showed double countered glomerular basement membrane. Immunofluorescence was available 3 cases. IgG positivity in 3 cases, 2 cases C1Q positive, 2 case show IgA positivity and C3 positive in 1 case. Electron microscopy was available in 6 cases with subepithelial deposits. Amyloidosis histology revealed glomerular capillary wall show irregular thickening with expansion of mesangium confirmed on PAS stain (Figure 5C). Silver stain showed irregular spikes. Congo red stain showed positivity which is apple green birefringence under polarized light in 8 cases (Figure 5D). Immunofluorescence was available in 5 cases and negative. Electron microscopy was available in 7 cases showing linear, non branching fibrils. A summary of other studies from India is presented in [Table 3], found MCD to be the most common cause of nephrotic syndrome. The study done from Vellore in 1970â&#x20AC;&#x2122;s by Date et al noted that MCD accounted for about 35% of all cases of nephrotic syndrome. Similarly, studies from Delhi by Agarwal S.K. et al found (37%) and Aggrawal et al from Rohtak found (33.3%), MCD cases responsible for more than one-third of nephrotic syndrome. The Das et al from Hyderabad also found similar observation with MCD in 21.8% cases of nephrotic syndrome for a study period

of 1990 to 2008. Rathi et al from Chandigarh showed increasing trend of FSGS, this trend has not been observed in our study. In a year 2010-12 study published from Kolkata, Golay et al [11] found that FSGS was underlying disease in 27.4% of their patients making it the most common one However, they found MCD in 27.1% of cases, making it the second most common cause of nephrotic syndrome. This figure is very similar to our present data, where MCD were responsible for 26.38% of cases. A comparative studies with Asian region is summarized in [Table 4 ], shows certain interesting and conflicting data. Study by Chang et al, [12] from Korea MCD as a most common in nephrotic syndrome similar to our present findings. However Zhou et al [13] from China observed 25.3% cases of MCD which is similar 26.38% cases of MCD of our present study. Studies from Nepal done by Zhou et al [13] , Garyal et al [14], from China and I. M. Onwubuya et al [15] from Nigeria shown that most common cause is MGN responsible for 29.5% ,42.3% and 30.6% cases of nephrotic syndrome respectively. Sabir S et al and Kazi et al [16] from Pakistan found FSGS to be the most common lesion among Primary glomerulonephritides .This may be due to demographical, geographical and racial characteristics, differences in indication of renal biopsy, analyzed clinical syndromes and variation in pathological classification. Therefore for drawing accurate conclusions were difficult by comparison with different data of Asian studies.

Table 1: Age and sex wise distribution of various histomorphological patterns of adults nephrotic syndrome. Histomorpho logical pattern MCD MPGN FSGS MGN DN AMYLOID SLE IgA ESRD DPGN RPGN APSGN TIN TOTAL Total of M and F

15-25 yrs M F 22 9 10 8 13 5 2 1 0 0 1 0 0 3 2 0 4 2 2 0 1 0 1 0 0 1 58 29 87

26-35 yrs M F 9 8 2 5 4 3 8 3 3 1 5 2 0 2 0 0 1 0 2 0 1 0 0 1 0 0 35 25 60

36-45 yr 46-55 yr 56-65 yrs M F M F M F 6 5 1 0 1 1 5 1 2 1 4 0 5 2 0 2 2 0 3 2 7 2 1 2 1 0 4 1 2 1 2 0 1 2 2 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 2 0 0 0 2 1 0 0 1 0 0 0 0 0 1 0 1 0 0 1 0 0 1 0 0 0 0 22 16 16 10 13 7 38 26 20

66-75 yrs M F 0 0 0 0 0 1 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 3

76-85 yrs M F 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1

Total 62 (26.38%) 38 (16.17%) 37 (15.74%) 31 (13.19%) 15(6.38%) 17(7.23%) 6(2.55%) 3(1.27%) 9(3.8%) 8(3.4%) 3(1.27%) 4(1.7%) 2(0.85%) 235

MCD: minimal change disease, MPGN: membranoproliferative glomerulonephritis, FSGS: focal segmental glomerulosclerosis, MGN: membranous glomerulonephritis, DN: diabetic nephropathy, SLE: Systemic lupus erythematosis, IgA: IgA nephritis, ESRD: end stage renal disease, DPGN: Diffuse proliferative glomerulonephritis, RPGN: Rapidly progressive glomerulonephritis, APSGN: Acute post streptococcal glomerulonephritis,TIN: tubulointersitial nephritis, M : male, F: female.

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Table 2: Histopathology and electron microscopy correlation. Histomorpho logical pattern (light microscopy)

No. of cases

Positive correlation

% of Positive correlation

Negative correlation

% of Negative correlation

100

MCD MPGN

100

FSGS

33.33

MGN

100

AMYLOID

100

SLE

100

IgA

66.67

33.33

ESRD

100

APSGN

100

TOTAL

91.86

8.14

Table 3: Comparison of glomerular lesions among nephrotic syndrome in adults in different Indian studies. Date et al.[8]

Agarwal et al.[9]

Rathi et al[2]

Goyal et al.[11]

Present study

Year

1971-85

1987-98

2000

Place

Vellore

Delhi

Rohtak

1990-2008

2002-07

2010-12

2000-2015

Hyderabad

Chandigarh

Kolkata

Mumbai

1532

2250

404

1615

364

410

235

Primary glomerular diseases

1276 (83.3%)

1316 (58.5%)

318 (78.7%)

1278 (79.1%)

324 (89%)

361 (88.1%)

184 (78.3%)

MCD

457 (35.8%)

487 (37% )

106 (33.3%)

279 (21.8%)

48 (14.8%)

98 (27.1%)

62 (26.38%)

MPGN

177 (13.9%)

153 (11.6%)

58 (18.2%)

73 (5.7%)

58 (17.9%)

24 (6.6%)

38 (16.17%)

FSGS

238 (18.6%)

263 (20%)

56 (17.6%)

195 (15.2%)

99 (30.6%)

99 (27.4%)

37 (15.74%)

MGN

174 (13.6%)

263 (20%)

54 (16.9%)

129 (10.1%)

79 (24.4%)

89 (24.6%)

31 (13.19%)

DPGN/ PSGN

32 (2.5%)

190 (14.9%)

9 (2.8%)

6 (1.6%)

12 (5.1%)

Secondary glomerular diseases

256 (16.7%)

934 (41.5%)

86 (21.3%)

337 (20.9%)

40 (11%)

49 (11.9%)

51 (21.7%)

Reference

Aggrawal et al.[10]

Das et al.[3]

Table 4: Comparison of glomerular lesions among nephrotic syndrome in adults in different Asian studies. Reference

Chang et al.[12]

Zhou et al.[13] Garyal et al.[14]

I.M.Onwubuya et al.[15]

Kazi et al.[16]

Present study

Country

Korea

China

Nepal

Nigeria

Pakistan

India

1818

1374

137

165

316

235

MCD (%)

15.5

25.3

10.2

19.7

14.8

26.38

MPGN(%)

4.0

1.5

21.9

19.7

4.3

16.17

FSGS(%)

5.6

6.0

8.0

15.9

39.9

15.74

MGN(%)

12.3

29.5

42.3

15.9

26.6

13.19

DPGN/ PSGN(%)

0.7

2.9

12.9

2.8

5.10

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Fig. 1: (1A), Minimal change disease, showing unremarkable glomerulus with patent capillary lumina & normal mesangial cellularity (H & E ,100X). Fig (1B), Electron microscopy: Flattening & fusion of the foot processes (arrows) of the visceral epithelial cells. (uranyl acetate lead citrate, 2500X).

Fig. 2 :(2A),Membranoproliferative glomerulonephritis, Showing mesangial cell proliferation. Lobular accentuation. Increase in mesangial matrix and capillary wall thickening, (PAS, 100X). Fig (2B),Showing splitting (arrow){tram tracking} of the capillary basement membrane, (Silver, 100X). Fig (2C),Immunofluorescence, Note the diffuse, broad capillary loop & mesangial deposits, (Anti-C3, 100X). Fig(2D), Electron microscopy: Shows widening of the glomerular capillary wall due to mesangial cell interposition (arrow). Note the increase in mesangial matrix & the electron dense deposits. (uranyl acetate, lead citrate, 2000X).

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Fig 3: (3A), Focal segmental glomerulosclerosis. Showing focal sclerosis of the glomerulus with collapse of the capillary tuft (top centre) while rest of the glomerulus is unremarkable, (H&E, 100X). Fig(3B) ,Sclerosed area in the glomerulus (peripheral sclerosis), (Silver stain, 1000X), Fig(3C) , Sclerosis in the perihilar region (arrow) of the glomerulus on the left, (Silver stain, 450X). Fig(3D), Electron microscopy: Note the focal area of sclerosis (arrow) and the flattening of the foot processes (arrow head), (uranyl lead citrate, 8000X).

Fig. 4: (4A), Membranous glomerulonephritis. Showing the thickening of the glomerular capillary walls without increase in the cellularity (PAS, 100X). Fig (4B), Spikes (arrows) on the sub epithelial surface of glomerular basement membrane (Methanamine silver stain ,200X). Fig (4C), Immunofluorescence: showing diffuse, finely granular deposits outlining the glomerular capillary walls (Anti-IgG, 100X). Fig (4D), Electron microscopy : Showing sub epithelial deposits (arrows) in contact with & indenting the visceral epithelial cells (10000X).

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Fig. 5: (5A), Diabetic nephropathy. Showing kimmelstiel - wilson nodules (arrows) (H&E, 40X). Fig(5B), Lupus nephritis, WHO Class IV lesion. Glomerular capillary walls are segmentally thickened by wire loop deposits (H & E, 400X). Fig (5C), Diffuse mesangial pattern of glomerular amyloidosis indicated by a cellular weakly PAS positive mesangial expansion (PAS, 400X).Fig(5D), Amyloidosis. Deposits of amyloid exhibiting - apple green birefringence under polarized light ( Congo red stain, 100X).

Conclusion

This study highlights that Minimal Change Disease is the most common cause of nephrotic syndrome in adult male patients while Membranous glomerulonephritis and Diabetic nephropathy are the most common lesions in more than 45 years of age. Electron microscopy was available in only 86 cases (36.6%) out of 235 cases and Immunoflurescence was performed in 31.5% of cases. Light microscopy findings are well correlated with electron microscopy findings in 91.86% of cases .Hence greater use of these advance dignostic methods can further change the adult nephrotic syndrome spectrum. Even though in past studies, the histological spectrum of nephrotic syndrome was similar in different parts of India except for the study carried out in Chandigarh showing increasing trend towards FSGS But, there has been considerable heterogeneity in histological spectrum of nephrotic syndrome in adjacent Asian countries. It is essential as well as necessary to maintain a central renal biopsy registry with increase participation of more nephrology centers of India for obtaining the accurate incidence, spectrum and distribution of adults nephrotic syndrome.

Acknowledgements

Department of Pathology, Jaslok Hospital and Research Centre , Mumbai.

Reference 1.

Berry D, Mackenzie C, Richard Bright. Physician in an Age of Revolution and Reform, London.1992; 1789-1858.

Rathi M, Bhagat R. L, Mukhopadhyay P, et al.Changing histologic spectrum of adult nephrotic syndrome over five decades in north India: A single center experience. Indian J Nephrol. Mar-Apr 2014; 24(2): 86–91.

Das U, Dakshinamurty K V, Prayaga A. Pattern of biopsyproven renal disease in a single center of south India: 19 years experience. Indian J Nephrol. 2011;21:250–7.

4. Sakhuja V, Jha V, Ghosh A K, Ahmed S, Saha T K. Chronic renal failure in India. Nephrol Dial Transplant. 1994; 9:871–2. 5.

Sabir S, Mubarak M, Ul-Haq , Bibi A. Pattern of biopsy proven renal diseases at PNS SHIFA, Karachi:A cross sectional survey. J Renal Inj Prev.2013 Oct 10;2(4):133-7.

Balakrishnan N, John G T, Korula A, et al. Spectrum of biopsy proven renal disease and changing trends at a tropical tertiary care centre 1990-2001. Indian J Nephrol. 2003; 13:29–35.

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Reshi AR, Bhat MA, Najar MS, et al. Etiological profile of nephrotic syndrome in Kashmir. Indian Journal of Nephrol 2008; 18(1): 9-12.

12. Chang J H, Kim D K, Kim H W, et al. Changing prevalence of glomerular diseases in Korean adults: A review of 20 years of experience. Nephrol Dial Transplant. 2009;24:2406–10.

Date A, Raghavan R, John T J, Richard J, Kirubakaran M G, Shastry J C. Renal disease in adult Indians: A clinicopathological study of 2,827 patients. Q J Med. 1987;64:729–37.

13. Zhou F D, Shen H Y, Chen M, et al. The renal histopathological spectrum of patients with nephrotic syndrome: An analysis of 1523 patients in a single Chinese centre. Nephrol Dial Transplant. 2011;26:3993–7.

Agarwal S K, Dash S C, et al. Spectrum of renal diseases in Indian adults. J Assoc Physicians India. 2000;48:594–600.

10. Aggarwal H K, Yashodara B M, Nand N, Sonia, Chakrabarti D, Bharti K. Spectrum of renal disorders in a tertiary care hospital in Haryana. J Assoc Physicians India. 2007;55:198–202. 11. Golay V, Trivedi M, Kurien A A, Sarkar D, Roychowdhary A, Pandey R. Spectrum of nephrotic syndrome in adults: Clinicopathological study from a single center in India. Ren Fail. 2013;35:487–91.

14. Garyal, Kafle R K et al. Hisopathological spectrum of glomerular disease in Nepal: A seven-year retrospective study. Nepal Med Coll J. 2008;10:126–8. 15. Onwubuya IM , Adelusola KA, Sabageh D, Ezike K. N, Olaofe OO. Biopsy proven renal disease in Ile-Ife, Nigeria: A histopathologic review.Indian J Nephrol. 2016 Jan-Feb; 26(1): 16–22. 16. Kazi J I, Mubarak M, Ahmed E, Akhter F, Naqvi S A, Rizvi S A. Spectrum of glomerulonephritides in adults with nephrotic syndrome in Pakistan.Clin Exp Nephrol. 2009;13:38–43.

*Corresponding author: Dr. Ganesh Ramdas Kshirsagar; 96/3403,C-wing,The Neharu Nagar Suryadarshan CHS Ltd, Neharu Nagar Kurla-East, Mumbai (India), 200024 Phone: +91 9821505456 Email: gkshirsagar31@yahoo.in Date of Submission : 09.02.2017 Date of Acceptance : 09.06.2017 Financial or other Competing Interests: None. Date of Publication : 19.12.2017

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Original Article DOI: 10.21276/APALM.1499

Histopathological Analysis and Correlation of Ki67 and Progesterone Receptor Status with WHO Grading In Meningiomas Tamilselvi Veeramani1* and J. Maheswari2 Department of Pathology, Karpagam Faculty of Medical Sciences and Research, Othakkalmandabam, Coimbatore. India 2 Department of Pathology, KAP DR. Vishwanathan Government Trichy Medical College, Trichy. India

ABSTRACT Background: Meningiomas are slow growing tumors that are among the most common of CNS neoplasms and form the most common CNS tumor to be reported above 35 years of age. Methods: This retrospective study was carried out in the Department of Pathology during the period of January2011 to May2013. A total of 50 cases were graded according to the WHO 2016 grading criteria. The biopsy specimens were fixed in 10% neutral buffered formalin, sections was stained with Hematoxylin & Eosin. Immunohistochemistry was done with Progesterone receptor and Ki67 antibodies for selected cases. Results: The incidence of meningiomas was 33.11% with a female sex predilection and most common in the 5th decade. Transitional meningioma was the most common variant to occur. The incidence of WHO Grade I, Grade II and Grade III meningiomas were 88%, 4% and 8% respectively. Comparison of Ki67 LI and PR score in various grades of meningiomas were done. The average Ki67 LI and PR score were 1.1%, 10.25; 6%, 3; 16%, 0 in grade I, II and III meningiomas respectively. p value showed a statistically significant difference between different grades of meningiomas with respect to PR and Ki67 status. Spearman correlation showed a clearly significant inverse relationship between the two antibodies. Conclusion: The use of immunohistochemical markers aids in determining the aggressive nature of the tumor, its recurrence potential and can be used as prognostic markers. Keywords: Meningioma, Progesterone Receptor, Immunohistochemistry, Prognosis.

Introduction

Meningiomas are a diverse group of neoplasms derived from the arachnoidal cap cells lining the meninges and their extensions of dura. Although most are benign, their intracranial location leads to fatal consequences.[1] According to the CBTRUS statistics 2012, meningiomas constitute to 35% of all CNS tumors and the 5yr survival rate being 70% for benign and 55% for malignant meningiomas.[2] Based upon the WHO 2016 grading, meningiomas are classified as grade I, grade II (atypical) and grade III (anaplastic meningiomas). The various parameters taken into consideration for histological grading include: Cellularity , Mitotic index , Sheet like or small cell pattern, Macronucleoli with nuclear pleomorphism and Tumor necrosis.[3]The Armed Forces Institute of Pathology declared that the criteria for malignancy in meningiomas are satisfied when the tumor displays either or both features of anaplasia and brain parenchyma invasion. Brain-invasive meningiomas are considered as equivalent to WHO Grade II neoplasms.[4] In the 2016

WHO classification of CNS tumors, brain invasion as a histologic criteria can alone suffice for diagnosing WHO grade II / Atypical meningioma.[5] About 10-15% of all meningiomas are considered to be malignant. Even though grade I meningiomas are considered as benign, recurrence is seen in about 7-20% cases despite complete resection.[6] The incidences of recurrence for atypical and anaplastic meningiomas are 29-40% and 50-78% respectively.[7] Thus, WHO grading based on the histopathological features alone has certain limitations in predicting the exact behavior of meningiomas. Hence, the use of immunohistochemical markers aids in determining the aggressive nature of the tumor and its recurrence potential. Ki67 & PR are the markers used to determine the nature of meningiomas. Ki67 is a non-histone intranuclear protein expressed within the proliferating cells of the cell cycle. Its expression is regarded as a specific marker for tissue proliferation. Hormonal receptor studies are not much done in our country. Various studies conducted in the developed

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Veeramani et al. countries points towards an inverse relationship between PR & Ki67 status in meningiomas. Hence, this study has been undertaken to evaluate the histological patterns along with WHO grading and to assess the role of IHC markers in determining the biological behavior of meningiomas.

Materials and Methods

A-715 3- 51-80% tumor nuclei show positivity 4- >80% positive tumor nuclei Intensity of staining 0-absent 1-weak 2-moderate

This prospective study was carried out in the Department of Pathology, Thanjavur medical college during the period from January 2011 to May 2013. Ethical committee clearance was obtained from the Institutional Ethical Committee Board.

3-strong

Sample size-50

Statistical analysis was performed using the SPSS software. Multivariate analysis was done using ANOVA and Kruskal-Wallis test to determine the significance of difference between various meningioma grades for both the markers Ki67 and PR. P value <0.05 was considered as statistically significant. Spearman correlation was done to determine the relationship between Ki67 and PR status in different grades of meningiomas.

Inclusion Criteria: Specimens sent with a clinical suspicion of meningiomas. All biopsy specimens sent with the clinical diagnosis of CNS tumors. Exclusion Criteria: CNS infections, Reactive lesions and Non-neoplastic cystic lesions of CNS Grading of tumors was done according to the WHO 2016 grading criteria.[5].The specimens were mostly biopsies. All specimens were fixed in 10% neutral buffered formalin followed by which routine tissue processing was done and were subjected to histopathological examination. Sections of 4-5 micron thickness were made and staining was done with Hematoxylin and Eosin. IHC with vimentin was done for 2 cases namely, rhabdoid and papillary variants to confirm their meningothelial nature. IHC with GFAP was done in a single case of Brain invasive meningioma. Immunohistochemistry was done using antibodies against Ki67 and PR for about 15 randomly selected cases of various grades of meningiomas. IHC was done based on the peroxidase method using standard horse radish peroxidase kit. Ki67 is considered a more specific marker for estimating the growth fraction and hence is the most widely used marker for determining the proliferation rate of neoplasms. [8] Determination of Ki67 LI was done by calculating the percentage of nuclei that stains positively from regions that show maximal intensity of nuclear staining among 1000 tumor cells at high power magnification. [9] PR status is determined by a semi quantitative scoring scale based on 2 parameters namely. [10] The percentage of positive tumor cells 0- Absence of positively stained nuclei 1- <10% positively stained cells in the entire section 2- 10-50% positivity www.pacificejournals.com/apalm

Immunoreactive score (IRS=0 to 15) was calculated by multiplying the staining intensity and the percentage of positively stained tumor cells. [10]

Results

The incidence of meningiomas in our study was 33.11% with female to male ratio being 4:1. The most common age group affected is 50-60yrs. The most common variant (Tab.1) reported in our study is the transitional subtype accounting for 48% and the second most common variant being meningothelial meningioma with an incidence of 20% (10 cases). A single case each of papillary, Rhabdoid, anaplastic and brain invasive meningiomas (Fig.1,2) were reported. Grade I meningiomas were the predominant subtype to occur with an overall incidence of 88% (44 cases). Grade II meningiomas constitutes to 4% (2cases) and Grade III meningiomas constitutes 4 of the total 50 cases with an incidence rate of 8%.A case of rhabdoid meningioma ( WHO Grade III ) was reported in a 10 year old female child. IHC with vimentin showed intense cytoplasmic positivity and confirmed the diagnosis.(Fig.3) Table 2 shows different grades of meningioma variants and their MIB-1 LI. 15 random cases were selected and immunohistochemistry was performed on paraffinembedded sections using standard HRP kit. The study reveals rhabdoid meningioma with the highest Ki 67 score of 21% (Fig.4 ) and the least score was observed with transitional meningioma. (0.8%) (Fig.5) Differences in the mean Ki67 labelling index was calculated using Anova test and the results were found to be statistically significant between various grade I, II and grade III meningiomas (P<0.001). eISSN: 2349-6983; pISSN: 2394-6466

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Table 3 displays PR scoring in selected cases of meningiomas. Highest PR score was observed with the transitional and meningothelial variants.(fig 6) Least PR score of 0 was observed with the Papillary(Fig.7)

and Rhabdoid (Fig.8) variants. Kruskal-Wallis test was performed to determine the difference in average PR score of various grades of meningiomas, which revealed a P value of < 0.04 and hence found to be statistically significant.

TABLE 1: Incidence of Various Types of Meningiomas S.NO 1 2 3 4 5 6 7 8 9 10

HPE Diagnosis Angiomatous meningioma Anapastic meningioma Atypical meningioma Brain invasive meningiomas Fibroblastic meningioma Meningothelial meningioma Papillary meningioma Psammomatous meningioma Rhabdoidmeningiomas Transitional meningioma Total

No of cases 6 2 1 1 1 10 1 3 1 24 50

Percentage ( % ) 12% 4% 2% 2% 2% 20% 2% 6% 2% 48% 100%

Table 2: Ki 67 Labelling Index in Various Grades of Meningioma Variants S.NO 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

PATHO NO 151/13 318/13 1907/13 1261/12 2334/13 3046/13 3060/12 3288/13 3286/13 3455/13 264/12 3209/13 3553/13 4113/13 1866/11

HPE DIAGNOSIS Transitional meningioma Fibroblastic meningioma Psammomatous meningioma RhabdoidMeningiomas Psammomatous meningioma Angiomatous meningioma Meningothelial meningioma Meningothelial meningioma Transitional meningioma Transitional meningioma Papillary meningioma Brain invasive meningiomas Psammomatous meningioma Angiomatous meningioma Transitional meningioma

WHO GRADE I I I III I I I I I I III II I I I

MIB INDEX 1.2% 1.0% 1.7% 21% 0.8% 2.2% 1.3% 1.1% 0.2% 1.4% 11% 6% 1.1% 0.9% 0.4%

Table 3: Pr Scoring in Different Grades of Meningioma Variants. S.NO

PATH NO

HPE DIAGNOSIS

WHO GRADE

1 2 3 4 5 6 7 8 9 10

151/13 318/13 1907/13 1261/12 2334/13 3046/13 3060/13 3288/13 3286/13 3455/13

Transitional Meningioma Fibroblastic Meningioma Psammomatous Meningioma RhabdoidMeningiomas Psammomatous Meningioma Angiomatous Meningioma Meningothelial Meningioma Meningothelial Meningioma Transitional Meningioma Transitional Meningioma

I I I III I I I I I I

PR (0-15) Intensity Score (IS) 3 2 2 0 3 2 3 2 3 2

Proportion score (PS) 3 5 4 0 4 4 4 5 5 4

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IRS= IS x PS 9 10 8 0 12 8 12 10 15 8

Veeramani et al. S.NO

PATH NO

264/12

12 13

A-717 WHO GRADE

HPE DIAGNOSIS

PR (0-15) Intensity Score (IS)

Proportion score (PS)

IRS= IS x PS

Papillary Meningioma

III

3209/13

Brain InvasiveMeningioma

3553/13

Psammomatous Meningioma

4113/13

Angiomatous Meningioma

1866/11

Transitional Meningioma

Table 4: Comparison of Ki 67 Li And Pr Score in Various Grades of Meningiomas. S.NO

WHO GRADE

Average Ki67 LI

Average PR score

NO OF CASES

1.1%

10.25%

III

16%

Fig. 1: Brain invasive meningioma showing irregular protrusion of tumor cells infiltrating the brain.

Fig. 2: Brain invasive meningioma- GFAP highlights entrapped fragments of brain parenchyma between the tumor cells, H & E, (10X)

Fig. 3: Rhabdoid meningioma showing strong cytoplasmic immunoreactivity for vimentin, (40X).

Fig. 4:Rhabdoid meningioma - Grade III, Ki67 LI of 21% (40X).

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Fig. 5: Transitional meningioma - Grade I with Ki67 LI of <0.1% (10X).

Fig. 6: Transitional meningioma- Grade I, PR score of 12 (10X).

Fig. 7: Papillary meningioma - Grade III with PR score of 0 (10X).

Fig. 8:Rhabdoid meningioma - Grade III showing PR score of 0 (40X).

Discusson

Konstantinos Violaris et al[15]). The study on receptors of sex hormones is of great interest, given the female preponderance in meningiomas. Studies suggest that the expression of PR is roughly inversely proportional to the WHO histological grade. In addition to this, PR scoring also helps in assessing those cases under grade I meningiomas that are likely to recur. [15]

Meningiomas are the most common non â&#x20AC;&#x201C; glial primary brain tumor. Our study showed a clear female predominance with an incidence of 80% which is similar to various other studies (CBTRUS statistical report 2012). [2] Transitional meningiomas are the most common subtype with an incidence of 48% (Table 1) whereas the study by Sanghamithra et al [11] showed meningothelialmeningiomas as the most frequent type of occurrence. According to Sameh Ahmed et al [12] and SashidharBabu et al [13], grade III meningiomas showed the least rate of occurrence. In contrast our study showed grade II to be the least common type (4%). Our study points out that Grade I meningiomas are the commonest to occur with a female sex predilection. This was found to be similar with majority of the studies (Sanghamithra et al[11],Intisar S.H.Patty et al[14],

Ki 67 -Grading based upon the histopathological features pose certain limitations in determining the exact biological behavior of meningiomas. Mitotic count alone cannot provide adequate details regarding the aggressive nature of a tumor. Identification of mitotic figures in H&E sections is hampered by various factors. Hence, use of Ki67 acts as an independent prognostic factor in predicting the biological behavior. Ki 67 is an intranuclear protein that is expressed within proliferative phases of the cell cycle. The monoclonal

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Veeramani et al. antibody against Ki 67, namely the MIB-I labelling index is used to predict the recurrence potential and aggressive nature of meningiomas. Generally, higher the Ki 67 index, higher is the grade of meningiomas and its tendency for recurrence[16]. Hence, PR when used in conjunction with Ki67 LI may act as useful ancillary markers in determining the prognosis and grading of meningiomas. Our study showed almost similar Ki67 labelling indices as in the studies of Sanghamithra et al[11] and Intisar Patty et al[14].. The Ki67 LI correlated well with the increasing grades of meningiomas and this is in accordance with the studies conducted by Amatya et al[16] and Nasrin Shyanfaret et al. [17]They showed that Grade I meningiomas are the one with highest PR expression seen in about 97%, Grade II and grade III meningiomas with 20% and 0% positivity respectively. Similar results were obtained with our present study too. Brandis et al[18] and Wolfsberger S et al[19] reported that malignant meningiomas are devoid of PR. This is in accordance with our study where rhabdoid and papillary meningiomas was PR negative with a score of 0. Meningiomas with higher proliferative index and negative PR are more likely to be of higher grade and hence carry an increased potential forrecurrence even after complete resection. Despite the limitations caused by the small study group chosen, Ki67 LI along with PR status in combination with WHO histopathological grading might help in identifying patients at a high risk of relapse and this early detection makes institution of more effective treatment as early as possible.

Conclusion

Progesterone receptor and Ki67 antibodies when used in addition to histopathological grading can aid in identifying the cases that are likely to recur in Grade I meningiomas. Study trials when conducted similar to our present one might help in evaluating the exact tumor burden in the country and would encompass the role of ancillary methods like immunohistochemistry in the prognostication of tumors.

Acknowledgment

We extend our gratitude to our institution departmental head for providing expertise and assistance. We thank all the references cited. We are grateful to our family who supported us in our work.

References

A-719 meningioma. Neurosurgery. 2005 Dec;57(6):1088-1095; discussion 1088-1095. 2.

Ostrom QT, Gittleman H, Fulop J, Liu M, Blanda R, Kromer C, et al. CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2008-2012. Neuro-oncology. 2015 Oct;17Suppl 4:iv1-iv62.

Commins DL, Atkinson RD, Burnett ME. Review of meningioma histopathology. Neurosurg Focus. 2007;23(4):E3.

Perry A, Brat DJ. Philadelphia: Churchill Livingstone; 2010. Practical Surgical Neuropathology: A diagnostic approach; pp. 185–218.

Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, et al. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. ActaNeuropathol. 2016 Jun;131(6):803–20.

Choi SJ, Chang ED, Kwon SO, Kye DK, Park CK, Lee SW, et al. Comparison of Proliferative Activity in Each Histological Subtypes of Benign and Atypical Intracranial Meningiomas by PCNA and Ki-67 Immunolabeling. Journal of Korean Neurosurgical Society. 2000 Sep 1;29(9):1215–21.

Yang S-Y, Park C-K, Park S-H, Kim DG, Chung YS, Jung H-W. Atypical and anaplastic meningiomas: prognostic implications of clinicopathological features. J NeurolNeurosurgPsychiatr. 2008 May;79(5):574–80.

Shrestha P, Shrestha I, Kurisu K. Usefulness of Ki-67 in the histological evaluation of neoplastic lesions of central nervous system. Journal of Institute of Medicine. 2008;30(1):68–71.

Karabağli P, Sav A. Proliferative indices (MIB-1) in meningiomas: Correlation with the histological subtypes and grades. Journal of Neurological Sciences [Turkish]. 2006; 23(4):279-86.

10. Roser F. The prognostic value of progesterone receptor status in meningiomas. Journal of Clinical Pathology. 2004 Oct 1;57(10):1033–7. 11. Chatterjee U, Mukherjee S, Ghosh S, Chatterjee S. Detection of progesterone receptor and the correlation with Ki-67 labeling index in meningiomas. Neurology India. 2011;59(6):817. 12. Sakr SA, Salem M. Atypical meningioma: Clinicopathological analysis of a new WHO classification. Pan Arab Journal of Neurosurgery. 2011;15(1):36–41. 13. Challa S, Babu S, Uppin S, Uppin M, Panigrahi M, Saradhi V, et al. Meningiomas: Correlation of Ki67 with histological grade. Neurology India. 2011;59(2):204.

1. Claus EB, Bondy ML, Schildkraut JM, Wiemels JL, Wrensch M, Black PM. Epidemiology of intracranial

14. Intisar s.h. Patty. Central nervous system tumors a clinicopathological study Kurdistan 1st conference on biological sciences j. dohuk Univ., 2008;11, no. 1,.

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15. Violaris K, Katsarides V, Sakellariou P. The Recurrence Rate in Meningiomas: Analysis of Tumor Location, Histological Grading, and Extent of Resection. Open Journal of Modern Neurosurgery. 2012;02(01):6–10. 16. Amatya VJ, Takeshima Y, Sugiyama K, Kurisu K, Nishisaka T, Fukuhara T, et al. Immunohistochemical study of Ki-67 (MIB-1), p53 protein, p21WAF1, and p27KIP1 expression in benign, atypical, and anaplastic meningiomas. Hum Pathol. 2001 Sep;32(9):970–5. 17. Shayanfar N, Mashayekh M, Mohammadpour M. Expression of Progestrone Receptor and Proliferative Marker ki 67,

in Various Grades of Meningioma. ActaMedicaIranica. 2010;48(3):142–7. 18. Brandis A, Mirzai S, Tatagiba M, Walter GF, Samii M, Ostertag H. Immunohistochemical detection of female sex hormone receptors in meningiomas: correlation with clinical and histological features. Neurosurgery. 1993 Aug;33(2):212-217; discussion 217-218. 19. Wolfsberger S, Doostkam S, Boecher-Schwarz H-G, Roessler K, van Trotsenburg M, Hainfellner JA, et al. Progesterone-receptor index in meningiomas: correlation with clinico-pathological parameters and review of the literature. Neurosurg Rev. 2004 Oct;27(4):238–45.

*Corresponding author: DR. V. Tamilselvi, Assistant Professor, Department of Pathology, Karpagam Faculty of Medical Sciences and Research, Othakkalmandabam, Coimbatore.-641032, Tamilnadu. INDIA, PINCODE-641032. Phone: +91 8903489000 Email: tamilselviaruna16@gmail.com Date of Submission : 26.04.2017 Date of Acceptance : 09.09.2017 Financial or other Competing Interests: None. Date of Publication : 19.12.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Original Article DOI: 10.21276/APALM.1504

Diagnostic Utility of Haematological Scoring System (HSS) with Clinicopathological and Bacteriological Evaluation in Early Diagnosis of Neonatal Sepsis Bhagyashree M. Ahirrao, Nandkumar V. Dravid, Mahesh H. Ahirrao, Arundhati S. Gadre and Shirish Gondane Department of Pathology, ACPM Medical college Morane, Dhule, Maharashtra, India

ABSTRACT Background: Neonatal sepsis is a major cause of mortality with high incidence of 11 to 24.5 per 1000 live births in India. Delay in diagnosis further increases the mortality to 30-40% of total neonatal deaths. Though blood culture is gold standard for diagnosis of neonatal sepsis, it has limitations. The comprehensive Hematologic scoring system (HSS) formulated by Rodwell et al is used for early diagnosis of sepsis. This comprises of Immature/Total neutrophil ratio, Total PMN count, Immature/Mature ratio, Immature PMN count, degenerative changes in PMN, platelet count. Methods: This is a prospective study of haematologic profiles of 303 neonates All neonates with congenital abnormalities diagnosed at birth admitted in neonatal care unit in our institute. Field stained blood smears were examined for HSS. Blood culture was done before administration of antibiotic treatment. Neonates with predisposing perinatal risk factors or if there was clinical suspicion of sepsis were included in this study from Nov 2014 to feb 2016. Result: Of the 303 neonates in the present study, 77 had positive blood cultures, The incidence of septicemia was higher in males (72.7%) than females (27.3%). Majority of the neonates presented with early onset type of sepsis (91%). Elevated I: T ratio and I:M ratio were seen in most cases of septicemia. Conclusion: HSS is the most sensitive indicator of sepsis. Use of HSS by peripheral smear study Blood culture can be used effectively as a sepsis screen for early diagnosis is useful to reduce neonatal morbidity and mortality. Keywords: Haematological Scoring System, Neonatal Sepsis, Peripheral Blood Smears

Introduction

Neonatal sepsis refers to a clinical syndrome characterised by systemic signs and symptoms due to generalised bacterial infection with a positive blood culture in the first 28 days of life. It is probably responsible for 30-50% of the total neonatal deaths each year. According to recent data from National Neonatal Perinatal Database (NNPD) 2002-03 collected from 18 centers from various parts of India, incidence of neonatal sepsis has been reported to be 29.9 per 1000 live births.[1] Neonatal septicemia with its high incidence and grave prognosis, in spite of adequate treatment with modern antibiotics, has been a challenge for all times. Optimal diagnosis and treatment strategies are difficult to define. The signs and symptoms are protean with a high mortality and thus there is urgent need to know whether the baby has sepsis to institute treatment as quickly as possible. Hematologic scoring system comprises of Immature/Total neutrophil ratio, Total PMN count, Immature/Mature ratio, Immature PMN count, degenerative changes in PMN, platelet count.[2] Current study was undertaken to assess

role of HSS in early diagnosis of neonatal sepsis with clinicopathological correlation.

Materials and Methods

The present study is a prospective analysis of the hematological profile of 303 neonates admitted to the neonatal intensive care unit at Tertiary care Medical College & Hospital, during the period from November 2014 to February 2016. Neonates born at any gestational age with weight between 1000-2500gm with predisposing perinatal risk factors or if there was clinical suspicion of sepsis with clinical features of neonatal sepsis of IMCI criteria ( WHO 0ct2011) were included in this study. All neonates congenital abnormalities diagnosed at birth, mothers receiving antibiotic treatment regimen , neonates having clinical features resembling sepsis but attributable to causes other than sepsis like birth asphyxia, hypoglycaemia, inborn errors of metabolism, undergoing surgeries were excluded. A detailed clinical history of each patient was recorded after informed and written consent from parents. Method of Collection of Data: The blood samples were sent to the Pathology laboratory in EDTA bulbs and had

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been collected by peripheral venipuncture using aseptic precautions. Neonates were divided into 3 groups : Group 1 (Proven sepsis): neonates with sepsis (with positive blood culture) Group 2 (Probable sepsis): neonates with probable infection (with clinical signs and negative blood culture) Group 3 (No sepsis): normal neonates (without signs of sepsis). The routine hematological investigations included hemoglobin, hematocrit, red blood cell indices (MCV, MCH and MCHC), total WBC count, band cell count, I:T ratio, I:M ratio degenerative changes and platelet count. These investigations were performed on multichannel automated cell counter with standard calibration. Blood film was stained with Leishmanâ&#x20AC;&#x2122;s stained and. analysis of the smear findings were done by the pathologists blinded to the infection status of the neonate. The WBC count was corrected for nucleated red cells and a differential count was performed manually. Immature neutrophils included promyelocyte, myelocyte, metamyelocyte and band forms.[fig:3] A band cell was defined as a neutrophil in which, the nucleus was indented by more than half, but in which the isthmus between the lobes was wide enough to reveal two distant margins with nuclear material between.[fig4] The polymorphonuclear leucocytes were also examined for degenerative morphological changes such as toxic granulation, toxic vacuolization and Dohle bodies. The hematological findings were analyzed according to the hematological scoring system of Rodwell et al. which includes the following 7 findings-Total leucocyte count, total Neutrophil (PMN) count, Immature PMN count, Immature to Total PMN ratio (I:T), Immature to Mature PMN ration (I:M),Platelet count and Degenerative changes in neutrophils. [table 1,2] C-reactive protein levels were also recorded. This test was done in the immunology laboratory by immunochromatography. Micro-ESR also noted. For culture 2ml blood in were taken before administering any antibiotic and sent to lab immediately. Cultures were observed after 24-48hours & 120 hours. If growth was observed, material was further analyzed for isolation of organism & antibiotic sensitivity. If no growth was observed after 7 days, culture was reported negative. Statistical Analysis: The data collected was statistically analyzed as per SPSS version 16, to find out the performance of the test individually and as a scoring

system under the following parameters. Special emphasis was given to Sensitivity, Specificity, Positive predictive value, Negative predictive value. The Chi-Square Test was utilized to compare the observed and expected frequencies in each category. The contingency coefficient was used to measure the strength of association. This research work was approved by the institutional ethical committee.

Result

Age group wise maximum neonates i.e 79.9% were upto 24 hours of life, while 8.6% were 48 to 72 hours old and 6.6% were more than 96 hours of age. Of 303 neonates in the study confirmed sepsis was noted in 25.4% i.e 77 neonates having culture positive and clinical symptoms present, 53.1% i.e 161 neonates were labelled to be having probable sepsis because they ad negative culture reports but clinically positive symptoms. 65 neonates i.e 21.5% were labelled to be not having sepsis because they had culture and clinical negative findings. In patients with â&#x2030;Ľ 2500 gms birth weight, 56.8% had probable sepsis and 16.9% had proven sepsis. Within 1500 to 2499 gms birth weight neonates, 34.8% had proven sepsis and 48.3% had probable sepsis. In 1000 to 1499 gms birth weight neonates 58% had probable sepsis and 27.2% had proven sepsis. While in neonates with upto 1000 gms birth weight 26.7% had either proven and probable sepsis. There was statistically significant (p<0.01) difference of the sepsis status in different birth weight groups of neonates. Of 15 neonates with less than 28 weeks gestational age 26.7% had proven or probable sepsis. Of 73 neonates with 28 to 32 weeks gestational age 21.9% had proven sepsis and 60.3% had probable sepsis. Of 15 neonates with 33 to 37 weeks gestational age 34.7% had proven and 50.5% had probable sepsis. Of 114 neonates with 38 weeks and above gestational age 19.3% had proven sepsis and 54.4% had probable sepsis. There was statistically significant (p<0.05) difference of sepsis status in different gestational age groups of neonates. Sepsis of proven or probable type was noted in 238 neonates in the study. Early onset sepsis was noted in 219 neonates and 19 had late onset sepsis. In proven sepsis neonates 90.9% had EOS while in probable sepsis also 92.5% had probable sepsis. Thus EOS was prominently noted in either of the sepsis status. In 189 neonates with preterm birth, 29.1% had proven sepsis and 52.4% had probable sepsis. While of 114 neonates with term birth 19.3% had developed proven sepsis and 54.4% had probable sepsis. There was statistically significant difference of Low birth weight (p<0.05), very low birth weight (p<0.05) and no significant difference (p>0.05) of mode of delivery, term of birth status of neonates with the sepsis status.

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Of 77 neonates being culture positive having proven sepsis, maximum i.e 41.6% had Klebsiella, 19.5% - E coli and Staph aureus was isolated in 15.6%. In 9.1% neonates pseudomonas, 3.9% coagulase negative staphylococci microorganism was isolated while 2.6% either had Acinetobacter B or Enterococcus or H influenza or Proteus on culture examination.

Of 161 neonates with probable sepsis 55.3% had raised CRP and in 16.9% with no sepsis CRP was raised. The difference of raised CRP in the three groups of sepsis status was statistically very highly significant (p<0.001) [Table:4] For diagnosis of sepsis (Culture positive and Culture negative but clinical positive) the HSS component - Sensitivity, specificity, Positive and negative predictive value of HSS component in sepsis and probable sepsis(n=238).[ Table:5]

Raised CRP level was notes in 144 neonates. Of 77 neonates with proven sepsis, 57.1% had raised CRP. Table 1: Hematologic Scoring System5. Criteria

Abnormality

score

Total WBC count

count ≤5,000/μL ≥25,000 (at birth) ≥30,000 (12-24hrs) ≥21,000 (day 2 onwards)

Total PMN count

No mature PMN seen Increased/ decreased

2 1

Immature PMN count

Increased

I:T PMN ratio

Increased

I:M PMN ratio

≥0.3

Degenerative changes in PMN

Toxic granules/cytoplasmicvacuoles

Platelet count

≤150,000/μL

The normal values are according to Manroe et al[3]: Total PMN count- 1800-5400/ul Immature PMN count – 600/ul Immature: Total PMN ratio- 0.12 Immature: Mature PMN ratio- ≥0.3

Table 2: Interpretation of HSS. Score

Intrepretation

Sepsis less likely

3-5

Sepsis probable

Sepsis very likely

Table3: Distribution of neonates according to sex. Sex

Frequency

Percent

Female

115

38.0

Male

188

62.0

Total

303

100.0

Table 4: Interpretation of HSS score (n=303). Interpretation

Frequency

Percent

Sepsis is unlikely (HSS ≤2)

19.1

Sepsis is very likely (HSS 3or 4)

153

50.5

Sepsis possible (HSS≥5)

30.4

Total

303

100.0

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Table 5: component in sepsis and probable sepsis (n=238) group. Sr.NO HSS component Sensitivity 1 Total WBC count 21.8% 2 Total PMN count 85.7% 3 Immature PMN 88.2% 4 Immature : Total PMN 92.4% 5 Immature : Mature PMN 96.2% 6 Degenerative Change 49.6% 7 Platelet count 39.9% Table 6: Relation of HSS score with sepsis category. Category Score (0-2)% Sepsis Probable sepsis 36(22.4%) No sepsis 22(33.8%)

Specificity 98.5% 23.1% 18.5% 15.4% 12.3% 92.3% 92.3% Score (3-4)% 54(33.5%) 38(58.46)

PPV 98.1% 80.3% 79.8% 80% 80.1% 95.9% 95%

NPV 25.6% 30.6% 30% 35.7% 47.1% 33.3% 29.6% Score (>5)% 77(100%) 71(44.1%) 5(7.69%)

Fig.1: Grouping the neonates according to sepsis status (n=303).

Fig. 2: Age group-wise distribution of neonates (n=303).

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Fig. 3: Peripheral Blood Smear Showing Neutrophilic Leucocytosis Leishman’s(x400).

Fig. 4: Leishman’s Stain (X100) Showing Immature Neutrophils.

Discussion

sequestration secondary to infections and decreased production secondary to bone marrow suppression. Total WBC count has a low predictive value because of a wide range of normal count from 5000 – 20000/mm3. In Rodwell’s study [2] a score of ≥ 3 identified 26 of 27 neonates in the sepsis group, this compared to the present study where it identified all 77 culture positive neonates. A score of ≥ 3 was predictive of sepsis and more reliable indicator of sepsis than the best performing single parameter, the I:M ratio. Thus HSS, which is a combination of 7 parameters, is more diagnostic then any single parameter.

Sepsis is the commonest cause of neonatal mortality. It is responsible for about 30-50% of the total neonatal deaths in developing countries. It is estimated that up to 20% of neonates develop sepsis and approximately 1% die of sepsis related causes.[4] The definite diagnosis of septicemia is made by a positive blood culture which requires a minimum period of 48-72hrs and yields positive result in 30-40% of cases.[5] An early and accurate etiological diagnosis is not always easy, especially since the disease may start with minimal or non-specific symptoms. Delayed treatment until clinical recognition of signs and symptoms of sepsis entails risk of preventable mortality, notwithstanding the fact that presumptive antibiotic therapy may result in overtreatment. In order to diagnose septicemia early, several rapid diagnostic tests have been described, which are easily performed and have the benefit of quick availability of reports. [5] [Table: 6] In the present study, score of >3 detected 202 out of 238 cases (84.9%) of culture positive proven sepsis and probable sepsis. Rodwell study [2] detected around 88% of neonates with sepsis and probable sepsis. Makkar et al[6] found 93.7%, of cases with microbiological and clinical evidence of sepsis. 36 out of 58 neonates had clinical evidence of sepsis though they had low HSS score. Thus high HSS score was more reliable predictor of sepsis than a low HSS score suggesting absence of disease. In the present study, I:T, I:M ratio has high sensitivity and specificity, high PPV and NPV comparable to many other studies.[5,6,7,8,9,10,11]

The higher the score, the greater was the certainty of sepsis being present. In Rodwell’s study and the current study, 85-87% of positive cases had score of ≥ 4. A score of ≥ 5 was even more predictive of sepsis. With a score which ranged between 0-2 there was a 99% probability that sepsis was absent. The HSS should improve the efficiency of the CBC as a screening test for sepsis and permits an objective assessment of hematological changes. Ghosh et al[6] analysed the hematological profiles in the light of the HSS and found that I:M ratio was the most reliable indicator in identifying infants with sepsis. The present study also showed that I:M ratio was most reliable.

Conclusion

Thrombocytopenia has poor sensitivity but high specificity. It is frequently associated with sepsis and indicates poor prognosis. This is due to increased platelet destruction,

The HSS is simpler, quick, cost effective and readily available tool in the early diagnosis of neonatal sepsis and could provide a guideline to decision regarding antibiotic therapy. We preferred to study efficacy of HSS alone as other parameters are costly to perform and require high technical lab support which would not have been relevant to rural setup like ours. Instead, a simple peripheral smear based test like HSS, which can distinguish between infected and non infected neonates with little laboratory backup is indeed a boon for peripheral pathologists and neonatologists.

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Reference

National Neonatal Perinatal Database 2002-2003 report. http://www.nnfi.org/images/NNPD2002-03.pdf. (Last accessed on Jan 22, 2016).

Makkar M, Gupta C, Pathak R, Garg S, Mahajan NC. Performance of evaluation of hematological scoring system in early onset neonatal sepsis. Journal of Clinical Neonatology 2013 Jan-Mar;2(1):25-29.

Rodwell RL, Leslie AL, Tudehope DI. Early diagnosis of neonatal sepsis using a Haematologic Scoring System. J Pediatr 1988;112:761-767.

Supreetha MS et al. Evaluation of Neonatal septicemia using Haematological parameters. International Journal of Recent and Scientific Research vol 6,Issue 2,pp 2775-2778,Feb 2015.

Manroe BL, Weinberg AG, Rosenfeld CR, Browne R. The neonatal blood count in health and disease. Reference values for neutrophilic cells. J Pediatr 1979;95(1):89-98.

Shankar MJ, Agrawal R, Deorari AK, Paul V. Sepsis in newborn. AIIMSNICU protocols 2008:1-18.

Chandna A, Rao MN, Srinivas M, Shyamala S. Rapid Diagnostic Tests in Neonatal Septicemia. Indian J Pediatr 1988;55:947-953.

Ghosh S, Mittal M, Jaganathan G. Early diagnosis of neonatal sepsis using a hematological scoring system. Indian J Med Sci 2001;55:495-500.

Narasimha A, Harendrakumar ML. Significance of hematological scoringsystem (HSS) in early diagnosis of neonatal sepsis. Indian J Hematol Blood Transfus 2011;27(1):14-17. 10. Gheibi S, Fakoor Z, Karamyyar M, Khashabi J, Ilkhanizadeh B, Sana FA, et al. Coagulase negative staphylococcus; most common causes of neonatal septicemia in Urmia, Iran. Iran J Pediatr 2008 Sept;18(3):237-243. 11. Khair KB, Rahman MA, Sultana T, Roy CK, Rahman MQ, Shahidullah M et al. Role of hematological scoring system in early diagnoses of neonatal septicemia. BSMMU J 2010;3(2):62-67

*Corresponding author: Dr. Bhagyashree Ahirrao, Department of Pathology, ACPM Medical college Morane, Dhule, Maharashtra, India Phone: +91 9823959415 Email: dr.bhagyashreeahirrao@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 06.05.2017 Date of Acceptance : 19.09.2017 Date of Publication : 21.12.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Original Article DOI: 10.21276/APALM.1506

Role of Immunohistochemistry in Trephine Biopsies of Bone Marrow: A 5-year Retrospective Study from A Tertiary Care Hospital Ravi Teja J, Lenna Dennis Joseph*, Febe Renjita Suman, Jerusha Samuela Jacob, Rithika Rajendran, Jesu Magdalene S and Sai Shalini CN Dept of Pathology, Sri Ramachandra Medical College & Research Institute, Chennai (India)

ABSTRACT Background: Bone Marrow Biospy (BMB) often needs ancillary tests like Immunohistochemistry (IHC) to confirm the morphological diagnosis, to categorize malignant conditions. Methods: A retrospective study was done on BMB with IHC done on the sections are reviewed and analysed correlating the clinical, Bone marrow aspirate(BMA) and BMB interpretations. Result: A 5 year study on 934 BMB required IHC as an adjunct on 16.2% of the biopsies. It was done on 10.6% of cases. The distribution of cases was 43% of Acute leukemia(AL), 19% multiple myeloma, 8& lymphomas, 9% metastasis. 20 % of other cases which included chronic lymphocytic leukemia(CLL), myelodysplastic syndrome (MDS), megaloblastic anemias, infective conditions and reactive marrows. Conclusion: IHC is a reliable method to confirm and categorize AL, to differentiate reactive and neoplastic plasmacytosis, to confirm, categorize and stage lymphomas, to detect metastasis. Keywords: Bone Marrow, Immunohistochemistry, Leukemia, Metastasis, Trephine Biopsy

Introduction

Bone marrow interspersed within the intertrabecular spaces of the medullary cavity in the bone, has complex anatomy and physiology which are valuable to support almost all the systemic functions. Its involvement in diseases varies either from within as primary marrow diseases or as reflected changes of systemic diseases. Bone marrow studies are indicated in various haematological and non-haematological disorders. Bone marrow trephine biopsy (BMB) had been standardized in 1943 after many trials and studies since it was first performed by Mosler.[1,2] It had been suggested that bone marrow aspirates (BMA) and BMB should be assessed by a competent person.[3] The need for histochemical and immunohistochemical stains depend on the clinical circumstances and the preliminary findings. Indications for immunohistochemistry (IHC) on BMB depend on the morphology and the panels are often classified as primary panel and secondary panel. In this era of immunophenotyping, IHC is almost always done for lymphoma diagnosis and categorization in lymph nodes. However International Council for Standardization of in Haematology (ICSH) had set guidelines for the standardization of bone marrow IHC.[4] The present study is aimed to discuss the diagnostic utility of IHC in BMB.

Materials and Methods

This is a retrospective study done on BMB reported in the department of pathology for a period of 5 years (Jan 2011Dec 2015). The bone marrow registers were reviewed and all bone marrow biopsies for which IHC was requested were selected. All the details regarding clinical scenario, relevant laboratory parameters and BMA reports available in the registers were tabulated. The IHC based diagnoses were grouped into acute leukemias (AL), multiple myeloma (MM), lymphoma, metastatic deposits and others. The demography, clinical details, peripheral blood findings, BMA morphology and BMB were studied. Standard descriptive statistics were used.

Result

A total of 934 BMB were received during the study period of 5 years (2011-2015). The pathologists required IHC as an adjunct to morphology in 151 (16.2%) cases. IHC was done on 99 (10.6%) biopsies and not done on 52 (5.6%) cases. IHC was done with the BioGenex Xmatrx automated staining with rabbit and mouse monoclonal antibodies CD45, MPO, CD20, CD3, CD5, CD10, Tdt, CD34, CD117, CD68, CD138, kappa, lambda, fascin, BCL2, cyclin D1, PAX5, CD10, ALK and EMA as primary

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antibodies. The chromogen used was 3,3 diaminobenzidine (DAB). The age of the patients ranged from less than 1 year to 83 years with a male: female ratio of 1.5:1. Table 1 shows the distribution of the cases with clinical diagnosis. AL was diagnosed on 43% of BMB of which 69.7% were clinically suspected as inflammatory conditions. The BMA was inadequate in 48.8% cases and 6.97% were false negative. Multiple myeloma was diagnosed on 19% of the biopsies by IHC (Fig 1A, 1B). 21% were not suspected and 26.3% BMA were inadequate. 10.5% BMA were reported as false negative. Lymphoma infiltration diagnosed on morphology was confirmed and categorized on 8% of biopsies by IHC. Among them only 25% were received for staging. Other 75% of BMB showed lymphoma infiltration which was not suspected clinically. 62.5% BMA were inadequate and false negative report was provided in 12.5% cases. Metastasis was identified in 9% of cases of which 33.3% cases were from known cases of primary solid tumors

(Fig 2B,2B). Malignant solid tumors were not suspected in 66.7% cases. BMA was inadequate in 33.3% of cases. The other 20% of cases included leukemic conditions (chronic myeloid leukemia-1, chronic lymphocytic leukemia-2, myelodysplastic syndrome (MDS-4), 15% anemia cases (megaloblastic anemia -2, aplastic anemia-1), inflammatory conditions (15%) and reactive marrows(33.3%).The reactive marrows were suspected to be MM, MDS and ALs. IHC was contributory to the diagnosis in 46% of the cases.

Discussion

BMA and BMB play an important role in the diagnosis of various haematological and non-hematological diseases. BMA are good samples for morphologic assessment, cell count, flowcytometry (FCM) and genetic workup. Sampling errors are common due to disease conditions or technical default. IHC studies are possible on BMB which aid in confirming or detecting various disease conditions. In this study done on the IHC work up of BMB, AL was confirmed and categorized in 43% of cases. FCM on BMA and peripheral blood if the blast count is high is

Table 1: Clinicopathological correlation by IHC. Category By IHC

CLINICAL DIAGNOSIS

No. of cases

BMB & IHC

ACUTE LEUKEMIA ANEMIA SUSPECTED MALIGANACY PANCYTOPENIA MDS RELAPSE INFLAMMATORY/INFECTIOUS OTHERS

13 8 8 6 4 2 1 1

AML 6 4 3 3 4 0 0 1

ALL 7 4 5 3 0 2 1 0

MULTIPLE MYELOMA (n=19)

Multiple myeloma Lymphoma MDS Anemia

15 1 2 1

Kappa 9

Lambda 10

LYMPHOMA (n=8)

Lymphoma Pancytopenia Inflammatory/infectious MDS Others

2 3 1 1 1

HL 5

NHL 3

1 1 1 2 1 1

1 Prostatic carcinoma Adenocarcinoma lung Metastatic carcinoma Rhabdomyosarcoma GIT metastasis

ACUTE LEUKEMIA (n=43)

METASTATIC BONE DISEASE (n=9)

KNOWN PRIMARY

Prostatic adenocarcinoma Retinoblastoma

UNKNOWN PRIMARY

MDS Multiple Myeloma Anemia PNET Gastric ulcer

Others(n=20)

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Fig. 1: A Photomicrograph shows sheets of plasma cells positive for CD138, IHC (20X); Fig 1B Photomicrograph shows sheets of plasma cells positive for Lambda light chain, IHC (20X).

Fig. 2: A Photomicrograph shows glands & sheets of tumor cells positive for PSA, IHC (20X); Fig 2B Photomicrograph shows tumor cells arranged in glands positive for CK7, IHC (20X).

the method of choice for immunophenotyping in AL.[5] However, this was not possible when samples were not collected for want of clinical suspicion or when BMA is a dry tap. The antibodies used commonly are CD45, MPO, CD20, CD3, CD5, CD10, Tdt, CD34, CD117. CD68 was used when monocyte lineage was suspected. This is in accordance to earlier studies.[6,7] A study on ALL in 2008 showed high concordance between FCM and IHC with CD3 and CD10 in ALL than CD20.[8] Approximate blast count could be done with CD34 and CD117.[9] The 6.97% false negative BMA were reviewed which showed scanty particles with less number of blasts. IHC could estimate the approximate blast percentage as both BMA and BMB were done simultaneously. However CD19 and CD79a were not included in our panel of markers.

BMB was done in almost all suspected cases of myeloma. [10] The antibodies used were CD138, kappa and lambda light chains. IHC had been used to detect plasma cells and monoclonality.[11] In our study IHC was useful in confirming the morphological diagnosis of plasma cell myeloma. Monoclonality for either kappa or lambda light chain was noted (0.9:1 ratio). 21% cases were not suspected clinically. 10.5% BMA gave false negative report of a reactive marrow. IHC done to assess the clonality also helped to diagnose these false negative as well as unsuspected cases. 10.5% cases were clinically suspected to be MM and BMA and BMB also showed high plasma cell count. By IHC these cases were found to be reactive.

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Among the lymphomas diagnosed by IHC on BMB 25% cases were received for staging. Only those BMB where

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there were diagnostic challenges were subjected to IHC. Identification of bone marrow involvement is highly essential from clinical point of view.[12] A minimum panel of antibodies positive in the lymph node biopsy were used. However, 75% of cases were clinically undiagnosed . As lymphoma was suspected, IHC was done which helped in differentiating lymphoma from non-malignant lymphoid aggregates, diagnostic confirmation and categorization. A study done in 2014 also identified 50% of lymphomas by IHC.[13] To differentiate Hodgkin lymphoma(HL) and non-Hodgkin lymphoma(NHL), the antibodies used were CD45, CD30, CD3, CD20, CD5. The NHL were further categorized by fascin, BCL2, cyclin D1, PAX5, CD10, ALK and EMA. Studies done in the past also preferred to use a panel of antibodies.[14] 55.6% of BMA in these patients were inadequate and 11% cases were false negative. Metastasis from solid tumors was confirmed in 9% of BMB by IHC. 2 BMB from known prostatic adenocarcinoma (PAC) and another clinically suspected MDS needed IHC confirmation of prostatic carcinoma. The antibodies used for PAC were CD45, vimentin and PSA. The BMB from clinically suspected MDS case was provisionally diagnosed as PAC and confirmed by IHC. In 2013 it was found micrometastasis in PAC in 11.2% of their patients by IHC.[15]The other metastasis were adenocarcinoma from lung, metastatic carcinomas, rhabdomyosarcoma, gastrointestinal tract metastasis and retinoblastoma. In our study primary tumor was identified in 44.4% cases, but in 22.2% cases further metastatic workup beyond IHC was not possible due to economic constraints. Studies have shown BM metastasis from breast cancer, gastric cancer and prostate cancer are common and are prognostically significant.[16] But in our study BMB were not received for many solid tumors. The other 20% BMB included chronic leukemias, MDS, anemic conditions, inflammatory conditions and reactive marrows. IHC was done in MDS for approximate blast count by CD34 and CD117. In two cases of megaloblastic anemia IHC was done with CD45, MPO and glycophorin to differentiate erythroblasts and myeloblasts as the BMA were inadequate with a peripheral pancytopenic picture. IHC was also done to rule out metastasis as isolated tumor cells.[17] IHC for CMV was done for the confirmation of inclusion bodies in one case. In our study, though IHC was requested in 16.2% it was done on 10.6 % of BMB.IHC helped to confirm AL, MM, lymphomas and solid tumor infiltration in the BM.IHC also helped to diagnose AL, MM and lymphoma which were falsely reported as negative by BMA (6%). IHC was utilized to trace the primary site for malignancy in unknown solid tumors metastasized into BM. IHC was also used to

distinguish benign and malignant proliferative conditions and inflammatory diseases. 52(5.6%) biopsies were not subjected to IHC due to economic constraints. In few cases flowcytometry confirmed and categorized AL minimizing the necessity for IHC. The morphological diagnosis in these cases included 73% acute leukemias (n=38), 17% plasma cell myelomas (n=9), 3.8% lymphomas(n=2), 3.8% MDS (n=2), and aplastic anemia(n=1). The MDS and aplastic anemia biopsies required IHC as there were few atypical cells suspicious of blasts. Hence a provisional diagnosis was given in all the above biopsies indicating the necessity for IHC.

Conclusion

IHC is a reliable, robust, technologically standardized procedure which can be performed in BMB. It is a valuable diagnostic tool to confirm and categorize AL especially when the BMA is inadequate for FCM. IHC is a useful technique to differentiate reactive and neoplastic plasmacytosis. IHC is also useful in approximately estimating blast percentage in post chemotherapy AL and MDS. It is better to perform IHC in all BMB with diagnostic challenge for better quality of confirmatory report and patient care.

Reference 1.

Mosler F. Klinische Symptome und Therapie der MedullalĂ¤ren Leukemi. Berl Klin Wochenschr 1876;13:233.

Turkel HE, Bethell FH. Biopsy of bone marrow performed by a new and simple instrument. J Lab Clin Med. 1943 Jul;28:1246-51.

Bain BJ. Bone marrow trephine biopsy. Journal of clinical pathology. 2001 Oct 1;54(10):737-42.

Torlakovic EE, Brynes RK, Hyjek E, Lee SH, Kreipe H, Kremer M, McKenna R, Sadahira Y, Tzankov A, Reis M, Porwit A. ICSH guidelines for the standardization of bone marrow immunohistochemistry. International journal of laboratory hematology. 2015 Aug 1;37(4):431-49.

Jennings CD, Foon KA. Recent advances in flow cytometry: application to the diagnosis of hematologic malignancy. Blood. 1997 Oct 15;90(8):2863-92.

Bavikatty NR, Ross CW, Finn WG, Schnitzer B, Singleton TP. Anti-CD10 immunoperoxidase staining of paraffinembedded acute leukemias: comparison with flow cytometric immunophenotyping. Human pathology. 2000 Sep 30;31(9):1051-4.

Orazi A, Cotton J, Cattoretti G, Kotylo PK, John K, Manning JT, Neiman RS. Terminal deoxynucleotidyl transferase staining in acute leukemia and normal bone marrow in routinely processed paraffin sections. American journal of clinical pathology. 1994 Nov;102(5):640-5.

Layla A, Al Gwaiz LA, Bassioni W. Immunophenotyping of acute lymphoblastic leukemia using immunohistochemistry

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in bone marrow biopsy specimens. Histology and histopathology. 2008;23(10-12):1223-8.

Morphological Diagnosis in Non Hodgkinâ&#x20AC;&#x2122;s Lymphoma. Blood. 2014 Dec 6;124(21):5393-5393.

Jain S, Mahapatra M, Pati HP. CD34 immunohistochemistry in bone marrow biopsies for early response assessment in acute myeloid leukemia. International Journal of Laboratory Hematology. 2015 Dec 1;37(6):746-51.

14. West RB, Warnke RA, Natkunam Y. The usefulness of immunohistochemistry in the diagnosis of follicular lymphoma in bone marrow biopsy specimens. American journal of clinical pathology. 2002 Apr 1;117(4):636-43.

10. Smith A, Wisloff F, Samson D. Guidelines on the diagnosis and management of multiple myeloma 2005. British journal of haematology. 2006 Feb 1;132(4):410-51.

15. Singh MK, Goel MM, Singh US, Dalela D, Shankhwar SN, Kumar A. Detection of bone marrow metastases in prostate cancer: Role of trephine biopsy and Immunohistochemistry. Clinical Cancer Investigation Journal. 2013 Oct 1;2(4):319.

11. Chang H, Yeung J, Qi C, Xu W. Aberrant nuclear p53 protein expression detected by immunohistochemistry is associated with hemizygous P53 deletion and poor survival for multiple myeloma. British journal of haematology. 2007 Aug 1;138(3):324-9. 12. Vinciguerra V, Silver RT. The importance of bone marrow biopsy in the staging of patients with lymphosarcoma. Blood. 1973 Jun 1;41(6):913-20. 13. Arami S, Svec A. Immunohistochemistry in Staging Bone Marrow Biopsy Specimens: a Useful Adjunct for

16. Kucukzeybek BB, Calli AO, Kucukzeybek Y, Bener S, Dere Y, Dirican A, Payzin KB, Ozdemirkiran F, Tarhan MO. The prognostic significance of bone marrow metastases: Evaluation of 58 cases. Indian Journal of Pathology and Microbiology. 2014 Jul 1;57(3):396. 17. Krishnan C, Twist CJ, Fu T, Arber DA. Detection of Isolated Tumor Cells in Neuroblastoma by Immunohistochemical Analysis in Bone Marrow Biopsy Specimens. American journal of clinical pathology. 2009 Jan 1;131(1):49-57.

*Corresponding author: Dr.Leena Dennis Joseph, Department of Pathology, Sri Ramachandra Medical College & Research Institute, Chennai-600116, India Phone: +91 9994081470 Email: febemd@gmail.com Date of Submission : 04.05.2017 Date of Acceptance : 04.09.2017 Financial or other Competing Interests: None. Date of Publication : 21.12.2017

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Original Article DOI: 10.21276/APALM.1562

Role of Renal Biopsy in Evaluation of Morphological Spectrum and Pathogenesis of Lupus Nephritis Archana Chirag Buch*, Rupali Bavikar, Shreya Rajesh Patel, Swapnil karnik and Jehan Ansari Dr. D.Y. Patil Medical College , Hospital And Research Center , Pune (India)

ABSTRACT Introduction: Lupus nephritis is a common complication in patients with systemic lupus erythematous (SLE) characterized by deposition of antibodies in glomerular, vascular and tubulointerstitial compartments of the kidney. International society of Nephrology/ Renal Pathology Society (ISN/RPS) 2003 classification plays an important role in the diagnosis and predicting prognosis in these cases. The aim of this study was to evaluate the morphological changes in renal biopsy and to correlate them with the pathogenesis of lupus nephritis. Methods: This was a cross sectional study of 50 renal biopsies of lupus nephritis from two tertiary care centers. Clinical data, lab findings, histopathology slides and immunofluorescence findings were retrieved and analysed by two senior pathologists. The activity and chronicity indices were calculated by each pathologist and were compared for inter observer variation. The activity index was correlated with immunofluorescence findings. Results: The mean age of patient was 29.75 years with M:F ratio of 1:9.The frequency of each class according to ISN/RPS classification was class I (0%),class II (20%),class III (2%),class IV (62%),class V (14%) and class VI (2%). All class IV cases were global and active with maximum activity indices. The mean of activity and chronicity indices of pathologist A was 8.83, 2.7 and pathologist B was 9.13, 1.9. High activity showed increased intensity (3+) of IgG and C3 staining on immunofluorescence. Conclusion: We found that activity and chronicity indices calculated on renal biopsy along with immunofluorescence findings can suggest the extent of pathogenesis of lupus nephritis. This can be useful for further management and follow up. Keywords: Lupus nephritis, SLE, Renal Biopsy,

Introduction Lupus nephritis is a major cause of morbidity and mortality affecting 70% of patients with systemic lupus erythematosus (SLE).[1] Lupus nephritis is manifested by proteinuria, active urinary sediments and progressive renal dysfunction. It follows relapsing, remitting pattern which may vary from patient to patient.[2] Lupus nephritis is characterized by anti dsDNA antibodies and immune mediated injuries which lead to variety of morphological changes in glomerular, vascular and tubulointestitial compartments of kidney. These changes vary depending on deposition of different subsets of antibodies at different phases of the disease which can be demonstrated by immunofluorescence.[3] Morphology of lupus nephritis is graded along with activity and chronicity by the modified International society of Nephrology /Renal Pathology Society (ISN/RPS) 2003 classification. This classification is useful for prognosis and therapeutic management. We aim to study the morphological spectrum including immunofluorescence findings of renal biopsies of lupus nephritis and correlate it with pathogenesis. The objectives of our study were to classify all the renal biopsies of lupus patients according to modified ISN/RPS classification,

to determine activity and chronicity indices, to study the interobserver variation in calculation of these indices and to correlate morphological and immunofluorescence findings with pathogenesis.

Material and Methods This was a cross sectional study enrolling 50 renal biopsies over a period of two years (2014-2016) diagnosed as lupus nephritis from two large tertiary medical centers in Pune (Western Maharashtra). All the patients with antinuclear antibody and anti dsDNA positive cases with adequate renal biopsy having both histopathology and immunofluorescence reports were included in the study. The patients with inadequate renal biopsy that includes absence of glomeruli and lack of tissue for immunofluorescence were excluded from the study. Slides, blocks and relevant clinical data along with immunofluorescence findings of the renal biopsies were retrieved from the archives of department of Pathology of both the institutes. Institutional ethical committee clearance was taken. The renal biopsy slides were studied by light microscopy using Haematoxylin and Eosin (H&E), Periodic acid Schiff

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(PAS), Masson trichome and Jones methenamine silver stains. Renal biopsies were classified based on modified ISN/RPS classification.4 The activity and chronicity indices were calculated independently by two senior pathologists. Active lesions were considered according to presence of cellular crescents, fibrinoid necrosis, endocapillary proliferation, leucocytic infiltration, large subendothelial deposits and interstitial inflammation. 5 The chronicity was graded based on glomerulosclerosis, tubular atrophy, interstitial fibrosis and fibrous crescents.5 Each activity and chronicity factor was graded on a scale of 0-3 depending on the percentage of involvement of glomeruli : 0 (absence of lesion), 1 (25% of glomeruli involved), 2 ( lesions involving 25-50% of glomeruli), 3 (lesions involving >50% of glomeruli). The activity factors, cellular crescents and necrosis were multiplied by factor 2. Total was taken as activity index out of maximum of 24. Total chronicity index was out of 12. The immunofluorescence for immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), C3, C1q findings were retrieved from the data. The intensity of fluorescence was graded from 0-3. The findings were analyzed by Microsoft Excel and statistical package Win pepi (version 11.65). Descriptive data was presented as mean and percentages and the agreement between two raters was done by interclass correlation coefficient.

Results The mean age of patient was 29.75 years with the range of 14 to 64 years. The male to female ratio was 1:9. The prevalence of various classes of lupus nephritis is as shown in Table 1.Twenty two patients (44%) were diagnosed as nephrotic syndrome on admission, six (12%) were diagnosed as acute nephritic syndrome, two patients (4%) presented as rapidly progressive nephritic syndrome according to the WHO criteria. The remaining 20 patients (40%) showed urinary abnormalities such as microscopic hematuria and /or proteinuria. Serum creatinine levels were raised in almost all cases ranging from 1.2- 8 mg/dl, higher values were seen in class IV, V and VI of ISN/RPN classification. The average number of glomeruli were 10.4 among all renal biopsies. The maximum number of cases belonged to class IV diffuse lupus nephritis. All cases of class IV were of active diffuse global proliferative type of lupus nephritis. Segmental lesions were not found. The different morphological findings of various classes are as shown in Figure 1. www.pacificejournals.com/apalm

The activity index was calculated on H&E stained slides on light microscopy according to the Austin et al scoring system. [5] We divided the wide range of activity into three groups. Mild activity when the activity index was between zero to eight, moderate activity when it was between nine to sixteen and severe activity when between seventeen to twenty four. The prevalence of activity and chronicity grading in our study is as shown in Table 2. In our study, Class II and class V cases had low activity and chronicity indices. We had only single case of class III and VI. Class III case had mild activity (6) and chronicity (4). Class VI had moderate activity and severe chronicity. This case was probably diffuse proliferative glomerulonephritis going into advanced sclerosis lupus nephritis. Most of our cases were diffuse proliferative lupus nephritis (n=31) which revealed high activity and low chronicity index. The activity in these cases was mainly due to endocapillary proliferation (96%), leucocytic infiltration (93.3%), interstitial inflammation (73.3%), subendothelial deposits (60%), fibrinoid necrosis (40%) and cellular crescents (11%). The presence of wire loop and hyaline thrombi formation indicates subendothelial form of immune deposits large enough to be detected by H&E, but better appreciated by PAS stain. These were mostly seen in class IV lupus nephritis. Various activity and chronicity are depicted in Figure 2, 3,4.The activity and chronicity indices were calculated independently by two pathologists. The results were analysed statistically as shown in Table: 3. Interobserver reliability of activity was 0.758 with 95% C.I.: 0.551 to 0.877. Interobserver reliability of chronicity was 0.897 with 95% C.I.: 0.796 to 0.950. Thus there was excellent correlation between the reporting of activity and chronicity index between the two pathologists. We found that all cases of lupus nephritis were showing full house granular positivity for IgG, IgA, IgM and C3 in the glomerular basement membrane and mesangium. (Chart 1) However it was noted that the intensity of immunofluorescence varied. IgG and C3 were more intensely stained as compared to IgA and IgM. We also found IgG and C3 deposits in the tubular basement membrane. (Figure 5) Corresponding histopathology of such cases revealed tubular atrophy and interstitial inflammation. We did not have C1q and C4 results in all cases; hence were not considered for the analysis. While analyzing activity with immunofluorescence results, we opine that renal biopsies with increased activity had high intensity of immunofluorescence staining. Whereas there was inverse relation with the chronicity. Table 4 eISSN: 2349-6983; pISSN: 2394-6466

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Table 1: Distribution of cases according to modified ISN/RNP classification. Class

Names

Minimal mesangial Lupus nephritis

Number ( %)

Mesangial proliferative lupus nephritis

III

Focal lupus nephritis

Diffuse lupus nephritis

31 (62%)

Membranous lupus nephritis

7 ( 14%)

Advanced sclerosis lupus nephritis

1 ( 2%)

0 ( 0) 10 ( 20%) 1 ( 2%)

Table 2: Prevalence of activity and chronicity grading in lupus nephritis Grade

Mild (I)

Moderate (II)

Severe (III)

Activity

32(64%)

18 (36%)

0 ( 0%)

Chronicity

43 (86%)

6 (12%)

1 (2%)

Pathologist

A (Mean +/- SD)

B (Mean +/- SD)

95% C. I.

Activity index

8.83+/- 2.73

9.13+/- 3.20

-0.47 to 1.07.

Chronicity index

2.7+/- 2.58

1.97+/- 2.72

0.28 to1.18

Table 3: Inter observer variation

Table 4: Association of activity and chronicity grading with intensity of IgG. No. of cases

1+ N(%)

2+ N (%)

3+ N (%)

Mild activity (0-8)

7 (21)

18 (56)

7 (21.8)

Moderate activity (9-16)

3(16.7)

5 (27.3)

10 (55)

Mild chronicity (0-4)

5 (11.7)

22 (51.1)

16 (37.2)

Moderate chronicity (5-8)

1(16.7)

3 (50)

2 (33.3)

Severe chronicity (9-12)

0(0)

1 (100)

0(0)

Chart 1: Correlation of severity of immunofluorescence with different antibodies.

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Fig. 1: A: Mesangial proliferative LN B: Focal segmental LN C : Diffuse LN D: Membranous LN E: Advance Sclerosis LN ( H&E,x400).

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Fig. 2: A: Cellular crescents, B: fibrinoid necrosis, C: endocapillary proliferation, D: leucocytic infiltration, E: large subendothelial deposits F: interstitial inflammation. ( H& E, X400).

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Fig. 3: A: Glomerulosclerosis B: Tubular atrophy and interstitial fibrosis C: Fibrous crescents. ( H& E, X400).

Fig. 4: A. Photomicrograph of hyaline thrombi (short arrow) ( H & E, x400) B. Photomicrograph of hyaline thrombi ( short arrow), Wire loop lesions (long arrow) ( PAS, x400) .

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Fig. 5: Lupus nephritis Immunofluorescence A: IgG (3+) B; C 3 ( 3+); C: IgM ( 2+); IgA ( 1+).

Discussion Systemic lupus erythematosus is a multisystem autoimmune disease characterized by development of antibodies towards chromatin material, particularly to dsDNA. There are several other antibodies such as anti nucleosome, antiRo, anti-Smith, anti-C1q, anti-alpha actinin, anti-annexin II and anti ribosomal P protein.[6, 7] These antibodies bind to various kidney structures like glomerular basement membrane, mesangial cells, glomerular epithelial cells, glomerular endothelial cell, podocytes and tubular basement membrane.[6,7] These antibodies have pathogenic potential causing lupus nephritis. Anti-dsDNA antibodies , actually forms a part of natural antibodies of IgM type. In lupus nephritis, these antibodies change to IgG type that increases the pathogenic potential. Anti-dsDNA antibodies are found in 70-96% of patients with lupus nephritis compared to 0.5% in patients with non lupus autoimmune disease or in healthy individual. [6]. Pathogenicity in the lupus nephritis is due to the passive entrapment of circulating dsDNA/anti-dsDNA immune

complexes in the glomeruli. Deposition of immunoglobulins and activation of complement within the mesangium is a cardinal feature in lupus nephritis. Complement activation plays an important role in the pathogenesis. 8 This leads to increased mesangial proliferation, apoptosis, activation of PKC and MAPK signaling pathways and increase in synthesis of proinflammatory and profibrotic mediators and cytokines. These ultimately contribute to progressive inflammatory and fibrotic process leading to various morphologic changes. The newer ISN/RPS classification grades these pathogenetic response in the form of activity and chronicity with important modifications concerning qualitative and/or quantitative differences between various subclasses. [9] We found maximum number of female cases with mean age of 29.75 years. This was in concordance with study done by Gomma W. et al. [9] However Nezhad ST et al had lower mean age of patients with lupus nephritis. This may be probably due to his lower sample size. Our clinical presentations of the cases were comparable with many international and national reports of lupus nephritis. [10-12]

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The findings regarding the distribution of cases in classes are similar to reports from different region continents. We found that class IV diffuse proliferative lupus nephritis is the most common pathological lesion in our study. This finding is similar to various previous studies.[ 11-19] However some studies from Arab countries showed higher incidence of Class III. [20,21] Different ethnic group and varied sample size may be the explanation for this difference. Global active lesions were maximum in our study which is similar to findings of others. [12,22] We had no case of Class I nephritis. Literature search also revealed no or less cases of class I nephritis. [9] This may be explained by the fact that electron microscopy is mandatory to diagnose this lesion, as on light microscopy, all glomeruli appear normal. [19] Renal biopsy is also performed in the patients in late stage, in most of the cases when renal functions are deranged. This also suggests that renal biopsy should be performed prior to appearance of renal symptoms in SLE patients for early detection of evidence of lupus nephritis. The activity and chronicity index were graded as mild, moderate and severe. These reports are higher as compared with those reported by Hiramatsu et al [23] The description of class III and IV depends on the activity and chronicity on the proportion of glomeruli with active and chronic lesions. Austin et al devised a semiquantitative scoring system for activity and chronicity by grading and adding different morphologic components in a given biopsy as a guide to treatment and prognosis.[12] Activity index can be a significant risk factor for secondary outcome like end stage renal failure leading to death. Activity index is related to circulating gamma interferon levels thereby reflecting immunological disease activities in patients with SLE. Activity index can also be a useful guide for response to the treatment.5 We identified wire loop and hyaline thrombi formation, which indicated sub endothelial form of immune deposits large enough to be detected by H&E, mostly in diffuse proliferative glomerulonephritis. It is believed that presence of wire loop in LM study is correlated with massive sub endothelial deposition on electron microscopy. [24]

mild activity and others (36%) showed moderate activity. We did not have follow up data; which was one of the major limitation of our study. Chronicity index is a measure of sclerosed glomeruli, fibrous crescents, interstitial fibrosis and tubular atrophy. Tubular atrophy and interstitial fibrosis are the main parameters related to renal function and responsiveness to therapy[25] Variable degree of tubulointerstitial inflammation and fibrosis are found in all forms of chronic progressive renal diseases. The severity of tubulointerstitial changes inversely correlates with renal prognosis. We also found immune aggregates along the renal tubular basement membrane in few cases. It is stressed that the persistence of interstitial inflammation after therapy may predict the renal failure. Chronicity index is a better index to determine or predict the prognosis of the patients. Grcevska L et al had a study with follow up data of 10 years which revealed that probability of renal failure 10 years after the diagnosis of lupus nephritis was higher when activity was >11 and chronicity of >3. There was no probability of renal failure in 90% in case of zero chronicity. When Chronicity was >/= 1, there was 50% chance of renal failure.[26] Our study revealed 21( 42%) cases with zero chronicity, 22 (44%) with 1-3 and 7 (14%) cases were >3 chronicity. Activity and chronicity indices are the major morphological parameters deciding the prognosis of the patient, it is essential to have least subjective variation. The newer classification has reduced the subjective variation by quantifying the grading criteria. We still studied the interobserver variation in identifying activity and chronicity indices by two senior nephropathologists. Interobserver variation in our study was statistically not significant. This was due to correct semiquantitative measurement for grading the activity and chronicity indices into 0-3 scale. Furthermore we found that grouping the indices in mild, moderate and severe grading almost nullifies the interobserver variation. [5]

We found that mean activity was maximum in Class IV lesions and least in class V. When graded into mild, moderate and severe, maximum biopsies (64%) showed

We found that the intensity of IgG was maximum, followed by C3 in cases of lupus nephritis. This confirmed that the pathogenicity is mainly because of anti ds/DNA, which is IgG type of antibody and the complement activation. These findings were also in concordance with Lai F et al who revealed that IgG immunofluorescence was predominant among other immunoglobulins which were associated with C1, C4 and C3 components.[27] We have attempted to correlate the activity index with the intensity of immunofluorescence findings. We found that 55% of biopsies with moderate activity had high intensity of staining (3+) with IgG. 27.7% had intensity 2+, and 16.6% had intensity 1+. Whereas mild activity index cases

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We believe that activity index might be reflecting the severity of the immunologic disease activities in the patients with lupus nephritis. Hence activity index can be a useful clinicopathologic guide for management of lupus nephritis.

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revealed maximum of 2+ intensity of IgG Staining. Similar findings were also noted with intensity of C3 staining. Intensity of IgA, IgM were not found very significant with the activity index. Ferluga et al., showed statistically significant association between increasing mean values of the activity index and glomerular deposit distribution patterns .28 However study done by Nossent showed no statistical significant correlation between activity index and any of the immunofluorescence intensity (all P values >0.3), while CI showed a negative correlation with IgA deposits only [29] The limitation of the study is lack of electron microscopic findings of the renal biopsies due to unavailability of electron microscope facility and unaffordability. However, many studies have agreed to the fact that lack of electron microscopic findings should not prevent the skilled pathologist from rendering a diagnosis of lupus nephritis using a combination of complete light microscopy and immunofluorescence studies.[18] We also insist on the fact that new classification of lupus nephritis with determination of activity and chronicity of the lesions, clinical data and immunofluorescence findings can help in understanding the pathogenesis and thereby helping in management and prognosis of the patient.

Conclusion We retrospectively analyzed 50 subjects with light microscopy and immunofluorescence findings proven lupus nephritis. We found maximum cases of diffuse proliferative glomerulonephritis ( Class IV) . The activity index co related well with the intensity of immunofluorescence findings. High intensity of IgG and complement C3 confirmed the fact that pathogenicity of lupus nephritis is mainly due to anti ds DNA and complement activation which induces proinflammatory and profibrotic pathways. These can be measured by activity and chronicity index on light microscopy. Thus not only clinicopathological correlation, but detailed light microscopy and immunofluorescence findings play a very important role in judging the immunological progression of the disease; which ultimately helps in deciding the treatment and prognosis.

References 1.

Cameron JS. Lupus nephritis. JASN.1999;10(2):413–24.

Sidiropoulos PI, Kritikos HD, Boumpas DT. Lupus nephritis flares .Lupus 2005;14(1):49-52

Hahn BH. Antibodies to DNA. NEJM 1998;338(19):1359– 1368.

Weening JJ, D’agati D, Schwartz M, et al: The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int 2004 ; 65:521–530,

Austin HA , Muenz LR, Joyce KM, et al: Diffuse proliferative lupus nephritis: Identification of specific pathologic features affecting renal outcome. Kidney Int 1984;25:689–95

Rahman A, Isenberg DA. Systemic erythematosus. NEJM. 2008;358(9):929-39.

Mannik M, Merrill CE, Stamps LD, Wener MH. Multiple autoantibodies form the glomerular immune deposits in patients with systemic lupus erythematosus. J of Rheumatology. 2003;30(7):1495–504.

Zhou W, Marsh JE, Sacks SH. Intrarenal synthesis of complement. Kidney International. 2001;59(4):1227–235.

Gomaa W, Bahlas S , HabhabW, .Clinicopathological characteristic of in western region of Saudi Arabia:An two tertiary medical centers.Journal of Ultrastructure 2014;2:12-19

lupus

MustaqM etal lupus nephritis experience from Microscopy and

10. Qari.f Clinical pattern of systemic lupus erthematosus in Western Saudi Arabia ,Saudi Med J 2002;23:1247–250. 11. Heller T, AhmedM., SiddiqqiA., WallrauchC., BahlasS, Systemic lupus erythematosus in Saudi Arabia: morbidity and mortality in a multiethnic population Lupus, 2007;16:908–14 12. YokoyamaH., WadaT., HaraA., YamahanaJ., NakayaI., KobayashiM., et al. The outcome and a new ISN/RPS 2003 classification of lupus nephritis in JapaneseKidney Int 2004;66:2382–88. 13. JallouliM., FriguiM., HmidaM., MarzoukS., KaddourN., BahloulZ.Clinical and immunological manifestations of systemic lupus erythematosus: study on 146 south Tunisian patients. Saudi J Kidney Dis Transpl 2008;19:1001–8 14. Arfaj A., Khalil N., Al SalehS. Lupus nephritis among 624 cases of systemic lupus erythematosus in Riyadh, Saudi Arabia Rheumatol Int 2009;29: 1057–1067. 15. Huong D., Papo T., Beaufils H., Wechsler B., Bletry O., Baumelou A., et al. Renal involvement in systemic lupus erythematosus. A study of 180 patients from a single center Medicine (Baltimore)1999;78:148–66 16. Neumann K., Wallace D., Azen C., Nessim S., Fichman M., Metzger A., et al. Lupus in the 1980: III. Influence of clinical variables, biopsy, and treatment on the outcome in 150 patients with lupus nephritis seen at a single center Semin Arthritis Rheum1995;25:47–55 17. Seedat Y., Parag K., Ramsaroop R. Systemic lupus erythematosus and renal involvement. A South African experience Nephron1994;66:426–30 18. Shayakul C., Ong-aj-yooth L., ChirawongP., NimmannitS., ParichatikanondP., LaohapandT., et al. Lupus nephritis in Thailand: clinicopathologic findings and outcome in 569 patients Am J Kidney Dis 1995;26:300–307 19. NezhadS., SepaskhahR. Correlation of clinical and pathological findings in patients with lupus nephritis: a fiveyear experience in Iran Saudi J Kidney Dis Transpl 2008 ;19:32–40

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20. Al AttiaH., Al AhmedY., Chandani A. Serological markers in Arabs with lupus nephritis Lupus 1998;7: 198–201

25. Howie A , Turhan N., Adu D. Powerful morphometric indicator of prognosis in lupus nephritis. QJM. 2003;96(6):411–20.

21. Al-JarallahK., Al-AwadiA., SiddiquiH., Al-SalimI., ShehabI., Umamaheswaran I. , et al. Systemic lupus erythematosus in Kuwait—hospital based study. Lupus 1998;7:434-8

26. Grcevska1 .L, Petrusevska2 .G, Dzikova. S, Polenakovic .M. Histopathological index of Activity and Chronicity :predictors of renal outcome in lupus nephritis .BANTAO journal 2003;1(2):138

22. LuF. Yu, TanY., WuL., ZhuN., LiuG., ZhaoM.. Class IV-G and IV-S lupus nephritis in Chinese patients: a large cohort study from a single center Lupus 2009;18:1073–10

27. Lai .F, Li .P, Choi .P, K. To, Wang .A, Leung .C, et al. Histological appraisal of lupus nephritis Hong Kong J Nephrol 2000;2:13–18

23. HiramatsuN., KuroiwaT., IkeuchiT., MaeshimaA., KanekoY., HiromuraK., et al. Revised classification of lupus nephritis is valuable in predicting renal outcome with an indication of the proportion of glomeruli affected by chronic lesions. Rheumatology (oxford) 2008;47:702-7

28. Ferluga D, Jerse M, Vizjak A, Rozman B, Kos-Golja M, Bren AF. Correlation among WHO classes, histomorphologic patterns of glomerulonephritis and glomerular immune deposits in SLE. Wien Klin Wochenschr.2000;112 :692-701.

24. Guillermo AH. The valve of electron microscopy in the diagnosis and clinical management of lupus nephritis. Ultrastuct Pathol 1999;23:63-77.

29. Nossent H, Berden J, Swaak T. Renal immunofluorescence and the prediction of renal outcome in patients with proliferative lupus nephritis. Lupus 2000;9(7):504-10.

*Corresponding author: Archana Chirag Buch, Dr. D.Y.Patil Medical College , Hospital And Research Center , Pune (India) Phone: 020 2780 5000 Email: drarchanabuch@yahoo.co.in

Financial or other Competing Interests: None.

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Date of Submission : 21.06.2017 Date of Acceptance : 05.09.2017 Date of Publication : 21.12.2017

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Original Article DOI: 10.21276/APALM.1592

Comparison of Papanicolaou and Acridine Orange stains in the Diagnosis of Trichomonas Vaginalis Infection in Vaginal Discharge Neena Piyush Doshi Dept of Pathology, GMERS Medical College, Gotri, Vadodara (India)

ABSTRACT Background: Trichomonas vaginalis is the most common cause of vaginitis. Diagnosis based on clinical presentation is often inaccurate. Clinical presentation with adjuvant laboratory evidence of trichomoniasis is required for the confirmation of the diagnosis and initiation of therapy. The aim of the present study was to compare the sensitivity and specificity of rapid staining Acridine Orange method for detection of trichomonas vaginalis infection with the routinely employed Papanicolaou staining. Materials and Methods: Vaginal swab smears were prepared from 300 patients attending gynaecology outpatients’ clinic of S.S.G. Hospital, Vadodara with complain of abnormal vaginal discharge. The prospective study was conducted over a period of two years (from January’ 98 to December’ 99). The Papanicolaou and Acridine Orange stained smears were screened for presence of Trichomonas vaginalis infection. Results: In the present study, the incidence of Trichomonas vaginalis infection was 12.3%. Considering Papanicolaou stain as standard, the sensitivity and specificity of Acridine Orange (AO) stain for detection of T. vaginalis was 92.8% and 93.01% respectively. The positive and negative predictive values were 74.2% and 99.2%, while false positive and false negative values are 3% and 0.66% respectively. Conclusion: Trichomonas vaginalis infection is a sexually transmitted disease of the reproductive age group, which needs to be timely diagnosed to prevent its adverse effects. The rapidity, ease and reliability of Acridine Orange stain justify its use in routine laboratory diagnosis of Trichomonas vaginalis infection. Keywords: Richomonas Vaginalis, Papanicolaou Stain, Acridine Orange Stain, Fluorescence

Introduction

Trichomonas vaginalis is the most common sexually transmitted disease (STD) worldwide and affects an estimated 170-180 or more million people annually.[1,2] The WHO estimates an incidence of 276 million new cases each year.[3] Trichomoniasis is almost entirely a disease of the childbearing era. There is no doubt that this infection is sexually transmitted. Nevertheless, the male carrier is generally symptom free, because a certain amount of oestrogenic activity is required for the trichomonads to reach full maturity. In some instances, the infection can be acquired by inadequate hygiene or the use of infected towels, toilet seats, douche nozzles, clothes, bed linen and contaminated swimming pool water.[1] Iatrogenic spread occurs through use of contaminated gloves and improperly sterilized surgical instruments especially vaginal speculum. Reinfection can occur from untreated sexual partner, extravaginal site lodgment of parasite, which is inaccessible to local therapy and, urinary tract. Incubation period of Trichomonas vaginalis (T. vaginalis) varies from 5 to 28 days.[4] 75% patients complain of profuse yellowish-white, greenish-yellow to creamy-white vaginal

discharge. It is usually thin, but up to 30% cases have thick discharge. 7-10% patients complain of foul smelling discharge. Pathognomonically the discharge is foamy or frothy due to fermentation of carbohydrates, which is seen in about only 10% of patients.[5,6] The presence of secondary infection may mask the self-diagnostic sign of frothy discharge. Burning and irritation of vulva becomes worse after periods due to increased levels of oestrogen, which favors T. vaginalis growth. Infection may be associated with genital pruritus and pain (dyspareunia, dysmenorrhea, lower abdominal pain and lower backache). It may cause urinary symptoms like dysuria, frequency and/or urgency. Since the introduction of metronidazole for the treatment of trichomoniasis, a major problem in the control of this disease has been the accuracy of diagnosis. Also number of strains resistant to metronidazole is on an increase.[5] And there is no alternative treatment currently.[3] It is so widespread and causes so much ill health among young women, that it has become an important social problem. In addition to abnormal vaginal discharge, it is increasingly associated with endometritis during pregnancy, pre-term labor, premature rupture of membranes, abortion, post-

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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cessarean infection, and human immunodeficiency virus (HIV) infection. [4,7] T. vaginalis is associated with 1.52fold increased risk of HIV-1 acquisition.[8] Therefore, the detection of T. vaginalis infection is of utmost importance. The elimination of trichomoniasis depends not only on treatment of the symptomatic cases, but also in detecting carrier patients who have mild symptoms or none, or who accept their vaginal discharge as ‘normal’ because every woman has a discharge anyway. Wet smear examination though simple is an insensitive method. A minimum concentration of 104 organisms per milliliter of vaginal discharge is necessary for wet mount to be positive.[2 7] The acknowledged insensitivity is in partly due to the rapid loss of the characteristic motility of the microorganism. A large number of culture and staining techniques have been designed to increase the reliability of laboratory diagnosis of T. vaginalis. Diamond’s medium is considered the gold standard. More recently culture method composed of liquid medium is used.[5] Culture is a sensitive method for detecting trichomonas as compared to wet smear preparation, however it is relatively slower, requiring 2-7 days for diagnosis.[4] Papnicolaou stain, Giemsa stain, and other Romansowsky stains are amongst the stains for vaginal smears commonly used for the diagnosis of T. vaginalis.[2] It is difficult to diagnose when trichomonads are scanty. Rounded forms of trichomonads can be confused easily with polymorphonuclear leukocytes or may look like dysplastic cells. Bare nuclei, degenerated cells and inspissated mucous plugs can be confused with trichomonads. The need for more sensitive, specific rapid and simpler method for detecting trichomonas vaginalis infection, to overcome the drawbacks of the existing methods, was recognized for a long time. More recent reports, describe the use of Acridine Orange stain using fluorescence microscope. The stain is differently adsorbed by the parasite and holds promise in improving the diagnostic ability of trichomonas vaginalis infection. The present study is undertaken to evaluate the usefulness of Acridine Orange staining in the diagnosis of vaginal trichomoniasis and compare the efficacy with Papanicolaou staining routinely employed on vaginal smears. To define the performance characteristic of Acridine Orange stain, Papanicolaou stain is considered as standard for the present study.

Materials and Methods

The present study was carried out in the Department of Pathology, Medical College and S.S.G. Hospital, Vadodara,

Gujarat, India over a period of two years (from January’ 98 to December’ 99). 300 patients attending gynaecology clinic of S.S.G. Hospital with complain of abnormal vaginal discharge formed the ‘target group’ of this prospective study. A detailed clinical history was taken. The findings were noted down in the performa. Smear Collection: Trichomonas vaginalis infection is essentially of the vaginal epithelium. The parasites shelter between rugae and acquire their food from glycogen rich vaginal epithelium. The specimen for the present study was thus collected from the posterior vaginal fornix pool. The vaginal swab containing the specimen was rolled onto two clean slides, one for Papanicolaou stain and other for Acridine Orange. The slides were immediately fixed by immersing in a coplin jar full of cytofix (a mixture of equal volumes of 95% ethanol and ether). Processing of Smears: The smears were fixed in cytofix for a minimum of 30 minutes and stained by Papanicolaou and Acridine Orange. In case of any delay in staining, the slides are removed from cytofix and air-dried. For Acridine Orange stain, the staining characteristics are better with cytofixed slides than air-dried smears. Papanicolaou (Pap) Staining Method: 90% ethyl alcohol 10 dips 70% ethyl alcohol 10 dips 50% ethyl alcohol 10 dips Running water 1 minute Harris’s haematoxylin 2 minutes Running water 2 minutes Acid alcohol 1 dip Running water 5 minutes 50% ethyl alcohol 4 dips 70% ethyl alcohol 4 dips 90% ethyl alcohol 4 dips Orange G. 6 (O.G. 6) 5 minutes 95% ethyl alcohol 4 dips 95% ethyl alcohol 4 dips Eosin azure 50 (EA 50) 2 minutes 95% ethyl alcohol 4 dips 100% ethyl alcohol 4 dips 100% ethyl alcohol 4 dips Xylol half an hour Xylol half an hour Blotted and mounted with Distrene 80 Dibutylphthalate Xylene (DPX) Microscopic Appearance of T. Vaginalis Infection in Pap Smear: In Papanicolaou smears, the parasites appear as gray-green, round or elliptical structures, varying in size

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A-751 Microscopic Appearance of T. Vaginalis Infection Using AO Fluorescence Dye: Prior to screening, the AO stained smears are mounted in buffer. The exciter filter of the fluorescence microscope was adjusted to 400 ηm and barrier filter was used as a combination of filters 44 (10% transmission at 440 ηm) and 53 (10% transmission at 530 ηm). Acridine Orange is a nucleic acid stain, staining differently DNA and RNA, the former fluoresces yellow to green and the latter bright red under ultraviolet illumination. Acridine Orange is very sensitive for picking up organisms. Trichomonas vaginalis trophozoites stain a characteristic brick red with a yellowish-green banana shaped or rounder nucleus (Fig. 6). The cytoplasm of leukocytes does not stain but the nuclei fluoresce bright green. Yeasts stain red. Bacteria including those within phagocytic cells stain bright red.[2] The likelihood that the additional positives detected by Acridine Orange stain would not be so, is slight, since nothing else in smears significantly resembles trichomonads to cause difficulty.

from 8 to 20 μm (Fig. 1). Large size trichomonads appear when growing conditions are unfavorable while smaller sizes prevail in favorable growing conditions. The smears from trichomoniasis are usually rich in leukocytes. There is excessive cytolysis. Debris and large plaques of necrotic cells are common. These make the background appear ‘dirty’. Clustering of leukocytes and macrophages around epithelial cells, called the ‘cannon ball or BB shot appearance’, may be observed. There is excessive eosinophilia involving the normally basophilic cells such as the intermediate and the parabasal (Fig. 2). Leptothrix, which are long filamentous bacterial structures, commonly occur in conjunction with trichomonads. All the above features are clue to the diagnosis of trichomonas vaginalis infection, however identification of the parasite is essential before the diagnosis can be established with assurance. It may be difficult to find T. vaginalis in severe inflammation (Fig. 3). Inspissated material (Fig. 4) and bare nuclei (Fig.5) may be confused with T. vaginalis.

Results

Acridine Orange (AO) Staining Method: 80% ethyl alcohol 4 dips 70% ethyl alcohol 4 dips 50% ethyl alcohol 4 dips Distilled water 4 dips 1% acetic acid 4 dips Distilled water 1 dip Working solution of AO (0.01%) 3 minutes Phosphate buffer (pH 6.4) 1 minute M/10 Calcium chloride 30 seconds Phosphate buffer (pH 6.4) wash M/10 Calcium chloride 30 seconds Phosphate buffer (pH 6.4) dip

In the present study, comparing results by both methods in each case, 26 cases were positive for T. vaginalis infection by both methods. There was disparity in 11 cases, in which 2 cases were positive by Papanicolaou stain and not by Acridine Orange, while 9 cases were positive by Acridine Orange but negative by Papanicolaou stain. Combining both, there were 37 positive cases in all as shown in table 1. Incidence of Trichomonas vaginalis infection was 12.3%. Considering Papanicolaou stain as the standard for present study, the sensitivity and specificity of Acridine Orange (AO) stain for detection of T. vaginalis is calculated as 92.8% and 93.01% respectively. Positive and negative predictive values are 74.2% and 99.2% respectively, while false positive and false negative values are 3% and 0.66% respectively.

Smears were allowed to dry. Stained slides keep no longer than 24 hours at room temperature and 2 months at 4° C. The stock solution does not keep for more than 6 months.

Table 1: Pap and AO stained smears positive for T. vaginalis infection. Papanicolaou stain

Acridine Orange

Positive

Negative

Positive

a=26

b=09

Negative

c=02

d=263

265

272

Total=300

a = true positive, b = false positive, c = false negative, d = true negative

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Table 2: Incidence of Trichomonas vaginalis in female vaginal discharge as observed in different studies. Sr. No. Work done by Incidence (%) 1. Karaman et al [9] 8.1 2. Klinger EV et al[10] 10.7 3. Rassjo EB et al[11] 8.0 4. Menezes CB et al[12] 15 5. Khamees SS [13] 26.8 6. Present study 12.3 Table 3: Sensitivity of Acridine Orange stain for detecting T. vaginalis infection.

Sr. No. 1. 2. 3. 4.

Work done by

Sensitivity (%)

Khatoon R et al [2] Radonjic IV et al[14] Hussein AH et al[15] Present study

100 71.43 81.8 92.8

Table 4: Specificity of Acridine Orange stain for detecting T. vaginalis infection. Sr. No. Work done by 1. Khatoon R et al [2] 2. Radonjic IV et al [14] 3. Hussein AH et al[15] 4. Present study

Specificity (%) 98 99.44 100 93.01

Fig. 1: (Pap stained vaginal smear, 40x view): Trichomonas vaginalis in Papanicolaou stained vaginal smears.

Fig. 2; (Pap stained vaginal smear, 10x view): Cannon ball appearance, dirty background and excess eosinophilia.

Fig. 3; (Pap stained vaginal smear, 40x view): Severe inflammation making it difficult to find the trichomonads.

Fig. 4: (Pap stained vaginal smear, 40x view): Inspissated material can be confused with T. vaginalis.

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Fig. 5: (Pap stained vaginal smear, 40x view): Bare nuclei can be confused with T. vaginalis.

Fig. 6; (AO stained vaginal smear, 40x view): T. vaginalis in Acridine Orange stained smears.

Discussion

but with low sensitivity and specificity. Besides it cannot be performed in clinics with heavy patient load. Cultures considered as gold standard are relatively slow. In order to identify T. vaginalis by culture, it is necessary to have an inoculum of 300â&#x20AC;&#x201C;500 trichomonas/ml.[14] Giemsa stain is not appropriated since it stains only the nucleus of the organism.[12] Papanicolaou stained smear examination needs competence and expertise. False-positives as well as false negatives are common with this technique.[3] Cellular picture of inflammatory vaginal discharge is similar particularly in trichomonas and Chlamydia. Acridine orange staining has the advantage of ease and rapidity to perform with higher sensitivity and specificity for diagnosis of Trichomonas vaginalis infection. The only disadvantage is the requirement of special microscope and availability of the dye. However, the cost of fluorescent microscope has decreased, so simplicity and speed of AO staining and diagnosis should encourage itsâ&#x20AC;&#x2122; use.[14] AO stained smears lose their fluorescence; hence it is not possible to have permanent record. Latex agglutination, enzyme-linked immunosorbent assay, immunochromatography-based tests, direct immunofluorescence assay, DNA hybridization probe test and polymerase chain reaction (PCR) are the newer tests.[14, 7, 3]

Long considered a minor sexually transmitted disease (STD) with few associated complications, T. vaginalis is now known to be associated with pregnancy related complications and morbidity in non-pregnant women. Besides, trichomoniasis can be asymptomatic in nearly 50% of the infected women or they may present with wide variety of symptoms. Once established, the infection may persist for long periods in women.[6] As per the published literature, the prevalence of T. vaginalis ranges from 0.4-27.4% in women.[4] In India, trichomoniasis accounts for 2-7% of all sexually transmitted infections.[6] In the present study, Acridine Orange stained smears picked up 11.6% positive cases while Papanicolaou stain picked up 9.3% positive cases. Combining both results there were 37 positive cases in all, thus an incidence of 12.3%. Table 2 shows the incidence of Trichomonas vaginalis infection as observed by different workers. The sensitivity of Acridine Orange stain in detecting Trichomonas vaginalis infection as observed by different workers is put in table 3. The sensitivity of Acridine Orange stain as observed by different workers varied between 3393.8%. The wide range of sensitivity could be attributed to sampling error, faulty staining technique and mild infection. Nassef NE et al showed 86.7% sensitivity by AO stain as compared to culture.[7] The specificity of Acridine Orange stain in detection of Trichomonas vaginalis as observed by workers is put in table 4.

Conclusion

Accurate diagnosis of T. vaginalis, whether symptomatic or asymptomatic, is affected by many variables like patient factors, clinicianâ&#x20AC;&#x2122;s experience, specimen sampling and processing and expertise of those who perform microscopy. Traditional wet mount smear is rapid method

High prevalence, increasing resistance to treatment and association with health complications are the burning issues related to T. vaginalis infection. A definitive diagnosis of trichomonas vaginalis infection cannot be made on clinical symptoms alone. Diagnosis must be laboratorial. Considering the higher sensitivity, specificity and the rapidity and ease of Acridine Orange staining, its use in routine laboratory diagnosis of trichomoniasis is justifiable. This will provide early diagnosis and prompt treatment to patients suffering from the infection. It will

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help in reducing disease associated morbidity and adverse health outcomes. Although, in gynaecologic practice the value of Papanicolaou stained smears cannot be under estimated because of its primary usefulness in screening women for cervical cancer, but Acridine Orange staining is advocated in cases suspected of trichomonas infection.

Acknowledgements

I express my gratitude to Dr. M.R. Desai, then Professor & Head, O/G, Medical College, Vadodara for allowing me to collect cytology smears. I sincerely thank Dr. R.Z. Patel and Dr. R.K. Pasle, Pathologists, Vadodara in helping me through the making of stains. The work wouldn’t have been possible without the help of Dr. Rajapurkar, MD, MPUH, Nadiad, who very kindly allowed me to avail the facility of the institutional fluorescence microscope.

Nassef NA, Afif AF, Bauni AA, Abo El-Nasr MF, Atia AF. Evaluation of microscopy and polymerase chain reaction for diagnosis of symptomatic and asymptomatic female trichomoniasis. 2014;7(1):37-46.

McClelland RS, Sangare L, Hassan WM, Lavreys L, Mandaliya K, Kiarie J, Ndinya-Achola J, Jaoko W, Baeten JM. Infection with Trichomonas vaginalis increases the risk of HIV-1 acquisition. J Infect Dis. 2007 Mar 1;195(5):698-702.

Karaman U, Karadag N, Atambay NB, Kaya A, Daldal NU. A comparison of Cytological and Parasitological Methods in the Diagnosis of Trichomonas vaginalis. Turkiye Parazitoloji Dergisi. 2008;32(4):309-12.

10. Klinger EV, Kapiga SH, Sam NE, Aboud S, Chen CY, Ballard RC, Larsen U. A Community-based study of risk factors for Trichomonas vaginalis infection among women and their male partners in Moshi urban district, northern Tanzania. Sex Transm Dis. 2006 Dec;33(12):712-8.

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Pereira-Neves A, Benchimol M. Trichomonas vaginalis: in vitro survival in swimming pool water samples. Exp Parasitol. 2008 Mar,118(3):438-41.

11. Rassjo EB, Kambugu F, Tumwesigye MN, Tenywa T, Darj E. Prevalence of sexually transmitted infections among adolescents in Kampala, Uganda, and theoretical models for improving syndromic management. J Adolesc Health. 2006 Mar;38(3):213-21.

Khatoon R, Jahan N, Khan HM, Rabbani T, Ahmad S. Evaluation of Different Staining Techniques in the Diagnosis of Trichomonas vaginalis Infection in Females of Reproductive Age Group. J Clin Diagn Res.. 2014 Dec;8(12):DC05-8.

12. Menezes CB, Mello MS, Tasca T. Comparison of permanent staining methods for the laboratory diagnosis of Trichomoniasis. REV Inst Med Trop Sao Paulo. 2016; 58:5.

Menezes CB, Frasson AP, Tasca T. Trichomoniasis - are we giving the deserved attention to the most common non-viral sexually transmitted disease worldwide? Microb Cell. 2016 Sep 5;3(9):404–19.

Sood S, Kapil A. An update on Trichomonas vaginalis. Indian J Sex Transm Dis. 2008;29(1):7-14.

Schwebke JR, Burgess D. Trichomoniasis. Clin Microbiol Rev. 2004 Oct;17(4):794–803.

Preethi V, Mandal J, Halder A, Parija SC. Trichomoniasis: An update. Tropical Parasitology. 2011;1(2):73-75.

13. Khamees SS. Prevalence of Trichomonas vaginalis among women in Albatnan District. Int. J. of Pharm. & Life Sci. 2012 Dec;3(12):2177-80. 14. Radonjic IV, Dzamic AM, Mitrovic SM, Arsic Arsenijevic VS, Popadic DM, Kranjcic Zec IF. Diagnosis of Trichomonas vaginalis infection: The sensitivities and specificities of microscopy, culture and PCR assay. Eur J Obstet Gynecol Repord Biol. 2006 May 1;126(1):116-20. 15. Hussein AH, Saleh MH, Nagaty IM, Ghieth KA, El-Azab NA. Prevalence, Clinical Criteria and Sociodemographic Predictors of Trichomonas vaginalis Infection in Suspected Egyptian Women, Using Direct Diagnostic Techniques. Iranian Journal of Parasitology. 2015;10(3):432-40.

*Corresponding author: Neena Piyush Doshi, Dept of Pathology, GMERS Medical College, Gotri, Vadodara (India)- 390021 Phone: +91 0265 239 8008 Email: neenapdoshi@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 06.07.2017 Date ofAcceptance : 29.07.2017 Date of Publication : 22.12.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Original Article DOI: 10.21276/APALM.1620

Fine Needle Aspiration Cytology of Solitary Thyroid Nodule with Histopathology Correlation Susmitha MS, Veena S*, Ramesh K Babu Department Of Pathology. Shimoga Institute Of Medical Science. Sagar Road. Shimoga (India)

ABSTRACT Background: Solitary thyroid nodule (STN) is a localized thyroid enlargement with apparently normal rest of the gland clinically. Preoperative Fine Needle Aspiration Cytology (FNAC) aids in distinguishing benign and malignant nodules and planning the treatment. Based on the cytology findings, patients can be followed in cases of benign diagnosis and thereby decreasing the rate of unnecessary surgery. However the accuracy of FNAC is found to be varied in different studies. The aim of the study was to study the cytological findings in STN and to correlate the FNAC and histopathology results. Methods: A retrospective study including FNAC of 107 patients presenting with solitary thyroid nodule from January 2015 to June 2017 was done at Department of Pathology. Cytology and histopathology correlation was available in 40 cases. Results: Solitary thyroid nodules involved females more commonly than males with a ratio of 10.8:1. Malignancy was present in 12.5% of cases and papillary carcinoma was the commonest malignancy. For malignancies, FNAC showed sensitivity of 40%, specificity of 97.1%, Positive predictive value of 66.6%, Negative predictive value 91.9% and Efficacy of 90%. Conclusion: FNAC of solitary thyroid nodules has high specificity and efficacy. Low sensitivity of FNAC in STN necessitates caution and patient follow up in cases with cytological benign diagnosis. Keywords: Thyroid, Nodule, Solitary, Cytology

Introduction

Solitary thyroid nodule (STN) is a localized thyroid enlargement with apparently normal rest of the gland clinically. The prevalence of thyroid nodules is 3-7% by palpation, 20-76% on basis of ultrasound and upto 50% at autopsy. Prevalence also depends on the regional iodine deficiency. It is up to 50% of adults in iodine deficient areas. It occurs more commonly in women than men. Malignancy is encountered more frequently in solitary nodules than multinodular goiters, with reported higher incidence in childhood STN than in adults. Malignancy occurs in approximately 5% of all the thyroid nodules.[1-6] FNAC is a rapid, cost effective and reliable diagnostic tool for detecting malignancy in STN. Preoperative FNA plays a pivotal role in distinguishing benign and malignant nodules and treatment plan. Unnecessary extensive surgery and its related adverse effects, such as hypothyroidism, hypocalcemia and recurrent laryngeal nerve injury could be avoided.[2] Radionucleotide scanning is the imaging technique useful in preoperative diagnosis of thyroid nodules, though unaffordable in many institutes. Ultrasound guided FNAC can be used for better sampling, especially in cystic and small lesions. The objective was to study FNAC findings in solitary thyroid nodules and correlate with histopathology.

Materials and Methods

A cross sectional study was done at Department of Pathology after obtaining approval from Institutional ethical committee. A retrospective analysis of cases and slide archives in the Department was done including cases from January 2015 to June 2017. Patients presenting with solitary thyroid nodule were included, irrespective of the thyroid profile status. Patients with diffuse thyroid enlargement and multiple nodules were excluded. FNAC was done using 23 guage needle and 5 ml syringe under aseptic precautions. Non aspiration technique or aspiration by minimal negative pressure was followed in majority of cases. Smears fixed in ethanol and stained with Haematoxylin and Eosin stain (H&E), Papanicolaou stain and air dried smears stained with Giemsa stain were analysed. Histopathological examination of hemithyroidectomy, subtotal or total thyroidectomy cases with preoperative cytology diagnosis were included in the study. Histopathology and cytology results were correlated and analysed. Sensitivity, specificity, efficacy, positive and negative predictive values for neoplasms and malignancies were calculated.

RESULTS

FNAC of thyroid was done on a total of 378 patients. 107 cases presented with solitary thyroid nodule. It included

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9 male and 98 female patients. Age group of patients ranged from 18 to 75 years. Adequacy criterion of atleast 6 clusters, with 10-15 cells per cluster, was followed. Of the 107 cases, 2 FNACs had inadequate material and no opinion was possible. FNA results were as shown in Table1. Colloid goiter (28.9%) (Fig 1.a,b), Colloid goiter with secondary changes(6.5%), Nodular colloid goiter with cystic change(21.5%), Nodular colloid goiter with adenomatous hyperplasia(14.7%), Hashimotos thyroiditis (3.7%).Follicular neoplasm constituted 8.4% of total cases (Fig 3). Papillary carcinoma (Fig.2), medullary carcinoma (Fig.1-c) and anaplastic carcinoma accounted for 3.7%, 1.9% and 0.9% respectively. The most common neoplasm was Follicular neoplasm. The commonest malignancy was papillary carcinoma.

follicular carcinoma showed microscopic , single capsular invasion. (Fig.3-e,f)

Histopathology correlation was available in only 40 cases (Table 2).Histopathologic evaluation showed Nodular colloid goiter in 27 cases(67.5%), Follicular adenoma in 8 cases (20%) (Fig.3-d), Papillary carcinoma in 4(10%), Minimally invasive follicular carcinoma in 1 case(2.5%).

Results of cytology and histopathology with correlation were as shown in Table 2. In 30 cases, same diagnoses were given in both. Distribution of cases in non neoplastic and neoplastic category with correlation revealed 7 False negatives and 2 False positives for neoplasms, 1 false positive and 3 False negatives for malignancy (Table 3). 32 cases were diagnosed as non neoplastic lesions on cytology. On histopathologic evaluation of these cases,25nodular colloid goitre, 5 follicular adenoma, 2 papillary carcinoma were reported.8 cases were diagnosed as neoplastic lesions by FNAC (4 follicular neoplasm, 1 hurthle cell neoplasm, 2 papillary carcinoma, and 1 medullary carcinoma). Among these, 2 non neoplastic lesions, 3 benign neoplastic lesions and3 malignancies were reported on histopathology. As per these results, 1 False positive and 3 false negatives for malignancy, 2 False positives and 7 False negatives for thyroid neoplasms were noted.

Papillary Carcinoma Thyroid (PCT) â&#x20AC;&#x201C; Tall cell variant showed Cells whose height were at least three times their width constituting 50% or more of papillary ca cells. Lesion was highly papilliferous with Invasion to skeletal muscle, showing PCT nuclear features .Nucleus located basally, with abundant oxyphilic cytoplasm and focal Sub nuclear clearing . (Fig.d,e,f) Minimally invasive

Statistical analysis was done using the formulae. FNAC of solitary thyroid lesions showed sensitivity 40%, specificity 97.1%, Efficacy 90%, Positive predictive value 66.6%, Negative predictive value 91.9% for malignancies. For Neoplasms, FNA had Sensitivity 46.2%, Specificity 92.6%, Efficacy 77.5%, Positive predictive value 75%, Negative predictive value 78.1%. (Table 4)

Table 1: Cytological Diagnosis with percentage. S. No. 1 2 3 4 5 6 7 8 9 10 11 12 13

Diagnosis on cytology Colloid goiter Colloid goiter with secondary changes Nodular colloid goiter with cystic change Nodular colloid goiter with adenomatous hyperplasia Hashimotos thyroiditis Follicular neoplasm Hurthle cell neoplasm Suspicious for papillary carcinoma Suspicious for thyroid malignancy Papillary carcinoma Medullary carcinoma Anaplastic carcinoma No opinion

No of cases(Total No = 107) 31 07 23 16 4 9 3 4 1 4 2 1 2

Percentage 28.9% 6.5% 21.5% 14.7% 3.7% 8.4% 2.8% 3.7% 0.9% 3.7% 1.9% 0.9% 1.9%

Table 2: Correlation of FNAC and Histopathology Diagnoses. FNAC Diagnosis

Colloid goiter

No. of cases

Histopathology diagnosis Nodular colloid goiter NCG with adenomatous hyperplasia NCG with sec change Follicular adenoma Papillary carcinoma

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No of cases 7 8 9 5 2

Veena S et al. FNAC Diagnosis Hashimoto thyroiditis

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Follicular neoplasm

Hurthle cell neoplasm Papillary carcinoma Medullary carcinoma

1 2 1 40

Histopathology diagnosis Nodular goiter Follicular adenoma Colloid adenomatous goitre Minimally invasive follicular carcinoma Papillary carcinoma Follicular adenoma

No of cases 1 2 2 1 2 1 40

Table 3: Distribution and correlation of cytology and histopathology lesions in STN. Histopathology

FNAC

Non neoplastic

Benign neoplasm

Malignant

Non Neoplastic

Follicular/Hurthleneoplasm

Malignant

Table 4: Statistical values for neoplastic and malignant lesions in STN. Statistical Index

Neoplasm

Malignancy

Sensitivity

46.2%

40%

Specificity

92.6%

97.1%

Positive predictive value

75%

66.6%

Negative predictive value

78.1%

91.9%

Efficacy

77.5%

90%

Fig. 1.a: colloid Goiter (H&E x 400). b: histopathology of colloid Goiter (H&E x400).c: shows cytology of medullary carcinoma thyroid with prominent paravacuolar granules (Giemsa x400).d â&#x20AC;&#x201C;Tall cell columnar varient of papillary carcinoma thyroid(PCT) with skeletal muscle invasion (H&E,x100), e - PCT (H&Ex400),f- PCT showing intranuclear inclusions (H&Ex400).

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Fig. 2: a,b – cytology of papillary carcinoma thyroid (H&Ex100), c- PCT (H&Ex400).d – PCT (H&E x100),e – PCT showing psammoma bodies &f – PCT with complex papillary branching pattern, grooving and inclusions (H&E x400).

Fig. 3: a – Follicular neoplasm (H&E x100) ,b,c – showing repetitive follicles in Follicular neoplasm (H&Ex400). d– Histopathology of Follicular Adenoma(H&Ex400). e,f – histopathology of minimally invasive follicular carcinoma with capsule on the right (H&E x100 and x400).

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Discussion

81.3% and 97.8%(Lopez et al) respectively. Efficacy of FNA in solitary thyroid nodule in the present study was 90%, which is comparable with the previous studies. It was 95.7% in Prakash et al, 92% in Gupta et al., 94.5% in Afroz et al., 87% in Kessler et al.[6,8,19-26]

FNAC is a simple, cost effective, rapid diagnostic test for thyroid nodules, with a significant role in determining the management of solitary thyroid nodules. FNA is useful in the detection of neoplasms in solitary nodules, that at times becomes challenging. Incidence of solitary thyroid nodules is about 5% in general population. STN are found to be more common in females compared to males. In our study female to male ratio was 10.8:1. Occurence of malignancy in the present study was 12.5%. It was found to be 14.8% in study of Prakash et al., 10.8% in SardaAK et al., 18% in Kaur k et al., 4.1% in Mundsad B et al, 46.2% in Jena et al, 18.5% in Anitha et al.[2,8-13] Statistics showed 7 false negatives and 2 false positives for neoplasms, 1 false positive and 3 false negatives for malignancy. These are of concern. False positives usually are seen with Atypical or Follicular adenoma and colloid nodules. Paucicellularity, degenerative changes simulating malignancy are the other causes.[8,14,15]1 follicular adenoma was misdiagnosed as medullary carcinoma on FNAC. 2 papillary carcinoma cases were misdiagnosed as non neoplastic on cytology(1 nodular colloid goiter and 1 nodular colloid goitre with adenomatous hyperplasia on FNA). Histopathology of one of these cases showed papillary carcinoma and nodular colloid goiter in adjacent thyroid. The papillae and pseudo papillae can be seen in Papillary carcinoma, Grave’s disease and hyperplastic nodule. Characteristic nuclear features such as nuclear grooves in more than 20% of follicular cells and presence of more than three intranuclear inclusions in the enlarged nuclei on single aspirate is pathognomonic of papillary thyroid carcinoma. Nuclear features and high cellularity are valuable in diagnosis of papillary carcinoma. But in cases with cystic change in papillary carcinoma, low cellularity and degenerative changes could be the causes for false negative result. Ultrasound guided FNA would help to reduce such false negatives by sampling the small foci of malignancy and avoiding nonmalignant parts of the lesion. Though only 10–15% of the cysts are neoplastic, FNA from solid area would reduce the misinterpretation and false negativity in cystic neoplasms. [16,17,18] For malignancies, FNAC showed sensitivity of 40%, specificity of 97.1%, positive predictive value of 66.6%, negative predictive value 91.9% and efficacy of 90%. Previous studies on FNAC of solitary thyroid nodule have showed sensitivity and specificity of 68.1% and 100%(Agarwal SK et al), 75% and 100% (Bapat et al), 89.4% and 99.2% (Gupta C et al), 40% and 100%(Prakash et al), 78.1% and 76.5%(Duek et al), 88.9% and 96.1%(Nnagada et al), 86.1% and 59.7% (Chao et al), www.pacificejournals.com/apalm

Akerman et al have quoted that the reasons for low sensitivity could be that tumor being missed at aspiration, misinterpretation of cytologic findings, cellular atypia, indeterminate diagnosis. [27]The difficulty in cytological diagnosis in certain cases is attributed to overlap of cytological patterns between neoplastic and non- neoplastic lesions. The differentiation between adenomatous nodule and follicular neoplasm is difficult. Increased nuclear size and pleomorphism, cellularity, the presence of microfollicles, scant and thick background colloid increase the likelihood of accurately detecting neoplasms. Haemorrhagic aspirate with microfollicles is seen in follicular neoplasms because of high microvessel density in the nodule. [1, 28]Although many studies suggest features to differentiate the two, the diagnostic dilemma in interpretation of adenomatous hyperplasia and follicular neoplasm continues to concern.

Conclusion

FNAC of solitary thyroid nodules has high specificity and efficacy.It is one of the rapid, reliable, cost effective procedures for the diagnosis of thyroid malignancy and effective management. In view of false negative results, in cases with high clinical suspicion of malignancy, a non neoplastic diagnosis on FNAC should be viewed with caution and advised periodic follow up.

Acknowledgement

special thanks to medical research unit – MRU , SIMS, shimoga for their technical support.

References 1.

Faquin WC. The Thyroid Gland.Recurring Problems in Histologic and Cytologic Evaluation. Arch Pathol Lab Med. 2008;132:622–632.

Jena A, Patnayak R, Prakash J, Sachan A, Suresh V, Lakshmi AY. Malignancy in solitary thyroid nodule. Indian J EndocrMetab. 2015;19:4:498.

Gupta M, Gupta S, Gupta VB. Correlation of fine needle aspiration cytology with histopathology in the diagnosis of solitary thyroid nodule. J Thyroid Res 2010:3.

Muratli A, Erdogan N, Sevim S, Unal I, Akyuz S. Diagnostic efficacy and importance of fine-needle aspiration cytology of thyroid nodules. J Cytol 2014;31:73-8.

Tai JD, Yang JL, Wu SC, Wang BW, Chang CJ. Risk factors for malignancy in patients with solitary thyroid nodules and their impact on the management. J Can Res Ther 2012;8:379-83.

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Gupta N, Goswami B, Hunjan PS, Shankar LR and Kakar A. Evaluation of the clinicopathological profile of solitary thyroid nodules: Our experience. Thyroid Res Pract.2016;1-12.

Nadgouda VG. Approach to a Patient with Solitary Thyroid Nodule. Medicine Update 2011;185-90.

8. Muddegowda PH, Lingegowda JB, Hiremath SS, Kishanprasad HL, Nagesh T, Joshua. Panorama of solitary thyroid nodule. Int J Med Health Sci. January 2012;1(1):19-26. 9.

Sarda AK, Gupta A, Jain PK, Prasad S. Management options for solitary thyroid nodules in an endemic goitrous area. Postgrad Med J 1997; 73:560-4.

10. Kaur K, Sonkhya N, Bapna AS, Mital P. A comparative study of fine needle aspiration cytology, ultrasonography and radionuclide scan in the management of solitary thyroid nodule: A prospective analysis of fifty cases. Ind J Otolaryngol head neck surg 2002 June; 54(2):96-101. 11. Mundasad B, Mcallister I, Carson J, Pyper PC. Accuracy of fine needle aspiration cytology in diagnosis of thyroid swellings. In: The internet journal of endocrinology 2006. 12. Anitha S, Ravimohan TR. A study of incidence of malignancy in solitary nodule of thyroid. International Journal of Contemporary Medical Research 2016;3(4):993-995. 13. Das DK, Khanna CM, Tripathi RP, Pant CS, Mandal AK, Chandra S et al. Solitary nodular goitre-Review of cytomorphologic features in 441 cases.ActaCytol 1999; 43:563-74. 14. Nguyen GK, Lee MW, Ginsgerg J, Wragg T, Bilodeau D et al. Fine needle aspiration of the thyroid: an overview. Cytojournal 2005; 2:12-16. 15. Hay ID. Thyroiditis: a clinical update. Mayo ClinProc 1985; 60:836-43. 16. Bommanahalli BP, Bhat RV, Rupanarayan R. A cell pattern approach to interpretation of fine needle aspiration cytology of thyroid lesions: A cyto-histomorphological study. J Cytol 2010;27(4):127-32. 17. Sukumaran R, Kattoor J, Pillai KR, Ramadas PT, Nayak N, Somanathan T et al. Fine Needle Aspiration Cytology of Thyroid Lesions and its Correlation with Histopathology in a Series of 248 Patients. Indian J Surg Oncol.2014;5(3):237–241.

18. Ersoz C, Firat P, Uguz A, Kuzey GM. Fine-needle aspiration cytology of solitary thyroid nodules: how far can we go in rendering differential diagnoses? Cancer 2004;102:302-7. 19. Aggarwal SK, Jayaram G, Kakar A, Guel GD, Prakash R, Pant CS. Fine needle aspiration cytologic diagnosis of the solitary cold thyroid nodule - comparison with ultrasonography, radionuclide perfusion and xeroradiography. ActaCytol 1989; 33(1):41-7. 20. Bapat RD, Shah SH, Relekar RG, Pandit A, Bhandarkar SD. Analyis of 105 uninodulargoitres. J Postgrad Med 1992; 38:60-1. 21. Duek SD, Goldenberg D, Linn S, Krausz MM, Hershko DD. The role of fine-needle aspiration and intraoperative frozen section in the surgical management of solitary thyroid nodules.Surg Today 2002; 32(10):857-61. 22. Nnagada HA, Musa AB, Gali BM, Khalil MIA. Fine needle aspiration cytology of thyroid nodules: A Nigerian tertiary hospital experience. The internet journal of Cardiovascular Research 2006. 23. Chao CT, Lin JD, Chao HH, Hsueh C, Chen MF.Surgical treatment of solitary thyroid nodules via fine needle aspiration biopsy and frozen section analysis. Ann SurgOncol 2007; 14:712-8. 24. Lopez H, Montano AS, Acosta TEM, Ramirez ZFR, Torres DRM, Ruiz AP et al. Combined use of fine-needle aspiration biopsy, MIBI scans and frozen section biopsy offers the best diagnostic accuracy in the assessment of the hypofunctioning solitary thyroid nodule. Eur J Nucl Med MolImag 2004 Sept; 31(9):1273-9. 25. Afroze N, Kalyani N, Hasan SH. Role of fine needle aspiration cytology in the diagnosis of palpable thyroid lesions. Indian J PatholMicrobiol 2002 July;45(3):241-6 26. Kessler A, Gavriel H, Zahav S et al. Accuracy and consistency of fine-needle aspiration biopsy in the diagnosis and management of solitary thyroid nodules,” Israel Medical Association Journal 2005;7(6):371–373. 27. Akerman M, Tennvall J, Biorklund A, Martennson H, Moller T. Sensitivity and specificity of fine needle aspiration cytology in the diagnosis of tumors of the thyroid gland. ActaCytol 1985; 29:850-5. 28. Lingegowda JB, Muddegowda PH, Rajesh KN, Kurpad RR. Application of pattern analysis in fine needle aspiration of solitary nodule of thyroid.J Cytol 2010;27(1):1-7.

*Corresponding author: Veena S, Department of pathology. Shimoga institute of medical science. Sagar road. Shimoga Karnataka 577201((India) Phone: +91 081822 29933 Email: drveenas82@gmail.com Date of Submission : 28.07.2017 Date of Acceptance : 17.08.2017 Financial or other Competing Interests: None. Date of Publication : 22.12.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Original Article DOI: 10.21276/APALM.1621

Rapid Cytodiagnosis By Different Staining Techniques in Comparison with Conventional Stains in Thyroid Cytology Veena Raja1*, Chinnaiya Subramaniyam Babu Rajendra Prasad2 and Lavanya Rajagopal1 Department Of Pathology, SRM Medical College and Research Institite, SRM University, Kattankulathur, Chennai (India) 2 Department Of Pathology, Sri Deveraj Urs Medical College (R.L. Jalappa University), Kolar, Karnataka (India)

ABSTRACT Background: Fine needle aspiration cytology (FNAC) is a very important component of pre-operative / pre-treatment investigation in combination with clinical, radiological and other laboratory data. In spite of its advances and advantages, conventional FNAC fails to achieve a 100% accuracy. To improve the accuracy of FNAC, a supravital stained Toluidine blue wet mount (TBWM) preparation of the aspirate and Methylene blue/Eosin (M/E) stained smears are studied which will give additional information regarding the lesion on which FNAC is done. Objective: To assess the diagnostic rapidity of the Toluidine blue wet mount preparation and Methylene blue/Eosin stain in FNAC and to compare the morphological features and results obtained from rapid stains with conventional PAP and H&E techniques in FNAC. Methods: The study was conducted on Fine needle aspirates from 100 patients presenting with thyroid swelling in R.L Jalappa hospital attached to Sri DevarajUrs Medical College, Kolar, Karnataka. TBWM, M/E stain, H&E stain and Pap stain was done for all 100 cases. Time taken for each stain is assessed and the morphology of rapid stains were compared with the conventional stain. Quality index was calculated for all the stains. Results: The rapidity of the stains was assessed for all four stains. Toluidine blue wet mount takes only 2 minutes for staining followed by Methylene blue/eosin which takes 5 minutes. M/E stain gave the same result as compared to the conventional stains [PAP and H&E]. In TBWM there was difficulty in diagnosing few cases due to three dimensional clusters. The sensitivity and specificity of TBWM were 66% and 90% whereas for M/E stain 98% and 100%. The quality index of PAP stain, H&E stain, M/E stain and TBWM were 0.85, 0.81, 0.71, 0.66. Conclusion: Thus, both stains can be routinely undertaken as a supplementary procedure for conventional stains to improve the cellularity and to reduce the time taken for re-sampling. Keywords: Toludine Blue Wet Mount, Methylene/Eosin, Rapid Cytodiagnosis, Quality Index

Introduction

Fine needle aspiration cytology (FNAC) technique has become more common and popular nowadays. Though it is not a substitute for conventional histopathology, it should be regarded as a very important component of pre-operative / pre-treatment investigation in combination with clinical, radiological and other laboratory data.[1,2]Since the majority of lumps are benign, FNAC plays a major role in making a diagnosis and planning appropriate management.[3,4] The diagnostic accuracy of FNAC depends on adequacy and representativeness of sample and good cytomorphological detail without much artifactual distortion. Conventional FNAC has its own advantages and limitations, which are well known to any cytopathologist. In spite of its advances and advantages, conventional FNAC fails to achieve a 100% accuracy.This is partly because of,(i) A lack of sufficient cellularity in desmoplastic lesions.(ii) Wastage of aspirated cells when they stick to the hub and lumen of the needles, (iii) Morphological distortion produced

when the cells are trapped in fibrin mesh, (iv) Distortion of fragile cells during smearing, (v) Loss of cell to cell and cell to stromal architecture.[1,2]Hence in an attempt to improve its accuracy, a supravital stained Toluidine blue wet mount (TBWM) preparation of the aspirate and Methylene blue/Eosin (M/E) stained smears are studied which will give additional information regarding the lesion on which FNAC is done. These supravital stains attains good definition of cell outline, cytoplasmic contents and nuclear details. Therefore this study is aimed at assessing the diagnostic rapidity of the Toluidine blue wet mount preparation and Methylene blue/Eosin stain in FNAC and to compare the morphological features and results obtained from rapid stains with conventional PAP and H&E techniques in FNAC.

Materials and Methods

The study was conducted on Fine needle aspirates from 100 patients presenting with thyroid swelling in R.L Jalappa hospital attached to Sri DevarajUrs Medical College, Kolar, Karnataka. Consent was taken from all the patients

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included in the study. Aspirates yielding very little material was excluded from the study.

Time taken for each stain is assessed and the morphology of rapid stains were compared with the conventional stain.

Toludine Blue Wet Mount Staining Step 1: A drop of fine needle aspirate is expressed on centre of slide

Assessment of Quality Index: The morphology of the cells was assessed using the following scoring system for all the four stains on all 100 cases using the following scoring system. [Table 1]

Step 2: In case of body cavity fluid cytology drop of fluid was placed in the center of slide if fluid was turbid or the fluid was centrifuged at 1500 rpm/min for 10 minutes. The supernatant fluid was discarded. Then a drop of well mixed sediment was placed in the center of slide. Step 3: Add drop of 0.5% Toluidine blue stain Step 4: Mix with needle Step 5: Add a drop of diluted Eosin stain and mix well (optional) Step 6: Cover with cover slip Step 7: Wet mount was examined under microscope and cytomorphology was observed. Alternative Method of Preparation of Wet Mount This method was tried whenever aspirates were very scanty and adhered to hub of the needle. Under such condition it was very difficult to express the aspirate over the slides. Step 1: A few drops of toluidine blue stain was aspirated using the same syringe and needle and rinsed. Step 2: Then the stain mixed material was expressed in the center of slide. Step 3: A drop of eosin solution was placed next to cell stain mixture and mixed well (optional) Step 4: Cover with cover slip Step 5: Wet mount was examined under microscope and cytomorphology was observed. Methylene Blue Alkaline/Eosin Staining Step 1: Air dry the smear Step 2: Fix smear in 95% methanol – 1 min Step 3: Wash in running tap water Step 4: 1 Dip in 1% eosin Step 5: Wash in running tap water Step 6: 1 dip in Loffler’s methylene blue alkaline Step 7: Wash in running tap water Step 8: Air dry and mount with DPX Toludine blue wet mount preparation, Methylene/Eosin stain, H&E stain and Pap stain was done for all 100 cases.

The maximum score for a single case, taking into account of all the four parameters, was 11. Thus, the maximum possible score in the study was calculated by multiplying the number of cases by 11 for each of four stains. The “Quality index” was obtained by finding out the ratio of actual score obtained to the maximum score possible. Quality Index = Actual score obtained / maximum score possible.[5] Statistical Analysis: We used the IBM SPSS Software (v.22) to perform the statistical analysis. Validation of rapid cytodiagnostic tests was done against the gold standard PAP and H&E stains using tests like Sensitivity, Specificity, Positive predictive value and ROC curve. The p value ≤ 0.05 was considered significant.

Results

100 cases with good cellularity where all stains can be performed were chosen for the study. For each case four stains have been done and were analysed by two pathologists for the morphological features and for final diagnosis. Pathologists were blinded for final diagnosis. Rapidity Assessment: The rapidity of the stains was assessed for all four stains. Toluidine blue wet mount takes only 2 minutes for staining followed by Methylene blue/ eosin which takes 5 minutes, H&E stain with artificial drying takes 8 minutes and PAP stain with artificial drying takes 10 minutes. Out of these 100 cases, M/E stain gave the same result as compared to the conventional stains [PAP and H&E] except for 2 cases of Lymphocytic thyroiditis are diagnosed as Benign thyroid lesion. This is due to lymphocytes in these two cases were identified as naked follicular epithelial cells. In TBWM there was difficulty in identifying Hyperplastic goiter due to three dimensional clusters and difficulty in identifying Hurthle cells and fire flares. The next difficulty we faced is to diagnose Papillary carcinoma of thyroid as the nuclear grooves and inclusions are not well appreciated in TBWM. Thus the total number of concordant cases by TBWM is 73. [Table 2] The sensitivity, specificity, positive predictive value and negative predictive value of TBWM and M/E stain were 66%, 90%, 70%, 100% and 98%, 100%, 98%, 100%

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Discussion

respectively. ROC Curve by comparing rapid stains with conventional stains are shown in figure 1.Area Under Curve for TBWM and M/E were statistically significant with p value <0.05. [Table: 3]

FNAC plays a vital role as a rapid diagnostic technique because of its simplicity, cost effectiveness, early availability of results, accuracy and minimal invasion. Chandler foot et al (1958), Silverman et al (1989) Verma et al (1991), Chang et al (1993), yang et al (1995), Tsou et al (1997) experimented various rapid stains such as Neutral red â&#x20AC;&#x201C; Janus green, Diff Quik, rapid MGG, Liuâ&#x20AC;&#x2122;s stain, ultra-fast

The morphology of the cells was assessed using the scoring system for all the four stains on all 100cases and the quality index were calculated[Table 4].The salient features of all four stains were shown in Table 5. Table 1: The scoring system to assess Quality Index. SCORE SLIDE QUALITY Background Overall staining Cell morphology Nuclear characteristics

Hemorrhage/Necrosis Bad Not preserved Smudgy chromatin

Clean Moderately good Moderately preserved Moderately crisp chromatin

Good Well preserved Crisp chromatin

Table 2: Distribution of concordant thyroid lesions based on stains. NATURE OF LESION

CONVENTIONAL STAINS (PAP AND H&E)

METHYLENE/EOSIN

WET MOUNT

Lymphocytic thyroiditis Colloid goiter

34 19

32 19

33 19

Hyperplastic goitre

Nodular goiter Benign thyroid lesion de-Quervains thyroiditis Acute suppurative thyroiditis

10 1 3 2

9 1 2

Suspicious for malignancy

Malignancy Papillary carcinoma thyroid Follicular neoplasm Anaplastic carcinoma

7 4 2

3 2

100

TOTAL Table 3: Area Under Curve. Test Result Variable(s)

Area

Std. Errora

p valueb

Asymptotic 95% Confidence Interval Lower bound

Upper bound

WM 0.679 0.092 <0.033* 0.499 0.858 M/E 1.000 0.000 <.0001* 1.000 1.000 The test result variable(s): WM has at least one tie between the positive actual state group and the negative actual state group. Statistics may be biased. a. Under the nonparametric assumption; b. Null hypothesis: true area = 0.5 Table 4: The results of estimating the efficacy of four stains (n=100). PARAMETER Hemorrhage/ necrosis Clean Bad Moderately good

PAP

SCORE

180

11 18

11 36

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H&E SCORE BACKGROUND

M/E

SCORE

90 180 OVERALL STAINING 15 15 28 56

178

176

21 61

21 122

39 52

39 104

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Rapid Cytodiagnosis by Different Stains

PARAMETER Good

PAP 71

SCORE 213

Not preserved Moderately preserved Well preserved and crisp

6 32

6 64

186

Smudgy chromatin Moderately crisp chromatin Crisp chromatin Actual score obtained Maximum score possible QUALITY INDEX

15 37 48

H&E SCORE 57 171 CELL MORPHOLOGY 6 6 38 76

M/E 18

SCORE 54

WM 9

SCORE 27

11 63

11 126

15 72

15 144

NUCLEAR CHARACTERISTICS 15 16 16

102

112

114

144

168

939

899

789

731

1100

0.85

0.81

0.71

0.66

Table 5: The salient features of all four stains. Feeatures Smearingtechnique Fixation Artifacts Cell loss

Cytoplasm Nucleus

pap Important Immediate fixation Delay in fixation Due to wet fixation Decreased, due to immediate fixation Well appreciated Excellent

H&E Important Immediate fixation Delay in fixation Due to wet fixation Decreased, due to immediate fixation Well appreciated Excellent

Nucleolus

Distinct

Excellent

Tissue fragment

Good

Advantage

Crisp Nuclear details

Good Very good nuclear details, similar to PAP

Disadvantage

Cell loss due to wet fixation

Slide preservation Cost Rapidity

Preserved Expensive 10 minutes

Preserved Expensive 8 minutes

Cell size

m/e Important Air drying followed by fixation Nil Minimal

TBWM Nil Nil Air bubbles Minimal

Increased due to air drying

Increased

Moderately appreciated Excellent

Poor Moderately good Excellent, appears Excellent immediately Moderate Moderate Immediate assessment, Good Obtain material from nuclear details needle hub Three dimensional Increased nuclear size due to clusters masking the drying cellular details Preserved Cannot be preserved Cost effective Cost effective 5 minutes 2 minutes

Table 6: Comparison of diagnostic accuracy of rapid stain with other studies. AUTHORS Chandler foot et al.6 Silverman et al.7 Kusumverma et al.8 Chang et al.9 Tsou et al.11 Joy MP et al.3 Sumathy C et al.2 SumathiS et all.1 Present study Present study

YEAR OF THE STUDY 1958 1989 1991 1993 1997 2003 2012 2013 2015 2015

RAPID STAIN USED Neutral red Janus green stain Diff-quik stain Rapid MGG stain Liu’s stain Riu’s stain Toludine blue stain Toludine blue wet mount Toludine blue wet mount Toludine blue wet mount Methylene blue/eosin

DIAGNOSTIC ACCURACY 80% 96% 97% 94.9% 93.5% 98.54% 89% 87% 76% 98%

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Fig. 1: ROC curve by comparing M/E and TBWM with conventional stains [PAP and H&E].

Fig. 2: Lymphocytic thyroiditis: A – M/E, B – TBWM.

Fig. 3: Hyperplastic goiter: A – M/E, B – TBWM.

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Fig. 4: Papillary thyroid carcinoma: A â&#x20AC;&#x201C; M/E, B - TBWM.

PAP, Riuâ&#x20AC;&#x2122;s stain respectively for immediate diagnosis.[6-11] This work was inspired by the earlier work of Joy, M.P. et al in 2003 where they applied toluidine blue as a rapid stain for quick diagnosis of ultrasound guided aspiration cytology.[3] The rapidity of the stains was assessed and found that toluidine blue wet mount stains in 2 minutes followed by methylene blue/eosin in 5 minutes. Rapid staining technique has an advantage of assessing the cellularity and adequacy of the material within few minutes and the reaspiration can be performed immediately. This will be of much help in USG or CT guided FNAC. Caya et al in 1984 reported that false negative reports were resulted from unrepresentative aspirates. False negative aspirates may include normal or reactive elements but necrotic material is an additional source of error.[12,13] This problem of sampling error cannot be eliminated entirely in FNAC but it is found reduced by rapid cytology assessment. This sampling error is reduced in our study by simultaneously doing rapid wet mount study. Cagle et al., in 1993 reported that inadequate sampling was solely responsible for 10% false negative report in lung FNAC.[14] In our study the needle and hub are rinsed with toluidine blue stain, which effectively washes all the cells collected in the lumen yielding an improved cellularity. Degenerated cells and neoplastic cells are more fragile and distorted easily during smearing which created confusion in diagnosis. Trapping of cells within fibrin meshwork also distorted the morphology of cell. Since cytomorphology forms the basis for the cytodiagnosis, artifactual morphological distortion influences the diagnostic accuracy of FNAC.[7] This smearing artifact is avoided in our study since we used wet mount preparations as one of the rapid stain. The disadvantage of this wet mount technique was

three dimensional clusters of cells which reduces the diagnostic accuracy in our study. One of the most important features in cytodiagnosis is the morphology of the nucleus.[12] The advantage of this supravital stain is that the cell structure is well preserved with toluidine blue stain.[15]Supravital stain has a high affinity for DNA and hence absorbed rapidly into the nucleus. As the dysplastic and anaplastic cells contain more nucleic acid, the nuclear stains of tumor cells are very prominent with toluidine blue.[16] Another advantage is the postfixation after air-drying facilitates chromatin staining in M/E stain which will improve the diagnostic accuracy in malignant cases. Colloid stains blue to purple and form thin membrane like coat often with folds and cracks.[17] In our study thick colloid stains as patchy dark blue colour material and thin colloid stains as granular purple material in both M/E and TBWM. The cyst macrophages are also well appreciated in both the stains. There was difficulty in identifying lymphocytic thyroiditis in M/E due to lymphocytes in two cases were identified as naked follicular epithelial cells[Fig 2].The major discrepancy in TBWM was found in the cases of hyperplastic goitre, as the fire flares are not well appreciated. There was no difficulty faced in diagnosing hyperplastic goitre in M/E [Fig 3].In cases of follicular neoplasm the arrangement of the cells in follicular pattern and the nuclear details was well appreciated in both M/E and TBWM. Anaplastic carcinoma of thyroid shows pleomorphic cells with prominent nucleoli and multinucleated cells which was well appreciated in both the rapid stains.Smear from papillary carcinoma of thyroid shows sheets of cell with enlarged ovoid nucleus, granular chromatin and prominent nucleoli with cytoplasmic inclusions and nuclear grooves. In our study papillary sheets of follicular cells showed nucleus with small prominent basophilic nucleoli and some

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with nuclear inclusion in M/E stain. But in TBWM the inclusions could not be identified [Fig 4]. The diagnostic accuracy of rapid stains were compared with other studies and shown in table 6. The slides of TBWM could not be preserved since the cells were not fixed and should be photographed immediately. This is the major disadvantage of this stain. But it can be preserved for few hours, by sealing the cover slip by applying melted Vaseline or DPX. By this improvement the cytomorphology can be retained for a period of 2 to 3 hours without any morphological distortion and the quick drying of wet mount can also be prevented. In most of the cases the material gets stuck in needle hub which is very difficult to obtain on the slide. This drawback was overcome by TBWM, where the toluidine blue is directly aspirated on the same needle used for FNAC and the material is expressed on the slide. This technique helps in obtaining the overall material from the needle and will improve the cellularity and helps in supplementing the conventional stains. M/E stain helps in assessing the cellularity within few minutes which will guide us whether to proceed with the staining or to do re-aspiration. This method helps in reducing the time consumption in cases of USG guided FNAC and in intra operative cytodiagnosis.

Conclusion

Thus Toluidine blue wet mount and Methylene blue/Eosin stain can be used as a rapid diagnostic test. It is also used to assess adequacy of sample especially for deep seated lesions and in USG guided FNAC. It can be used for intra operative cytodiagnosis as an adjunct to frozen section diagnosis. Thus, both stains can be routinely undertaken as a supplementary procedure for conventional stains to improve the cellularity and to reduce the time taken for re-sampling. This work gives a new dimension to the art of FNAC and also opens a new door for further researches in this regard.

References 1. 2.

Sumathi S, Mrinalini VR. Supravital stained wet film study of fine needle aspirates: A reliable supplementary diagnostic procedure. Clin Cancer Investig J 2012;1:135-39. Sumathy C, Durai SJ, Swaminathan K, Vallimanalan S, Munavarah SA. Supravital stained rapid wet mount preparation of fine needle aspirates-A cytomorphological study. TEJMS 2012;3:62-66.

Joy MP, Iyer VK, Aron M, Kapila K, Verma K. Rapid staining using toluidine blue: A reliable method for quick diagnosis in ultrasound guided aspiration cytology. Indian J PatholMicrobiol 2003;46:589-92.

Erkilic S, Kocer NE. Diagnostic accuracy of toluidine blue stained wet films in effusion cytology. ActaCytol 2006;50:407-9.

Idris AAA, Hussain MS. Comparison of the efficacy of three stains used for the detection of cytological changes in Sudanese females with breast lumps. Sudanese J Pub Health 2009;4:275-77.

Foot CN, Nelson D, Quist H. Supravital staining of sediments of serous effusions, a simple technique for Rapid Cytological diagnosis. Cancer 1958;11:151-157.

Silverman JF, Finley JL, O’Brien KF, Dabbs DJ, Park HK, Larkin EW, et al. Diagnostic accuracy and role of immediate interpretation of fine needle aspiration biopsy specimen from various sites. ActaCytol1989;33:791-796.

Verma K, Tiwari MC, Agarwal J, Kapila K. Diagnostic accuracy of immediate interpretation of fine needle aspiration. Indian J Med Res 1991;94:197-199.

Chang MC, Chan RD, Ho WL. Intra operative cytology, the use of Liu’s stain for immediate diagnosis. Zhonghua Yi XueZaZhi (Taipei) 1993;51:368-375.

10. Yang GC, Alvaraz ll. Ultra fastpapanicolaou stain. An alternative preparation for fine needle aspiration cytology. ActaCytol1995;39:55-60. 11. Tsou MH, Lin HH, Ko JS. Riu’s Stain and the cytologic diagnosis of thyroid fine needle aspiration a single cancer center experience. DiagnCytopathol1997;16:543-547. 12. Caya JG, Clower LJ, Wollenberg NJ, Tiev TM. Transthoracoc fine needle aspiration cytology, Analysis of 82 patients with detailed verification criteria and evaluation of false negative cases. Am J ClinPathol1984;82:100-103. 13. Winning AJ, Seed MJ, Husain WA, Metaxas OAN. Interpretation of Negative results in fine needle aspiration of discrete pulmonary lesions. Thorax 1986;41:875-9. 14. Cagle PT, Kovach M. Ramzy I. Causes of false results in transthoracic fine needle aspirates. ActaCytol1993;37:16-20. 15. David J, Henry CK, Feider, Splittgerber GF. Toluidine blue dye as a breast localization marker. Am J Roentqenol1989;153:261-263. 16. Dudgeon LS, Patrick CV. A new method for the rapid microscopical diagnosis of tumors. Br J Surg1927;15:250-261. 17. Orell SR. Fine needle aspiration cytology. 5th ed. New Delhi: Elsevier; 2013.

*Corresponding author: Dr. Veena Raja, No. 1, Arasamara Pallam Street, Krishnapuram, Ambur – 635802, Vellore District, Tamilnadu, (India) Phone: +91 9600385030 Email: drveenaraja88@gmail.com Date of Submission : 28.07.2017 Date of Acceptance : 12.08.2017 Financial or other Competing Interests: None. Date of Publication : 22.12.2017

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Original Article DOI: 10.21276/APALM.1637

Diagnostic Value of Immunohistochemistry in Soft Tissue Tumors Sridevi. V*., Susruthan Muralitharan., and Thanka. J Dept of Pathology, SriMuthukumaran Medical college and Hospital, Chennai (India)

ABSTRACT Background: Diagnosis of soft tissue tumors is a great challenge to pathologist but with the help of immunohistochemistry (IHC), proper analysis and diagnosis of soft tissue tumours can be made easy. The main use of immunohistochemistry in soft tissue neoplasms especially in sarcomas is to identify differentiation in the neoplastic cells.IHC are used as a panel or as single marker depending on the tumor. Methods: A total of 513 soft tissue tumor (STT) cases were collected and reviewed. The cases were separated into as benign, intermediate and malignant cases. In 90 cases of STT in which immunohistochemistry (IHC) were used was further analysed and classified depending on the various positivity and negativity of the marker used .The significance of IHC was also analysed. Result: In our study of 513 cases of STT there were 380 benign cases, 90 malignant cases and 43 intermediate cases. A total of 90 cases of sarcomas were present out of which 54% cases required IHC, 20 % cases required IHC to support the diagnosis but 26% of cases did not require IHC, the diagnosis was made on haematoxylin and eosin (H&E). The IHC markers helped in correct diagnosis of STT cases. Conclusion: Immunohistochemistry plays an important role in grading and giving precise diagnosis of soft tissue tumors. So it is important to use IHC to diagnose STT where haematoxylin and eosin did not give a precise diagnosis. Perfect diagnosis of STT helps in the correct therapeutic management of patients. Keywords: Soft Tissue Tumors, Immunohistochemistry Markers, Soft Tissue Sarcomas, Diagnosis.

Introduction

Soft tissue tumours (STT) are composed of numerous different tumour types that are classifiedaccording to the type of mesenchymal tissue which they resemble.[1] Immunohistochemistry (IHC) plays an important role in STT diagnosis.[2] Immunohistochemistry has been introduced in the 80’s and it is the application of immunologic principles to the study of cells and tissues.[3] IHC helps to rule out a non-mesenchymal tumor, followed by trying to define mesenchymal cell lineage. As there are many complex diagnostic entities recognized of an intermediate malignant category including some tumors with a deceptively bland histological appearance, it is important to diagnose these tumours.[4,5,6] The use of progressively small amounts of tissue for diagnosis highlights the importance of IHC. Immunohistochemistry will not be a substitute for skilled interpretation of conventionally stained microscopic specimens. As STT have several line of differentiation, numerous pseudo benign lesion and non mesenchymal malignant tumor are present which has to be differentiated for therapeutic reasons. So IHC is the major tool and plays an important role in determining the diagnosis of most of the soft tissue sarcomas. Panel of markers has to be used and quality of IHC technique and interpretation is also important.[2] But wide “random” panels can be misleading.

Because of the lack of sensitivity or specificity of markers, and of frequent “aberrant” immunoreactivities, the use of a single immunostain can lead to misdiagnoses. It is difficult to recommend panels as the choice of antibodies will change according to the specific clinicopathological differential diagnosis. However, a few “basic” panels can be suggested, depending on the morphological category of the tumor. Histogenesis

Markers

Mesenchymal(general)

Vimentin

Epithelial

Cytokeratins (CK), epithelial membrane antigen (EMA)

Smooth Muscle

Desmin, smooth muscle actin (SMA)

Skeletal Muscle

Myoglobin

Fibrohistiocytic

CD68

Endothelial

CD34,CD31

Lipomatous

Immunohistochemistry generally not used

Neuroendocrine, Ewing’s sarcoma/ PNET

Neuron-specific enolase, CD99

“Aberrant positivities”, such as the frequent expression of cytokeratins in leiomyosarcomas, Ewing sarcomas or

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epithelioidangiosarcomas should be known.[7] The type of positivity whether nuclear, cytoplasmic, membranous and expected extent like diffuse or focal positivity should also be known.The extent of the immunoreactivity is also important e.g. S100 will be diffusely positive in benign nerve sheath tumors but only focally positive in MPNSTs. As the treatment is different, it is extremely important to always rule out a non-sarcomatous tumor: carcinoma, melanoma, and lymphoma using adequate immunostains. Some of the commonly used markers are discussed below. Epithelial markers Cytokeratins and EMA should be included in the immunohistochemical panel of most spindle and pleomorphic cell malignant tumors. Lymphoid markers LCA, TdT should be included in the differential diagnosis of round cell neoplasms. Desmin is positive in rhabdomyosarcomas and in 70-90% of smooth muscle tumors. Caldesmon it is relatively specific for smooth muscle differentiation.[8,9]Myogenin is very specific in smooth muscle differentiation. CD31is the most sensitive and specific vascular marker is, which is expressed by 95-100% of benign and malignant vascular tumors. However, the expression of CD31 by macrophages is a potential pitfall. CD68 is useful in the identification of histiocytic proliferations or reactive lesions. CD99 is expressed by most Ewing sarcomas, in a membranous fashion. FLI-1 expression in Ewing sarcoma / PNET is characteristic. Vimentin that is still widely used, although it does not provide any reliable diagnostic information. Vimentin is an important marker to detect the preservation of antigenicity of tissues. Ki 67 is the most commonly used proliferation marker. Ki-67staining is a more useful procedure than counting mitotic figures for assessing prognosis of the patients with sarcoma.

Materials and Methods

From January 2006 to June 2011 all soft tissue tumor diagnosed in the department of pathology, Sri Ramachandra

Medical College and ResearchInstitute were retrieved from the surgical pathology files and analysed. A total of 513 cases were collected and reviewed. Out of these cases benign, intermediate and malignant cases were identified. The cases which lacked details of important clinical and pathological features were excluded from the study. The gross characteristics of the tumor which included the tumor location, size, necrosis, circumscription, cut section and secondary changes were obtained from the pathology report registers. Hematoxylin and eosin (H&E) stained sections of all the STT were reviewed and analysed. All the STT cases were analysed on the basis of cases which needed IHC for confirmation of the diagnosis, tumors which were diagnosed based only on H&E without IHC and cases which needed IHC for diagnosis. Most of the cases which needed IHC were malignant soft tissue tumor.

Result

Our study was a retrospective study in which we had analysed 513 cases of soft tissue tumours and retrieved cases which needed IHC in the period between January 2006 to June 2011, in the Department of Pathology, SRMC and RI. There were 380 benign cases, 90 malignant cases and 43 intermediate cases. A total of 90 cases of sarcomas were present. 16 % of synovial sarcomas were common among STT sarcomas followed by 14% fibrosarcoma and 13 % leiomyosarcoma. (Fig: 1, Table: 1) Out of these, many STT cases needed IHC for diagnosing and confirmation. So the analysis of usefulness of IHC in sarcomas showed that it was not needed for the diagnosis of sarcomas in 26% of cases which was done on H&E. IHC was done to support the haematoxylin and eosin diagnosis in 20% of case and diagnosing 54% of sarcoma cases was possible only with IHC.

Discussion

Soft tissue sarcomas (STSs) are heterogeneous group of malignant tumors with a wide spectrum in terms of

Table: 1: Total number of histological types of STT. S.NO

Histological type

Total cases

Benign

380

Intermediate

Malignant

Total cases

513

Table 2: Frequency of various histological types of sarcomas. HISTOLOGICAL TYPE

FREQUENCY

PERCENTAGE

Synovial Sarcoma

16.5%

Liposarcoma

8.2%

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IHC in Soft Tissue Tumors

HISTOLOGICAL TYPE

FREQUENCY

PERCENTAGE

Fibrosarcoma

14.4%

Leiomyosarcoma

13.4%

Rhabdomyosarcoma

5.2%

MPNST

4.2%

Ewingâ&#x20AC;&#x2122;s/PNET

5.2%

Angiosarcoma

Alveolar sarcoma

DSRCT

Pleomorphic sarcoma

8.3%

ExtraskeletalChondrosarcoma

1.0%

SclerosingEpitheloidFibrosarcoma

2.1%

Low Grade Fibromyxoid Sarcoma

DFSP

7.2%

High Grade STS

10.3%

Total

100%

Fig. 1: Various histological subtypes of sarcomas.

Fig. 2a: H&E.10x-Pleomorphic Leiomyosarcoma: Highly pleomorphic cells with giant cells.; 2b: 20X - Immunohistochemical stains for Vimentin and 2c: SMA shows diffuse cytoplasmic positivity in the tumour cells.

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Fig. 3a: H&E. 10x -Rhabdomyosarcoma :Small round blue cells with moderate amount of eosinophilic cytoplasm. 3b: 20x -Immunohistochemical stains for Vimentin and 3c :Desmin shows cytoplasmic positivity in the tumor cells.

Fig. 4a: H&E. 100x - DSCRT : Sheets of small round cells surrounded by desmoplastic stroma.4b: 100x :Desmin shows diffuse cytoplasmic. 4c: EMA membrane positivity in the tumour cells.

Fig. 5a: H&E. 100X-DFSP :Spindle shaped cells arranged in storiform pattern. 5b:200x CD34 shows cytoplasmic and membrane positivity in the tumour cells.

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IHC in Soft Tissue Tumors

histologic type and prognosis.[1] STT has been classified according to the WHO as benign, intermediate and malignant. It can occur in any age group. Histological grade of sarcomas is the most important prognostic factors. A total of 513 cases were studied, of which 380 cases were benign, 43 cases were intermediate and 90 cases were malignant. The study was undertaken to analyze the importance of immunohistochemistry (IHC) in soft tissue tumours. Among the immunohistochemical analysis of malignant tumours 54% of cases required immunohistochemistry (IHC) for the diagnosis, were as in 20% of cases IHC was useful to support the diagnosis. Immunohistochemistry is the most valuable adjuvant to hematoxylin and eosin staining in diagnostic histopathology. It is important in diagnosis of STT because of their variety with several lines of differentiation and the frequent difficulty of diagnosis with numerous psedosarcomatous benign lesion and non mesenchymal tumours. JM Coindre et al has shown that IHC is part of the definition of the tumours and is determinant for the diagnosis of rabdomyosarcoma (RMS), desmoplastic small round cell tumour (DSRCT) and synovial sarcoma.(Fig:2,3,4) Desmin and myogenin will be positive in the diagnosis of RMS.[10] Our study also showed that these sarcomas needed IHC for the diagnosis. The desmin marker was useful in our study to identify all the RMS. Truong lo et al has also shown the same utility of desmin in RMS. As myogenin is more specific to skeletal muscle differentiation it is useful in diagnosing alveolar RMS.[11] Malignant vascular tumours like angiosarcoma also need IHC to diagnose. CD 31 is the best marker for vascular lesion. In our study synovial sarcomas with biphasic pattern, IHC was useful to confirm the diagnosis. But in monophasic and poorly differentiated subtype IHC has to be done to diagnose the sarcomas. Folpe Al and Lopes JM et al studies showed that IHC is useful for the diagnosis of monophasic and poorly differentiated synovial sarcomas[12,13] which correlated with our study. For the synovial sarcoma cases for which Bcl-2 was needed, it was done and positive reaction was present. Suster S et al has also shown that the Bcl2 is positive in synovial sarcomas but it is a non specific marker.[14]A DSRCT was present among the sarcomas in our study which showed desmin, CK and EMA positive with SMA positivity in the stroma. Ordonez NG et al IHC finding of DSRCT with 90% positivity for CK, EMA and desmin with dot like positivity. JM Coindre et al studies also showed that IHC may be helpful in the diagnosis of sarcomas like Ewing’s sarcoma, Leiomyosarcoma, Dermatofibrosarcoma protuberance (DFSP) and myxoid liposarcoma (Fig:5) and it is also useful to support the diagnosis. For low grade sarcomas

like DFSP IHC CD34 is positiveand it is done to rule out benign fibrous histiocytoma.[15] The DFSP cases in our study showed storiform pattern of cell arrangement but CD 34 stain was done to support the diagnosis. Del Tos AP et al study showed the diagnostic utility of S100 in myxoid Liposarcoma which can be positive in some cases. Few of our myxoid liposarcoma (LPS) also showed S100 focal positivity. Sarcomas with no specific immmunohistochemical profile is seen in fibrosarcoma, pleomorphic malignant fibrous histiocytoma (MFH), these sarcomas will not display any specific marker.JM Coinder study showed IHC may be useful to rule out other sarcomas with a specific line of differentiation. The nuclear proliferative antigen Ki67 is identified to correlate with the prognosis of the patients to predict the outcome and distant metastasis of sarcoma patients. A significant correlation has been reported between Ki67 expression and mitotic rate in sarcomas.[16] A correlation between ki67 reactivity and tumour grade in sarcomas has been detected in different retrospective studies. The findings in our study indicate that precise use of immunohistochemical stain can be a valuable adjunct to routine histopathology, exact grading of the sarcomas and in treatment management of patients.

Conclusion

Our study concludes that immunohistochemistry is an important tool in giving precise diagnosis of soft tissue tumours and it should be done as a panel to diagnose cell of origin.IHC can be done on routine basis to diagnose undifferentiated STT, confirm the diagnosis and to accurately grade the sarcomas so that it will be beneficial for the management of the patient and preoperative and postoperative treatment planning would be possible.

Reference 1.

Fisher C. Immunohistochemistry in Diagnosis of Soft Tissue Tumours. Histopathology. Wiley, 2010; 58 (7):1001.

Heim-Hall J, Yohe SL. Immunohistochemistry of Soft Tissue Neoplasms. ArchPathol Lab Med. 2008; 132:476-489.

Coindre JM. Immunohistochemistry in the diagnosis of soft tissue tumours. Histopathology. 2003; 43: 1-16.

Fisher C. The comparative roles of electron microscopy and immunohistochemistry in the diagnosis of soft tissue tumours. Histopathology. 2006;48:32-41.

Hornick JL. Novel uses of immunohistochemistry in the diagnosis and classification of soft tissue tumors. Modern Pathology. 2014; 27: 47–63.

Suster S. Recent advances in the application of Immunohistochemical markers for the diagnosis of soft tissue tumours. Semin. Diagn. Pathol. 2000; 17: 225-235.

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de Saint N, Somerhausen A. Immunohistochemistry in the diagnosis of soft tissue tumors. Path 2005: 2-12.

Ceballos KM, Nielsen GP, Selig MK, O’ Connell JX. Is antih-caldesmon useful for distinguishing smooth muscle and myofibroblastic tumors? An immunohistochemical study. Am. J. Clin. Pathol. 2000; 114: 746-753.

Yves-Marie Robin et al. Transgelin is a novel marker of smooth muscle differentiation that improves diagnostic accuracy of leiomyosarcomas: a comparative immunohistochemical reappraisal of myogenic markers in 900 soft tissue tumors. Modern Pathology.2013; 26: 502–510

10. Kumar S, Perlman E, Harris CA et al. Myogenin is a specific marker for rhabdomyosarcoma: an immunohistochemical study in paraffinembedded tissues. Mod. Pathol. 2000; 13: 988-993. 11. Dias P Chen B, Dilday B et al. Strong immunostaining for myogenin in rhabdomyosarcoma is significantly associated with tumors of the alveolar subclass. Am. J. Pathol. 2000; 156: 399-408.

A-773 12. Olsen SH, Thomas DG, Lucas DR. Cluster analysis of immunohistochemical profiles in synovial sarcoma, malignant peripheral nerve sheath tumor, and Ewing sarcoma. Mod Pathol. 2006;19:659–668. 13. Yang Let al.Clinical pathological analysis of synovial sarcoma. Chinese Journal of clinical oncology.2007;4:246-249. 14. Cunha KS et al. Evaluation of Bcl-2, Bcl-x and Cleaved Caspase-3 in Malignant Peripheral Nerve Sheath Tumors and Neurofibromas .Annals of the Brazilian Academy of Sciences. 2013 March. 15. Li N, McNiff J, Hui P, et al. Differential expression of HMGA1 and HMGA2 in dermatofibroma and dermatofibrosarcomaprotuberans: potential diagnostic applications, and comparison with histologic findings, CD34, and factor XIIIa immunoreactivity. Am J Dermatopathol.2004;26:267–272. 16. Gupta, et al., Typing and Grading of Soft Tissue Tumors and their Correlation with Proliferative Marker Ki-67. J CytolHistol. 2015; 6:3.

*Corresponding author: Dr Sridevi. V, 52(Old No:39), Gangadeeshwaran Koil Street, Nutech-Sreenivas Apartment, Flat:C-9, Purasaiwalkam, Chennai (India):600084 Phone: +91 09444952854 Email: drsridevi78@yahoo.co.in Date of Submission : 06.08.2017 Date of Acceptance : 30.08.2017 Financial or other Competing Interests: None. Date of Publication : 22.12.2017

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Original Article DOI: 10.21276/APALM.1658

Glioblastoma Multiforme: A clinicopathological analysis Asha Shenoy and Shruti Shribhagwan Singhal* Dept. of Pathology, Topiwala National medical college and B.Y.L. Nair Hospital, Mumbai. India

ABSTRACT Background: Aim of the study was to classify Glioblastoma Multiforme (GBM) and its variants on histology and analyze their clinicopathological features. Better understanding of the heterogeneous nature of GBM and its variants may provide improved treatment paradigms, prognostic classification and approaches towards molecular targeted treatments. Methods: 40 cases of GBM were analyzed retrospectively and prospectively, covering a period of 8 years (January 2008- July2015). Cases were analyzed on the basis of clinical presentation, histopathological features and radiological reports. Results: Of the 40 cases of GBM, the Conventional GBM, GBM with oligodendroglial component, Giant Cell GBM accounted for 26, 9 and 3 cases respectively. While there was 1 case each of Gliosarcoma and P-NET variants. Conventional GBM most commonly presented with Headache (69.2%) and paresis (57.7%) whereas GBMO patients presented with convulsions. On radiology, the variants of GBM cannot be distinguished as the findings were similar in all of them. Non-palisading necrosis was predominantly present in Conventional GBM (61.5%) and palisading necrosis in GBMO (66.7%). Conclusion: Histopathology remains the main tool for differentiating the variants as radiological differentiation is not possible due to similar appearing features. Keywords: Glioblastoma Multiforme, Glioblastoma Multiforme Variants.

Introduction

GLIOBLASTOMA synonym (Glioblastoma Multiforme - GBM) is the most malignant neoplasm of the central nervous system with predominant astrocytic differentiation, affecting adults and preferentially located in the cerebral hemispheres [1] . Glioblastoma can be primary (De Novo), or secondary on the background of pre-existing astrocytic neoplasm. This is a morphologically diverse neoplasm with a dismal prognosis. Though three morphologically different variants have been recognized, additional variants which have significant morphology overlap with tumors having more favourable prognosis and treatment response rates, have been described. Even though Glioblastoma Multiforme is a quite rare tumor with a global incidence rate of only 3.17 per 100,000 [2] it significantly impacts the life of the affected patients due to its poor prognosis with a median survival time of only 12-15 months from the time of diagnosis. [3]

Materials and Methods:

After approval from the institutional ethics committee for this retrospective and prospective study, 40 cases from January 2008 to July 2015 were analyzed. Retrospective study was done using the blocks and slides available. For prospective study biopsy tissue was fixed overnight in 10% buffered formalin and submitted entirely for processing.

Intraoperative tissues sent were processed as squash preparation and frozen section on cryostat while still in an unfixed state and H&E staining was performed. After frozen section diagnosis, remaining tissue was transferred to a fixative and processed routinely. Paraffin sections were cut 4 to 6 microns in thickness and routine H&E staining was performed, special stains were performed wherever necessary. The clinical and radiological data was obtained from patientâ&#x20AC;&#x2122;s proforma available in the Pathology Department. Anatomical location of the tumor was based on radiological imaging and or operative findings. GBM was diagnosed and classified as per the 2007 WHO.

Results

In the 8 years study period (January 2008 to July 2015), there were 433 cases of central nervous system space occupying lesions, out of which 40 were diagnosed as GBM. The overall incidence of GBM was 9.2%.The average number of GBM cases received was 5 per year. The maximum number of cases were diagnosed in the 4th and 5th decade, 9 cases each (22.5 % each). In our study, a male predominance was found (Male: female=2.07:1). The youngest patient was 5 years old while the oldest one was 73 years old. GBM was found to be more common in males

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in the 41-50 years age group and in females in the 51-60 years age group. Headache and paresis were the commonest symptoms encountered, 24 cases (60%) and 21 cases (52.5%) respectively. Majority of the patients (22.5%) had duration of symptoms from 15-30 days. 29 cases (72.5 %) presented in less than 3 months. 95% of cases had supratentorial GBM and 5 % had infratentorial. The most common locations of the tumour were frontal lobe and temporoparietal region. (17.5% each). 95 % (38/40) of the tumors were present in cerebral hemisphere whereas 2.5 % tumor involved cerebello-pontine angle and brain stem. Out of 38 Glioblastomas in cerebral hemisphere 18 involved only single lobe whereas 17 involved 2 lobes and 3 involved thalamocapsular ganglion. CT findings were available in 25 cases and MRI findings in 15 cases. On CT scan, tumors were iso to hypodense (84%), heterogeneous enhancing (95%) and showed hemorrhage and necrosis (100%), peritumoral oedema (97.5%). On histopathology majority of the cases showed marked cellularity (72.5%), moderate pleomorphism (90%), microvascular proliferation (95%), non-palisading necrosis (55%) and mitosis (100%). These are the defining histological features of GBM. [Table-1, Figure-1] Predominant types of cells were small cells (72.5%). [Table-2] 5 pediatric cases were seen. Histologic features of pediatric GBM are similar to adult GBM. [Table-3] Of the 40 cases, 34 cases were analyzed on the frozen section .All these were given the diagnosis of high grade glioma on the basis of micro vascular proliferation and necrosis. On further histopathological examination, these

were given the final diagnosis as Conventional GBM (26 cases) or GBMO (9 cases). [Figure-2] In the present study, 5 variants of GBM were obtained. [Table-4, Figure-3] Conventional GBM (69.3%) and Glioblastoma with oligodendroglial component (GBM-O) (44.4%) were present in the elderly age group of 40-70 years. Only single case of Gliosarcoma presented in 50-60 year of age group. In contrast to these, the only case of Glioblastoma with Primitive Neuroectodermal Tumor(GBM-PNET) and 66.6% of Giant Cell GBM (gcGBM) presented in a younger age group (10-30 years). Both Conventional and GBMO had male predominance which was more marked in Conventional (76.9%) as compared to GBMO (66.6%). Headache (69.2%) and paresis (57.7%) were the main presenting symptom in most cases of conventional GBM. GBMO (33%) more commonly presented with convulsions than Conventional GBM (7.7%). Both Conventional GBM (76.9%) and GBMO (66.7%) presented acutely i.e. within 3 months of onset of symptoms. Conventional GBM was present predominantly in frontal region (19.2%) and GBMO was equally present in fronto-temporal, temporoparietal and parieto-occipital region (22.2%). CT findings were available in 25 cases (16 of Conventional GBM, 6 of GBMO and 3 of Giant-cell GBM). MRI findings were available for 15 cases (10 of Conventional GBM, 3 of GBMO and 1 each of GBM-PNET and Gliosarcoma). Conventional GBM and GBMO showed iso to hypodensity on CT (87.5% and 66.5% respectively). Hemorrhagic necrosis was present in all the cases. Non-palisading necrosis was predominantly present in Conventional GBM (61.5%) and palisading necrosis in GBMO (66.7%). [Table-5]

Table 1: Defining histological features in GBM. Histology

Total n=40

Percentage%

Cellularity n=40

Marked

72.5

Moderate

27.5

Marked

Moderate

Present

100

Absent

Palisading

Non palisading

P +np

Present

100

Absent

Pleomorphism n=40 Microvascular proliferation n=40 Necrosis n=40 Mitosis n=40

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Table 2: Additional histological features in GBM. Microscopy

Total

large cell type

small cell

72.5

giant cell

12.5

Oligocomponent n=40

Present

22.5

Absent

77.5

Secondary structure n=40

Present

7.5

Absent

92.5

Gemistocytes n=40

Present

27.5

Absent

72.5

Perivascular lymphocytes n=40

Present

7.5

Absent

92.5

Thick walled blood vessels n=40

Present

7.5

Absent

92.5

Thrombosed vessel n=40

Present

12.5

Absent

87.5

Type of cells n=40

Percentage%

Table 3: Histology of Pediatric vs. Adult GBM cases. Histology Pleomorphism Microvascular proliferation Necrosis Mitosis

Pediatric

Adult

n =5

n=35

marked

4 (11.45%)

moderate

5 (100%)

31(88.6%)

present

5(100%)

33(94.2%)

absent

present

5(100%)

35(100%)

absent

present

5(100%)

35(100%)

absent

Table 4: Variants of GBM found in the study (n=40): Variant

Number

Percentage

Conventional

GBMO

22.5

Giant Cell GBM

7.5

Gliosarcoma

2.5

GBM PNET

2.5

Total

100

Table 5: Immunohistochemical findings â&#x20AC;&#x201C; GBM-PNET: IHC stains

GFAP Synaptophysin CD 56 Vimentin

Small Cell GBM Astrocytic Small cell component component + + + + + +

GBM- PNET Astrocytic Small-cell component component + + + + + -

*sPNET are Supra-tentorial PNET which lack glial component.

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sPNET Small-cell component + + -

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Fig. 1: Frozen section: (a) Squash smear of glioblastoma showing glial cells with hyperchromatic and pleomorphic nuclei. (HE X 400) (b) Squash smear of Glioblastoma showing microvascular proliferation. (HE 400X) (c) Paraffin section of Glioblastoma showing microvascular proliferation (HE 400X) (d) Paraffin section of Glioblastoma showing pseudopalisading necrosis. (HE 400X).

Fig. 2: GBM Histopathology: (a) GBM showing marked pleomorphism and gemistocyte (HE 400X) (b) GBM showing pseudopalisaded necrosis (HE 400X) (c) GBM showing glomeruloid appearance of microvascular proliferation. (HE 400X) (d) GBM showing perivascular lymphocytes . (HE 400X).

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Fig. 3: GBM Variants :(a) GBMO showing Oligodendroglial component on right side (HE 400X) (b) GBM PNET showing small cells in sheet. (HE 400X) (c) Giant cell GBM showing multinucleated giant cells (HE 400X) (d) Gliosarcoma showing spindle cells in fascicles in lower part of section. (HE 400X).

Discussion

GBM or astrocytoma grade IV ( WHO classification) is the most aggressive and the most frequent of all primary brain tumors.[4] Incidence rate of GBM at our institute was 9.2% which is almost similar to the incidence stated by Ghosh et al who reported an incidence of 7.9% for GBM. WHO (2007) states an incidence of around 12-15% for GBM of all intracranial neoplasms[5]. In our study, 45 % of CNS tumors were encountered in the fourth to sixth decade. The mean age was 46.4 years. The youngest patient was 5 year old while the oldest one was 73year old. The finding is similar to study done by Manisha Khanna et al, who found majority of the tumors in age group of 51-60 years [6]. Childhood glioblastomas are extremely rare compared to their adult counterparts [4].Of the 40 cases, 27 were male and 13 were female. The male: female ratio in our study was found to be 2.07:1 which was comparable with Manisha Khanna et al [6] (2.38:1). Ghosh et al. observed a male/female ratio of 3.9:1[5]. Although the duration of symptoms was more than 3 months in 11 cases, there were no histological proven or past clinical history of high grade gliomas. Hence no case of secondary GBM was found in the study. According to the literature, GBM is preferentially located supratentorially [4]. .In our study too about 95

% of cases were supratentorial and 5% cases showed infratentorial GBM. In our study, the commonest site of GBM was frontal region and temporo-parietal region (17.5% each). These findings were comparable to study by Manisha Khanna et al [6]. In our study, 10% tumors were present in frontal lobe involving corpus callosum but none extended to other lobe or across the midline. In literature [7] it is said that the corpus callosum is relatively resistant to infiltration by edema or infection. Any lesion seen extending across the midline in this way, whether symmetric or asymmetric, should always be suspected of being a diffuse astrocytoma. MRI Brain mainly revealed extensive white matter edema (97.5%) noted with significant midline shift. Non enhancing areas corresponded to haemorrhage and necrosis on histopathology were seen in 100 % of the cases. These findings is in accordance to study by Gabriel Iacob et al [4]. In our study, morphological analysis included the degree of cellularity, pleomorphism, mitosis, type of necrosis and vascular proliferation. 29 cases of GBM (72.55%) were markedly cellular. Moderate cellularity was seen in 11 cases (27.5%). 29 cases (72.5%) of the tumors showed cells of small size. Non palisading necrosis was present in majority of the cases (55%) whereas 35% of the cases showed palisading necrosis and in 10 % both type of necrosis was

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Shenoy A. et al. present. Variable numbers of mitotic figures were seen in almost all cases (100%) Microvascular proliferation was seen in all cases (100%) . Of the 40 cases, 34 cases were analyzed on the frozen section .All these were given the diagnosis of high grade glioma on the basis of micro vascular proliferation and necrosis. On further histopathological examination, these were given the final diagnosis as Conventional GBM (26 cases) or GBMO (9 cases). All the cases of glioblastoma diagnosed on squash smear and frozen section showed significantly increased population of glial cells in a fibrillary background with mitoses with microvascular proliferation and necrosis. The study by Chandrasoma PT et al provides evidence that, with careful target placement, stereotactic biopsy can provide biopsy material that represents the entire lesion with an accuracy that is sufficient for clinical management [8] . In present study, we had 26 cases (65%) of Conventional GBM. It was prevalent in 3rd to 5th decade (69.3%). Out of 26 cases of Conventional GBM , 20 were found in males and 6 in females. The ratio in Conventional GBM was 3.3:1. Headache and paresis was the main presenting symptom in most cases of conventional GBM. Most frequent site affected in patients with Conventional GBM was frontal. Majority of the cases showed marked cellularity (69.2%), moderate pleomorphism (92.3%), presence of small cells (73.1%), non-palisading necrosis (61.5%), microvascular proliferation (92.3%) and presence of gemistocytes (30.7%). Mitosis was present in all the cases. Giant cells were seen in 7.7% of the cases. These correlates with findings mentioned in literature[9,10]. Clinical and pathological studies of GBMO are currently scarce. Its exact incidence is thus largely unknown and has ranged from 4% to 27% of all GBMs in previous studies[9, 11-13] with an incidence of 22.5% (9 cases) in the current study. In present study, it was noted that GBMO was prevalent in elderly patientâ&#x20AC;&#x2122;s i.e.in age group of 60-70 years. The youngest patient of GBMO was a 16 year old male and the oldest patient was also a male of 72 years. Out of 9 cases of GBMO , 6 were found in males and 3 in females. So GBMO was more common in male just as Conventional GBM, but the M: F ratio of GBMO was 2:1, compared to 3.3:1 in Conventional GBM. Patients with GBMO (33%) more commonly presented with convulsion than Conventional GBM (7.7%). Sites commonly affected by GBMO were frontotemporal, temporo-parietal, and parieto-occipital (22.2%). www.pacificejournals.com/apalm

A-779 In a study of Yongzhi Wang et al., 40 (18.3%) of the 219 primary GBMs selected fulfilled the criteria for GBMO. Fourteen patients (35%) had seizure attacks as presenting symptoms. The patients with GBMO were more likely to present with seizures than were patients with conventional GBM. There were no significant differences in sex, tumor location, tumorrelated seizures were more frequent in the GBMO patients (35%) than in conventional GBM patients (19.7%). Microscopic examination of GBM-O showed Oligodendroglial component intermingled with or in different foci of glioblastomatous tissues (figure-19). Majority of the cases showed marked cellularity (66.6%), moderate pleomorphism (88.9%), and presence of small cells (88.9%), palisading necrosis (66.7%) and thrombosed blood vessels (22.2%). All the cases showed microvascular proliferation (100%) and Mitosis (100%). These findings were similar to findings mentioned in literature[14, 15] Giant cell GBM is a rare variant of GBM thought to encompass 2-5% of GBM diagnoses [1], while in our study incidence is about 7.5% which is slightly higher. In the present study, patients with Giant cell GBM presented mainly with headache (66.7%).This finding was almost similar to Conventional GBM (69.2%). The other symptoms were loss of consciousness, aphasia and paresis (33.3 %) each. In contrast to the Conventional GBM, majority of the cases (66.7%) presented with duration of symptoms of more than 3 months. This finding is in contrast with the findings of Valle et al. where they found the duration of symptoms to be short (and similar to Classical GBM)[16]. The difference noted may be due to small sample of the gcGBM in the present study. As the name implies, the tumor cells are markedly enlarged and bizarre, often appearing multinucleated. All the cases showed marked cellularity, moderate pleomorphism , Giant cells , palisading, non-palisading and pseudo palisading necrosis were found in 33.3% respectively. All cases showed mitosis and microvascular proliferation. Gemistocytes were seen in 66.7% and 33.3% showed perivascular lymphocytes. In this study, patients with Conventional GBM and gcGBM showed similar gender and racial distributions as well as insignificant tumour size and location differences. However, age at diagnosis was significantly younger in gcGBM vs. GBM (51 vs. 62 years) and gcGBMs were more likely to undergo complete resection. [17] In the present study gliosarcoma accounted for 2.5% of all GBM case (1/40). In various series, the incidence of GS has been reported to vary from 2% to 8 % [18]. This corroborates with our study findings. eISSN: 2349-6983; pISSN: 2394-6466

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In the present study, the only case of Gliosarcoma, a 55 year old female presented with psychiatric symptoms since 8 days. Tumour was located in the left fronto-parietal region. Study by Morantz [19]and colleague described that great percentage of patients with GS were older than 60 years of age and in study by Manisha et al [6] most of the patients were in the age group of 51 to 70 years and found GS most commonly in the temporal region, followed by frontal and parietal. Common site of occurrence was cerebrum and the common clinical symptoms were muscle weakness, headache and mental changes.

differentiating the variants as radiological differentiation is not possible due to similar appearing features. Newer diagnostic technique like immunohistochemistry and molecular analysis has great prognostic significance and help to decide the line treatment. 1.

Kleihues P, Burger PC, Aldape KD, Brat DJ, Biernat W, Bigner DD. Glioblastoma. In: Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, eds. WHO Classification of Tumours of the Central Nervous System. Lyon, France: IARC Press; 2007:33–49.

Microscopic examination of GS showed sarcomatous tissue intermingled with or in different foci of pre-existing glioblastomatous tissues . The case showed marked cellularity, moderate pleomorphism, small cells, nonpalisading necrosis, mitosis, microvascular proliferation and presence of secondary structure of Scherer. These findings were similar to Manisha khanna et al[6]. Narendra Kumar et al.[20] reviewed 27 gliosarcoma patients and found that all the tumors were having the biphasic histologic pattern consisting of gliomatous and sarcomatous components which is in accordance to our study.

Dolecek TA, Propp JM, Stroup NE, Kruchko C. CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2005-2009. Neuro Oncol 2012; 14 Suppl 5: v1-49.

Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ et al. European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group. N Engl J Med. 2005 Mar 10;352(10):987-96.

Iacob G, Dinca EB. Current data and strategy in glioblastoma multiforme. J Med Life. 2009;2:386-93.

Ghosh A, Sarkar S, Begum Z, Dutta S, Mukherjee J, Bhattacharjee M, et al. The first cross sectional survey on intracranial malignancy in Kolkata, India: reflection of the state of the art in southern West Bengal. Asian Pac J Cancer Prev. 2004;5(3):259-67.

Khanna M, Mendiratta P, Roy S: Clinicopathological study of 115 cases of Glioblastoma multiforme with special reference to Gliosarcoma – An original research article. Int J Pharm Sci Res 2013; 4(6); 2384-2392

Rees JH, Smirniotopoulos JG, Jones RV et-al. Glioblastoma multiforme: radiologic-pathologic correlation. Radiographics. 1996;16 (6): 1413-38.

Chandrasoma PT, Smith MM, Apuzzo ML. Stereotactic biopsy in the diagnosis of brain masses: comparison of results of biopsy and resected surgical specimen. Neurosurgery. 1989 Feb;24(2):160-5.

Miller CR , Perry A. Glioblastoma. Arch Pathol Lab Med 2007; 131:397-406.

In our study we found 1 case of GBM-PNET in a 26 year old female who presented with convulsions since 15 days. The tumor histo-morphologically consisted of undifferentiated cellular areas alongside classic GBM areas. The undifferentiated cellular areas composed of small undifferentiated cells with scant cytoplasm and oval round hyper chromatic nuclei. Immunohistochemistry revealed astrocytic component of the tumor strongly positive for GFAP and undifferentiated area stained strongly for synaptophysin. This concluded the diagnosis of GBM-PNET. [Table6] As the clinical and histopathological properties of those tumors have been described only recently, the number of cases reported in the literature is limited. The largest series published on these tumors belong to Varlet et al[ 21](n=40) and Perry et al. (22) (n=53). These studies highlighted the clinical, radiological, and histopathological differences of GB-PNET from classic GBM. They are encountered more commonly among adults and 52.5% of tumors are localized in the temporal lobe, whereas they are rarely seen with infratentorial localization. Having a well circumscribed character facilitates the surgical excision.

Conclusion

The most common histological findings of GBM included marked cellularity, moderate pleomorphism, microvascular proliferation, non-palisading necrosis and mitosis. Clinical and histopathology remains the main tool for

References

10. Karsy M, Gelbman M, Shah P, Balumbu O, Moy F, Arslan E. Established and emerging variants of glioblastoma multiforme: review of morphological and molecular features. Folia Neuropathol. 2012;50(4):301-21. 11. Kraus JA, Lamszus K, Glesmann N, et al. Molecular genetic alterations in glioblastomas with oligodendroglial component. Acta Neuropathol. 2001;101:311–320. 12. Salvati M, Formichella AI, D’Elia A, et al. Cerebral glioblastoma with oligodendrogliomal component: analysis of 36 cases. J Neurooncol. 2009;94:129–134. 13. Homma T, Fukushima T, Vaccarella S, et al. Correlation among pathology, genotype, and patient outcomes in glioblastoma. J Neuropathol Exp Neurol. 2006;65:846–854.

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Shenoy A. et al. 14. Wang Y, Li S, Chen L, You G, Bao Z, Yan W, Shi Z, Chen Y, Yao K, Zhang W, Kang C, Jiang T. Glioblastoma with an oligodendroglioma component: distinct clinical behavior, genetic alterations, and outcome. Neuro Oncol. 2012 Apr; 14(4):518-25. 15. Salvati M, Formichella AI, Dâ&#x20AC;&#x2122;Elia A, Brogna C, Frati A, Giangaspero F, Delfini R, Santoro A. Cerebral glioblastoma with oligodendrogliomal component: analysis of 36 cases. J Neurooncol 2009;94: 129-134. 16. Valle-Folgueral JM, Mascarenhas L, Costa JA, Vieira F, Soares-Fernandes J, Beleza P, et al. Giant cell glioblastoma: review of the literature and illustrated case. Neurocirugia (Astur ) 2008;19(4):343-9. 17. Kozak KR, Moody JS. Giant cell glioblastoma: a glioblastoma subtype with distinct epidemiology and superior prognosis. Neurooncology 2009; 11: 833-841.

A-781 18. Dohrmann GJ, Farwell JR, Flannery JT. Glioblastoma multiforme in children. J Neurosurg 1976; 44 :442-8. 19. Morantz RA, Feigin I, Ransohoff J. Clinical and pathological study of 24 cases of gliosarcoma. J Neurosurg 1976; 45: 398-408. 20. Kumar N, Kumar P, Angurana SL, Khosla D, Mukherjee KK, Aggarwal R, et al. Evaluation of outcome and prognostic factors in patients of glioblastoma multiforme: A single institution experience. J Neurosci Rural Pract 2013, 4(Suppl 1):s46-55. 21. Varlet P, Soni D, Miquel C, Roux FX, Meder JF, Chneiweiss H, Daumas-Duport C. New variants of malignant glioneuronal tumors: a clinicopathological study of 40 cases. Neurosurgery. 2004 Dec;55(6):1377-91. 22. Perry A, Miller CR, Gujrati M, et al. Malignant gliomas with primitive neuroectodermal tumor-like components: a clinicopathologic and genetic study of 53 cases. Brain Pathol. 2009;19:81â&#x20AC;&#x201C;90.

*Corresponding author: Dr. Shruti Shribhagwan Singhal, C-wing Flat No. 701, Sai Shradha Bldg ,Phase-1,Ashokvan , Borivali East Mumbai (India)400066 Phone: +91 7709207631 Email: shrutisinglasep@yahoo.co.in Date of Submission : 24.08.2017 Date of Acceptance : 09.09.2017 Financial or other Competing Interests: None. Date of Publication :22.12.2017

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Original Article DOI: 10.21276/APALM.1538

Dermatological Manifestations In Human Immunodeficiency Virus Infected Patients: Histopathology and CD4 Cell Count Correlation Poonam Radadiya* and Sheetal Kher Dept of Pathology, GMERS Medical College, Gotri, Vadodara (India)

ABSTRACT Background: More than 90% of the Human immunodeficiency virus (HIV) infected patients develop skin lesions at some point throughout the course of the disease. Although these conditions may be seen in general healthy population, their occurrence in patients with acquired immunodeficiency syndrome is often atypical, more severe and explosive. Histopathology study of skin biopsies from these lesions is very useful for the final diagnosis of these lesions. Methods: Total 60cases were studied. Punch biopsies from the skin lesions were studied with routine histopathology examination and special stains were used as and when required. The results were divided into infectious and non-infectious categories. Results were correlated with the CD4 counts of the patients. Results: Out of that22 (36.66%) patients had infectious lesions and 38 (63.33%) patients had non-infectious lesions. The spectrum of various non-infectious and infectious lesions such as viral, bacterial, fungal, protozoa, dermatitis, popular lesions and their association with CD4 counts is discussed. Conclusion: In present study mean CD4 cell count was found to be low (<350) in individuals with infectious skin lesions, where as noninfectious lesions were associated with higher (>350) CD4 cell counts. Keywords: CD4, Histopathology, Human Immunodeficiency Virus, Punch Biopsy, Skin Lesions

Introduction

India has the third largest HIV epidemic in the world. In 2015, HIV prevalence in India was estimated 0.26%. [1] Diseases of skin and mucous membranes are common clinical manifestations of acquired immunodeficiency syndrome (AIDS). Cutaneous disorders may be the initial signs of HIV-related immunosuppression. Cutaneous disorders are not only associated with terminal immunodeficiency, but also occur throughout the course of human immunodeficiency virus (HIV) infection. Cutaneous manifestations of human immunodeficiency virus (HIV) may result from HIV infection itself or from opportunistic disorders secondary to decline in immunocompetence from the disease.[2]More than 90% of patients develop skin lesions at some time during the disease. In some patients, skin is the first organ affected.[3] Skin diseases have proved to be sensitive and useful measures by which HIV progression can be monitored. Impaired skin immune system occurring early in HIV disease is believed to be responsible for the frequent occurrence of both infectious and non-infectious skin diseases even before the development of full blown HIV infection. [4] Although skin lesions may be seen in the general healthy population, their

occurrence in HIV infected patients is often atypical and more severe, explosive, extensive or resistant to therapy. The unusual histology of some of the diseases in AIDS may contribute to misdiagnosis. Thus, proper histological diagnosis of skin manifestations is very important as it may serve as the earliest manifestation to suspect a case of HIV infection. Infectious agents can produce skin lesions even though the classic organ of involvement for that agent does not include the skin.

Materials and Methods

This was a prospective observational study of 1 year duration carried out in the Department of Pathology of a tertiary referral center. Total 60 known HIV positive patients of all ages with symptomatic skin lesions attending skin and venereal disease out-patient department and Anti Retroviral Therapy Clinic at this center were included in the study. Patientâ&#x20AC;&#x2122;s HIV positivity was confirmed by three different sets of Ag systems (HIV comb-AIDS Rapid test, Rapid spot test and Tridot). The complete clinical details, in particular skin lesions were noted along with CD4 counts. Irrespective of any other systemic involvement or presence of other STDs, only skin lesions were sampled after taking informed written consent. The lesions were sampled using

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the punch biopsy and the diagnosis was made with routine histopathology examination and with the help of special stains as and when required. Pertinent clinical history like age, duration of the lesion, site of the lesion, significant family and personal history, history of associated diseases and any drug intake was taken and entered in the Performa. After detailed general and local examination, the site of the biopsy was selected. All universal aseptic precautions according to National AIDS Control Organization guidelines were followed. The selected patientâ&#x20AC;&#x2122;s consent was taken after explaining the details of the biopsy procedure. The three dimensional size and shape of the skin biopsy was assessed including the circular or elliptical shape of the biopsy for gross examination. The biopsy obtained was processed by standard formalin fixing paraffin embedding method. Serial sections and special stains were studied as and when required. The diagnosis was correlated with the CD4 counts.

Results

Out of total 60 known HIV infected patients, 43 were males and 17 were female patients. Maximum numbers of cases were seen in between 31 and 40 years of age group [Table 1]. CD4 counts were correlated in all 60 cases. Out of 60 HIV infected patients, 18 (30%) patients had CD4 counts <350, 33 patients showed CD4 counts between 350 to500, 09 patients had CD4 count >500. Out of this

60 cases, 22 (36.66%) had infectious pathology, whereas 38 (63.33%) patients had non-infectious pathology. Variety of infectious skin lesions were observed such as viral, bacterial, fungal and parasitic (Arthropod) infections. Total 07 (11.6%) patients showing viral pathology included Molluscum contagiosum, human papilloma virus (HPV), herpes zoster and herpes simplex virus (HSV). Total 08 (13.3%) patients had bacterial infections, which included leprosy, cutaneous tuberculosis, folliculitis, syphillis, donovanosis and furunculosis. One (1.6%) case of parasitic infections was seen which was of leishmaniasis. Total fungal infections were 06 (10%), which included candidiasis, dermatophytoses-tinea, cryptococcosis and histoplasmosis. Infectious results were correlated with the CD4count of the patients [Table 2]. Which shows most (15 out of 22) of the patients with infectious lesions had CD4count less than 350. Out of 38 non-infectious patients, majority of patients (10) had papular lesions including pruritic papular eruptions (PPE), seborrheic dermatitis, psoriasis, eosinophilic folliculitis and lichen planus. Total four patients had vascular lesion and three patients had malignant/premalignant lesions, whereas two patients had other non-specific pathology including follicular keratosis and perforating keratosis. These results were correlated with CD4counts of the patients [Table 3]. Which shows most of the patients (35 out of 38) with non-infectious lesions had CD4count more than 350.

Table 1: Age distribution in the present study. Sr.no. 1. 2 3 4

Age in years 21-30 31-40 41-50 51-60 Total

Number 20 25 11 4 60

% 33.3 41.6 18.3 6.6 100.0

Table 2: Infectious lesions observed in the study with CD4 count correlation. Sr no 1

3 4

Histopathological diagnosis Viral lesions:- Warts, Condyloma Lata, Molluscum contagiosum Bacterial infections:- Chanchroid, Hansens, Tuberculosis verrucosa cutis, Folliculitis, Pustular lesion, Lupus vulgaris, Syphilis, Pyoderma gangrenosum. Fungal infections:- Candidiasis, Cryptococcus, Histoplasma, TineaCorporis Parasitic &Protozoal infections:Leishmaniasis, Scabies Total

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Numb er (n=60)

Mean CD4

CD4 Count <350

350-500

>500

11.6

343.28

13.3

394.5

144.66

1.6

256.0

36.66

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Table 3: Non infectious lesions observed in this study with CD4 count correlation. Sr no. Histopathological diagnosis

Numb er (n=60)

CD4 Count

Mean CD4

<350

350-500

>500

Dermatitis:- Atopic Dermatitis, Ashy Dermatitis, Urticaria, Hyperpigmented patch, chronic non-specific dermatitis.

13.3

421.62

Papular lesion:- Pruritic papular, Psoriasis, Seborrhoic Dermatitis, Eosinophilic folliculitis, Scaly lesion, Lichen planus

16.6

428.7

Epidermal Lesion:- Epidermoid cyst, Seborrhoic keratosis

5.0

438.0

Drug reaction

Vascular lesion:- Leukocytoclastic vasculitis, Granuloma Pyogenicum

6.6

427.25

Connective tissue lesion:- DLE, Granuloma Annulare

6.6

422.75

Malignant/Premalignant lesion:- Verrucous Carcinoma, Squamous cell carcinoma, Bowenoid papulosis

5.0

273.66

Vesiculobullous lesion:- Pemphigus vulgaris, Bullous pemphigoid, Bullous impetigo, Erythema Multiforme

6.6

448.25

Others:- Follicular keratosis, Perforating folliculitis

3.3

475.0

Total

63.33

377.01

Fig. 1: Photograph showing cutaneous leishmaniasis: LD bodies are seen intracellularly in macrophages and extracellularly [H&E stain, 100x].

1 1

Fig. 2: Cutaneous candidiasis showing budding hyphae (red stained) [PAS stain, 100x].

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Fig. 3: Leprosy: Hypopigmented patch over forearm and microscopic picture showing atrophic epidermis with compact granuloma along neurovascular bundle [H&E stain, 10x].

Fig. 4: Lepromatous Leprosy: Rod shaped Lepra bacilli stained pink in blue background [Fite-Faraco stain, 100x].

Discussion

Although a lot of literature regarding the etiology of cutaneous manifestations in HIV patients is available in Western world and some parts of Asia, very few case studies in Indian patients are available. In the present study the most affected age group was 21-40 years constituting 74.9%and a second peak in the age >40years with 24.9%. In the study conducted by Sanjay M. Chawhan et al. most of the patients were from 31 to 40 years age group.[5] In the study conducted by Nirja jindal[6] most affected age group was 21-40 years constituting 73.7% and a second peak in the age group >40 years with 21%. A study by Kumarasamy et al. had 50% patients between 18 and 30 years age group.[7]In the present study, we found 71.6% male patients as compared with 28.3% females, with 2.5: 1, male to female ratio. The study conducted by Sanjay M. Chawhan et al. showed 67% male patients as compared with 33% females.[5] A study done by Nirja jindal showed female preponderance with a male to female ratio of 0.9:1. [6] This difference may be due to difference in geographical distribution. In our study of 60 HIV infected patients, CD4 correlation was done in all patients. Maximum patients, i.e., 33 (55%) had CD4 count between350 to 500, followed by 18 (30%) patients with CD4 counts below350and 09 (15%) patients had CD4 counts above 500. Maximum number of infective lesions and malignancy were seen in patients with CD4 counts below 350 [Table 2].Whereas patients with CD4 count above 350 showed minimum infective, but more of the non-infectious lesions [Table 3]. Which is similar to the results of the study done by Sanjay M. Chawhan et al. which show Maximum number of infective lesions in patients with CD4 counts below 350 whereas patients with CD4 count above 350 showed minimum infective, but most of the non-infectious lesions.[5]Previous

studies showed that CD4 counts <200 cells/cumm were associated with more number of infectious lesions.[4,8,9,10,11] Muñoz-Pérez (1998) stated that various dermatoses such as genital herpes, tinea, Kaposi’s sarcoma, xerosis, HSV, drug eruptions, candidial folliculitis, M. contagiosum, psoriasis, abscess, verruca vulgaris, PPE, oral hairy leukoplakia and seborrheic dermatitis could be used as clinical markers of disease progression due to their strong association with CD4 counts.[12].We found that 22 out of 60 (36.66%) patients had infectious lesions. Unusual clinical presentations of common skin infections or florid, unusual forms had been described in these patients by various authors.[9] In these patients, infectious agents can produce skin lesions even though the classic organs of involvement for that agent do not include the skin, e.g., cryptococcosis, Cytomegalovirus and histoplasmosis. We found 07 (11.6%) patients with viral lesions, 08 (13.3%) patients with bacterial lesions including leprosy, 06 (10%) patients with fungal infection and 01 (1.6%) patient with parasitic lesion containing leishmaniasis [Figure1]. In the study by Sanjay M. Chawhan maximum infectious lesions were of viral etiology.[5]Maximum patients showed CD4 counts <350 as studied by other authors.The HPV related lesions were verruca vulgaris, verruca plana and bowenoid papulosis and condylomata accuminata. Muñoz-Pérez et al. found no significant difference between the incidence of condyloma acuminata or verruca vulgaris in stage III and stage IV disease or with CD4 counts.[12] Muñoz-Pérez et al. in their study mentioned that HIV infection itself predisposes to an increased risk of HPV infection that is not directly related to the degree of immunosuppression.[12] Nichols et al. stated that bacterial infections in AIDS were often under represented.[13] In our study we found 08 (13.3%) cases of bacterial infection including Mycobacterium infections as

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compared to 14 (12.72%) cases of bacterial infection in the study by Sanjay M Chawhan et al.[5] Dermatological lesions of tuberculosis (TB) infection are rarely found in Western countries.[14] Various mycobacterium lesions in our study were leprosy (three cases of borderline tuberculoid and one case of tuberculoid leprosy), papulonecrotic tuberculid, scrofuloderma and TB cutis orificialis one each. In this study, six cases of fungal lesions were found which includes cases of dermatophytoses and candidiasis, histoplasmosis and cryptococcosis. All the patients with Fungal infections had CD4 counts below 350 cells/cumm. Which is similar to the study by Sanjay M Chawhan et al.[5] We found 1 (1.6%) case of parasitic infection, which is of dermal leishmaniasis. Sanjay M Chawhan et al. reported 7 (6.36%) cases of parasitic infection, which included six cases of demodicidosis and one case of scabies.[5]Kaplan et al. reported four cases of scabies who presented with pruritic dermatitis. Clinically, the lesions of scabies may resemble psoriasis vulgaris or Darier’s disease.[9,15] The most common non-infectious skin manifestation found in our study was PPE. The study by Sanjay M Chawhan et al. showed similar results.[5]They were intensely pruritic, papular lesions more on the trunk and extremities with a predominance of eosinophils as described by Francis. The severe form of similar eruptions is seen in African and Haitian patients.[8] Hevia et al. (1991) mentioned histological and clinical criteria for the diagnosis of these lesions.[16] Most of the cases of PPE in our study were seen with CD4 counts more than 350 cells/cumm. We have found three cases of eosinophilic folliculitis. Rosenthal et al. found its association in patients with CD4 counts between 200 and 500 cells/cumm.[17] It could be an important clinical marker of HIV infection, particularly in patients at increased risk of developing opportunistic infection. The clinical and histological differential diagnoses of eosinophilic folliculitis include demodicidosis and PPE. We found three cases of psoriasis. Incidence of psoriasis as high as 70% had been reported by Duvic et al.[18] We found one case of seborrheic keratosis. Although, it is mentioned that the incidence of seborrheic dermatitis is very high from 40% to 83% in Western literature; and in some other studies.[19] Two cases of granuloma pyogenicum are noted and four cases of connective tissue lesions are identified. In Malignant and premalignant conditions, three cases were reported; verrucous carcinoma, squamous cell carcinoma and bowenoid papulosis, one of each. Miscellaneous group included 2 cases of perforating folliculitis and follicular keratosis. Total eight cases of dermatitis were reported. In this study, no any case of Kaposi’s sarcoma or lymphoma were found. Wiwanitkit (2004), D. N. Lanjewar (2011) and Sanjay M Chwhan (2013) also found striking low prevalence of cutaneous and other malignancies in these

patients.[10,20,5]No any cases related to drug reactions are reported. Histological variations from normal were noted in certain lesions. In many biopsies, Periadnexal and perivascular inflammatory infiltrate, i.e., lymphocytic infiltration was seen in most of the lesions irrespective of histological diagnosis. Epidermal hyperplasia in the form of acanthosis, irregular parakeratosis or hyperkeratosis was seen in most of the lesions. None of the lesions showed normal epidermis. To summarize, in the present study on skin lesions in HIV infected individuals, infectious skin lesions were seen more commonly with CD4 counts below 350 and non-infectious skin lesions were seen more commonly with CD4 counts more than 350. The most common infectious lesion was because of viral infections followed by bacterial infections and most common noninfectious lesion was PPE. Strikingly low occurrence of cutaneous malignancies was seen in the present study.

Abbreviations

HIV- Human immunodeficiency virus PPE- Pruritic papular eruption HSV- Herpes simplex virus HPV- Human papilloma virus

References 1.

NACO (2015) ‘Annual report 2015-16’.www.naco.gov.in

Cedeno-Laurent F, Gomez-Flores M, Mendez N, AncerRodriguez J, Bryant JL, Gaspari AA, et al. New insights into HIV-1-primary skin disorders. J int AIDS soc.,2011 jan 24. 14:5.

Coldiron BM, Bergstresser PR. Prevalence and clinical spectrum of skin disease in patients infected with human immunodeficiency virus. Arch Dermatol. 1989;125:357–61.

Tschachler E, Bergstresser PR, Stingl G. HIV-related skin diseases. Lancet. 1996;348:659–63.

Chawhan SM, Bhat DM, Solanke SM. Dermatological manifestations in human immunodeficiency virus infected patients: Morphological spectrum with CD4 correlation. Indian J Sex Transm Dis. 2013 Jul-Dec; 34(2): 89–94.

Jindal N, Aggarawal A. HIV seropositive and HIV associated dermatosis among patients presenting with skin and mucocutaneous disorders. Indian journal of dermatology, venerology andleprology. 2009;75:283-286.

Kumarasamy N, Vallabhaneni S, Flanigan TP, Mayer KH, Solomon S. Clinical profile of HIV in India. Indian J Med Res. 2005;121:377–94.

Francis N. Non-neoplastic, cutaneous and mucocutaneous manifestations of HIV infection. Histopathology. 1993;23:297–305.

Kaplan MH, Sadick N, McNutt NS, Meltzer M, Sarngadharan MG, Pahwa S. Dermatologic findings and manifestations of

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acquired immunodeficiency syndrome (AIDS) J Am Acad Dermatol. 1987;16:485–506.

15. Inserra DW, Bickley LK. Crusted scabies in acquired immunodeficiency syndrome. Int J Dermatol. 1990;29:287–9.

10. Wiwanitkit V. Prevalence of dermatological disorders in Thai HIV-infected patients correlated with different CD4 lymphocyte count statuses: A note on 120 cases. Int J Dermatol. 2004;43:265–8.

16. Hevia O, Jimenez-Acosta F, Ceballos PI, Gould EW, Penneys NS. Pruritic papular eruption of the acquired immunodeficiency syndrome: A clinicopathologic study. J Am Acad Dermatol. 1991;24:231–5.

11. Kumarasamy N, Solomon S, Madhivanan P, Ravikumar B, Thyagarajan SP, Yesudian P. Dermatologic manifestations among human immunodeficiency virus patients in south India. Int J Dermatol. 2000;39:192–5. 12. Muñoz-Pérez MA, Rodriguez-Pichardo A, Camacho F, Colmenero MA. Dermatological findings correlated with CD4 lymphocyte counts in a prospective 3 year study of 1161 patients with human immunodeficiency virus disease predominantly acquired through intravenous drug abuse. Br J Dermatol. 1998;139:33–9. 13. Nichols L, Balogh K, Silverman M. Bacterial infections in the acquired immune deficiency syndrome. Clinicopathologic correlations in a series of autopsy cases. Am J Clin Pathol. 1989;92:787–90. 14. Dover JS, Johnson RA. Cutaneous manifestations of human immunodeficiency virus infection. Part II. Arch Dermatol. 1991;127:1549–58.

17. Rosenthal D, LeBoit PE, Klumpp L, Berger TG. Human immunodeficiency virus-associated eosinophilic folliculitis. A unique dermatosis associated with advanced human immunodeficiency virus infection. Arch Dermatol. 1991;127:206–9. 18. Duvic M, Johnson TM, Rapini RP, Freese T, Brewton G, Rios A. Acquired immunodeficiency syndromeassociated psoriasis and Reiter’s syndrome. Arch Dermatol. 1987;123:1622–32. 19. Vasudevan B, Sagar A, Bahal A, Brig AP, Mohanty VS. Cutaneous manifestations of HIV – A detailed study of morphological variants, markers of advanced disease, and the changing spectrum. Med J Armed Forces India. 2012;68:20–7. 20. Lanjewar DN. The spectrum of clinical and pathological manifestations of AIDS in a consecutive series of 236 autopsied cases in mumbai, India. Patholog Res Int 2011. 2011 547618.

*Corresponding author: Poonam Radadiya, GMERS Medical college, Gotri, Vadodara (India)-390021 Phone: 0265 239 8008 Email: drsheetalkher@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 04.06.2017 Date of Acceptance : 11.09.2017 Date of Publication : 22.12.2017

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Original Article DOI: 10.21276/APALM.1645

Clinico Histopathological Correlation Study of Leprosy Bharat Ankush Ghodke, Arvind G Valand, Aradhana Bhubaneswar Deka*, Sushma Nagsen Ramraje and Zeba Shamshad Ali Department Of Pathology, Grant Government Medical College and Sir JJ group of Hospitals, Mumbai (India)

ABSTRACT Background: Aim of the study was to study the histomorphological changes in leprosy, the clinicopathological correlation of different types of leprosy lesions and cases of clinically diagnosed lepra reaction. Methods: One hundred and twelve patients of clinically diagnosed leprosy were chosen. Skin biopsy was performed, fixed in 10% buffered formalin, processed, sectioned and stained with Hematoxylin and Eosin as well as Modified Fite Faraco stain. Result: Of the one hundred and twelve patients enrolled in the study (age range from 7 to 70 years) 90 patients were males and 22 were females. All cases were classified according to the Ridley Jopling Scale. Correlation between clinical diagnosis and histopathological type of leprosy and lepra reactions was done. Conclusion: Leprosy is more common in males than in females having a ratio of 4:1.The commonest histopathological subgroup was tuberculoid leprosy. Keywords: Tuberculoid, Histopathology, Leprosy, Granuloma

Introduction

Leprosy is a chronic infectious, communicable disease caused by mycobacterium leprae, which expresses itself in different clinic – pathological forms depending on the host immunological status.[1]Leprosy is a major public health problem, which still has a social stigma and myths attached to it.[2]It has been correctly said that some diseases do not take away the life, but they just ruin it. This holds true for leprosy because of the social stigma and complications like deformities associated with it. Though effective and simpler treatments are available, it is still difficult to identify early cases. Leprosy is a very ancient disease dating back many centuries. Possibly it originated in Africa and spread very early to India. References of leprosy are found in Indian, Chinese & Egyptian medical literature. Mention about Leprosy has been made in Manu smriti and was referred to as “Kushtha” in the ancient Vedic writings.[3] The prevalence rate is 2-4 per thousand population. India accounts for 1/3 of the leprosy cases in the world. [4] 2,13,899 leprosy cases reported in 2014 globally. India is still the country contributing largest number of new leprosy cases which account for 2/3rd of the new leprosy cases detected annually. In India, a total of 1,27,000 new cases were detected during 2013-2014. Annual new case detection rate (ANCDR) was 9.98/100000 population which decreased from 10.79 in 2012-2013. 33 states and

union territories have achieved the level of elimination, i.e PR less than 1 case per 10,000 population. However, some areas still have high endemicity rate.(PR more than 1 case per 10,000 population) .[5] Early diagnosis of leprosy is important to reduce the morbidity. Ridley and Jopling in 1974 suggested a classification system which employed correlation of clinical & histopathological status. Similar attempt to judge the utility of this method in the present study was undertaken.

Materials and Methods

A prospective study of 112 cases was carried out from April 2001 to June 2003.Clinical Examination was thoroughly done. Type of lesions such as hypopigmented anesthetic patch / plaque / papule / nodule / infiltration was noted. A representative lesion was chosen for the present study and skin biopsy was performed on O.P.D. basis. Biopsy was fixed immediately on removal in 10% buffered formalin and processed routinely and stained by Hematoxylin & Eosin Stain and Modified Fite - Faraco Stain.

Result

An attempt has been made to correlate clinically suspected cases of leprosy with histopathological findings and classifying them according to Ridley and Jopling Scale.[6]

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Amongst 112 clinically diagnosed cases, 48 cases (42.85%) were of Borderline Tuberculoid Leprosy followed by 18 cases (16.11%) of Borderline Lepromatous Leprosy and only 1 case of Type I Lepra Reaction (0.89%). Histopathologically out of the 112 cases, Tuberculoid leprosy was diagnosed with highest number of 36 cases (32.14%), followed by 31 cases of Borderline Tuberculoid Leprosy(27.68%), 14 cases of (12.5)%, 11 cases of Indeterminate Leprosy Lepromatous Leprosy (9.82%), 8 cases of Borderline Leprosy (7.14%), 6 cases of Borderline Lepromatous Leprosy (5.36%), 3 cases of Histoid Leprosy (2.68%), 2 cases of Type II Reaction (1.75%) and 1 case of Type I Reaction (0.89%). There were 90 (80.35%) male patients and 22 (19.65%) female patients. Age group varied from 1st decade to 7th decade and the maximum number of cases found in between the age group of 11 to 40 years. The youngest patient was of 7 years old while the oldest was 70 years old. Majority of Leprosy lesions were situated on forearm. The remaining lesions were scattered over the body. Out of 16 clinically diagnosed Tuberculoid leprosy cases, histopathological diagnosis was confirmed with 13 cases (81.25%), while 2 cases turned out to be of Borderline Tuberculoid Leprosy and 1 case of Indeterminate Leprosy. Out of 48 clinically diagnosed cases of Borderline Tuberculoid Leprosy only 22 cases (45.83%) showed histopathological agreement and of the remaining cases 13

cases were diagnosed to be TT, 3 cases as BB, 1 case of Borderline Lepromatous Leprosy, 8 Indeterminate cases and 1 case showed Type I Reaction. Out of the 4 clinically diagnosed Borderline Leprosy cases only 1 case showed agreement (25%), other 2 cases turned out to be of TT and 1 case of Borderline Lepromatous Leprosy. Out of 18 clinically diagnosed Borderline Lepromatous Leprosy cases, only 2 cases showed histopathological agreement (11.11%). 5 cases were diagnosed to be Tuberculoid Leprosy, 4 cases of Borderline Tuberculoid Leprosy, 2 cases of Borderline Leprosy, 4 cases of LL and only 1 case was diagnosed as Indeterminate type of leprosy. 8 cases were clinically diagnosed as Lepromatous leprosy, of which 6 cases were confirmed with histopathology (75%), 1 case was diagnosed as BTH and other case was of Histoid Leprosy There were 11 clinically suspected leprosy cases (clinically not divided in any group). In histopathological diagnosis, 3 cases turned out to be TT, 1 of BTH, 2 of BB, 1 of BLH, 1 of LL and 3 cases were diagnosed as indeterminate. Out of two clinically diagnosed Histoid Leprosy showed 100% agreement on histology. Out of 2 clinically diagnosed indeterminate cases one case(50%) showed agreement and the other case turned out to be BTH. Clinically One case of Type I Reaction showed 0% agreement which turned out to be a case of BLH. Clinically 2 cases of Type II Reaction showed 100% agreement on histopathology.

Table 1: Distribution of 112 clinically Diagnosed Cases of Leprosy: Sr. No. 1 2 3 4 5 6 7 8 9 10 Â

Clinical Diagnosis TT BTH BB BLH LL INTD Hansen Histoid Type I Type II Total

No. of Cases 16 48 4 18 8 2 11 2 1 2 112

Percentage (%) 14.29% 42.86% 3.57% 16.07% 7.14% 1.79% 9.82% 1.79% 0.89% 1.79% 100.00%

Table 2: Distribution of 112 histopathologically Diagnosed Cases of Leprosy: Sr. No. 1 2 3 4 5 6

Histopathological Diagnosis TT BTH BB BLH LL INDT

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No. of Cases 36 31 8 6 11 14

Percentage (%) 32.14% 27.68% 7.14% 5.36% 9.82% 12.50%

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Histopathological Diagnosis Histoid Type I Type II (ENL) Total

No. of Cases 3 1 2 112

Percentage (%) 2.68% 0.89% 1.79% 100.00%

Table 3: Age wise distribution of Leprosy cases. Age 0-10 11-20 21-30 31-40 41-50 51-60 61-70 Total

T.T 3 5 7 8 7 3 3 36

B.T.H 9 12 4 1 4 1 31

B.B 3 2 1 1 1 8

B.L.H 2 1 2 1 6

L.L 2 5 2 1 1 11

INDT 5 2 4 1 1 1 14

Hist 1 2 3

Type1 1 1

Type2 1 1 2

Total 3 24 26 25 14 10 10 112

% 2.67 21.43 23.22 22.32 12.51 8.93 8.92 100

Table 4: Correlation between Clinical Diagnosis and Histopathological Type of Leprosy and Reactions. Clinical Type T.T. B.T.H. B.B. B.L.H. L.L. INDT Histoid Type I Type II Suspected Hansen Total

No. of Cases 16 48 4 18 8 2 2 1 2 as 11 112

Histopathological Type T.T.

B.T.H

13 13 2 5 – – – – –

2 22 – 4 1 1 – – –

– 3 1 2 – – – – –

– 1 1 2 – – – 1 –

– – – 4 6 – – – –

3 36

1 31

2 8

1 6

1 11

Histoid

Type I

Type II

Percentage of Agreement / Disagreement

1 8 – 1 – 1 – – –

– – – – 1 – 2 – –

– 1 – – – – – – –

– – – – – – – – 2

81.25 / 19.75 45.83 / 54.17 25 / 75 11.11 / 88.89 75 / 25 50 / 50 100 / 0 0 / 100 100 / 0

3 14

– 3

– 1

– 2

0/0

B.B. B.L.H. L.L INDT

Fig. 1: Tuberculoid Leprosy (TT) – Showing multiple epithelioid cell granuloma involving superficial and deeper dermis. (H&E, 10X).

Fig. 2: Borderline Tuberculoid Hansen – Showing multiple granulomas in deeper dermis along with giant cells. (H&E, 10X).

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Fig. 3: Borderline Tuberculoid Hansen (BTH) â&#x20AC;&#x201C; Low power view showing ill formed epithelioid cell granulomas in superficial dermis with sparing of ssubepidermal zone.

Fig. 4: Lepromatous Leprosy (LL) - Higher power view showing collection of foamy macrophages in superficial dermis with thinned out epidermis. (H&E,)10X).

Discussion

25 cases (22.32%) on face, ear and neck. 17 case (15.17%) on lower extremities (foot, ankle, knee and thigh), 14 cases (12.5%) on chest, abdomen and buttocks, 10 cases (8.9%) had lesion all over body and 6 cases (5.35%) were on back. However, variation in site of involvement was seen in other studies.

A total of 1723 biopsies were received in our department during April 2001 to June 2003, of which Skin biopsies were 1099. Out of these, 112 cases were diagnosed as Leprosy (6.53%). Since[7]Leprosy has a wide spectrum of Clinical manifestation; hence a good classification is an effective means of understanding and communicating concepts regarding a difficult case. Ashok S.K. et al (1995) [8] studied 27 clinically diagnosed cases of Leprosy, in which they found 1 case (3.7%) of TT, 20 cases (74.07%) of BTH, 1 case (3.7%) of BB, 2 cases (7.40%) of BLH and 1 case of (3.70%) of LL. Surinder et al in 1993[9] in their study found of 60 clinically diagnosed Leprosy cases, 25 cases (41.66%) were of BTH, 28 cases (46.66%) were of BLH and 7 cases (11.68%) were of LL. In the present study variation of age group was found between 7 years to 70 years. 89 cases (79.46%) were between age group of 11-50 years. In the study of Rao P.S.S. et al. [10], adults were found to be affected twice than children. Mathur et al[11] in 1978 found that majority of cases were in the age group of 21- 50 years. This emphasise that although Leprosy is borne at an early age, but because of relatively long incubation period the symptomatic cases appear at later age. In the present study there were 90 males (80.35%) and 22 females (19.65%). Male to female ratio was 4:1. Mathur et al[11] in 1978 and showed male preponderance of cases (3:1)

Tuberculoid leprosy (TT): There were 36 cases (32.14% ) of TT in our study which correlates well with study done by Sehgal VN et al. [7] The age involvement varied from 7 years to 65 years; with a peak incidence in 2nd 4th decade. Male to female ratio was found to be 3:1, this finding correlates well with findings of Rao P.S.S. [10]. The epidermis was unremarkable in 24 cases, 8 cases showed stratification of epidermis with hyperkeratosis. The dermis showed well formed epitheloid granulomas located both in superficial and deeper dermis. The granulomas seen in superficial dermis hugging the base of epidermis without any clear zone and involving the neurovascular bundle. Giant cells were present in 16 cases. Periadnexal dense infiltration of lymphocytes seen in all cases. Fite Faraco stain was negative in all cases.

Coming to the site of Involvement leprosy lesions almost occur all over body. In our study, upper extremities (palm, forearm, arm and shoulder) was the commonest site of involvement accounted for 40 cases (34.82%) followed by

Borderline tuberculoid Hansen: Out of 112 cases of leprosy, 31 cases (27.68%) were of BTH. 25 cases falls in age group of 11-40 years. The youngest patient diagnosed as BTH was of 12 yrs old male child. The male to female ratio was 5:1 in our series. This correlates well with study conducted by Kar PK. [12]having 38 cases of BTH most of the cases were adult and had male to female ratio of 3:1. Epidermis was unremarkable in 23 cases and 8 cases showed thinning of epidermis. The granulomas were few. 10 cases showed ill formed granulomas with involvement

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of nerve. However the giant cell number exceeded as compared with the lesions of TT. Involvement of adnexae and neurovascular bundles was seen. Fite Faraco stain was also negative. Borderline Leprosy [BB]: 8 cases of borderline leprosy were detected [7.14%], this correlates well with the study by Bhatia AS et al. [13] (8.25 %).All 8 cases were male patient with a peak incidence between 11-30 yrs. 5 cases of male preponderance was also noted by Bhatia AS et al. [13] with peak incidence in age group of 20 - 40 years. The epidermis was flat in 2 cases and unremarkable in the remaining. The dermis shows presence of foamy macrophages, which were uniformly activated to epitheloid cells. Lymphocytes were scanty and dispersed out around adnexal structures. Fite Faraco showed +1 positivity in 6 cases. Borderline Lepromatous Hansen (BLH): 6 cases of BLH were found making an incidence of 5.36%. Majority of cases presented in 3rd to 5th decade. All the six cases were male patients. .This is also a borderline group which has a tendency to move in both upper and lower polar form. Shenoi SD[14] has similar findings. 4 Out of 6 cases showed thinned out epidermis. One case showing clear zone underneath it. Foamy macrophages were present in the dermis in all 6 cases. Lymphocytes were prominent and dispersed. Fite Faraco stain showed +2 to +3 positivity in all 6 cases. . Lepromatous Leprosy (LL): 11 cases of LL making an overall incidence of 9.82%, which compares well with the study of Rao P.S.S[10] showing incidence of 11.13%. 7 cases were in the age group of 11- 40 yrs; earlier presentation may be because of early awareness in patients who attend O.P.D. immediately.8 cases classically showed presence of thinned out epidermis and sub epidermal clear zone. The underlying dermis showed presence of foamy macrophage in all 11 cases along with lymphocytes. Fite Faraco stain showed globi of AFB +3 to +4 in all the cases. Indeterminate Leprosy (INDT): 14 cases of indeterminate leprosy seen making an incidence of 12.5%. Age group varies from 11-40 years, which correlate well with the study conducted by Shenoi SD.[14] Epidermis was Unremarkable in 10 cases while four cases showed thinning. Sparse mononuclear infiltrate involving adnexae by lymphocytes was seen in 12 cases. Fite Faraco stain was negative in 10 cases. Histoid Leprosy: 3 cases of Histoid leprosy detected making an incidence of 2.68%. Desikan KV et al. [15]studied

109 cases of clinically diagnosed Histoid, of which only 25 cases were confirmed to be of Histoid Leprosy. All 3 of our cases were in the age group of 21-40 years. Epidermis was thinned out with a sub epidermal clear zone and a localized mass of polyhedral to spindle shaped histocytes oriented in a storiform pattern. Fite Faraco stain showed + 4 to +5 positivity. Type I Reaction: A single case of type I reaction in a 64 years male was found in our study making an incidence of 0.89% which is slightly lower as compared as other published literature. Epitheloid differentiation of macrophages, with a heavy mixed inflammatory infiltrate comprising of neutophils, lymphocytes and plasma cells was seen. There is also seen oedema, giant cells and necrosis. Type II Reaction (ENL): 2 Cases of Type II were detected (33 years male and 66years female) with incidence of 1.79% which is correlated well with the study done by Petit J.H.S.et al.[16]Heavy acute inflammatory reaction located in the deeper dermis and the subcutaneous tissue along with marked oedema. Fite Faraco stain was positive. The Histopathological classification[17] has advantage over clinical classification and it gives a better indication of any recent shift of patientâ&#x20AC;&#x2122;s condition in a spectrum. (Ridley, 1974). To confirm a case of Leprosy from a suspected lesion, histopathological examination must be carried out not only to make a definite diagnosis of leprosy but also to classify the type of the disease. Classification of the type of leprosy is essential for the treatment. Many workers (Shenoi et al 1988[14], Desikan KV et al 1975[15]) have conducted clinical and histopathological correlative studies in leprosy lesions and disparities between the clinical and histopathological features have been observed. In this study out of 16 clinically suspected cases of TT cases on only 13 (81.25%) cases showed correlation. Kar P. K. et al.[12] found Histopathological correlation (87.5%). (14 out of 16 cases). Bhatia AS et al.[13] found 100% correlation between Clinical and Histopathological Diagnosis of TT case. The correlation in our study between clinical and histopathological diagnosis in cases of BB was only 25%. Shenoi S.D et al.[14] found (54.5%) correlation i.e. out of 11 clinically diagnosed BB cases only 6 cases showed Histopathological correlation . In our study Histopathological correlation in cases of BLH was possible in only 2 cases out of the 18 clinically suspected cases (11.11%). Bhatia A.S et al.[13] showed that out of 109 clinically diagnosed cases of BLH only

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Valand et al. 47 cases (43.11%) showed histopathological correlation. In our study, out of 8 clinically suspected cases of LL only 6 cases showed Histopathological correlation(75%). Dubey G.K et al.[19]mentioned 93.54% cases with histopathological agreement out of 62 clinically diagnosed Lepromatous Leprosy cases.100% agreement was found in a study conducted by Shenoi S.D. et al.[14] In our study, out of 2 clinically suspected of Indeterminate Leprosy, 1 case showed Histopathological correlation (50%). Study conducted by Kar PK et al,[12] out of 32 clinically diagnosed cases of indeterminate case 22 cases showed Histopathological correlation (81.2%). We found 2 clinically suspected cases of Histoid Leprosy which showed 100% agreement with histopathological features. Desikan KV et al.[15] showed out of 109 clinically suspected Histoid Leprosy cases 67 cases showed histopathological agreement. In our study, 1 Histologically diagnosed case of Type I reaction which was clinically suspected as BTH. Sehgal VN et al.[22]diagnosed 11 cases of Type I reaction ( out of 11 cases 5 belong to upgrading and 6 to downgrading reaction). We found 2 Clinically suspected cases of ENL both showed Histopathological features of Type II reaction (100% agreement). Sehgal VN et al.[22] detected 11 cases of ENL in their study. The salient features of ENL showed vasculitis of dermis and subcutaneous tissue along with edema of dermis, and endothelial cell proliferation.

Conclusion

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Reference 1.

Panday AN, Tailor HJ. Clinicogistopathological correlation in leprosy. Ind J Dermatol Venerol Leprol 2008; 74: 174-76

Marcy-Rovenour; Public Health & Preventive Medicine. Jaipee Medical Publisher; Pg: 218-222; 1998.

Dharmendra; History of Spread and Decline of Leprosy; 24-26; Classification and Clinical Manifestation of Leprosy; 319-346; Textbook of Leprosy; Kothari Medical Publishing House; 1978.

Park K. Textbook of preventive and social medicine.14 th edition, Jabalpur, Bhanot publishers.1999; 223-233.

World Health Organisation. Global burden of leprosy at the end of 2012. Wkly Epidemiol Rec 2012;86:389-400.

Ridley DS, Jopling WH. Classification of leprosy according to immunity: a five group system, Int J Lepr Other Mycobacterial Disease, 1966,34:255-73.

Sehgal V.N, Rege V.L, Reys M; Correlation between clinical and histopathological classification in Leprosy; International Journal of Leprosy; Vol 45; 278 - 280.

Ashok SK., Reddy SN, Ratnakar C.; Correlation of Skin and Nerve Histopathology in Leprosy; Leprosy Review; 1996:67; 119 - 125;

Kaur S, Sharma VK; Concurrent skin and nerve histology in Leprosy and its role in the classification of Leprosy; Leprosy Review; 1993;110-16;.

10. Rao P.S.S, Karat A.B.A, Kaliaperumal V.G and Karat S; Prevalence of Leprosy in Gudiyatham Taluk, south India Part I. Specific rates with reference to Age, Sex and Type; 1772:40; 157-161;

In the Present study constituted 6.53 % of Leprosy cases from 1723 biopsies. Age predominantly affected was between 11 to 50 years. Males were affected more than female having a ratio of 4: 1. Commonest site of involvement being Upper Extremities. The commonest sub group of Leprosy diagnosed on Histopathology ground was tuberculoid leprosy. It was concluded that the Borderline Spectrum of Leprosy contributed to the highest number of cases followed by the polar type, Indeterminate and lastly the Reactions.

11. Mathur N.K, Kanwar A.J, Kalla Gand Ujwal J.J;Leprosy in Jodhpur; Clinical and epidemiological study; Leprosy in India; 1978;204 - 209;.

This study was useful as it had advantage over clinical classification and it gives a better indication of any recent shift of patientâ&#x20AC;&#x2122;s disease position in the spectrum and thus proper treatment could be imparted.

14. Shenoi S.D and Sidappa K; Correlation of Clinical and Histopathological Features; 1998;202-206;.

Acknowledgements

Heartfelt gratitude to Dr R.Ganapati, Eminent Leprologist from Bombay Leprosy project, situated at SionChunabhatti, Mumbai-22. Department of Dermatology, Sir J J Hospital and G T Hospital. All Leprosy Patients. www.pacificejournals.com/apalm

12. Kar P.K, Arora P.N, Ramashastry C.V, Sayal S.K and Dhaka R.S; A Clinico-Pathological Study of Macular Lesions in Leprosy; 1994;66:435-442. 13. Bhatia A.S, Katoch K, Narayan R.B, Ramu G, Mukherjee A and Ravinder K. Lavania; Clinical and Histopathological Correlation in the Classification of Leprosy; Vol-61, 433-438.

15. Desikan K.V and Iyer C.G.S; Histoid Variety of Lepromatous Leprosy - A Histopathologic Study; Vol-40; 149-156. 16. Petit J.H.S and Waters M.F; The etiology of erythema nodosum leprosum; 1967:35(1); 1-9;. 17. Ganapati R., S. S. Pandya, S. S. Naik, V. V. Dongre and N.G. A. Desouza; Assessment of School Surveys as a method of case detection in an urban area endemic for Leprosy; 1977:732-736;.

eISSN: 2349-6983; pISSN: 2394-6466

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Clinico Histopathological Correction Study of Leprosy

18. Verma KC, Ganguli DD, Jain VK; Applicability of Ridley Jopling Scale in clinical practice, Leprosy in India 1981;556-560;. 19. Dubey G.K, Joglekar V.K, Grover S and Chaubey B.S; Correlation of Clinical and Histopathological studies in classification of Leprosy : Leprosy in India; 1981:53; 562-565;

20. Jerath VP, Desai SR. Diversities in clinical and histopathological classification of leprosy. Lepr India 1982; 54:30. 21. Singh K, Iyengar B, Singh R; Variation in Clinical and Histopathological Classification of Leprosy â&#x20AC;&#x201C; A Report and a plausible explanation; 1983;55; 472-479;. 22. Sehgal VN, Gautam RK, Korane RV, Beohar PC; The Histopathology of Type I (Lepra) and Type II (ENL) reactions in Leprosy; Indian Journal Of Leprosy; 1986:58; 240-243;

*Corresponding author: Dr. Aradhana Deka, Regional Blood Bank, St. Georgeâ&#x20AC;&#x2122;s Hospital Mumbai- 400001 India Phone: +91 8108120630 Email: aradhana.deka@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 14.08.2017 Date of Acceptance : 08.09.2017 Date of Publication : 22.12.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017

Original Article DOI: 10.21276/APALM.1661

Spectrum of Cervical Cytological Lesions in Premenopausal and Postmenopausal Women Vaishali Baburao Nagose*, Nirvana Rasaily Halder, Shruthi Amit Deshpande, Shivanand Shriram Rathod and Varsha Ashok Jadhav Department of Pathology, Mamata Medical College, Rotary Nagar, Khammam, Telangana. India

ABSTRACT Background: Screening with Pap smear initially targets women with higher prevalence of high grade precancerous cervical lesions [Cervical Intraepithelial Neoplasia 2/3 (CIN2/CIN3)] - women mostly in their third and fourth decade. But different conditions affect uterine cervix, including non neoplastic & neoplastic diseases, at different age. Thus, the Pap smears findings should vary in premenopausal & post menopausal groups. Methods: A prospective study of two years was conducted to screen Pap smears in women who were categorised as premenopausal age group <46 years & postmenopausal ≥46years. Result: A total of 6647 cases were analysed within age ranging from 18 to 85 years. 5369 (80.77%) patients were in premenopausal & 1278 (19.23%) in postmenopausal age group. Premenopausal group showed interpretation as “Negative for Intraepithelial Lesion or Malignancy” (NILM) in 97.56%. Here, two of the three cases of Squamous Cell Carcinoma (SCC) were of < 40 years. Postmenopausal age group showed maximum cases of Atypical Squamous Cells of Undetermined Significance (ASCUS), Low Grade Squamous Intraepithelial Lesion (LSIL) & SCC. Maximum cases of High-Grade Squamous Intraepithelial Lesion (HSIL) belonged to >60 years. Conclusion: It is suggested that the Pap screening should not be ceased, but be continued beyond 60 years of age. In premenopausal age, along with SIL, the possibility of malignancy should not be neglected & infections should also be paid more attention. Thus, irrespective of the age of female after 30 yrs, it is highly recommended for them to undergo PAP screening. Keywords: Pap Smear, Premenopausal, Postmenopausal, ASCUS, HSIL, SCC.

Introduction:

Cervix, the gatekeeper of the uterus, gets its name from the Latin word meaning “neck” due to its role as the narrow connection between the larger body of the uterus above & the vagina below. Various conditions can affect uterine cervix, including non neoplastic & neoplastic diseases. The incidence of these varies according to the different age groups. Cervical inflammatory lesions are very common in sexually active females all over the world; with non specific cervicitis, the most common of them, found commonly in third decade, also on histopathalogy.[1] Otherwise it can be said to occur rarely before menarche or after menopause. [2] The etiology is variable and is of importance because it may lead to considerable morbidity as endometritis, salphingitis, pelvic inflammatory disease, chorioamnionitis and also it may have a role in the initiation or promotion of cervical neoplasia.[3] The most important neoplastic condition – carcinoma cervix - the second most common carcinoma in females worldwide - has peak age of incidence at 47 years world over &55–59 years in India. It has a long latent phase

during which it can be detected as identifiable and treatable premalignant lesions which precede the invasive disease by upto 10 years.[4]The transformation zone is the most common site of initiation of the neoplastic process in it,[5],[6] which is easily amenable to screening by PAP smear. The success story of the Pap smear, in bringing down the incidence of invasive cervical carcinoma is already known. Also it has caused a shift toward earlier stages at the time of diagnosis which has brought down the mortality. This reduction in the incidence and mortality of invasive cervical cancer is dramatic & observed worldwide.[7] The Bethesda System (TBS) for reporting cervical cytology has been used for almost three decades now guiding the proper management by clinicians based on the cytological interpretation clearly giving the terms for premalignant & malignant lesions. Pap smears findings should thus vary in premenopausal & postmenopausal groups in terms of infections, premalignant & malignant lesions, as sought after in this study.

Materials and Methods:

This study was conducted in the Department of Pathology, at a Medical College in South India; over a period of two

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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years, from July 2015 to June 2017. A total of 6647 cases were selected who were above 18 years and sexually active for > 3 years. Relevant history, presenting complaints (like discharge per vaginum, bleeding per vaginum, pain in lower abdomen, backache) and examination findings including per speculum findings were entered in a proforma. On the day of procuring Pap smear care was taken that no local douche, antiseptic cream and no local internal examination was done. The patient was placed in dorsal lithotomy position. After proper positioning, gently a sterile Cusco’s bivalve speculum was introduced through vagina and cervix was visualized. The longer projection of the Ayre’s spatula was placed in the cervix near squamo‑columnar junction and rotated through 360°. The cellular material thus obtained was quickly, but gently smeared on a clean glass slide. The glass slide was then immediately put into the Coplin jar containing 95% ethyl alcohol as a fixative. The prepared smears were then stained according to Papanicolaou’s technique. The cytological interpretation of the smears was made according to The Bethesda System 2014. For analysis purpose they were subdivided into premenopausal (<46 yrs) & postmenopausal (≥46 yrs or history of attaining menopause) groups. [8]

Result

Total 6647 patients fulfilling the inclusion criteria in the above mentioned period with ages ranging from 18 to 85 years were analysed. Out of them, 5369 (80.77%) patients were in premenopausal & 1278 (19.23%) in postmenopausal age group. The most common presenting complaint was discharge per vaginum, present in 5296 (77.68%) patients. Post menopausal bleeding was the chief complaint in 14 patients (0.21%). Only 5 (0.08%) patients presented with post-coital bleeding. Most common per speculum finding was white discharge at external os in 5127(77.13%) patients. Ulcerative or fungating growth was observed in only 5 patients (0.08%). This line should not be there in maintext. The categorization of all the patients according to The Bethesda System 2014 is shown in Table 1. Total 180

(2.71%) smears were unsatisfactory. Various organisms were seen, some common ones like Bacterial vaginosis, Trichomonas vaginalis (Figure 1) and Candida, and few less common organisms like Herpes Simplex Virus. Epithelial abnormalities included - Atypical squamous cells (ASC) - of Undetermined Significance (ASCUS) & cannot exclude HSIL (ASC-H); Squamous Intraepithelial Lesion (SIL) - Low-grade (LSIL) & High-grade (HSIL); Squamous cell carcinoma (SCC); Atypical Glandular cells of Undetermined Significance (AGUS) & Adenocarcinoma.This line should not be there in maintext. (Figure 2 a-d). Premenopausal Age Group: Of the smears obtained from 5369 premenopausal patients of this study, 96 (1.79%) were unsatisfactory. The major finding in them was obscuration of the cellular details due to either dense acute inflammatory infiltrate or blood. The majority of the smears (5238 - 97.56% of premenopausal age group) belonged to NILM category, of them the maximum (2418 - 46.16% of this age group) had interpretation as ‘Inflammatory smear without underlying pathology’. Most common interpretation in organism subcategory was ‘Shift in flora suggestive of bacterial vaginosis’ (1428 – 26.6% of premenopausal age group). Only 0.65% of patients showed epithelial abnormalities. In this group, most cases of HSIL (six out of 11) fell between 30 – 39 years age, this decade was also the second most common affected age group for it overall. Two of the 3 cases of SCC of this group were < 40 years of age. (Table2) One case each of Adenocarcinoma & ASC-H were found. Postmenopausal Age Group: Out of the 1278 post menopausal cases, 84 (6.57%) smears were unsatisfactory, main cause being scant cellularity. 1149 (89.91%) were reported as NILM & 3.44% (44) showed epithelial abnormalities. Total 18.31% women of this age group showed ‘Shift in flora suggestive of bacterial vaginosis’, which was the largest of organism subcategories. Maximum patients with epithelial abnormality belonged to ASCUS subcategory – 18 smears, followed by 11 belonging to HSIL. Maximum cases of HSIL (7) were in ≥ 60 years of age (Table 2).

Table 1: General Categorization of PAP smears according to The Bethesda System 2014. Category Unsatisfactory NILM

PremenopausalNo of cases (% of Premeno-pausal group)

PostmenopausalNo of cases (% of Postmenopausal group)

Overall No of cases (%)

96 (1.79%)

84 (6.57%)

180 (2.71%)

5238 (97.56%)

1149 (89.91%)

6387 (96.09%)

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Nagose et al.

A-797 PremenopausalNo of cases (% of Premeno-pausal group)

PostmenopausalNo of cases (% of Postmenopausal group)

Normal

645

132

777

Inflammatory smear without underlying pathology

2418

437

2855

232

118

164