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Annals of Pathology and Laboratory Medicine July-August 2017; Vol. 4, Issue 4

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Contents

Original Article Endometrial hyperplasia: Emergence of the EIN system

V Rajalakshmi, Rajeswari Kathiah, Meenakshi Sundaram, Sathish Selvakumar. A Importance of Perlsâ&#x20AC;&#x2122; stain as a routine test in anaemia in adults Riju Rani Deka, Deba Kumar Saikia, Sanjeeb Kakati, Bipul Kumar Das Dysfunctional Uterine Bleeding: A Clinico-Pathological Study at a Tertiary Care Centre Ramesh B.H, Rajeshwari Karibasappa Kumbar A tertiary care centre experience with periampullary and pancreatic neoplasm in pylorus preserving pancreaticoduodenectomy specimens. Limi Mohandas, Sheela Vasudevan, Kalaranjini KV Fine needle aspiration cytology of cervical lymph nodes: Our experience Aditi Dharmesh Vasavada, Dimple Hardik Darad, Dharmesh Girish Vasavada, Hardik Darad Pediatric Liver Biopsy: A clinicopathologic study Pragati Aditya Sathe, Dipali Eknath Mahale Histopathology of gastrointestinal tract malignancies: A two year retrospective study. Meera Shantaram Mahajan, Neha Amrut Mahajan, Shrinivas Shankarrao Kale, Chandrashekhar Prabhakar Bhale Morphometric changes in jejunal mucosa in HIV positive patients presenting with enteropathy: An Indian study Dibyajyoti Boruah, Jasvinder K Bhatia, Kiran Deep Kamal, Ajay Malik Clinicopathological Analysis of Undifferentiated Malignant Neoplasms of the Sinonasal tract Ashokkumar Sundaramoorthy, S Srigayathri, Geetha Devadas An Analysis of Quality Control in Pap Cytology in A Tertiary Care Centre By Using ASC to SIL Ratio Hemalatha J, Deepak Kumar B, Srinivasa Murthy V, Vani BR Correlation of Histopathological Study of Breast Lesions with cytology and mammography as a measure of internal quality and diagnostic accuracy Anusha Mulka, Vaishali Dhananjay Kotasthane, Rajendra Dhaka, Dhananjay Shrikant Kotasthane Cytological Evaluation of Two Methods of Effusion Cell Block Preparations Deepa Siddappa Masur, Shilpa Somashekhar Biradar The Histomorphological Spectrum of Renal Lesions in an Autopsy Study Vaneet Kaur Sandhu, Arun Puri, Navtej Singh Study of Lipid Profile in Patients of Polycystic Ovarian Syndrome Before and After Metformin Therapy Noora Saeed, Nishat Afroz, Sheelu Shafiq Siddiqi, Aaliya Ehsan, Mohd Rafey Estimation and correlation of different hemoglobin levels in HbE hemoglobinopathies in Indian population using capillary electrophoresis method. Abhijit Kalita, Avinanda Mahanta A Prospective & Multicentric Study of RBC Parameters in Patients of Sickle Cell Disorder Prateek Pradeep Umrikar, Alpesh Prahladpuri Goswami Determination of an optimum cut-off point for %fPSA/tPSA to improve detection of prostate cancer Vineeth G Nair, M H Shariff Study of Congenital Malformations in Fetal and Early neonatal autopsies Pradnya Pandurang Kale-Jain, Sujata R Kanetkar, Dhirajkumar B Shukla, Atul Bhanudas Hulwan, Pramod Borade, Nikita Vinod Vohra Cytological Study of Pleural Effusions and its Utility in Clinical Approach Pravin Gojiya, Alpeshpuri Goswami, Shaila Shah HbA2 and Fetal haemoglobin in the diagnosis of Thalassemia and Hemoglobinopathies Sandhya Venkatachala, Manjula Rajendran

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Case Report

Letter to editor

Clinical and Histopathological Correlation in Hansenâ&#x20AC;&#x2122;s Disease A454-A459 Muruganantham Arunagirinathan, Vennila Muniswamy, Sivaraman Jeevirathinam Fine Needle Aspiration Cytology Of Cervical Lymphadenopathy: Is There Anything A460-A465 Different At South Coastal Region Of India? Priya R, Dhananjay Shrikant Kotasthane, Vaishali Dhananjay Kotasthane, Koteeswaran G Role of Mean platelet Volume as an adjunct in evaluation of acute inflammation. A466-A469 Nabila Afsar, Idrees Akhtar Afroze, Habeebunnisa Tahniath, Zakia Abid Benign lymphoepithelial cyst in an adolescent female mimicking lymphoma: A C98-C101 diagnostic dilemma in a retrovirus negative patient Somshankar Chowdhury, Sufian Zaheer, Preeti Sharma, Ashish Kumar Mandal Cutaneous Rhinosporidiosis mimicking soft tissue tumour: A Case report with C102-C105 review of literature Richa Bhartiya, Pallavi Agrawal, Rakesh Mehra Leiomyosarcoma with unknown primary C106-C109 Manika Khare, Ashish Airun, Umesh Babu Sharma, S K Jain Touch Imprint Cytology Of Renal Tumors: Report Of Three Cases Ratnakar M Potekar, Ambica Chalmeti Inflammatory Myofibroblastic Tumour of Ovary Simulating Malignancy with Review of Literature Ruchi Sinha, Shuchi Smita, Iffat Jamal, Shashikant Kumar, Nishi Sharma, Mamta Kumari Synchronous Primary Ovarian Mucinous Carcinoma and Endometrioid Endometrial Carcinoma : A rare case report. Mohan Krishna Pasam, S Rajendiran, M Susruthan, Usha Rani Prerectal mucinous cystadenoma. A case report and considerations about its origin Jaime Zuloaga, Javier Arias-Diaz, Maria Jesus Fernandez-AceĂąero, Cristina Diaz-del-Arco, Julio Mayol, Antonio Torres Corynebacterium amycolatum causing breast abscess: An infecting Diphtheroid with a difference Hena Butta, Feroz Pasha, Reetika Dawar, Vikas Kashyap, Leena Mendiratta, Upasana Bora, Raman Sardana Lipoma Arborescens Preethy Murthy, Puvitha Rajeswari Duraisami, M Murthy Cytology of Adenomatoid Tumour of Epididymis Meghna Singh, Jitendra Chaudhary

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Original Article DOI: 10.21276/APALM.1026

Endometrial Hyperplasia: Emergence of The EIN System Rajalakshmi.V*, Rajeswari .k, MeenakshiSundaram and Sathish Selvakumar.A Department of Pathology, ESIC Medical College & PGIMSR, Chennai, Tamilnadu, India

ABSTRACT Background: The diagnosis of precancerous lesions of the endometrium remains unstandardized because, the existing World Health Organization classification categories do not correspond to distinctive biologic groups and are inadequately supported by reproducible histopathologic criteria. The objective of our study is to simplify the diagnosis of endometrial hyperplasia, make it more reproducible and stratify patients in to two risk groups instead of four using the recent EIN system. Materials & Methods: All cases diagnosed as endometrial hyperplasia classified according to the WHO system in patients with abnormal uterine bleeding, during the 3 year period 2014-2016 were reclassified as benign hyperplasia and Endometrial intraepithelial neoplasia according to the EIN system. Results: 38 out of the 46 cases (82.6 %) of simple hyperplasia were reclassified as Benign hyperplasia. 8 out of the 46 cases (17.4%) of simple hyperplasia were reclassified as EIN.1 case of complex hyperplasia without atypia was reclassified as EIN. All the 11 cases of complex hyperplasia with atypia were reclassified as EIN. Conclusions: Application of the criteria for EIN successfully segregates patients into high and low cancer risk subgroups with better reproducibility than WHO classification. Keywords: Endometrial intra epithelial neoplasia, Endometrial hyperplasia, EIN.

Introduction

Endometrial intraepithelial neoplasia (also known as ‘EIN’) is a precursor to endometrioid endometrial adenocarcinoma characterized by monoclonal growth of mutated cells, a distinctive histopathologic appearance, and 45-fold elevated cancer risk.[1,2,3]EIN arises through complex interactions involving the sequential accumulation of genetic damage in endometrial glands and the positive selective pressure of unopposed estrogen. EIN is to be distinguished from adenocarcinoma and the diffuse hormonal changes of EH seen in anovulation. [4,5] Endometrial hyperplasia is a common disease with incidence of 15 % in patients with abnormal intrauterine bleeding. Because only 1–28% of hyperplasias actually progress to malignant disease, depending on the degree of severity, it is important to stratify patients into high-risk and low-risk groups before initiating therapy. The World Health Organization (WHO) 1994 classification system for endometrial hyperplasias (WHO94) is used widely for this purpose. The most recent WHO classification system, the EIN system, acknowledges the shortcomings of WHO94 and, on this basis, has introduced the alternative molecular genetics-based and morphometric-based Endometrial Intraepithelial Neoplasia (EIN) classification system.[6]

The two classification systems differ in their foundations. The WHO94, which is based entirely on histologic findings, uses four subcategories based on architectural and cytologic alterations. In practice, the diagnostic criteria are difficult to apply reproducibly, because they largely are subjective. Even acknowledged experts experience substantial differences in reporting. Data from the Gynecologic Oncology Group showed that the WHO94 diagnostic criteria were misinterpreted easily in the community, because 30% of specimens that were submitted as complex atypical hyperplasia (CAH) were diagnostic of malignancy on expert review, whereas another 40% of specimens were a lesser degree of hyperplasia or were entirely benign.[7] The EIN system, in contrast, has a molecular genetic basis; can be implemented by morphometric analysis; and, even with hematoxylin and eosin (H&E) slides alone, has semiquantifiable features.[7] The present study was aimed at reclassifying the previously diagnosed endometrial hyperplasia cases according to the WHO system in to the better reproducible EIN system.

Materials &Methods

This is a retrospective study in which the endometrial curetting specimens done for patients with abnormal uterine bleeding, that were previously diagnosed as endometrial

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hyperplasia using the WHO94, from January 2014 to June 2016 were collected. Slides from all the patients were reviewed and recategorized according to the EIN system by the senior pathologist and the results were compared with the initial diagnosis. Exclusion Criteria: Cases diagnosed as endometrial carcinoma, disorderly proliferative endometrium and complex hyperplasia with atypia in fractional curettage which were later diagnosed as endometrial carcinoma were excluded from the study.

Results

The total number of slides included for the study is 60. The age group of patients was 32 to 63 years. Total number of fractional curettage specimen done on patients with abnormal uterine bleeding during this period was 708 and the incidence of benign hyperplasia among the patients with AUB is 5 % and of EIN is 3 % in our study. Table 1shows the comparative diagnosis based on WHO and the EIN system with the number of patients.38 out of the 46 cases (82.6 %) of simple hyperplasia were reclassified as Benign hyperplasia.8 out of the 46 cases (17.4%) of simple hyperplasia, 1 case of complex hyperplasia without atypia and all the 11 cases of complex hyperplasia with atypia were reclassified as EIN. Table 2 shows the no of patients affected according to the age group with the youngest patient being 32 year old and the oldest being 63 years and most of the patients (66.7%) were in their fifth decade.

Discussion

Endometrial intraepithelial neoplasia (EIN) is a monoclonal premalignant endometrial glandular lesion that precedes the development of endometrioid-type endometrial adenocarcinoma. EIN arises through complex interactions involving the sequential accumulation of genetic damage in endometrial glands and the positive selective pressure Table 2: WHO classification Simple hyperplasia Simple hyperplasia with atypia Complex hyperplasia Complex hyperplasia with atypia Total no of cases : 60

No of cases 46 2 1 11

of unopposed estrogen. Recent data have revealed a preclinical latent precursor lesion composed of mutated but morphologically nondescript glands that may persist for years in normal-appearing premenopausal cycling endometrium. This latent precursor shares many of the molecular features of EIN and endometrial adenocarcinoma, including frequent inactivation of both the tumor suppressor gene PTEN and the paired boxâ&#x20AC;&#x201C;containing gene PAX2. Upon progression to EIN, the distinctive appearance of crowded and cytologically altered glands heralds a 45-fold increased risk of developing endometrial adenocarcinoma. To preserve the high predictability of EIN for concurrent/ subsequent adenocarcinoma, strict adherence to defined diagnostic criteria is essential.[8] The diagnosis of EIN must meet 5 criteria in a single fragment, including architectural gland crowding, altered cytology, minimum size of 1 mm, exclusion of carcinoma, and exclusion of mimics. The diagnosis of EIN can be summarized as a focus of clustered endometrial glands exceeding a gland to stroma ratio of 1:1, which have altered cytology from the background endometrium, and which comprise a sufficient volume of 1 mm.[9] The new architectural criterion for EIN diagnosis, diminution of stromal volume to less than approximately half of the total sample volume, will also assist in discriminating between EH and EIN. Implementation of this proposal will bring diagnostic terminology into agreement with current concepts of premalignant endometrial disease and facilitate more uniform patient management. Benign endometrial hyperplasia involves the entire endometrial compartment and, with protracted estrogen exposure, shows the progressive development of cysts, remodeled glands, vascular thrombi, and stromal microinfarcts. They are best construed as a sequence of changes whereby the appearance at any single time point is uniquely dependent on the preceding combination and the duration of hormonal exposures. In contrast, the

EIN classification Benign hyperplasia Endometrial intraepithelial hyperplasia

No of cases 38 22

Table 3: Age group 31-40 years 41-50 years 51-60 years 61-70 years

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No of patients 13 40 6 1

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Emergence of The EIN System.

FIG 1A

FIG 1B

FIG 1C

FIG 1D

Fig. 1 A : Photomicrograph shows a focus of EIN with crowding of glands with cytological atypia (circled area )in contrast with the background benign glands( arrow),H&E,X100; 1 B : Photomicrograph shows a focus of EIN with crowding of glands ,H&E,X400; C &D : Photomicrographs shows a focus of EIN with cytological atypia,H&E,X400.

premalignant clone of an EIN lesion is characteristically offset from the background endometrium by its altered cytology and crowded architecture. The use of an internal standard for cytology assessment, combined with the distinctive topography of a clonal process, enables the diagnosis of EIN lesions with a long-term cancer risk 45-fold greater than that of their benign endometrial hyperplasia counterparts. The resolution of hormonal and premalignant subsets of traditional â&#x20AC;&#x153;endometrial hyperplasiasâ&#x20AC;? is possible using redefined diagnostic criteria, enabling patient therapy to be appropriately matched with the underlying disease mechanisms.[10] The EIN system, in contrast, has a molecular genetic basis; can be implemented by morphometric analysis; and

even with hematoxylin and eosin (H&E) slides alone, has semiquantifiable features.[11] From the table 1, it is very clear that categorization of endometrial hyperplasia in to two tier using the EIN system is much simpler and easily reproducible by the clinicians to stratify patients in to low risk and high risk groups for much easier follow up. In a Cox regression analysis, EIN was the strongest prognostic index of future endometrial carcinoma. A longterm follow-up study of 477 women with hyperplasia done by Baak et al, suggest that the EIN classification scheme predicts the development of future malignancies more accurately than the WHO system.[11]

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Rajalakshmi.V et al. One of the major strengths of the EIN system is its correlation to outcome data. As discussed, a biopsy diagnosis of EIN imparts a 45-fold increased risk of progression to carcinoma after the first year. Hysterectomy following the diagnosis of EIN is appropriate because there is a high rate of concurrent, as well as future, endometrioid endometrial carcinoma in women with EIN. In circumstances in which the patient desires fertility or is not a surgical candidate, progestin therapy is an increasingly offered alternative. Progestin regimens are not standardized, and clinical outcomes are primarily available from anecdotal series rather than controlled randomized clinical trials. A common practice following progestin administration is a follow-up biopsy every 6 months following withdrawal until a minimum of 3 negative biopsies areobtained.[1]

Conclusion:

The results of this study show that the EIN classification scheme is superior to the WHO94 scheme in discriminating lesions with the highest risk for conversion to malignant disease. Furthermore, a large group of women who initially are diagnosed with “hyperplasia” but are classified later without EIN have a near-negligible risk of developing malignant disease. Application of criteria for diagnosis of EIN successfully segregates patients into high and low cancer risk subgroups with better reproducibility than atypical hyperplasia diagnosis based on WHO system. Acknowledgement: I thank Dr. Arulmalar Neeyam , tutor of our department for helping me in this study.

References 1.

Owings RA,; Quick CM, Endometrial Intraepithelial Neoplasia, Archives of Pathology & Laboratory medicine, 2014, 138, 138:484–491 Hecht JL, Ince TA, Baak JPA, Baker HE, Ogden MW, Mutter GL. Prediction of endometrial carcinoma by subjective

A-341 endometrial intraepithelial neoplasia diagnosis. Mod Pathol. 2008;18(3):324–330. 3.

Baak JP, Mutter GL, Robboy S, et al. The molecular genetics and morphometry-based endometrial intraepithelial neoplasia classification system predicts disease progression in endometrial hyperplasia more accurately than the 1994 World Health Organization classification system. Cancer.2005;103(11):2304–2312.

Orbo A, Baak JPA, Kleivan I, et al. Computerized morphometrical analysis in endometrial hyperplasia for the prediction of cancer development: a longterm retrospective study from northern Norway. J ClinPathol.2006;53(9):697–703.

Baak JP, Orbo A, van Diest PJ, et al. Prospective multicenter evaluation of the morphometric D-score for prediction of the outcome of endometrial hyperplasias. Am J SurgPathol.2007; 25: 930–935.

Marie-Claude Renaud, Quebec QC, Tien Le, MD, Ottawa ON. Epidemiology and Investigations for suspected Endometrial Cancer, JointSoGC-GoC-SCC Clinical practice Guideline, 2013 291,

Silverberg SG, Kurman RJ, Nogales F, Mutter GL, KubikHuch RA, Tavassoli FA. Epithelial tumors and related lesions of endometrium. In: TavassoliFA, StrattonMR, editors. Tumors of the breast and female genital organs. Lyon: IARC Press, 2003: 221–232.

Zaino RJ, Kauderer J, Trimble CL, et al. Reproducibility of the diagnosis of atypical endometrial hyperplasia: a Gynecologic Oncology Group study. Cancer.2006;106(4):804–811.

Marotti JD, Glatz K, Parkash V, Hecht JL. International internet-based assessment of observer variability for diagnostically challenging endometrial biopsies. Arch Pathol Lab Med. 2011;135(4):464–470.

10. Allison KH, Reed SD, Voigt LF, Jordan CD, Newton KM, Garcia RL.Diagnosing endometrial hyperplasia: why is it so difficult to agree?Am J SurgPathol.2008; 32(5):691–698. 11. Mutter GL, Zaino RJ, Baak JP a, Bentley RC, Robboy SJ. Benign endometrial hyperplasia sequence and endometrial intraepithelial neoplasia.Int J GynecolPathol. 2007;26(2):103–114.

*Corresponding author: Rajalakshmi.V, Department of Pathology, ESIC Medical College & PGIMSR, Chennai, Tamilnadu,India. Email: raji_path@rediffmail.com

Financial or other Competing Interests: None.

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Date of Submission : 12.08.2016 Date of Acceptance : 14.04.2017 Date of Publication : 31.08.2017

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Original Article DOI: 10.21276/APALM.1067

Importance of Perls’ Stain as A Routine Test in Anaemia in Adults Riju Rani Deka1*, Deba Kumar Saikia2, Sanjeeb Kakati3 and Bipul kumar Das4 1 Department of Pathology,Tezpur Medical College, Tezpur, Assam,India Department of Pathology, Gauhati Medical College, Guwahati, Assam India 3 Department of Medicine, Assam Medical College, Barbari, Dibrugarh, Assam India 4 Department of Paediatrics, Gauhati Medical College, Guwahati, Assam India 2

ABSTRACT Background: Anaemia is one of the frequent finding in patients of all age. Proper diagnosis is of utmost important to avoid fatal morbidity in untreated chronic cases. Aims: This study is meant to observe the role of Perls’ stain in routine practice in etiological evaluation of anaemia in adults. Methods: Prospective cross sectional study from June 2007 to July 2010. 128 cases of anaemia, alone or as a part of bicytopenia and pancytopenia, in 20-80 years of age, who were recommended by clinicians for bone marrow aspiration analysis after initial routine haematological investigation, were collected as samples. Perls’ stain was done on bone marrow smears to estimate marrow iron store in fragments and ring sideroblasts in all the smears. Result: Anaemia was most common haematological presentation(49%), mainly in males (62%) and majority in 20-30 years of age (31.3%). Iron deficiency anaemia was most common cause of anaemia(59.6%) with 0 to1+ iron store. Megaloblastic anaemia, aplastic anaemia, thalassemia and other haemoglobinnopathies, anaemia of chronic disease(AOCD) , myelodysplastic syndrome(MDS) and secondary sideroblastic anaemia(SA) were other causes of anaemia found. 3.2% cases had ring sideroblasts of which 0.78% was MDS and 2.3% was secondary SA. Conclusion: Perls’ stain is a cheap and relatively simple test that can be used as a routine test in all bone marrow aspirates to provide provisional diagnosis of some relatively infrequent causes of anaemias which otherwise could be missed when only non invasive methods are relied upon for assessing body iron content. Keywords: Anaemia in Adults, Bone Marrow Iron Store, Myelodysplastic Syndrome, Perls’ Stain, Sideroblastic Anaemia

Introduction

Clinicians frequently identify anaemia in the older patients but data on the prevalence in this part of the country are unavailable. According to WHO worldwide prevalence of anaemia to be 24.8%.[1] The common causes of anaemia in adults are anaemia of chronic disease(AOCD), iron deficiency anaemia(IDA) due to gastrointestinal bleeding, B12 deficiency anaemia and myelodysplastic syndrome(MDS).Identification of the causes can decrease the morbidity of the patients and hence should be diagnosed at an earliest. Usually clinical and non-invasive haematological investigations can diagnose most of the anaemia but few conditions require demonstration of ring sideroblasts under light microscope as one of the criteria for diagnosis. Again, AOCD may complicate the interpretation of hematinic markers when IDA coexist. [2] Therefore the gold standard for diagnosis of IDA is to asses bone marrow iron status by Perls’ stain.[3] A study found bone marrow iron staining to be more reliable to exclude IDA rather than its diagnosis.[4]

The study here is to find out the prevalence of these conditions in the adult patients presenting with anaemia and hence the role of Perls’ stain as a routine test in evaluation of bone marrow aspirates in cases of anaemia in adults.

Materials and Methods

\This prospective pathological study was conducted in the Department of Pathology, Assam Medical College and Hospital, Dibrugarh for 3 year from June 2007 to July 2010. Anemia was defined according to World Health Organization (WHO) criteria as a hemoglobin concentration below 12 g/dL in women and below 13 g/ dL in men Irrespective of sex, all cases of anaemia alone or as a part of bicytopenia and pancytopenia, in an age of 20-80 years, who were recommended by clinicians for bone marrow aspiration analysis after initial routine haematological investigation were collected as samples. Exclusion Criteria: 1. Diagnosed case of anaemia in pregnant women. 2. Patients diagnosed to have malignancy of any kind.

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3. Patients receiving chemotherapy or radiotherapy. Sample Size: A total of 136 cases were recommended for bone marrow evaluation of which 128 cases could be evaluated . 5 samples were highly hemodiluted for evaluation while 3 cases showed dry tape. In all the cases, samples were collected in EDTA vial with proper anticoagulant ratio. Samples for Prothrombin time were collected in 32gm/l trisodium citrate anticoagulant solution in proper ratio corrected for haematocrit. Reticulocyte count was done manually using 1% new methylene blue solution. Complete blood count was done in sysmax800i cell counter quality control of which was maintained daily. Peripheral blood smears were stained with Leishmenâ&#x20AC;&#x2122;s stain. Written consent from the patients after explaining the procedure was taken. Under all aseptic and antiseptic conditions, bone marrow aspirations were done from posterior superior iliac spine, 0.2-0.5 ml of samples were collected , smears were made to get minimum 7-10 particles for assessment. MGG staining was used for morphological evaluation of bone marrow aspirates. Perlsâ&#x20AC;&#x2122; stain with working pararosalinine solution as counter stain was used. Diagnosis of iron deficiency anaemia was made on the basis of PBS finding of microcytic hypochromic anaemia and mild to severe poikilocytosis, micronormoblastic erythropoiesis and absent iron store in marrow fragments. Diagnosis of megaloblastic anaemia was based on history, anaemia to pancytpenia, macrocytosis and hypersegmented neutrophils in PBS, megaloblastic reaction in bone marrow along with giant metamyelocytes, mild dyserythropoiesis. Inconclusive cases were confirmed by serum Vitamin B12 and S. Folic acid level.Diagnosis of aplastic anaemia was made by marrow hypocellularity to acellularity in bone marrow aspirates..Thalassemia and other haemoglobinopathies were diagnosed from HPLC findings. Anaemia of chronic disease was diagnosed from history, increased marrow iron content and S. Ferritin, CRP in the light of the underlying disease. Sideroblastic anaemia was diagnosed cumulatively from a dimorphic blood picture, ring sideroblasts in bone marrow and history of an underlying cause. Before labelling as sideroblastic anaemia, other causes of dimorphic anaemia were excluded.

6+. Normal iron store was graded from 1+ to 3+. 0 grade indicated absent iron store while grade 4+to 6+ indicated increased iron store.

Result

Work up on 128 cases of anaemia led us to discover iron deficiency anaemia (IDA), megaloblastic anaemia (MA), aplastic anaemia (AA), thalassemia and other haemoglobinopathies , sideroblastic anaemia(SA), myelodysplastic anaemia(MDS) and anaemia of chronic disease(AOCD)) as causes of anaemia in adults. In our study, maximum number of cases were in 20-30 years of age (31.3%) and a minimum in elderly age group (71-80years) (6.25%) [fig.1]Prevalence of anaemia was more in males (62%). [fig.2] Anaemia (49%) was most common type of haematological presentation. [fig.3]. IDA was most common anaemia to present with anaemia only .Bicytopenia was main presenting feature of megaloblastic anaemia (MA) (61.5%) while sideroblastic anaemia (SA) and aplastic anaemia (AA) presented with pancytopenia with equal (44.4%) frequency. [tab.1] Irrespective of initial haematological presentation, IDA was most common cause of anaemia with male preponderance. [tab.2]. Peak incidence was in 20-30 years of age with absent or low normal (0 to 1+ ) iron store in bone marrow fragments.[tab.3] Megaloblastic anaemia was second major cause of anaemia (28.2%) with a female preponderance and presenting mainly with bicytopenia.[tab.2,tab.1] Young age (20-30 years) was the most common effected group(55.5%) with a normal (2+ to 3+) iron store.[tab.3] Least common finding was myelodysplastic syndrome (MDS) (0.8%) followed by sideroblastic anaemia(SA)(2.3%), both with a female preponderance. [tab.2] The only case of MDS was recorded in 61-70 years of age with increased(5+) iron store while SA was seen in 51-70 years of age with increased(4+ to 5+) iron store.[tab.3]

Provisional diagnosis of MDS was made from a history of refractory anaemia, unilineage to multilineage dysplasia in bone marrow and presence of significant number of (>15%) ring sideroblasts in bone marrow.

Thalassemia and other haemoglobinopathies (sickle cell trait and disease, E-beta thalassemia trait) made up 12.5% of total findings presenting mainly as anaemia followed by bicytopenia and pancytopenia. Majority of cases belonged to 20-30 years of age (75%) with male preponderance and found to have high normal to increased (3+ to 4+) iron content in bone marrow. [tab.3]

Iron store was graded according to the criteria laid down by Gale et al in 1963. Iron store was graded from 0 to

AA constituted 2.3% of all cases, all presented with pancytopenia with a male preponderance and an equal age

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distribution in most of the groups. All the cases had normal to mildly increased (2+ to 4+) marrow iron content. [tab.3]

systemic illness was referred for bone marrow aspiration analysis. Bone marrow was hypercellular, megaloblastoid erythropoiesis, and ring sideroblast was significantly increased. Exact enumeration of ring sideroblast was not possible due to diluted smear. These findings led to the diagnosis of the case as MDS (refractory anaemia with ring sideroblast, RARS) (0.78%). 3 cases showed few ring sideroblasts and history of chronic alcoholism in 2 cases and antitubercular therapy (isoniazid) in one. On the basis of these findings and the history, these cases were labelled as secondary sideroblastic anaemia (2.3%) due to alcoholism and antitubercular therapy.[tab.4]

Anaemia of chronic disease (AOCD) constituted 12.5% of total findings. AOCD was recorded in 41-70 years of age with male preponderance and presenting with anaemia and bicytopenia. They had high normal to increased (3+ to 5+) marrow iron content. [tab:1,tab.3] Perlsâ&#x20AC;&#x2122; stain revealed presence of ring sideroblast in only (n=4) 3.12% and few cases showed increased number of sideroblasts in bone marrow. A 62 years old lady with refractory anaemia not responding to therapy and with no

Table 1: distribution of anaemias with initial haematological finding. cytopenia

IDA

THAL & Others

MDS

AOCD

Total

Anaemia Bicytopenia

pancytopenia

Table 2: sex distribution of different types of cases. anaemia

Male(%)

Female(%)

Total

IDA

32 (72.7)

12 (27.3)

16 (44.4)

20 (55.6)

8 (66.6)

4 (33.3)

Thalasemia & other haemoglobinopathies

12 (75)

4 (25)

1(33.3)

2 (66.7)

MDS AOCD

1 (100)

10 (62.5)

6 (37.5)

Table.3: Iron content in marrow in different types of anaemias in various age groups:Anaemia IDA MA AA

20-30 yrs 8 (0-1+) 20 (2+-3+) 0

31-40 yrs 20 (0-1+) 4 (2+-3+) 4 (2+-3+)

41-50 yrs 4 (0) 8 (2+-3+) 4 (2+-4+) 4 (3+-4+)

51-60 yrs 4 (0) 4 (2+-3+)

61-70 yrs 4 (0)

71-80 yrs 4 (0)

4 (2+-3+)

Total cases 44 (34.4%) 36 (28.1%) 12 (9.4%) 16 (12.5%) 3 (2.3%)

12 (3+-4+)

2 (4+-5+)

1 (5+)

MDS

1 (5+)

1 (0.8%)

AOCD

4 (4+-5+)

6 (3+-5+)

2 (4+-5+)

16 (12.5%)

Thalassemia & others

Table 4: presence of ring sideroblast in bone marrow aspirates. Ring sideroblast

Number of Cases(n)

Present

3.12

absent

124

96.87

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Fig. 1: age distribution of cases:

Fig. 2: sex distribution of total cases.

Fig. 3: cases (%) according to initial haematological presentation.

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Fig. 4: Megaloblastoid reaction in bone marrow aspirate. Leishmen’s stain.100X.

Fig. 5: Absence of sideroblasts in bone marrow aspirate in iron deficiency anaemia. 1%Pararosalinine counterstain.40X.

Fig. 6: Perl’s stain in bone marrow aspirates.1% Pararosalinine counterstain.(A) grade0 iron store.100X (B) grade2 iron store.40X

Fig. 7: Perl’s stain in bone marrow aspirates.100X.(C) grade3 iron store.1% Pararosalinine counterstain (D) grade6 iron store. 1%Neutral red counterstain.

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Discussion

Anaemia is commonly encountered sign and symptom in adults but indication for bone marrow aspirate examination was relatively less and cases were referred for the same only when non-invasive techniques could not give a definitive diagnosis. In our study, maximum number of cases were in 20-30 years of age(31.3%) followed by 31-40 years (21.3% ) which was consistent with the finding in Pujara et al who also recorded 30% and 27% of cases in these age group respectively .[5] IDA was the most common finding in our study with a iron store of 0 to 1+. This finding was similar to the finding in Pujara et al.MA was second most common finding recorded in our study with a normal iron store(2+ to 3+). While Pujara et al found increased store , Dharwadkar et al found normal (1+to 3+) iron store in bone marrow fragments.[5,6] This dissimilarity with Pujara et al may be due to the grading followed by them who considered 1+ to 2+ iron as normal store while grades above that was recorded as increased store. AA in our study constituted 44.5% of cases of pancytopenia with a male preponderance. Our study showed a normal to mildly increased iron store of 2+ to 4+. Similar finding was noted in Halder et al who found AA to be the most common cause of pancytopenia(79%) with a male preponderance. Their study recorded 78.95% cases with increased and 21.05% with decreased iron store.[7] Pujara et al also found absent to low normal marrow iron content in the cases of aplastic anaemia.[5] This dissimilarity may be due to lesser number of cases of AA recorded in our study. Increased iron store in AA may be due to associated autoimmune disorder which causes AA. In our present study, out of the 16 cases, 50% had sickle cell trait , 43% had beta-thalassemia trait ,6.25% had E-beta thalassemia trait. They had 3+ to 4+ iron store. Kaduri et al in his review article mentioned that iron deficiency is more common in sickle cell disease. According to him, deficient iron state is beneficial as it reduces sickling by decreasing MCHC-S amount in RCBs.[8] Oluboyede et al found iron deficiency in sickle cell anaemia to be due to increased urinary iron loss.[9] Normal to increased iron content in our study may be due to transfusion in few of our cases of sickle cell anaemia. Corwin et al in their study found coincidence of thalassemia minor with homozygosity for haemochromatosis as a cause of iron overload in non transfused cases of thalassemia minor.[10] E-beta thalassemia minor is a non transfusion dependent state and further evaluation could establish such coincidental finding which is beyond the scope in our setup. www.pacificejournals.com/apalm

In our study, cases of sideroblastic anaemia showed high normal to increased iron store which is consistent with already established fact. In our study, it was observed that majority of cases (62.5%) of AOCD had high normal and rest with increased marrow iron content. It was on contrary to the finding in Pereira et al who found majority of cases with diminished to absent iron content (73.3%).[11] In our study, 3.2% cases showed ring sideroblasts in their marrows. Of these cases, 0.78% had myelodysplastic syndrome (RARS) and 2.3% had secondary siderolastic anaemia. Dharwadkar et al found 1.8% cases of refractory anaemia with 10% marrow sideroblast.[6] This difference may be due to their study sample which included cases of neoplasia also. Andrew et al found 3.4% cases of MDS which included 2(1.14% ) cases of RARS.[12] Lower incidence in our study may be due to the wider age range in which the study had been conducted unlike the study of Andrew et al who carried out the study in age 65 years and above. Similar finding was recorded in Aul et al who recorded 3.2% cases of MDS in their study and also concluded that with increasing age the incidence of MDS increases.[13]

Conclusion

Proper evaluation of anaemia in adults is important for proper management on failure of which may lead to fatal consequences in long run. In our study, it is seen that Perlsâ&#x20AC;&#x2122; stain is a cheap and relatively simple test that can be used as a routine test in all bone marrow aspirates to provide provisional diagnosis of some relatively infrequent causes of chronic anaemias which otherwise could be missed when only non invasive methods are relied upon for assessing body iron content. However studies on using Perlsâ&#x20AC;&#x2122; stain in broader spectrum of haematological disorders may lead to more conclusive outcome.

Reference 1.

Worldwide prevalence of anaemia 1993â&#x20AC;&#x201C;2005 : WHO global database on anaemia / Edited by Bruno de Benoist, Erin McLean, Ines Egli and Mary Cogswell.

Gale E, Torrance J, Bothwell T. The Quantitative Estimation of Total Iron Stores in Human Bone Marrow. Journal of clinical investigation 1963;42:1076.

Burns E, Goldberg SN, Lawrence C, Wenz B. Clinical utility of serum tests for iron deficiency in hospitalized patients. Am J Clin Pathol 1990;93:240-45.

Koca E, Cetiner DA, Buyukasik Y, et al. Bone Marrow Iron staining is a Reliable Test for Elimination of Iron Deficiency Anaemia Rather than its Diagnosis. Int J Haematol oncol 2013;23:260-63.

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Pujara KM, Bhalara RV, Dhruva GA. A study of bone marrow iron storage in haematological disorders. Int J Health Allied Sci 2014;3:221-24.

Dharwadkar A,Vimal S, Panicker Narayanan K, Chandanwale Shirish S, Vishwanathan V, Kumar H. Study of sideroblast and iron stores in bone marrow aspirates using Perls’s stain. Medical journal of Dr. D.Y. Patil university 2016;9:181-85.

Haldar B, Partha PP, Sarkar TK, Sharma S, Goswami KB, Aikat A. Aplastic Anemia:A Common Hematological Abnormality Among Peripheral Pancytopenia. N Am J Med Sci 2012;4:384–88.

Kaduri P. R. Iron in Sickel cell disease: A review why less is better. Am. J Haemtol 2003;73:59-63.

Oluboyede OA, Ajayi OA, Adeyokunnu AA. Iron studies in patients with sickle cell disease. Afr J Med Med Sci 1981;10:1-7.

10. Edwars CQ, Skolnick MH, Kushnew JP. Coincidental nontransfusional iron overload and thalassemia minor: Association with HLA-Linked Haemochromatosis. Blood 1981;58:844-48. 11. Pereira M R M, Veloso E R P, Menezes Y, Gualandro S, Vassalo J Yosinaria. Bone Marrow Finding In Systemic Lupus Erythematosus Patients With Peripheral Cytopenias. Clinical Rheumatol 1998;17:219-22. 12. Artz AS, Thirman MJ. Unexplained Anemia Predominates Despite an Intensive Evaluation in a Racially Diverse Cohort of Older Adults From a Referral Anemia Clinic. J Gerontol A Biol Sci Med Sci 2011;66:925–32. 13. Aul C, Gattermann N, Schneider W. Age related incidence and other epidemiological aspect of myelodysplastic syndromes. British journal of Haemtol 1992;8:358-67.

*Corresponding author: Dr. Riju Rani Deka, C/O P. C. Deka,Bishnu Nagar, Bishnu Rabha Path, P.O- Tezpur.Dist-Sonitpur,Assam, Pin-784001, India Phone: +91 9435180795 Email: dr.rijurdeka82@gmail.com Date of Submission : 09.08.2016 Date of Acceptance : 10.04.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Original Article DOI: 10.21276/APALM.1169

Dysfunctional Uterine Bleeding: A Clinico-Pathological Study at A Tertiary Care Centre Ramesh. B.H1* and Rajeshwari K2 1 Department of Pathology, Raichur Institute of Medical Sciences, Raichur, Karnataka 584102, India Department of Pathology, Gulburga Institute of Medical Sciences, Gulburga, Kalaburagi, Karnataka 585101,India

ABSTRACT Background: Dysfunctional uterine bleeding (DUB) is a form of abnormal uterine bleeding and it is one of the most common presenting complains in gynecology outpatient department in all age groups. Aim and Objectives: The present study aimed to study the clinical features and various histological patterns of endometrium in DUB and correlating histopathology of endometrium with clinical presentation in clinically diagnosed cases of DUB. Methods: Present study is a descriptive type of study conducted over 111 cases over a period of one year from June 2015- June 2016 in the department of pathology at a tertiary care centre Results: Majority of patients with DUB were in 3rd to 4th decade (40.35%) and were multiparous (93.3%). Of the 111 cases, 51.35% of cases showed histological features associated with DUB. Menorrhagia was the most frequent presenting complaint (56.14%) followed by metrorrhagia in 14.04%. Proliferative phase (50.88%) was the most common endometrial pattern seen with DUB cases in age group from 21-50 years. Endometrial hyperplasia occurred between the age group of 30-50years. Conclusion: Menorrhagia is the most common presenting complaint with proliferative phase endometrium. Age has definite influence on endometrial histology. Histopathology remains the gold standard method of diagnosis of DUB and its types and to exclude the local causes, so as to plan the accurate management in DUB patients. Keywords: Dysfunctional Uterine Bleeding, Histopathology, Endometrium.

Introduction

The normal menstrual cycle consists of length of 21-35 days, the duration of blood flow is 2-7 days and the amount of blood loss is 20-80ml. The deviation from this normal pattern is recognized as abnormal. [1] Abnormal uterine bleeding (AUB) can be further classified into two broad groups first due to organic causes with pathology and the second is the dysfunctional uterine bleeding (DUB) where there is absence of organic disease of the genital tract or according to Novak et al. DUB can be described as AUB from the uterus unassociated with the tumour, inflammation or pregnancy.[2,3] DUB can be classified into primary, secondary and iatrogenic groups. [3] Primary DUB is due to dysfunction in hypothalamo-pitutary-ovarian axis or dysfunction in the endometrium itself. Secondary DUB is due to endocrinopathies, haematological, vascular diseases and liver diseases. Iatrogenic DUB may occur due to drugs, exogenous harmone administration and intrauterine contraceptive devices. [4] Abnormal uterine bleeding is one of the most common clinical problems in gynaecology [5]. The cause for the

bleeding in women is related to hormonal disturbances, pregnancy complications, bleeding diathesis and more importantly local pathology including benign, malignant tumors and infection [6]. Dysfunctional uterine bleeding (DUB) is the term used to describe abnormal uterine bleeding not associated with organic lesions of uterus[7]. In most cases, it is associated with anovulatory or oligoovulatory cycles, leading to a shoot in the estrogen levels, which are unopposed due to absence of progesterone[8]. During adolescence, it may be due to a failure of the hypothalamo-pituitary system to respond to the positive feedback of estrogen. In the perimenopausal years, the anovulatory bleeding may be due to the declining functional capacity of ovary or a careful screening for malignancy is imperative and should be treated promptly [9,10]. Giving the importance of DUB in gynaecopathology, the present study was undertaken to study the endometrial patterns in clinically diagnosed DUB cases and to correlate the histopathological findings of endometrium with clinical behavior presented by the patient in DUB and also to know the prevalence of DUB cases at our tertiary care centre.

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Materials and Methods

The common age group encountered in our study ranged between 21-70 years. DUB was most commonly observed in the age group of 31-40years accounting for 40.35% of the cases as shown in table1.

Present study is a descriptive type of study conducted over 111 cases over a period of one year from June 2015- June 2016 in the department of pathology at a tertiary care centre.

DUB was more commonly seen in multiparous (93.3%) than in nulliparous women.The most common menstrual disorder in women presenting with DUB was menorrhagia in 56.15% of the cases followed by metrorrhagia in 14.05% of the cases, post menopausal bleeding in 8.77% of the cases, menometrorrhagia and polymenorrhagia in 7.01% of the cases as shown in table 2.

Following inclusion and excusion criteria were adopted in our study â&#x20AC;&#x201C; Inclusion Criteria: 1.All patients who were diagnosed clinically as DUB cases. 2. None of these patients has a organic lesions detected either by clinically or ultrasonographycally.3. The clinical diagnosed DUB cases with only hysterectomy specimens were included in the present study.

Normally the duration of symptoms ranged from a minimum of four days to a maximum of seven years. In our study the most common duration of symtoms we encountered was one year in 42.11% of the cases (24 cases).

Exclusion Criteria: 1. Adoloscent age group with clinical diagnosis of DUB. 2. Inadequate and improperly preserved specimens were excluded. 3. Hysterectomy specimens with incidental organic pathology diagnosed on gross were excluded.

In the present study, we also observed that most of the patient with DUB were anemic with low haemoglobin< 10grams% accounting for 89.47% of the cases(51 cases). Histopathological features:Out of total 57 cases of DUB without organic pathology, majority of the cases had anovulatory pattern of endometrium accounting for 84.21% of the cases. Ovulatory pattern of endometrium was detected in 15.79% of the cases.

Hysterectomy specimens received to the department of pathology were subjected to detailed gross examination and fixed in 10% buffered neutral formalin and processed. The sections of 3-4 microns were cut and stained with haematoxylin and eosin for histopathological diagnosis. A detailed clinical history, gynaecological examination and radiological findings were recorded. The clinical and histopathological findings were analyzed and following observations were made.

Among the anovulatory endometrium most common histological pattern observed in our study is proliferative phase accounting for 50.88% of DUB cases (Figure 1) followed by simple endometrial hyperplasia accounting for 17.54% of the cases (Figure 2) , complex hyperplasia in 10.53% of the cases, atrophic endometrium in 3.50% of the cases and complex hyperplasia with atypia in 1.765 of the cases as shown in table 3. Ovulatory pattern of endometrium showed secretory phase in 15.79% of the cases (Figure 3).

Statistical Analysis: The collected data was tabulated, analyzed and subjected for statistical analysis using SPSS 17.0. Results are presented as range for quantitative data and number and percentage for qualitative data.

Results

Our study also noted the correlation of clinical symptoms with histological patterns of endometrium and found that menorrhagia was the most common type of clinical bleeding in patient with proliferative phase contributing to 50.88% of the cases (29 cases) followed by secretary phase accounting for 15.79% of the cases ( 9 cases) and in endometrial hyperplasia in 29.83% of the cases (17 cases).

A total of 622 hysterectomy specimens were received to the deprtment of pathology out of which clinically diagnosed DUB cases with hysterectomy accounted for 17.85% (111 of cases) of the total specimens received. Among the clinical diagnosed DUB cases 51.35% (54 cases) showed uteri without any organic pathology and 48.65% (54 cases) showed uteri with organic pathology as shown in (Graph1). Table 1: The age distribution of patients presenting with DUB. Sl. No.

Age (in years)

No. of cases (%)

Percentage (%)

21-30

15.09

31-40

40.35

41-50

36.82

51-60

07.01

61-70

01.90

TOTAL

100.0

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Table 2: The salient clinical features in DUB Sl. No.

Symptoms

No. of cases

Percentage (%)

Menorrhagia

56.15

Metrorrhagia

14.05

Postmenopausal bleeding

08.77

Menometrorrhagia

07.01

Polymenorrhage

07.01

Continuous bleeding

01.75

Amenorrhoea followed by bleeding

03.50

Dysmenorrhoea

01.75

Total

100.0

Table 3: The distribution of various histologic pattern of endometrium in DUB Sl. No

Type of Endometrium

No. of cases

Percentage (%)

Proliferative Phase

50.88

Secretory Phase

15.79

Atrophic endometrium

03.50

Simple endometrial hyperplasia

17.54

Complex hyperplasia

10.53

Complex hyperplasia with atypia

01.76

Total

100.00

Table 4: The age distribution of various endometrial lesions in DUB Age (yrs)

Proliferative Phase

Secretary Phase

Atrophic Phase

Simple Hyperplasia

Complex Hyperplasia

Complex with atypia

Total

21-30

31-40

41-50

51-60

61-70

Total

Table 5: The comparison of endometrial patterns in DUB cases without organic pathology Mehrotra et al.(%)

Dass & Chugh (%)

Patel et al.(%)

Purandhare& Jhalam(%)

Katuwal study (%)

Present study(%)

Proliferative phase

62.5

41.5

63.3

66.3

48.6

50.88

Secretory phase

5.3

22.5

7.6

20.6

35.3

15.79

Atrophic phase

2.6

1.8

6.1

6.8

03.50

Endometrial hyperplasia

19.4

30.6

29.1

7.0

2.7

29.83

3.6

Endometrial pattern

Others

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Graph 1: Distribution of cases clinically diagnosed as DUB

Fig. 1: Microphotograph of proliferative phase showing tubular glands with columnar cells surrounded by dense stroma with normal gland to stroma ratio.

Fig. 2: Microphotograph of simple hyperplasia phase showing glands of varying sizes and shapes with cystic dilatation and increased gland to stroma ratio.

Fig. 3: Microphotograph of Secretary phase showing torturous glands with subnuclear vacuoles surrounded by oedematous stroma .Our study showed, proliferative phase was the most common histological pattern among adult reproductive age group and perimenapausal group accounting for 50.88% of the DUB cases as shown in Table-4

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Disscussion:

Dysfunctional uterine bleeding is defined as abnormal uterine bleeding unrelated to anatomic lesions of the uterus. It is largely caused by aberrations in the hypothalamo-pituitary-ovarian hormonal axis [8]. DUB can occur in any type of endometrium both normal and abnormal [10]. Most of the cases occur during adolescence and perimenopausal years [7]. The evaluation of AUB patient is achieved by identifying the DUB cases through various investigations like history, physical examination, laboratory diagnosis, ultrasonography and endometrial sampling. The diagnosis of DUB remains as a diagnostic challenge to both Gynaecologists and Pathologists. Hence the present study was taken to highlight the DUB causes, its histopathological patterns of endometrium and its clinicopathological correlation in clinically diagnosed DUB patients. Shergill et al and Patel et al reported the incidence of DUB among hysterectomies to be 26% and 49.65% respectively which is high compared to the present study of 17.85% [11,12] . Sucheta et al reported the commonest indication for hysterectomy was DUB in 33% of cases. The incidence is seen to be variable depending on the geographic regions [13] . Dass et al and Bhattacharji reported that majority of DUB cases occurred in 4th decade which correlated well with our study [14,15]. Whereas Pilli et al, Patel et al and Ghosh et al reported most cases in 4th& 5th decades [10, 12, 16]. The most common clinical symptoms in DUB cases were menorrhagia followed by metrorrhagia were observed by katuwal et al , Pilli et al, Patel et al, Mehrotra et al, Muzaffar et al and Bhosle et al studies. [ 4,10.12.17,19,20] Similar findings were observed in the present study and the results are in concordance with the above studies mentioned. Among the duration of symtoms in DUB cases, Joshi et al observed that maximum number of cases had duration of 1-6 months (37%) and 27.16% of patients had more than one year.[18] Whereas Ghosh et al observed the maximum number of cases had more than one year of symptoms in DUB cases [16] and similar findings were seen in our study and the results correlated with Ghosh et al study. Majority of the cases in the present study were multiparous (93.33%) and correlated with the findings of Pilli et al and Joshi et al [10,18].Patel et al reported 100% of patients were multiparous [12]. The incidence in the present study among nulli and uniparous was negligible as hysterectomy was performed only on patients who had www.pacificejournals.com/apalm

A-353 completed their families except on one nulliparous patient due to the severity of her symptoms who was also in the perimenopausal age group. Menorrhagia is one of the commonest causes of iron deficiency anemia in women of the reproductive age group. Comparison of the hemoglobin percentage shows a similar pattern of prevalence of varying degrees of anemia in DUB patients. The development of iron deficiency anemia initiates a compensatory mechanism which tends to reduce blood loss [16]. The endometrial pattern in DUB cases (Table-5) without any organic pathology reveals a predominance of proliferative phase in most of the studies, suggesting that anovulation to be the main cause of DUB [9,12,14].The incidence of proliferative phase in present study (50.88%) is in concordance with Katuwal et al, Dass et al (41.5%) and Mehrotra et al. (62.5%) study (48.6%)[4,14,17].While Bhattacharji (19.6%) and Joshi et al (31.7%) reported lower incidence of proliferative phase [15,18]. Similarly, the percentage incidence of secretary phase varied from 5.3% to 43.9% [9,12,14,17]. The incidence of secretary phase (15.79%) in present study is in concordance with Joshi et al study (16.7%) [18] .The highest incidence of secretary phase was reported by Bhattacharji ( 43.9%) and lowest incidence of 5.3% in Mehrotra et al. [15,117] studies. The discrepancy may be due to the fact that unlike D&C, which is performed in the premenstrual phase, performing hysterectomy is not bound by the phase of menstrual cycle. The percentage of atrophic endometrium (3.50%) in the present study has correlated with the study of Dass et al (1.8%) [14]. Bhattacharji reported higher incidence of 7.3% [15] . Atrophy of the endometrium may be associated with large dilated venules situated superficially under a thin endometrium. These venules may rupture and are probably the commonest cause of postmenopausal bleeding [21]. The incidence of endometrial hyperplasia varies in different studies from 7% to 31.4% [14,15,18] However, the percentage of endometrial hyperplasia obtained in this study (29.83%) is in concordance with Patel et al. ( 29.1%) [12 ]and Dass et al (30.6%) [14] studies and lowest in the study of Purandhare et al (7%) [9].Samal et al reported hyperplastic endometrium was the commonest finding in 32.3% of DUB cases in postmenopausal women[21]. Study of Pilli et al reported the incidence of endometrial hyperplasia to be 44% high compared to the present study of 29.83% which partly reflects hyperestrogenic activity [10].

Conclusion

In this study, Dysfunctional uterine bleeding occurred in both the reproductive and the perimenopausal age groups eISSN: 2349-6983; pISSN: 2394-6466

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and majorities were multiparous. Menorrhagia was the most common presenting complaint with predominance of proliferative endometrium. Besides this, it is observed that the age of the patient had a strong bearing on the type and pattern of endometrium seen on histopathological examination. Age has definite influence on endometrial histology. Histopathological examination of endometrium remains the gold standard method of evaluating the DUB cases and its subtypes and to exclude the local causes which helps in early diagnosis and to determine the plan and mode of management.

References 1.

Algotar KM, Nalawade A. Physiology of menstruation. In: Purandare CN editor: Dysfunctional uterine bleeding -An update. JAYPEE: New Delhi; 2004;1-15.

Kumar P, Malhotra N, editors. Abnormal and excessive uterinebleeding. Jeffcoate’s Principal of Gynecology. 7th ed. New Delhi: JAYPEE;2008;598-616.

Davey DA. Dysfunctional uterine bleeding. In: Whitfield CR editor: Dewhurst’s textbook of Obstetrics and Gynecology for postgraduates. 4th ed. London: Blackwell science; 1995;624-45.

Katuwal N ,Gurung G,Rana A,Jha A. A clinicopathological study of dysfunctional uterine bleeding.Journal of Pathology of Nepal. 2014;4:635- 38.

Munro MG.Abnormal uterine bleeding in the reproductive years. J Am Assoc Gynecol Laparoscop.1999;6:391-428.

Ash SJ, Farrell SA, Flowerden G. Endometrial biopsy in DUB. J Reprod Med.1996;41:892–896.

Fraser IS.Treatment of ovulatory and anovulatory dysfunctional uterine bleeding with oral progestogens.Aust NZ J Obstet Gynecol.1990;30:353-6

Emanuel MH, Verdel MJ, Wamsteker K, Lannes FB.A prospective comparison of transvaginal ultrasonography and diagnostic hysteroscopy in the evaluation of patients with abnormal uterine bleeding.Am J Obstet Gynecol 1995;172:547-52.

Purandare S, Jhalam L. Pathological picture in Hysterectomy done for abnormal uterine bleeding. J Obstet Gynaecol India. 1993;43:418-21.

10. Pilli GS, Sethi B, Dhaded AV , Mathur PR. Dysfunctional Uterine Bleeding-Study of 100 cases .J Obstet Gynaecol India 2002;52(3):87-9. 11. Shergill SK, Shergill HK, Gupta M, Kaur S. Clinicopathological Study of Hysterectomies. JIMA. 2002;100(4):238-9. 12. Patel SR, Sheth MS, Rawal MY. Dysfunctional Uterine Bleeding- Place of Hysterectomy in its Management. J Postgrad. 1986;32(3):150-3. 13. Sucheta KL,Mallikarjuna M,Madhu KP,Arun BJ,Niranjan N.Hysterectomy:Clinical profile,indications and postoperative complications.Int J Reprod Contracept Obstet Gynecol.2016 Jul;5(7):2093-96. 14. Dass A, Chugh S. Dysfunctional Uterine Bleeding: A clinicpathological study. J Obstet Gynaecol India. 1964;14:34854. 15. Bhattacharji SK. Dysfunctional uterine hemorrhage: correlation of endometrial pattern with clinical behavior. J Obstet Gynaecol India. 1964;14:372-9. 16. Ghosh BK, Sengupta KP. A study of the endometrium and cystohormonal pattern in functional uterine bleeding. J Obstet Gynaeol India.1968;310-6. 17. Mehrotra VG, Mukerjee K, Pandey M, Samanth V. Functional uterine bleeding -A review of 150 cases. J Obstet Gynaecol India. 1972;22:684-9. 18. Joshi SK, Deshapande DH. Clinico - pathological study in 274 cases of dysfunctional uterine hemorrhage. J Obstet Gynaecol India.1964;14:360-71. 19. Muzaffar M, Akhtar K, Yasmin S, Rehman M, Iqbal W, Khan M. Menstrual irregularities with excessive blood loss: a Clinico- Pathological correlation. J Pak Med Assoc 2005;55:1-4. 20. Bhosle A, Fonseca M. Evaluation and histopathological correlation of Abnormal uterine bleeding in Perimenopausal women. Bombay Hospital J 2010;52:69-72. 21. Samal S, Gupta U, Agarwal P, Samal N. Clinico- pathological Study of Dysfunctional Uterine Bleeding in Postmenopausal Women. J Obstet Gynaecol India. 2000;50(6):100-1.

*Corresponding author: Dr. Ramesh B. H., No.17, 3rd Cross, Triveni Road, KN Extension, Yeshwantpur, Bangalore-22. India Phone: +91 7411159287 Email: rameshpath@rediffmail.com

Financial or other Competing Interests: None.

Date of Submission : 17.11.2016 Date of Acceptance : 20.04.2017 Date of Publication : 31.08.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Original Article DOI: 10.21276/APALM.1190

A tertiary Care Centre Experience with Periampullary and Pancreatic Neoplasm in Pylorus Preserving Pancreatico-Duodenectomy Specimens Limi Mohandas, Sheela Vasudevan and Kalaranjini KV Department of Pathology, Sree Gokulam Medical College and Research Foundation, Venjaramoodu, Trivandrum , India

ABSTRACT Background: Periampullary neoplasms are of pathologic and clinical significance, out of proportion to the small size of the structure. These neoplasms have different long term follow up following resection despite having similar histology. Methods: A prospective study was done in the department of Pathology, Sree Gokulam Medical College and Research Foundation from Dec 2013 to May 2015 for a period of 18 months. This cross-sectional study included a total of eighteen cases. Study was commenced after approval from institutional ethical committee. Results: In the present study, females predominated M:F of 1:3.5. The median age for the malignant lesions ranged from 55 yrs to 60yrs and for benign lesions were 30-39years. The most common clinical presentation were neoplastic lesions accounted for 80% of the total lesions and non neoplastic lesions such as chronic calcific pancreatitis for 20%. Pancreatic adenocarcinoma accounted for most number of lesions. Tumor variables such as perineural invasion and margin status positivitiy were seen in pancreatic carcinomas. Keywords: Periampullary, Pancreaticoduodenectomy, Perineural Invasion, Lymphnodes Metatases

Introduction

Periampullary neoplasms are of pathologic and clinical significance, out of proportion to the small size of the structure. These neoplasms have different long term follow up following resection despite having similar histology.[1] It was Dr William Halstead who performed successful local resection of periampullary neoplasm in a 58year old woman with obstructive jaundice.[2] Dr Allen O Whipple revolutionised and popularised the procedure and which was later named as “Whipples procedure”.[3] This procedure proved effective for many patients who had carcinomas arising in the terminal portion of the common bile duct, in the ampulla or periampullary region in the duodenum, as well as for endocrine and other tumors. This procedure has been palliative in patients with infiltrative tumors in reducing the tumor burden.[4,5] The objectives of our study were studying the demographics, clinical presentation, distribution and pathology of various lesions of periampullary region and pancreas in a tertiary care centre like ours.

Materials and Methods

A prospective study was done in the department of Pathology, Sree Gokulam Medical college and Research

Foundation from Dec 2013 to May 2015 for a period of 18 months. This cross-sectional study included a total of eighteen cases . Preoperative evaluation was standardised and a CT/MRI abdomen were requested in all cases. Percutaneous transhepatic cholangiography, angiography , upper GI endoscopy and CT angiography were optional. Study was commenced after approval from institutional ethical committee. Inclusion Criteria: Cases with suspected pancreatic/ periampullary cancer that were assumed to be resectable according to preoperative diagnostic work up were included. Exclusion Criteria: Patients with distant metastasis or locally unresectable tumors, as indicated by preoperative workup and intraoperative findings were excluded. Pathologic Review: Specimens were dissected according to CAP protocol. Primary Pathology and extent of the disease were determined on gross and microscopic examination. TNM staging was done. Resection margins of the specimens were inked with Indian ink and considered positive if the neoplasm was present at the neck, uncinate processus, CBD, Duodeum/gastric resection, SMA, SMV and retroperitoneal margin. Statistical Analysis: All variables were entered in excel sheet and analysed by SPSS16. P value was obtained in few variables.

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Results

In the present study, out of the eighteen cases there were fourteen females (77.77%) and four males(2.23%), with M:F of 1:3.5. The median age for the malignant lesions ranged from 55 yrs to 60yrs and for benign lesions were 30-39years. The most common clinical presentation were jaundice (70%) followed by vague abdominal pain and abdominal discomfort (55%). Neoplastic lesions accounted for 80% of the total lesions and non neoplastic lesions such as chronic calcific pancreatitis for 20% (See Table 1). Among the neoplastic lesions adenocacinoma of periampullary region accounted for 75% and others were pancreatic endocrine tumors, IPMN and Mucinous cystadenoma. Pancreatic adenocarcinoma(50%)was the most common neoplasm arising from the periampullary region, followed by duodenal (33.34%) and gall bladder(8.34%). One case was given a broad diagnosis of periampullary carcinoma alone, because its origin was discernible on gross and microscopic examination. Tumor Characteristics (Table 2) Patients with pancreatic adenocarcinoma (Fig 1) had a mean age of 57yrs and a mean diameter of 39mm. Majority (5/6) showed moderate differentiation, perineural invasion(4/6) and lymphnode

metastases in two out of six cases. These neoplasms accounted maximum for positive resected margins. Duodenal adenocarcinomas (fig 2) had a mean diameter of 33mm and mean age of 57 years similar to pancreatic adenocarcinoma. These tumors also showed moderate differentiation in most cases. They had positive correlation with lymph node metastases. Lymphatic and vascular invasion was seen in a single case and was not statistically significant. The sample size of gall bladder origin was quite low and their characteristics were not statistically significant. Among other neoplastic lesions, were two cases of pancreatic endocrine neoplasm (fig 3), one case of mucinous cystadenoma and one case of intraductal papillary mucinous neoplasm (fig 4) which showed characteristic histological features. Among the non-neoplastic lesions, four cases of pancreatitis were included. However several neoplastic lesions showed adjacent pancreas with chronic calcific pancreatitis owing to a total number of 11 cases of chronic pancreatitis. These patients also had a similar clinical presentation of jaundice and vague abdominal pain as presenting symptom. On histopathology, calcification, fibrosis and islet cell preservation were noticed in all cases.

Table 1: distribution of lesions. DISTRIBUTION OF LESIONS

NUMBER OF CASES

Pancreatic adeno. Ca

Duodenal adeno. Ca

Pancreatic endocrine

Gall bladder adeno. Ca

Periampullary adenocarcinoma

Intraductal Papillary Mucinous Neoplasm

Mucinous cyst

Chronic calcific pancreatitis

Table 2: Tumour characteristics. MEAN AGE

MEAN DIAMETER

DEGREE OF DIFFERN-TIATION

LYMPHNODE INVOLVEMENT

PERINEURAL INVASION

MICRO VASCULAR INAVSION

PAC (6)

MODERATE

2/6

4/6

DAC (4)

56.88

WELL(1/4) MODERATE (3/4)

3 /4

0/4

1 /4

GB (1)

59.1

WELL

0/1

1/1

MODERATE

1/1

0/1

TUMOR TYPE

PERIAMPULLARY (1)

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Fig. 1: Pancreatic adenocarcinoma â&#x20AC;&#x201C; moderately differentiated (H&E 40x). small glandular proliferation separated by dense desmoplastic stroma. Perineural invasion by the neoplastic glands are seen.

Fig. 2: Duodenal adenocarcinoma (H&E 40x) Neoplasm arranged in a complex glandular and papillary pattern, lined by tall columnar epithelium with dysplasia(nucleomegaly, pleomorphism, loss of mucin and high N:C ratio).

Fig. 3: Pancreatic endocrine neoplasm(H&E 40x). solid tumor showing nests, insular and trabecular arrangement of cells. The cell showing moderate cytoplasm with enlarged nuclie showing salt and pepper chromatin.

Fig. 4: Intraductal papillary mucinous neoplasm(H&E 10x). A branch duct showing a neoplasm composed of glands arranged in complex papillary and villous pattern with moderate dysplasia.

Discussion

Periampullary and ampullary region has always been a site of interest due to its varied pathology and early symptomatic presentation.[1] The confluence of CBD and main pancreatic duct at the ampulla means that the tumors arising from this location have the ability to obstruct two major organs

and can present as billiary obstruction and pancreatitis.[1] Early symptoms help in early detection of these neoplasms and hence appropriate management can be rendered to the patient. Therefore we studied the clinical and pathological features which help in ascertaining the type and origin of the tumors and variables associated with it.[5]

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In our study series of periampullary and pancreatic pathology, the demography variable such as mean age(57yrs) were concordant with Yeo et al[6],Cameron et al[7] and Tran et al.[10] However our study showed a female predominance unlike other studies which showed male predominance.[6,7,8,9,10] Among the neoplastic lesions, maximum lesions were noticed in pancreas which was comparable to Yeo et al 6,9and Cameron Et al7,2. Pancreatic adenocarcinomas are the most common pathology presenting as mass in periampullary region. In the present study these adenocarcinomas had a mean diameter of 39mm which was comparable to Yeo et al series.[6,9] On gross examination these neoplasms was seen involving head and body of pancreas, firm to hard in consistency and grey white in appearance. Microscopic examination revealed proliferation of small glands separated by desmoplastic stroma, with cytologically deceptive benign features. Few subtypes of pancreatic adenocarcinoma was seen, among them colloid carcinoma (fig 3a&3b) being one. These neoplasms were distinctive with their gross and microscopy features. They had an average diameter of 54mm, which was comparable to Adsay NVet al.[11] Gross examination revealed soft to firm mass with gelatinous glistening like appearance, similarly microscopy showed abundant pools of mucin with sparse glands. In a study done by Adsay et al [12] these neoplasms were composed of well-defined pools of mucin with sparse malignant cells in various patterns of distribution. Immunohistochemical and histochemical mucin stains suggested luminalization of the basal aspects of the cells. Perineural invasion is often considered as a prognostic factors and a characteristic histological feature in pancreatic adenocarcinoma.[12]This is attributed to the acute neurotropism of pancreatic cells along with innervation of the gland. Some authors have demonstrated that perineural invasion is the first step before nodal invasion.[12] The characteristic perineural invasion was lacking in colloid carcinoma this was comparable with Ake Andre Sandberg et al.[13] In the present study several other pathologies were seen, which included pancreatic endocrine neoplasm(2), and other cystic neoplasms i.e., mucinous cystadenoma and intraductal papillary mucinous neoplasm. Pancreatic endocrine neoplasm are uncommon tumors with an annual incidence <1 /100000 persons per year. [14] In our study it accounted for 11.1% of the cases,

these neoplasms were encapsulated with grey white appearance and areas of haemorrhage. One of the case showed predominant vascular proliferation with areas of haemorrhage.[15]The usual histologic appearance is that of a solid nesting architecture; the tumor cells are round to ovoid with eosinophilic, slightly granular cytoplasm and dispersed nuclear chromatin resembling â&#x20AC;&#x2DC;salt and pepper.â&#x20AC;&#x2122; Immunohistochemistry plays a significant role in the diagnosis of endocrine neoplasms. They are synaptophysin and chromogranin positive, with synatpophysin being a more specific marker.[16] Cystic neoplasm of pancreas include an exhaustive list of conditions. However very often encountered and chief lesions include serous cystic neoplasm, mucinous cystic neoplasms, intraductal papillary mucinous neoplasm and solid pseudopapillary neoplasm.[15] In our study we came across IPMN and Mucinous cystic neoplasm. Intraductal papillary mucinous neoplasms of the pancreas (IPMN) display differing degrees of dysplasia and vary in terms of their malignant potential.[17] IPMN are noninvasive precursor lesions reflective of an underlying field defect and genetic instability, placing the entire gland at increased risk for cancer development.[18] A single case of intraductal mucinous neoplasm was obtained in this study. In the present study it presented as cystic neoplasm involving the head of pancreas, involving the branch duct. On microscopy it had histological features of intestinal type with moderate degree of dysplasia. According to the study done by Julie. N. Leal IPMN was most common in head of pancreas, showing involvement of branch duct however on histology gastric type of mucinous epithelium was more common as opposed to our study.[19] Mucinous cystic neoplasm of pancreas are often seen in younger age with female predilection. Most often found in the body and tail of pancreas. They show a standout characteristic ovarian stroma on microscopy and these stromal cells show oestrogen and progesterone sensitivity. [20] Very often IPMN forms the differential diagnosis of mucinous cystic neoplasms. Mucinous neoplasm can be differentiated by its ovarian stroma and lack of characteristic duct involvement.[21] Duodenal adenocarcinoma was the second common neoplasm involving periampullary region, which was least common in other series. Duodenal adenocarcinoma often presents as a polypoidal mass (fig involving in and around the ampulla.[15] These adenocarcinomas were composed

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Mohandas et al. of proliferated glands in complex papillary and villous pattern with areas of necrosis and calcification. Duodenal adenocarcinomas showed maximum number of node positivity which was comparable with Yeo et al.[6,9] Majority of the adenocarcinomas (pancreatic, duodenal and Gallbladder) showed moderate differentiation which according to Yeo et a[6,9] and Cameron et al [7,2] were features of favourable long term prognosis. Few features such as node positivity and margin positivity, considered as poor prognostic variables according to Yeo et al [6,9] were seen in our series in duodenal and pancreatic adenocarcinomas respectively. Non neoplastic lesions of pancreas included only cases of pancreatitis. 4/6 cases of pancreatic adenocarcinoma showed chronic calcific pancreatitis. Several data exist to indicate association between chronic pancreatitis and pancreatic adenocarcinoma.[22] Two theories has been proposed regarding this association, one being carcinoma arising from chronic pancreatitis[23] and the other is chronic pancreatitis developing secondary to carcinoma. The second theory can attributed to obstruction of the ducts by tumours leading to chronic pancreatitis.[20] Majority of the cases showed fibrosis, calcification and islet preservation as the characteristic features.[21]

Conclusion

In our study we have concluded that there is a change in gender predilection and possible emergence of duodenal adenocarcinomas in future era. The association of chronic pancreatitis and pancreatic adenocarcinoma still debatable. Our study also showed several prognostic variables similar to other studies with no significant change in the recent past.

References 1.

3. 4.

Klimstra D, Volkan AN. Tumors of pancreas and ampulla of vater. Odze Robert D, and. Goldblum John R, Surgical Pathology of the GI Tract, Liver, Biliary Tract and Pancreas, 3rd Edition Elsevier. 2009: 909-60 Cameron JL, Riall TS, Coleman J, Belcher KA. One Thousand Consecutive Pancreaticoduodenectomies. Annals of Surgery. 2006;244(1):10-15. Whipple AO. A reminiscence: pancreaticoduodenectomy. Rev Surg.1963;20:221-25 Jones BA, Langer B, Taylor BR, Girotti M. Periampullary tumors which ones should be resected? Am J Surg 1985; 149:46-51.

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Grace PA, Pitt HA, Tompkins RK, et al. Decreased morbidity and mortality after pancreaticoduodenectomy. Am J Surg 1986; 151:141-149.

MH van Roest, A.S. Gouw, S, H. Annette Results of pancreaticoduodenectomy in patients with periampullary adenocarcinoma: perineural growth more important prognostic factor than tumor localization.Ann Surg. 2008 Jul; 248(1):97-103

Yeo CJ, Sohn TA, Cameron JL, Hruban RH, Lillemoe KD, Pitt HA. Periampullary adenocarcinoma: analysis of 5-year survivors. Annals of Surgery. 1998; 227(6):821-831.

Cameron JL, Pitt HA, Yeo CJ, Lillemoe KD, Kaufman HS, Coleman J. One hundred and forty-five consecutive pancreaticoduodenectomies without mortality. Annals of Surgery. 1993; 217(5):430-438.

Yeo, C. J., Cameron, J. L., Sohn, T. A., Lillemoe, K. D., Pitt, H. A., Talamini,, et al. Six hundred fifty consecutive pancreaticoduodenectomies in the 1990s: pathology, complications, and outcomes. Annals of Surgery. 1997;226(3):248-260

10. Tran KTC, Smeenk HG, van Eijck CHJ, et al. Pylorus Preserving Pancreaticoduodenectomy Versus Standard Whipple Procedure: A Prospective, Randomized, Multicenter Analysis of 170 Patients With Pancreatic and Periampullary Tumors. Annals of Surgery. 2004;240(5):738-745. . 11. Adsay NV, Pierson C, Sarkar F et al. Colloid (mucinous noncystic) carcinoma of the pancreas. Am J Surg Pathol. 2001 Jan;25(1):26-42. 12. Fouquet, T., Germain, A., Brunaud, L. Bresler L, Ayav A. Is perineural invasion more accurate than other factors to predict early recurrence after pancreatoduodenectomy for pancreatic head adenocarcinoma? World J Surg (2014) 38: 2132. 13. Andrén-Sandberg Å. Prognostic Factors in Pancreatic Cancer. North American Journal of Medical Sciences. 2012;4(1):9-12. 14. Verbeke CS . Endocrine tumours of the pancreas. Histopathology 2010, 56, 669–682. 15. Kloppel G, Heitz U P. Tumors of the endocrine Pancreas. Fletcher Christopher D. M. Diagnostic Histopathology of Tumors. 3rd edition volume 2 Elsevier.2007: 1124-34 16.

Asa Sylvia L. Pancreatic endocrine tumors. Modern Pathology (2011) 24, S66–S77

17. D’Angelica M, Brennan MF, Suriawinata AA, Klimstra D, Conlon KC., Intraductal papillary mucinous neoplasms of the pancreas: an analysis of clinicopathologic features and outcome. Ann Surg, 2004. 239(3): p. 400–8.

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18. Hruban RH, Takaori K, Canto M, Fishman EK, Campbell K, Brune K,et al., Clinical importance of precursor lesions in the pancreas. J Hepatobiliary Pancreat Surg, 2007. 14(3): p. 255–63. 19. Julie N. Leal. T. Peter Kingham. Michael I. D’Angelica. Intraductal Papillary Mucinous Neoplasms and the Risk of Diabetes Mellitus in Patients Undergoing Resection Versus Observation. J Gastrointest Surg (2015) 19:1974–1981 20. uan R. Pancreas and ampullary region. Rosai and Ackerman’s Surgical pathology. Tenth edition volume 2 Elsevier 2012. 1021-22

21. Cunningham SC, Hruban RH, Schulick RD. Differentiating intraductal papillary mucinous neoplasms from other pancreatic cystic lesions. World Journal of Gastrointestinal Surgery. 2010;2(10):331-336. 22. Dhar P , Kalghatgi S, Saraf V. Pancreatic Cancer in Chronic Pancreatitis. Indian J Surg Oncol 2015; 6(1):57–62 23. Buckminster F, Yuko S, Chen A, BS,Ekong Uffort, et al. Inflammatory Mechanisms Contributing to Pancreatic Cancer Development Ann Surg 2004;239: 763–771

*Corresponding author: Dr. Limi Mohandas, Department of Pathology,S ree Gokulam Medical College and Research Foundation Venjaramoodu, Trivandrum 695607,India, Phone: +91 8592929206 Email: limi.mohandas@gmail.com Date of Submission : 04.12.2016 Date of Acceptance : 28.04.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Original Article DOI: 10.21276/APALM.1197

Fine Needle Aspiration Cytology of Cervical Lymph Nodes: Our Experience Aditi Dholakia1, Dimple Darad1* and Dharmesh Vasavada2 Dept of General Pathology, Gotri Medical College & Hospital, Vadodara, Gujarat, India Dept of Oral Pathology, M P Dental College & Hospital & Oral Research Institute, Vadodara, Gujarat,India 1

ABSTRACT Background: Fine Needle Aspiration Cytology (FNAC) is simple, quick, inexpensive and minimally invasive technique used to diagnose different types of swellings located in the head and neck. Methods: A prospective study was performed in a tertiary health care centre from January 2016 to September 2016 in Vadodara, Gujarat. Fine Needle Aspiration Diagnosis was correlated with detail of relevant clinical findings and investigation. Results: In this study, total of 260 cases of suspected cervical lymphadneopathy were subjected to cytological evaluation. The lesions cases were broadly categorised as inflammatory lesions, cystic lesions, benign tumor & tumor like conditions, salivary gland lesions and malignant lesions. Out of these 260 cases, 184 cases were given the diagnosis of either inflammatory or benign conditions and rest 76 cases were diagnosed positive for malignancy. Out of 76 cases positive for malignancy, 65 cases gave the diagnosis of Squamous cell carcinoma metastasing to lymphnode. Conclusion: Fine Needle Aspiration Cytology is easy, simple, safe and non invasive procedure for diagnosis of head and neck swelling and easy way for surgeon whether to decide surgery or not. Keywords: Head and Neck Swellings, Fine Needle Aspiration Cytology

Introduction

Fine needle aspiration cytology is routinely being used for the diagnosis of various neoplastic and non neoplastic lesions of head and neck region.1It is a valuable procedure for the initial evaluation of swelling in head and neck region and it can easily differentiate between benign and malignant lesion.[1] The use of fine needle aspiration cytology (FNAC) for the diagnosis of metastatic malignancies in the lymphnodes is an established method as lymphadenopathy may be the first sign of malignancy in a patient.[2] FNAC not only confirms the presence of metastatic disease, but also gives clues regarding the nature and origin of the primary tumor.[3] Moreover, the procedure is very cost effective, simple and free of complication, well tolerated by the patient on OPD basis and repeatable.[4] It almost offers an accurate diagnosis for reactive lymphoid hyperplasia, infectious disease, granulomatous lymphadenitis, and metastatic malignancy.[5] FNAC can avoid the need for excisional biopsy in most cases and allow rapid onset of therapy.[6] Masses in the head and neck are especially good targets for needle aspiration because many are superficially located or otherwise accessible to puncture.[7] Head and

Neck sites account for approximately one half of all body sites aspirated. The largest numbers of aspirates are from cervical lymph node enlargements, metastatic squamous carcinoma being the most common lesion encountered.[7] The prime objective of study was to assess the diagnostic accuracy of FNAC in the Head and Neck tumors, to assist the surgeon in selection of the patient for surgery and palliative therapy and to help the surgeon in detecting the metastasis and staging of the certain tumors.

Materials and Methods

The study was performed in a tertiary health care centre from January 2016 to September 2016 in Vadodara, Gujarat after obtaining the necessary permission for the same. Patients referred to the Dept of Pathology with the chief complaint of swelling in head and neck region and who required FNAC procedure as a primary investigation were considered for the study. The procedure was explained to the patient in the language he/she understood and written consent was obtained from the patient. Detailed personal and medical history was taken for all the patients in the Performa prepared. For taking FNA supine position was preferred. All the FNAC were performed using a 23-gauge needle after

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sterilizing the part with alcoholic disinfectant. An average of 2 passes was performed and minimum 5 slides were prepared. Two slides were air dried and stained by Giemsa stain and the remaining 3 slides were fixed in alcohol and stained with H & E stain. The FNA results were reviewed and the morphology of the individual cells and their patterns in the smears were studied in detail. In this study the FNAC diagnosis were correlated with the histological findings, wherever available, or the clinical correlation was obtained.

Result

During the period of one year, a total of 550 of FNACs from various sites were performed. Out of these 260 aspirates were obtained from suspected enlarged lymph nodes in the head and neck region. All the haematological malignancy was excluded. Out of 260 patients, 101 patients were female and 159 male patients. Out of these 159 male patients 82 patients and out

of 101 female patients 2 patients were addicted to some form of tobacco.(Table 1) Out of these 82 tobacco abusers with cervical lymphnode enlargement under investigation, 58 patients were diagnosed as metastatic squamous cell carcinoma, 2 patients diagnosed as metastatic epithelial malignancy with nueroendocrine differentiation; 11 patients were having metastatic epithelial malignancy and remaining 12 patients were negative for malignancy were given the cytological diagnosis of inflammatory condition. Two female patients were diagnosed as metastasis of squamous cell carcinoma. Out of total 78 cases of malignant lesions, histological correlation was possible in 50 cases. In all the 50 cases were positive for malignancy in histological examination also; thus making FNAC 100% sensitive for detection of malignancy.

Table 1: Distribution of Various Head & Neck Pathologies Benign Conditions

Female Patients

Male Patients

Non Tobacco Users Tobacco Users Non Tobacco Users

Tobacco Users

Inflammation: Acute Suppuative Inflammation

Reactive Lymphadenopathy

Granulomatous

Saliadenitis

Fungal Infection

Benign Cystic Leison Of Salivary Gland

Branchial Cyst

Keratinous Cyst

Thyroglossal Cyst

Benign Tumor And Tumor Like Conditions

Ectopic Thyroid Tissue Lipoma

Benign spindle cell tumor

Benign Salviary Gland Tumor Pleomorphic Adenoma

Warthins Tumor

Basal Cell Adenoma

Malignant Conditions Squamous Cell Carcinoma

Adenocarcinoma

Neuroendocrine Differetiation

Metastatic Epithelail Malignancy

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Benign Conditions

Female Patients

Male Patients

Non Tobacco Users Tobacco Users Non Tobacco Users Malignant soft tissue tumorRhabdomyosarcoma

Tobacco Users

Malignant Salivary Gland Tumor Acinic Cell Carcinoma Mucoepidermoid Carcinoma

0 99

Fig. 1: Granuloma formation in the background of acute on chronic inflammation. (10X).

Fig. 2: cluster of malignant salivary epithelial cells- having small, monomorphic nuclei, arranged in acinar pattern. An aspirate from a nasal mass (10X).

Fig. 3: A papillally arrangement of thyroid follicular cells in the lymphoid background. An aspirate from cervical lymphnode â&#x20AC;&#x201C; metastasis of papillary carcinoma of thyroid. (10X).

Fig. 4: A Cluster of moderately differentiated malignant squamous cells along with few dysplastic sqamous cells in the background (40X).

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Fig. 5: Scattered highly pleomorphic malignant epithelial cells in lymphoid background- Metastasis from epithelial malignancy to the cervical lymphnode (10X).

Discussion

Enlarged lymph nodes in head and neck region are most common indication for FNAC.[8] Over the years, FNAC has been found to be very useful, simple, cost effective and accurate in work up of various neoplastic & non neoplastic lesions, especially in head neck region.[1] Lymph nodes that are clinically suspicious for metastasis are the most common indications for FNAC.[9] FNAC has a significantly no risk of any subsequent complications. Except for minimal bleeding and some pain at the aspiration, FNA is an uneventful procedure. FNAC is having a high diagnostic rate to differentiate benign and malignant lesions. It is particularly useful for confirming metastasis of a known malignant disease or recurrence of already cured malignancy.[10] In this study, total of 260 cases of suspected cervical lymphadneopathy were subjected to cytological evaluation. The cases with insufficient material were not taken in the consideration. All the haematological malignancies & Thyroid lesions are not included in the present study. All the 260 cases were broadly categorised as inflammatory lesions, cystic lesions, benign tumor & tumor like conditions, salivary gland lesions and malignant lesions. Among these 260 patients, 101 patients were female and 159 patients were male. Out of total 260 patients, 82 male patients had given the history of tobacco addiction and only 2 female patients were having positive history of tobacco abuse.

Out of these 260 cases, 184 cases were given the diagnosis of either inflammatory or benign conditions and rest 76 cases were diagnosed positive for malignancy. Among the benign conditions, most frequent pathology presented as cervical lymphadenopathy was Chronic granulomatous inflammation- Tuberculosis.[7,11] Gupta AK reported the lymph node involvement of cervical site (62%), supraclavicular (16%) and others (22%). Present study revealed that cervical lymph node was commonest site of involvement. Out of 76 cases positive for malignancy, 65 cases gave the diagnosis of Squamous cell carcinoma metastasing to lymphnode, making it most frequent malignancy presenting as cervical lymphadenopathy. Out of these 65 cases of squamous cell carcinoma, 58 male and 2 female patients gave the history of some form of tobacco abuse. This confirms strong association between tobacco abuse and squamous cell carcinoma. In the present study, total of 260 cases were further categorised in different age groups and frequency of occurrence of Tuberculosis and malignant conditions were assessed. That showed the peak incidence of tuberculosis is seen in 2nd and 3rd decades of life; and incidence of malignancy shows its peak in 5th and 6th decades. The incidence of malignancy rose through the age group to greater than 85% in patients above the age of 60 years. Hence there is a pressing need for FNAC of enlarged lymphnodes in head & neck region in elderly age groups.

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Dholakia et al. Only one case of malignancy which falls in paediatric age group was actually of embryonal Rhabdomyosarcoma, which is a paediatric malignancy and commonly occurs in 1st and 2nd decades of life. Out of total 260 cases, histological correlation was available in 97 cases. Among the malignant lesions, histological correlation was possible in 50 cases in the present study. One very important aid in working up the neck mass and head and neck oncology in general is the looking at the neck level of involvement. For example, cancers of oral cavity will involve level 1 nodes (node in the submandibular and submental triangles of neck); where as metastases from nasolaryngeal region involves level 2 nodes (nodes along the upper one third of the sternocleidomastoid muscle) more commonly.

A-365 3.

Modi P, Oza H, Bhalodia J. Utility and Adequacy of Fine Needle Aspiration Cytology in Head and Neck Lesions: A Hospital Based Study. Int J Sci Stud 2014;2(8):100-105.

Pandey P, Dixit A, Mahajan N C. The diagnostic value of FNAC in assessment of superficial palpable lymph nodes: a study of 395 cases Al Am een J Med Sci 2013; 6(4):320-327.

Nesreen H, Neveen S Reliability of fine needle aspiration cytology (FNAC) as a diagnostic tool in cases of cervical lymphadenopathy. Journal of the Egyptian National Cancer Institute 2011; 23: 105–114.

Howlett DC, Harper B, Quante M, Berresford A, Morley M, Grant J. Diagnostic adequacy and accuracy of fine needle aspiration cytology in neck lump assessment: results from a regional cancer network over a one year period. J Laryngol Otol 2007;121(6):571–9

Solanki P, Patel A, Taviad P, Chaudhari V, Patel S Fine Needle Aspiration Cytology As A Diagnostic Procedure In Head And Neck Swellings. National Journal of Community Medicine 2012; 3(3):433-436.

Steel BL, Schwart MR, Ramzy I. Fine needle aspiration biopsy in the diagnosis of lymphadenopathy in 1103 patients. Role, limitations and analysis of diagnostic pitfalls. Acta Cytol. 1995;39(1):76–81.

Bagwan I, Kane S, Chinoy R. Cytologic Evaluation of the Enlarged Neck Node: FNAC Utility in Metastatic Neck Disease. The Internet Journal of Pathology 2006; 6(2):1-7.

Conclusion

FNAC is very useful for pre-operative diagnosis of lymph node, thyroid gland, salivary gland, orbit and other swellings in head and neck region, and thus helps surgeon in selecting the patient for palliative or surgical management. In the present study also FNAC of head and neck proved to be a useful tool in diagnosis.

Reference 1.

Khan N, Afroz N, Haider A, Hassan MJ, Hashmi SH, Hasan SA. Role of fine needle aspiration, imprint and scrape cytology in the evaluation of intraoral lesions. Journal of cytology/ Indian Academy of Cytologists. 2013; 30(4):263-269.

Ghartimagar, Ghosh, Ranabhat, Shrestha M , Narasimhan , Talwar Utility of fine needle aspiration cytology in metastatic lymph nodes. Journal of Pathology of Nepal 2012;1:92-95.

10. Singal P, Bal MS, Kharbanda J, Sethi PS. Efficacy of fine needle aspiration cytology in Head and Neck lesions. Int J Med and Dent Sci 2014; 3(2):421-430. 11. Sumit M, Suchandra R, Pradip MK. Fine needle aspiration cytology of supraclavicular lymph nodes: Our experience over a three-year period. Journal of Cytology 2011; 28 (3):108-110.

*Corresponding author: Dr Dimple Darad, Associate Professor, Dept of General Pathology, Gotri Medical College & Hospital, Vadodara, Gujarat, India Email: dimpledarad@yahoo.com

Financial or other Competing Interests: None.

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Date of Submission : 08.12.2016 Date of Acceptance : 17.05.2017 Date of Publication : 31.08.2017

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Original Article DOI: 10.21276/APALM.1220

Pediatric Liver Biopsy: A Clinicopathologic Study Pragati Aditya Sathe* and Dipali Eknath Mahale Department of Pathology, Seth GS Medical College& KEM Hospital, Mumbai, India

ABSTRACT Introduction: The spectrum of liver diseases in children differs from that in adults. Different diagnostic modalities which are available are not capable of diagnosing the liver disease individually unless they are supplemented by a liver biopsy. Hence liver biopsy is an invaluable tool in the diagnosis of liver diseases. Aims and Objectives: The aims of this study were to study the spectrum of liver diseases in pediatric age group at our institute, to correlate the liver biopsy findings with clinical findings and laboratory investigations and to review the literature on this topic. Results: We received a total of 130 liver biopsies during the five year study. Out of these, 65 biopsies were performed in children below one year of age while the remaining 65 liver biopsies belonged to children aged more than one year. The common indications for performing liver biopsies included prolonged jaundice, clay colored stools, abdominal distension, abdominal pain, suspected inborn error of metabolism and for a suspected hepatic tumor. Extrahepatic biliary atresia was the commonest pathology found in children below one year of age whereas metabolic disease was the most common pathology found in children above one year of age. Special stains helped in refining the diagnosis further. Laboratory investigations were correlated to offer a complete diagnosis. Conclusion: Paediatric liver biopsy is of value in diagnosis of disorders where clinical and laboratory parameters overlap. At the same time, clinical and laboratory correlation is a must due to overlapping histologic features of many liver diseases. Keywords: Pediatric, Liver, Biopsy, Metabolic Diseases, Cholestasis

Introduction

The spectrum of pediatric liver diseases is different than in adults. In pediatric age group, liver suffers from metabolic, infective, cholestatic and neoplastic disorders resulting in abnormal liver function tests, jaundice and hepatomegaly.[1] Various diagnostic modalities are available such as liver function tests, serum tumor markers and radiologic imaging techniques like ultrasonography (USG), computed tomography (CT) and Hydroxy iminodiacetic acid (HIDA) scan.[1, 2] All these cannot individually suggest an accurate diagnosis in liver diseases. Liver biopsy, though invasive, is therefore an important diagnostic tool for liver diseases.[1]

clinicopathologic correlation and comparison with similar studies in literature was done.

Results

A total of 130 liver biopsies belonging to paediatric age group (less than 12 years) were received in the department of pathology during the study period comprising 0.23% of total surgical workload (55,020 specimens) and 4.3% of pediatric surgical workload (3010 specimens) received in the surgical pathology laboratory during this period.

Materials and Methods

This is retrospective analysis of liver biopsies performed in patients below 12 years of age received at our department over a five year period.

Table 1 summarizes the spectrum of the liver lesions received according to their frequency. As the spectrum of liver diseases differs between infants and children more than one year old, the cases have been categorised into two groups. We received equal number of cases in both groups (65 cases in each).The most common liver disease affecting infants was EHBA (38.46%) followed by neonatal hepatitis (24.61%). Metabolic disorders (12.3%) were commonest liver pathology seen in children aged more than 1 year.

Clinical features, laboratory investigations (liver function tests, serum copper levels, serological markers for viral infections, tumor markers), radiologic findings (USG, CT, HIDA scan) and histologic findings (including the findings on routine as well as special stains) were noted from the hospital and department records of patients. Further

Extrahepatic Biliary Atresia: (Fig 1 a, b) - A total of 26 cases of EHBA were found in our study. Among these, 25 were infants. The commonest symptom was icterus followed by clay colored stools. Liver function test results were available in 17 cases. Liver enzymes were elevated in the following range -SGOT (131-405 U/L), SGPT (51-

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158 U/L), and Total bilirubin (2.6-15 mg/dl), D.bilirubin (1.6-9.6 mg/dl).HIDA and USG were available in all cases. In 24 cases, radiology was diagnostic of EHBA. In three cases, a differential diagnosis of neonatal hepatitis was given. Features overlapping with NH were seen in one case which was also positive for cytomegalovirus (CMV) Immunoglobulin G (IgG). Hence a differential diagnosis was given in this case. All patients except one underwent Kasaiâ&#x20AC;&#x2122;s procedure. Neonatal Hepatitis: (Fig 1 c, d) - A total of 18 cases of neonatal hepatitis were found in our study. Among these, 17 were infants and one case was a child aged 2.5 years.The most common symptom was icterus followed by abdominal distension.Liver function test results were available in 11 cases. Liver enzymes were elevated in the following range -SGOT (100-724 U/L), SGPT (66-367 U/L), Total bilirubin (4-22 mg/dl), Direct bilirubin (3.2-17.2mg/dl).Four cases showed raised titres for CMV. TORCH titre was done in one case which was negative. Test for Alpha -1- antitrypsin deficiency was done in one case which was negative. Cirrhosis: A total of eight cases of cirrhosis without identifiable etiology were found in our study. All eight cases were children aged above one year of age.The most common symptom was abdominal distension. Liver function test results were available in seven cases. Liver enzymes were elevated in the following range -SGOT (60181 U/L), SGPT (68-129 U/L), total bilirubin (9-19 mg/dl), direct bilirubin (5-8.4 mg/dl).In one case, the cirrhosis was of micronodular type. Special stains such as PAS, Orcein and Prussian blue were negative. Glycogen Storage Disease: A total of 12 cases of glycogen storage diseases were found in our study (Figure 2e). Among these, only two patients were infants and the remaining nine cases were children aged above one year of age.Most common symptom was abdominal distension (11 cases). Others were delayed milestones (4 cases), dolls facies (1 case), hypotonia (1 case) and convulsions (2 cases). Five cases had progressed to cirrhosis. All 12 cases showed PAS positivity and diastase sensitivity indicating presence of glycogen. None of the cases had PAS positive diastase resistant globules that would have suggested Type IV disease. Two cases showed the morphology of glycogen storage disease type III whereas in two cases there was overlap morphology of type 3 with type II and type I respectively. The remaining cases showed morphology of Type I disease. Confirmation was not possible as the enzyme studies were not available in our study.

with abdominal distension, lethargy, vomiting and icterus. One patient also had developmental delay and puffy face. Liver function test ranged as follows - SGOT (92-450 U/l), SGPT (82-342U/L), total bilirubin (10.6-10mg/dl), direct bilirubin (4.8-5.48 mg/dl) Further workup was not available and hence further categorization was not possible. Secondary Hemosiderosis: was seen in two patients, six and 11- year -old respectively (Figure 2 a, b). Both patients suffered from Thalassemia major. SGOT and SGPT were raised in one patient. Both cases had progressed to cirrhosis. Gaucher Disease: was noted in a four- month- old female child who presented with abdominal distension, fever and delayed milestones. Though the histology was characteristic, enzyme studies could not be performed. Chronic Hepatitis: A total of 11 cases of chronic hepatitis were found in our study. Among these, only two patients were infants and the remaining nine cases were children aged above one year of age. Most common symptom was abdominal distension. Liver function test results were available in six cases. Liver enzymes were elevated in the following range -SGOT (Normal-117 U/L), SGPT (Normal- 95 U/L), total bilirubin (1.4-1.6 mg/dl) and direct bilirubin (0.6-0.8 mg/dl). Five cases had progressed to cirrhosis. Grading and staging was done using Ishak scoring system. The histology showed features of grade 1 to 3 and stage 1 to 4. Viral markers were negative in all cases. Reticulin stain showed features of incomplete cirrhosis in one case and cirrhosis in four cases. Biliary Disorders: A total of 13 cases were found in our study in this group. Among these, nine patients were infants and the remaining four cases were children aged above one year of age. We had progressive intrahepatic cholestatic disorder (1 case) and two cases each of paucity of bile ductules, choledochal cyst and sclerosing cholangitis. Most common symptom was icterus followed by abdominal distension. Liver function test results were available in seven cases. Liver enzymes were elevated in the following range -SGOT (100-209 U/L), SGPT (67-109 U/L), total bilirubin (Normal- 13.4 mg/dl), and direct bilirubin (Normal- 9.2 mg/dl) Orcein was positive for copper associated protein in the case of PFIC. Seven cases could not be classified into any particular disorder.

Lipid Storage Disorder: was seen in two patients. Both cases belonged to the first group. Clinically they presented

Wilson Disease: A total of three cases of Wilson disease were found in our study. Among these, all patients were above one year of age.

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The symptoms were abdominal distension, fever, drooling of saliva, ataxia and tremors. Kayser-Fleischer (KF) ring was present in three cases. Liver function test results were available in three cases. Liver enzymes were elevated in the following range -SGOT (Normal -233U/L), SGPT (Normal-134U/L). Total protein was normal. High urine copper level was found in two cases and low serum ceruloplasmin was found in one of these cases. Histology in all cases was that of chronic hepatitis with cirrhosis. Dry weight of copper could not be performed in any case. Granulomatous Hepatitis: A total of four cases of granulomatous hepatitis were found in our study. Among these, two patients were infants and two cases were children above one year of age. The symptoms were fever, abdominal distension and pain, icterus and delayed development. USG was available in three cases. Histology showed features of granulomatous hepatitis favoring tuberculosis etiology in three cases. There was associated vertebral tuberculosis in one case. One out of four cases did not have demonstrable etiology for granulomas. Autoimmune Hepatitis: A total of two cases of autoimmune hepatitis were found in our study. Both cases were children above one year of age. The symptoms were abdominal distension, icterus, fever and vomiting. Liver function test results were available in both cases. Liver enzymes were elevated in the following range-SGOT (234-300 U/L), SGPT (184-280 U/L), total bilirubin (3.2-5mg/dl), direct bilirubin (1.4-4 mg/dl).

Antibody studies were available in one case and showed antinuclear antibody positive. Congenital Hepatic Fibrosis (CHF): was seen in two cases (Figure 2c, 2d). Both were six year old and presented with abdominal distension. Total bilirubin, SGOT, SGPT were normal. USG showed hepatosplenomegaly, features of liver parenchymal disease and portal hypertension. There was no associated organ disease. Budd Chiari Syndrome (BCS): was seen in two patients who presented with abdominal distension and right hypochondriac pain. SGOT, SGPT, total bilirubin, direct bilirubin were normal. CT abdomen showed liver cirrhosis. The diagnosis was based on clinical correlation. Hepatic Tumors: A total of four cases of liver tumours were found in our study (Figure 2 f). Among these, only one case was an infant and three cases were children above one year of age. The symptoms were abdominal distension, fever and abdominal pain. AFP levels were available in three cases and showed normal to high value. Histology in the four cases showed features of hepatoblastoma fetal type, Non Hodgkinâ&#x20AC;&#x2122;s lymphoma (two cases), and high grade malignant tumour possibility of sarcoma (one case). The tumor cells were positive only for Vimentin in the last case confirming the diagnosis of sarcoma without further categorization. Three liver biopsies did not fulfill criteria of adequacy on histology and three biopsies were autolysed.

Table 1: Age wise distribution of cases (n=130). No. of cases

Diagnosis EHBA Neonatal hepatitis Metabolic disorders

<1 year (n=65) 26 16 4

% of total cases 20% 12.3%

No. of cases >1 year (n=65) 1 2 16

Glycogen storage disorder

Wilson disease

Lipid metabolic disorder

Hemosiderosis

Gaucher Disease

Carbohydrate Metabolic disorder Hepatitis

0 4

1 13

Chronic hepatitis

Autoimmune hepatitis

Granulomatous hepatitis

%of total cases 0.76% 1.53%

9 3.07%

3.07%

3 1

9 2 2

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10%

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Diagnosis Cirrhosis (No etiology demonstrated) Biliary diseases Progressive familial intrahepatic cholestasis

<1 year (n=65) 0

% of total cases 0%

No. of cases >1 year (n=65) 8

Paucity of bile ductules

6.9%

Choledochal cyst

Sclerosing cholangitis

Intrahepatic cholestatic disease due to intrahepatic biliary disease

(Not classifiable) Malignancy Congenital Hepatic Fibrosis Budd Chiari syndrome Steatosis and steatohepatitis Hepatic necrosis (focal and diffuse) Non cirrhotic portal fibrosis Nonspecific portal inflammation No significant pathology Inadequate Autolysed Total

1 0 0 1 1 0 0 0 1 2 65

0.76% 0% 0% 0.76% 0.76% 0% 0% 0% 0.76% 1.53%

3 2 2 2 1 1 3 4 2 1 65

%of total cases 6.15%

3.07%

2.30% 1.53% 1.53% 1.53% 0.76% 0.76% 2.30% 3.07% 1.53% 0.76%

Fig 1: a. Extrahepatic biliary atresia: Proliferating bile ductules in the expanded portal tracts. (HE,x 400) b. Extrahepatic biliary atresia: Bile ductular plugging. (HE, x400) c. Neonatal hepatitis: Giant cell transformation. (HE, x400) d. Neonatal hepatitis: Inflammation and extramedullary hematopoiesis in the portal tracts. (HE, x 400).

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Fig. 2: a. Case of hemosiderosis showing brown pigment in the Kupffer cells. (HE, x 400) b. Prussian Blue stain highlighting the hemosiderin. (HE, x 100) c. Congenital hepatic fibrosis –Portal tracts show angulated bile ductules at the periphery of the tracts. (HE, x 40) d. Bile plugging of the abnormal ductules. (HE, x 400) e. Glycogen storage disease – Distended pale hepatocytes. Few glycogenated nuclei are seen. (HE, x400) f. A case of hepatoblastoma – Cells show hyperchromatic nuclei and scant cytoplasm. Vague ductular arrangement was appreciated. (HE, x400).

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Discussion

The pattern of liver diseases in paediatric age group differs in infancy and in children beyond infancy. We found equal number of cases in children below and above one year of age. The common indications for performing liver biopsies included prolonged jaundice, clay colored stools, abdominal distension, abdominal pain, suspected inborn error of metabolism and for a suspected hepatic tumor. We also found a wide spectrum of liver diseases ranging from congenital to neoplastic diseases in both age groups. Cholestatic disease was found to be commonest in infancy. EHBA and neonatal hepatitis were the most common causes. Metabolic disorders, chronic hepatitis and cirrhosis were commonly seen in children aged more than one year. These findings were consistent with studies published by Ahmed M et al, Ramkrishna et al, and Muthuphei, et al.[1, ,3] Some diseases like Reyeâ&#x20AC;&#x2122;s syndrome, fulminant hepatitis, schistosomiasis, echinococcus, hemophagocytic lymphohistiocytocytosis, and alpha-1-antitrypsin deficiency were not seen in our study. The commonest clinical presentation of both NH and EHBA is icterus. EHBA in addition is associated with clay colored stools.[ 4 ]The clinical features and laboratory parameters and even histologic features in EHBA and NH can overlap.[5 ]Presence of jaundice at birth, rise in alkaline phosphatase over other liver enzymes, HIDA scan and histologic features like proliferating biliary ductules with bile plugging is suggestive of EHBA (Figure 1 a-d). [6, 7.]Frequently, portal ductal plate is sent to opine on the diameter of the largest bile ductule which helps in deciding the success of the surgical procedure in EHBA. [5 ] The distinction between NH and EHBA is important as management differs. Neonatal hepatitis can be diagnosed as idiopathic only after etiology is not identified by serologic tests especially infections. [8] Neonatal hepatitis can progress to cirrhosis rarely and was seen in two of our cases. Findings of periportal fibrosis, moderate to severe portal inflammation, and/or diffuse giant cell transformation appear to be major factors predictive for poor outcome in cases of neonatal hepatitis as concluded by Chang et al and were seen in three of our cases. [10]Association with other congenital anomalies was reported in about one-fifth of cases by Carmi et al.[9] No such association was found in our study. Liver biopsy is important in cases where radiology has overlapping features.

cholestasis. Presentation and laboratory parameters in cases of cholestasis are similar to EHBA and NH. Histologic features should be observed carefully for presence, absence or reduction of bile ductules. PFIC, especially type 3, can in fact have very similar histologic features to EHBA and sometimes even neonatal hepatitis. Elevated levels of GGT and free passage of dye into the intestine on HIDA scan favor a diagnosis of PFIC (Type 3). [10] We diagnosed eight cases of cirrhosis on microscopy. Out of these, all cases were seen in children above one year of age. The commonest cause of cirrhosis in children is infectious disease followed by metabolic diseases. [11] None of the cases showed evidence of hepatitis B infection on histology. Jia-An-Zhu et al compared results of liver biopsy and USG in 28 children with liver cirrhosis concluding that USG showed abnormal characteristics which were not specific to the disease thereby reinforcing the necessity of USG guided liver biopsy in diagnosis of children with liver cirrhosis. [12 ]However, at the stage of cirrhosis, it may not always be possible to identify the etiology even on liver biopsy. Ishak scoring system should be used for grading and staging of chronic hepatitis.[13] None of cases in our study was positive for HbS Ag. The causes are variable and include chronic hepatitis B and C and autoimmune hepatitis. Serology is important for etiology. Liver biopsy helps in staging in these cases.[5, 14] Autoimmune Hepatitis: (AIH)has broad clinical spectrum including asymptomatic individuals with abnormal laboratory results, clinical symptoms similar to those of acute viral hepatitis, and hepatic insufficiency or even cirrhosis.[15, 14 ]

Causes of Intrahepatic cholestasis other than EHBA and NH were PFIC, cholestatic liver disease with paucity of ductules and inflammatory biliary tract pathology. TORCH titres were raised in two cases probably explaining

The cases were diagnosed based on characteristic histologic features like interface hepatitis and dense infiltration of portal tracts by mononuclear cells and predominance of plasma cells. In a study by Jimenez-Riviera et al, of 200 cases, the median age range was 12 years for type 1 AIH and 10 years for Type 2 AIH. One hundred and nine patients had anti-nuclear and/or smooth muscle antibody (ANA/SMA) titres positive and 18 patients had liver/kidney microsomal antibody (LKM-1) titres positive. [16] Similar histologic findings were seen in the study by Dehghani et al.[17] They studied 87 children of AIH. Mean age of presentation was 10 years with female predominance. Antinuclear, anti-smooth muscle, and anti LKM antibodies were positive in 14/62, 22/53 and 6/40 patients respectively. Twenty six patients were seronegative, and autoantibodies were not available

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in 19 cases. Seronegative AIH has typical appearance of AIH on histology, responds to immunosuppression, but lacks detectable autoantibodies.[18] This is a rare form of AIH in adults, but its prevalence and clinical characteristics remain to be defined in children. Histologic features favored a tuberculosis etiology in all cases of granulomatous hepatitis. Other causes need to be ruled out like fungal or parasitic infection by using special stain for fungus such as GMS.[5]Monajemzadeh et al evaluated prevalence and etiology of granulomatous hepatitis in liver biopsies of 33 Iranian children. [19] Etiological agents identified were mainly mycobacterial infection followed by sarcoidosis, HCV infection and kala-azar suggesting that mycobacterial infection is an important cause of granulomatous hepatitis. Wilson Disease: was found in three cases. Clinically, they presented with abdominal distension and neurological symptoms. Histologic features seen in our cases were cirrhosis with glycogenated nuclei and occasional focus of hepatocyte necrosis. The characteristic histologic features. may not seen in all cases. The findings on a liver biopsy are very variable and depend on the extent of liver damage. [5, 20 ] Hence a diagnosis of Wilson Disease can be confidently made only after correlation with serum ceruloplasmin, urinary copper levels and dry weight of copper. [20]In our study, three cases were diagnosed cases of Wilson disease. Two out of these cases had high urine copper and low serum ceruloplasmin levels. PAS positivity and diastase sensitivity was noted in seven out of 11 cases of Glycogen storage disease which supports a diagnosis of Glycogen storage disease (all types except Type IV). Presence of diastase resistant PAS positivity favours a diagnosis of Glycogen storage disorder Type IV. Enzyme levels were not available in these cases. However, the histology was quite classical. Presence of distended hepatocytes due to glycogen obliterates the sinusoids and shows a mosaic pattern. Presence of glycogenated nuclei in glycogen storage disease differentiates it from carbohydrate metabolic disorder. Sub-typing of these disorders in difficult without enzyme studies. [5, 14] The wrinkled tissue paper appearance of the cytoplasm of distended Kupffer cells is diagnostic of Gaucher disease. PAS stain makes the wrinkles prominent and should be performed. Secondary hemosiderosis was seen in cases of Thalassemia major. Liver biopsy is usually performed in neonates when neonatal (hereditary) hemochromatosis is suspected.[ 14] It

is usually not performed for secondary causes except to confirm fibrosis. Hepatic tumors: Hepatoblastoma showed fetal histologic type which is the commonest sub type (Figure 2f). Alpha fetoprotein was high in this case. The fetal type of hepatoblatoma may resemble normal liver parenchyma. In such a case, correlation with serum AFP levels is very important as it is a sensitive marker for hepatoblastoma. We reported one rare case each of Non Hodgkins Lymphoma and Hepatic Sarcoma. There is a definite role of liver biopsy in diagnosing hepatic masses as radiology may be overlapping.[14] To conclude, paediatric liver biopsy is of value in diagnosis of disorders where clinical and laboratory parameters overlap. At the same time, clinical and laboratory correlation is a must due to overlapping histologic features of many liver diseases. Thus, liver biopsy assisted by a good clinical examination, radiology and routine as well as specific laboratory evaluation can succeed in diagnosis and follow up of majority of liver diseases.

References 1.

Ahmad M, Afzal S, Roshan E, et al. Usefulness of Needle Biopsy in the diagnosis of Paediatric Liver Disorders. J Pak Med Assoc 2005 ;55:24-8.

Marion AW, Baker A J, Dhawan A. Fatty liver disease in children. Arch Dis Child. 2004;89:648–52.

Muthuphei M N. Childhood liver diseases in Ga- Rankuwa hospital, South Africa. East Afr Med J 2000 ;77:508-509.

Chardot C. Biliary atresia. Orphanet Journal of Rare Diseases 2006 ;1:1-9.

Hicks J, Mani H, Stocker J T.The Liver, Gallbladder, and Biliary Tract. In: Husain AN,Stocker J T, Dehner L P, editors. Pediatric Pathology. 4th edition. Wolters Kluwer; 2015.640-742.

Kahn E. Biliary atresia revisited. Pediatr Dev Pathol. 2004 ;7:109-24.

Shah I, Bhatnagar S, Rangarajan V, Patankar N. Utility of Tc99m-Mebrofenin hepato-biliary scintigraphy (HIDA scan) for the diagnosis of biliary atresia. Trop Gastroenterol. 2012;33:62-4.

Torbenson M, Hart J, Westerhoff M, et al. Neonatal giant cell hepatitis: histological and etiological findings. Am J Surg Pathol. 2010;34:1498-503.

Gupta L, Bhatnagar V.A study of associated congenital anomalies with biliary atresia. J Indian Assoc Pediatr Surg. 2016;21:10-3

10. Srivastava A. Progressive Familial intrahepatic cholestasis. J Clin Exp Hepatol 2014;4:25-36

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11. Pinto RB, Schneider ACR, da Silveira TR. Cirrhosis in children and adolescents: An overview. World J Hepatol 2015;7:392-405.

16. Jiménez-Rivera C , Ling SC , Ahmed N. Incidence and Characteristics of Autoimmune Hepatitis. Pediatrics 2015;136:e1238-48.

12. Jia-An Zhu, Bing Hu. Ultrasonography in predicting and screening liver cirrhosis in children: A preliminary study. World J Gastroenterol 2003 ;9:2348-9.

17. Dehghani S, Haghighat M, Imanieh M, et al. Autoimmune Hepatitis in Children: Experiences in a Tertiary Center. Iran J Pediatr 2013; 23:302-8.

13. Alvarez G, Wang H L. The Liver. In: Humphrey P A, Dehner L P, Pfeifer J D, editors. The Washington Manual of Surgical Pathology, first edition. Wolters Kluwer: Lippincott Wiliams and Wikins;2010. 210-11.

18. Krawitt EL. Clinical features and management of autoimmune hepatitis. World J Gastroenterol. 2008;14 : 3301-5.

14. Tannapfel A, Dienes HP, Lohse AW: The indications for liver biopsy. Dtsch Arztebl Int 2012; 109: 477–83.

19. Monajemzadeh M, Haghi M, Najafi M, et al. Granulomatous hepatitits : A 10 year study in Iranian children. Research journal of biological sciences 2009;4:503-5.

15. Mieli-Vergani G, Heller S, Jara P, et al. Autoimmune Hepatitis. JPGN 2009;49:158–164.

20. Roberts EA, Schilsky ML. Diagnosis and Treatment of Wilson Disease: An Update. Hepatology 2008;47:2089-111.

*Corresponding author: Dr.Pragati Aditya Sathe, A/7, Jeevan Sudha CHS, C D Barfiwala Road, Andheri West, Mumbai-400058,India. Email: pragativk@yahoo.com

Financial or other Competing Interests: None.

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Date of Submission : 19.12.2016 Date of Acceptance : 10.04.2017 Date of Publication : 31.08.2017

eISSN: 2349-6983; pISSN: 2394-6466

Original Article DOI: 10.21276/APALM.1246

Histopathology of Gastrointestinal Tract Malignancies â&#x20AC;&#x201C; A Two Year Retrospective Study Meera Shantaram Mahajan*, Neha Amrut Mahajan, Shrinivas Shankarrao Kale and Chandrashekhar Prabhakar Bhale Department of Pathology, MGM Medical college and hospital, Aurangabad (Maharashtra), India

ABSTRACT Background: Incidence of gastrointestinal malignancies is gradually increasing. The aim of the study is to determine age, sex and relative frequencies of various malignancies. Methods: Gastrointestinal malignancies diagnosed between January 2014 to December 2015 in MGM Medical College and Hospital, Aurangabad. In this descriptive retrospective study, total of 180 cases of gastrointestinal malignancies were diagnosed in our institution. Demographic and histopathological data were retrieved from the case files and histopath request forms. The slides were microscopically reviewed to confirm the diagnosis. Result: The mean age of the patients was 56 years (SD =13). Peak incidence was in between 40-60years. Male to female ratio of 1.5:1 was recorded. The common GI malignancies were esophageal(50.8%) followed by large intestine (50%).Other anatomic sites affected are stomach(36.6%), perianal region(9.1%) and small intestine(3.3%).Microscopically, adenocarcinoma was the commonest malignancy(67%) followed by squamous cell carcinoma(28%). Conclusion: Gastrointestinal tract malignancies are one of most common cancers in our country. . The incidence has been increasing due to life style changes and these cancers are usually diagnosed late when the disease has already invaded the lamina propria and musculature, because in early stages, the patient usually complains of trivial and nonspecific symptoms. Hence a new look to epidemiological studies in gastric cancer is necessary along with necessary focus on lifestyle modification. Keywords: Gastrointestinal Malignancy, Adenocarcinoma, Squamous Cell Carcinoma

Introduction

The gastrointestinal system has been reported the second common site for the non cutaneous cancer and second reason of the mortality resulted from cancer.[1] The pattern and distribution of gastrointestinal malignancies differ in different geographical areas, carcinoma esophagus being most frequent in Iran, carcinoma stomach in eastern Asian countries and colorectal carcinomas in developed world.[2] The geographic difference depends on the food, environment and genetic predisposition. People having low fiber intake in diet have high incidence of colorectal carcinomas whereas those consuming hot beverages have high incidence of esophagus carcinoma. Young people have less GI neoplasms as the dose and time of exposure of carcinogenic agent is less.[3]This study is aimed at determining age, sex and relative frequencies of various gastrointestinal biopsies in MGM Medical College and hospital, Aurangabad.

Materials and Methods

This is a descriptive retrospective histopathological study conducted at MGM Medical College and hospital,

Aurangabad. All records are from 180 consecutive patients who were documented pathologically to have gastrointestinal malignancy from Jan 2014 to Dec 2015.Demographic and histopathological data were retrieved from the case files and histopath request forms. Slides and paraffin embedded blocks of pathological specimens of gastrointestinal tissues were retrieved from archives and new sections were cut and processed with haematoxylin and eosin stain. The slides were microscopically reviewed to confirm the diagnosis.

Result

Our study included various gastrointestinal malignancy received in our department. Total malignancy cases were 180 during the year 2014(82 cases) to 2015(98 cases). Out of 180 cases, 108 were males and 72 were females. The male to female ratio was 1.5:1.The distribution of GI malignancy cases in both the sexes is shown in Figure1. Age average of the patients with GI cancer was 56 years (SD=13) and minimum and maximum age of the patients were 17 years and 92 years respectively. The incidence of different GI malignancies based on different age

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groups is depicted in Table1.Maximum number of cases were in 40 to 60 age groups. Majority of GI malignancies were found in esophagus (50.8%) followed by large intestine (50%).Other anatomic sites affected are stomach(36.6%),small intestine(3.3%) and perianal region(9.1%) shown in Table 2. Histologically adenocarcinoma was main type comprising 67% followed by squamous cell carcinoma which makes up 28% of all malignancies.Other histological variants found in our study were round cell tumor 2%,GIST 2% and adenosquamous carcinoma 1% as shown in figure 2 and 3.

Discussion

All over the world, GI tract malignancies form a significant proportion of malignant tumors in both sexes.[2] It is not uncommon and its incidence varies from region to region. Our study reveal the total number of GI malignancy received from 2014 and 2015 were 180.In our study,

majority malignancy was detected after age of 30 year and maximum number were seen between 41 to 60 years. In various studies major GI malignancy is seen after age of 60 year.[2,4]. Early presentation of malignancy is observed due to several reasons including change in dietary habits due to urbanization, upsurge of confectionary food outlet rich in refined carbohydrate low fiber content and fresh fruit, lead to increase in increased transit time so GI malignancies are not rare as previous studies.[5,6] Out of 180 cases, commonest malignancy is adenocarcinoma 120(67%) case followed by squamous cell carcinoma 50( 27.7%) cases. Most of the squamous cell carcinoma were presented from esophagus whereas adenocarcinoma from colon, that correlate well with other study.[5,6,7] Esophageal cancer is the 15th common cancer in developed countries and 4th in developing world.[8]Squamous cell carcinoma is more common worldwide, but adenocarcinoma is on the rise in the United states and other western countries. In our study most common malignancy is esophageal

Table 1: Agewise distribution of GI malignancies. Age

Adenoca

Round cell ca

SCC

GIST

Adenosquamous ca

1-15

16-30

31-45

46-60

61-75

120

>75 Total

Table 2: Distribution of different histological subtypes in different locations.

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Histopath of GI Malignancies-A 2Year Study.

Fig. 1: Distribution of GI malignancy cases in both the sexes.

Fig . 2: Figure showing H & E of tumor A)Adenocarcinoma, B)Gastrointestinal stromal tumor(GIST), C)Squamous cell carcinoma, D)Adenosquamous carcinoma, E)Round cell tumor.

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Fig. 3:Histological distribution of malignancies.

cancer (50.8%).Our findings correlate with study of Jijo V Cherian et al in Tamil Nadu( South India).Higher rate of squamous cell carcinoma in indian subcontinent may be due to tobacoo chewing, smoking, alcohol intake, ingestion of hot and spicy food.[9] About 60% of stomach cancer occurs in the developing countries. The areas of highest incidence are eastern Asia, Eastern Europe and some parts of South Africa, whereas in northern America and Europe the incidence is low. In our study 44 cases of stomach malignancy were seen, out of which 40 were adenocarcinoma and 4 were of Gastrointestinal stromal tumors (GIST).GIST is a heterogenous group of tumors frequently located in stomach. In our study all the cases of GIST were located in stomach. This finding correlates with studies that found that stomach is the commonest location for gastrointestinal tumors.[10,11] Though small intestine represent maximum surface area in gastrointestinal tract small bowel neoplasm is rare compared to esophagus and colorectal carcinoma and account for only 1-2% of all gastrointestinal neoplasms.[12] The incidence of colon and rectal cancer is higher in developed country than in developing countries.[13]In developed countries, it is among the third most common cancer and colorectal carcinoma is commonest gastrointestinal neoplasm. This geographic difference represent the effect of different dietary habbit. In a study by Fatima et al in south Nigeria shown colorectal carcinoma was the most common malignant gastrointestinal neoplasm accounting for 59 percent. Rate of colorectal carcinoma is comparable to other studies in India except that mean age is decreasing to 45years.[14] Screening studies should be considered to determine possible risk factor by introducing digital rectal examination and occult blood test at an early age. www.pacificejournals.com/apalm

Conclusion

Gastrointestinal tract malignancies are one of most common cancers in our country. The incidence has been increasing due to life style changes and many variables as smoking, alcoholism, tobacco, altered diet habits etc. The diagnosis of these cancers are usually late when the disease has already invaded the lamina propria and musculature, because in early stages, the patient usually complains of trivial and nonspecific symptoms. Hence a new look to epidemiological studies in gastric cancer is necessary. And also it is mandatory to focus on lifestyle modification such as reduced salt intake and increased vegetable and fruits consumption, together with avoidance of smoking and alcoholism.

Reference 1.

Yasemi M, Ahmadi MRH, Peyman H, Yasemi MR, Khajavikhan J, Hemati K. A 7 years retrospective study of gastrointestinal cancers incidence in the western Iran. Journal of clinical and diagnostic research. 2015;9(7):EC01EC05.

Jamal S, Mamoon N, Mushtaq S, Luqman M. Analysis of Gastrointestinal malignancies at Armed forces institute of Pathology, Ravalpindi, Pakistan. Asia Pacific Journal of Cancer prevention, 2005;6: 497-500.

Patel MM, Gamit B, Patel PR. Analysis of Gastrointestinal Malignancy: A five years study. National Journal of Community Medicine. 2012;3;555-557.

Pauna C, Lazar E. Colorectal Carcinoma - Epidemiological and Histopathological aspect. Cercetari Experimentale and Medico-Chirurgicale. 2006; anuIXIII:53-56.

Khurshed A, Ahmed R, Yasmin Bhurgri.Primary Gastrointestinal malignancies in childhood and Adolescence - an Asian Perspective. Asian Pacific Journal of Cancer prevention. 2007;8:613-617.

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Abdulkareem FB, Abudu EK, Awolola NA, et al. Carcinoma in Lagos and sagamu, Southwest Nigeria: A histopathological review. World Journal of Gastroenterology 2008;14(42):6531-6535.

Chandra N, Khan AR, Romana M, Lateef S. Histopathology of Gastric cancer in Kashmir - A five year retrospective Analysis. JK SCIENCE. 2007;9(1):21-23.

Jijo V.Cherian, Ramalingam Sivaraman, Arun K. Muthusamy, Venkataraman Jayanthi. Carcinoma of the Esophagus in Tamil Nadu(South India):16 years trend from Tertiary Center. J Gastrointestin Liver Dis September 2007;16(3):245-249. Kollarova H, Machova L, Horakova D, Janoutova G, Janout V.Epidemiology of Esophageal Cancer - An overview Article. Biomed Pap Med Fac Univ Palacky Olomouc Czech Rpub.200;151(1):17-28

10. Mieittinen M, Sobin LH, Lasota J. Gastrointestinal stromal tumors of stomach:A clinicopathologic,

immunohistochemical and molecular genetic study of 1765 cases with long term follow up. Am J Surg Pathol (2005) 29:52-68 11. Berman J, O`Leary TJ. Gastrointestinal stromal tumor workshop. Hum Pathol 2001;32:578-582. 12. Hatzaras I, Palesty JA, Abir F, et al. Small Bowel Tumors Epidemiologic and Clinical Characteristics of 1060 Cases from the Connecticut Tumor Registry.(Reprinted)ARCH SURG. 2007;14:229-235. 13. Parkin DM, Pisani P, Ferlay J .Global Cancer Statistics. Cancer Journal for Clinicians. 1999; 49(1):33-63. 14. Laishram RS, Kaiho N, Shimray R, et al. Histopathological Evaluation of Colorectal Carcinoma Status in Manipur, India. International Journal of Pathology; 2010;8(1):5-8. 15. Pishbijari HF, Rad MA, Ghofrani H, Shafaghi A, Toosi MN, Dolatshahi S. A retrospective study of Gastrointestinal Cancers in Theran, Medical Journal of the Islamic Republic of Iran, 2006, 20;.107-110\

*Corresponding author: Dr Neha Amrut Mahajan, 41, Atharva, besides water tank. Vedant Nagar. Railway station road. Aurangabad(431005).Maharashtra. INDIA Phone: +91 9405107982, Email: nehaamahajan@yahoo.com Date of Submission : 02.01.2017 Date of Acceptance : 14.05.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Original Article DOI: 10.21276/APALM.1302

Morphometric Changes in Jejunal Mucosa in HIV Positive Patients Presenting with Enteropathy: An Indian study Dibyajyoti Boruah1*, Jasvinder K Bhatia1, K D Kamal2 and Ajay Malik3 Department of Pathology, Armed Forces Medical College, Pune-Maharashtra, INDIA. Department of Internal Medicine, Armed Forces Medical College, Pune, Maharashtra, INDIA. 3 Department of Pathology, Army College of Medical Sciences, New Delhi, INDIA. 1

ABSTRACT Aims: Villous length, villous area, villous separation, villous tortuosity and crypt depth were evaluated in thejejunal mucosa of HIV positive cases with chronic diarrhea and correlated with controls and CD4 counts. Methods: Endoscopic biopsies of jejunal mucosa from 30 confirmed HIV positive cases with enteropathy and 21 HIV negative controls were used.Villous parameters were assessed by computerized digital image morphometry on hematoxylin-eosin-stained histological sections. Results: There were no significant differences in the mean villous length(p=0.9858, case:269µm, control:270µm) and villous area (p=0.0610, cases:30905µm2, controls:25864µm2)between cases and controls. The mean villous separation (cases:188µm, controls:139µm) and crypt depth (cases:237µm, control:193µm) were significantly larger and mean villous tortuosity was significantly lower (cases:3.04, controls:3.92) in cases. In HIV cases, CD4 counts showed negative correlation with crypt depth (r=-0.524), villous area (r=-0.228), villous separation (r=0.276) and positive correlation with villous tortuosity (r=0.297) in contrast to villous length (r=-0.012). Conclusion: Villous tortuosity was significantly reduced in the HIV cases; unlike villous length and villous area. Due to larger villous separation in HIV cases, villous surface area is likely to be lowered and hampered nutrient absorption. Crypt depth increased significantly in HIV positive cases and exhibited the best correlation with CD4 counts. Keywords: HIV, Enteropathy, Villous Morphometry, Crypt Depth, CD4 counts

Introduction

Chronic diarrhea, malabsorption and weight loss are frequently observed in the human immunodeficiency virus (HIV) positive/AIDS cases[1-3]. Several pathogenic mechanisms have been identified or hypothesized as causing diarrhea in HIV-infected individuals, including decreased mucosal surface area, altered secretion of inflammatory mediators, destruction of intestinal mucosa and opportunistic infections due to the lack of an immune reaction[4,5].A reduction in villous surface area and increase in crypt length in HIV infected patients had been reported by few studies using image morphometry[6, 7]. Evaluation of villous features is vital to assess the severity of modification of villous structure in enteropathy cases and to evaluate the effectiveness of a therapy[8-16]. Both histological changes and clinical symptoms in HIV infected patients can be resolved by highly active antiretroviral therapy (HAART)[3]. A recent study confirmed on the assessment of histological sections, that the crypt structure in HIV cases can be restored by HAART[8]. So morphometric evaluation of the intestinal mucosa will help in quantifying the histological changes in HIV infected patients with gastrointestinal (GI) symptoms.

In this study we attempted an objective evaluation of the characteristics of intestinal villi in HIV positive cases presenting with enteropathy using morphometric techniques on hematoxylin & eosin (H&E) stained sections. We studied morphometric parameters i.e., villous length, villous area, villous separation, villous tortuosity and crypt depth in jejunal mucosa of HIV patients with enteropathy and compared with the HIV negative controls. Correlations of evaluated morphometric parameters with CD4 counts in peripheral blood taken at the time of biopsy for all cases were also studied. We were unable to find any previous reports of such an objective evaluation in Indian scenario. Though number of HIV patients presenting GI symptoms has decreased in develop countries, in our country patients continue to present with diarrhea. Therefore, study of changes in jejunal mucosa by morphometry as an adjunct to histology helps in evaluation and better planning of therapy.

Methods

Thirty confirmed HIV positive cases who presented with chronic diarrhea, malabsorption and weight loss being managed at this tertiary care institute during 2013 to 2014

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Morphometry of Jejunal Mucosa in HIV

were included in this study. Endoscopic biopsies ofjejunal mucosa were received for histopathological examination of these cases. Patients, in whom upper gastrointestinal endoscopy was contraindicated and age less than 18 years were excluded from this study. The status of the opportunistic infections in the cases was not investigated in this study. Permission from Institutional Ethical Committee was obtained for this study (Oct 2012). Twenty one numbers of endoscopic biopsies of intestinal mucosa done for other GI symptoms with unknown HIV status received at this centre and had showed normal histology were used as controls. Histopathological Examination and Villous Morphometry: H&E stained histological sections of 5 micron thickness of formalin fixed paraffin embedded tissue of jejunal mucosa were analyzed. Morphometric analysis was performed by using a computerized digital photomicrograph system (Dewinter Optical Inc. with Digi Eye 330 digital photomicrography camera and Biowizard 4.2 Image analysis software). The measuring scale of the image analysis software was properly calibrated. For each sample at least five fields at 100X magnification, having optimal villous details, were recorded for the study[Figure.1A-C]. For each case, at least 15 villi and crypts were assessed. We evaluated villous length and villous area for each villus, crypt length for each well oriented crypt, villous tortuosity in each captured field and villous separation between the each pair of adjacent villi for every sample. The definitions of these parameters are depicted schematically in Figure2. The length of the line traced through the middle of the villus from the crest to the midpoint of its base line was considered as villous length. The area within the villous outer boundary and base line was considered as villous area. Linear distance between the midpoints of the base lines of two adjacent villi was considered as villous separation. Ratio of the total length of outer boundary of well defined villi to the total length of base lines of the villi in a field was considered as villous tortuosity; and an average of all five fields was calculated. All these measurements were carried out using the image analysis software. Statistical Analysis: SPSS17.0 (Statistical Package for Social Sciences) software programme was used in statistical analysis. Mean values with standard deviation (SD) for each sample of all studied parameters were calculated. Mean values of each parameter was considered as the parameter of the sample. The mean values with SD and range of these parameters were evaluated for both cases and controls. Unpaired Student’s t-test was performed

and ‘p’ values were calculated to evaluate significance in difference between the cases and the controls. The Pearson correlation coefficients ‘r’ for all pairs of studied parameters for the cases and controls were calculated.

Results

The mean age at the time of biopsy for the cases was 39 years (range:26 - 53 years) and comprised of 19 males and 11 females; the mean age for the controls was 42 years (range:18 - 68years) and comprised of 11 males and 10 females. At the time of biopsy, average duration of HIV infection for the cases was 4.5 years (range: 0 - 15 years) and average duration of antiretroviral therapy (ART) was 2.1 years(range: 0-9 years).H&E stained histological sections with villous atrophy and normal villous found in HIV positive cases are shown in Fig1A& B respectively; Fig1Cshows mucosa inHIV negative controls. On examination of H& E sections, villous atrophy of varying degrees was found in most of theHIV positive cases. Few cases showed almost normal villi with mild inflammatory infiltrate; while some showed mild abnormalities. The enterocytes were normal; mild increase in lymphocytic inflammatory infiltrate in the lamina propria was seen in some of the cases. No granulomas, dysplasia or evidence of malignancy were found. Evaluated morphometric parameters of the cases and controls are shown inFigure3(A-E). These figures represent the box plots of villous length, villous area, villous separation, villous tortuosity and crypt depth for the both groups. The mean values of the studied parameters with standard deviation (SD) and range for cases and controls are presented in the table 1. The p values to predict the difference of each pair of these two groups for all the parameters are also presented in this table. There was no significant difference in the villous length between HIV cases and controls (p=0.9858). Though, the mean villous area in cases (30905 µm2) was higher than the controls (25864 µm2), the difference was not statistically significant (p=0.0610). The mean villous separation in cases (188 µm) was significantly higher than the controls (139 µm).The mean villous tortuosity in cases (3.04) was significantly lower than the controls (3.92). The mean crypt depth in cases (237µm) was significantly greater than the controls (193µm). Correlation of Studied Parameters HIV cases: Pearson correlation coefficients between each pair of studied parameters analyzed for 30 HIV positive cases are shown in the Table2. Crypt depth showed significant positive correlation with villous length(r=0.504) and villous area(r=0.588). Villous length showed strong positive correlation with villous area(r=0.710) and villous

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A-381 Correlation of CD4 Counts with Morphometric Parameters: The mean value of CD4 counts of the cases was 333 mm-3 (range: 24-1048 mm-3). Scatter plots of villous length, villous area, villous separation, villous tortuosity and crypt depth versus CD4 counts for the all HIV positive cases with linear regressions are presented in Figure4(A-E). CD4 counts showed weak negative correlation with villous area (r=-0.228), mild negative correlation with villous separation (r=-0.275), moderate negative correlation with crypt depth (r=-0.524) and mild positive correlation with villous tortuosity (r=0.295). Whereas, villous length (r = -0.012) showed no meaningful correlation with CD4 counts.

tortuosity showed significant negative correlation with villous separation (r = -0.478). Controls: Pearson correlation coefficients between each pair of studied parameters analyzed for 21 HIV negative controls are shown in the Table3. Crypt depth showed mild negative correlation with villous length(r=-0.386); did not show meaningful correlation with other parameters. Villous length showed significant positive correlations with villous area(r=0.682) and villous tortuosity (r=0.455). Villous separation showed significant positive correlation with villous area(r=0.526) and negative correlation with villous tortuosity (r=-0.482).

Table: 1 Mean values with SD and range of the studied parameters of 30 cases and 21 controls with p values. Sl No.

Parameters (unit)

Cases (n=30) Male=19, Female=11

Controls (n=21) Male=11, Female=10

p value

Villous length (µm)

269 ± 53 (180 - 379)

270 ± 62 (176 - 408)

0.9858

Villous area (µm 2)

30905 ± 10192 (16259-57866)

25864 ± 7653 (9680 – 41396)

0.0610

Villous separation (µm)

188 ± 26 (148 – 251)

139 ± 23 (88 – 179)

<0.0001*

Villous tortuosity

3.04±0.41 (2.26 – 3.90)

3.92 ± 0.81 (2.53 – 5.40)

<0.0001*

Crypt depth(µm)

237±43 (139-270)

193±38 (139-270)

0.0004*

CD4 counts (mm-3)

333 ± 270 (24-1048)

Not available

Results are expressed as: Mean value ± SD(Minimum – Maximum), *- difference is significant Table2: Correlations between each pair of studied parameters: 30 HIV positive cases. Crypt depth Villous length Crypt depth

Villous area Villous separation Villous tortuosity

CD4 Counts

1.000

Villous length

0.504**

1.000

Villous area

0.588**

0.710**

1.000

0.275

0.194

0.327

1.000

-0.279

-0.010

0.105

-0.478**

1.000

- 0.524**

-0.012

-0.228

-0.276

0.297

Villous separation Villous tortuosity CD4 Counts

1.000

2-tailed Pearson ‘r’. *p<0.05, **p<0.01. Table3:Correlations between each pair of measured parameters: 21 HIV negative controls. Crypt depth

Villous length

Villous area

Villous separation

Crypt depth

1.000

Villous length

-0.386

1.000

Villous area

-0.048

0.682**

1.000

Villous separation

0.161

0.132

0.526*

1.000

Villous tortuosity

-0.017

0.455

0.184

-0.482*

Villous tortuosity

1.000

2-tailed Pearson ‘r’. *p<0.05, **p<0.01.

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Fig. 1(A-C): Images of H&E stained histological sections used for morphometric evaluation of parameters:(A)villous atrophy and (B) almost normal villusin HIV infected cases; and (C) HIV negative control. Original magnification is 100X and the scale put in the images.

Fig. 2: Measurement scheme of studied parameters: villous length, villous area, villous separation, villous tortuosity and crypt depth.

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Fig. 3(A-E): Box plots of cases and controls with p value of t-test between them for: (A) villous length, (B) villous area, (C) villous separation, (D) villous tortuosity and (E) crypt depth.

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Fig. 4(A-E): Scatter plots of CD4 count with (A) villous length, (B) villous area, (C) villous separation, (D) villous tortuosity and (E) crypt depth of the 30 HIV positive cases. Linear regression between these parameters and CD4 count are shown by the solid linesand Pearson correlation coefficient â&#x20AC;&#x2DC;râ&#x20AC;&#x2122; were shown in respective plot.

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Discussion

Histological changes of the gastrointestinal mucosa and opportunistic infections due to the decrease of immunity are commonly observed in HIV positive cases [1-6]. Increased levels of inflammation and decreased levels of mucosal repair and regeneration have been seen in enteropathy with HIV infection[17]. Reduction of villous surface area and increased crypt depth with less mitotic figures was observed in HIV patients with gastrointestinal complaints compared to the controls by Ullrich et al[5]. Batman et al reported that infection of cells in the lamina propria of the jejunum stimulates an increase in crypt length and a fall in villous surface area in HIV patients[6]. Villous atrophy and flatness indicates reduction of villous surface area in the intestine, through which nutrients are absorbed. This reduction of villous surface area decreases transportation rate of nutrients through the villi which may leads to malabsorption and weight loss. A quantitative assessment of the villi and crypts and their comparison with suitable controls gives a greater perception about such disease conditions. Correlations of these parameters with CD4 counts provide important information about disease progression. Many researchers have studied the histological changes in villous structure in cases of HIV with enteropathy; however few quantitative studies using image morphometry on villous alterations have been reported[5-14]. Morphometry is a useful tool to assess the effectiveness of a treatment by quantifying histological abnormalities in enteropathy [8, 16]. Significant increase in crypt depth was found inthe HIV positive cases compared to the controls in our study; like previous research [5]. No significant difference of mean villous length was found between HIV infected cases and controls in our study. We also observed that, crypt depth showed positive correlation with villous length in HIV positive cases; but negative correlation in controls. The villous area is related to its volume in three dimensional scenarios; though its mean value was larger in cases than the controls, but difference was not significant. Kelly et al also reported that mean villous area, which was called villous compartmental volume by them, were 16659 µm2 in HIV seronegative controls, 16733 µm2 and 18195 µm2 in HIV seropositive cases with CD4 count ≥200mm-3 and <200mm-3respectively, and the differences were not significant [13]. Though our findings in this respect were similar to their study, the size of the villous found by us was larger for studied samples. The disparity might arise due to www.pacificejournals.com/apalm

A-385 the geographical variation of populations or difference in choosing the region/criteria of villi for size evaluation or measurement techniques. The inverse of villous separation gives a measure of villous concentration. In HIV cases the mean of villous separation was found significantly larger than the controls. This translates that the villous concentration decreases in such cases. Decrease in repair and regenerating ability of villous in such condition leads to the lower villous concentration. In our study we defined a parameter named villous tortuosity, which was the ratio of total length of outer boundary of well defined villi to the total length of base lines of the villi in a field. Villous tortuosity is related to the villous surface area per unit area of mascularis mucosa in three dimensional scenario. Higher villous tortuosity is likely to have more villous surface area in intestine. In this study we found that mean villous tortuosity was significantly reduced in the HIV cases than the controls. Hence, total villous surface area is to be expected to decrease in HIV cases, which contributes towards the reduction of rate of diffusion of nutrients through the intestine and leads to malabsorption. Kelly et al also reported that mean value of epithelial surface area was larger in HIV seronegative controls than the HIV positive cases[13]. Peripheral blood CD4 counts have been used for monitoring HIV patients and deciding antimicrobial prophylaxis and antiretroviral therapy[17-18]. HIV infection leads to loss of CD4 T-cells and the risk of complications, especially potentially lethal opportunistic infections increase with the decline of this count [18-19].In our study, we found that the meanCD4 count was 333 mm-3 (median 311 mm-3)in the HIV cases and correlated CD4 count with the evaluated villous morphometric parameters. CD4 counts in controls of our study were not recorded. We found that villous length did not show any meaning full correlation with the CD4 counts. Villous area showed a weak negative correlation with the CD4 counts; i.e. possibility of finding a larger villous is slightly more when CD4 counts decrease. This result supported the finding of larger mean villous area in cases than in the controls. Villous separation showed mild negative correlation with the CD4 counts. It suggests that the villi are far from each other and have lower villous density when CD4 count decreases. Similarly, villous tortuosity demonstrated mild positive correlation with the CD4 counts. It hints that the villous surface area, through which nutrients are transported, increases with the enhancement of CD4 eISSN: 2349-6983; pISSN: 2394-6466

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counts. Crypt depth, which is related to crypt hyperplasia, showed moderate negative correlation with the CD4 counts. It suggests that the crypt hyperplasia may be resolved with improvingCD4 counts. A recent study has showed that using HAART treatment in HIV patient was able to restore the crypt structure[8]. In this study we observed that, the differences of villous structure and crypt depth in HIV cases in comparison to controls increase with decline of CD4 counts. Among the all morphometric parameters, crypt depth showed the best correlation with the CD4 counts. Small sample size and unknown HIV status of the controls were the limitations of this study.

Conclusion

Quantitative assessments of crypts and villi provide a greater understanding about disease progression and patients’ outcome in cases of HIV with enteropathy. Crypt depth increased significantly in HIV cases and showed negative correlation with CD4 counts. Villous separation was significantly larger and villous tortuosity was significantly smaller in HIV cases than the controls. Villous tortuosity showed weak positive correlation with villous length and moderate negative correlation villous separation. Villous area showed weak and villous separation showed mild negative correlations of CD4 counts of HIV cases. Crypt depth and villous tortuosity are better parameters for the morphometric assessment of enteropathy in HIV positive cases.

Acknowledgements

Authors acknowledge Professor and Head, Department of Internal Medicine and Professor and Head, Department of Pathology of the institute performing this study for their overall support to complete it.

Conflict of Interest

We declare that we have no conflict of interest.

Reference 1.

Sharpstone D, Gazzard B. Gastrointestinal manifestations of HIV infection. Lancet 1996;348:379–83.

Owens SR, Greenson JK. The pathology of malabsorption: current concepts. Histopathology 2007; 50: 64–82.

Bhaijee F, Subramony C,Tang S, Pepper DJ. Human Immunodeficiency Virus Associated GastrointestinalDisease: Common Endoscopic Biopsy Diagnoses. Pathology Research International 2011; Article ID 247923, doi:10.4061/2011/247923.

Kotler DP, Giang TT, Thiim M, Nataro JP, Sordillo EM , Orenstein JM,et al. Chronic bacterial enteropathy in patients with AIDS. J Infect Dis1995;171:552-558.

5. Ullrich R, Zeitz M, Heise W, L’age M, Hofken G, Riecken EO. Small intestinal structure and function in patients infected with Human Immunodeficiency Virus: evidence for HIV- induced enteropathy. Ann Intern Med 1989; 111: 15-21. 6.

Batman PA, Kapembwa MS, Miller ARO, Sedgwick PM, Lucas S, Sewankambo NK,et al.HIV enteropathy: comparative morphometry of the jejunal mucosa of HIV infected patients resident in the United Kingdom and Uganda. Gut 1998;43:350–355.

Cummins AG, LaBrooy JT, Stanley DP, Rowland R, Shearman DJC. Quantitative histological study of enteropathy associated with HIV infection. Gut1990;31:317–321.

Batman PA, Kapembwa MS, Belmonte L, Tudor G, Kotler DP, Potten CS et al. HIV enteropathy: HAART reduces HIVinduced stem cell hyperproliferation and crypt hypertrophy to normal in jejunal mucosa. J Clin Pathol 2014;67:14–18.

Batman PA, Miller AR0, Forster SM, Harris JRW, Pinching AJ, Griffin GE. Jejunal enteropathy associated with human Immunodeficiency virus infection: quantitative histology. J Clin Pathol 1989;42:275-281.

10. Keating J, Bjarnason I, Somasundaram S, Macpherson A, Francis N, Price AB et al. Intestinal absorptive capacity, intestinal permeability and jejunal histology in HIV and their relation to diarrhea. Gut 1995; 37: 623-629. 11. Kelly P, Davies SE, Mandanda B, Veitch A, McPhail G, Zulu I, et al. Enteropathy in Zambians with HIV related diarrhea: regression modeling of potential determinants of mucosal damage. Gut1997; 41: 811–816. 12. Pires ALG, da Silveira TR, da Silva VD. Digital morphometric and stereologic analysis of small intestinal mucosa in well-nourished and malnourished children with persistent diarrhea. J Pediatr (Rio J) 2003; 79:329-336. 13. Kelly P, Menzies I, Crane R, Zulu I, Nickols C, Feakins R etal. Responses of small intestinal architecture and function over time to environmental factors in a tropical population. Am J Trop Med Hyg2004; 70: 412–419. 14. Batmana PA, Kotlerb DP, Kapembwac MS, Boothd D, Pottend CS, Orenstein J M et al. HIV enteropathy: crypt stem and transit cell hyperproliferation induces villous atrophy in HIV/Microsporidia-infected jejunal mucosa. AIDS 2007; 21:433–439. 15. Cummins AG, Alexander BG, Chung A, Teo E, Woenig JA, Field JB, et al. Morphometric Evaluation of Duodenal Biopsies inCeliac Disease. Am J Gastroenterol 2011; 106:145–150. 16. Louis-Auguste J, Greenwald S, Simuyandi M, Soko R, Banda R and Kelly Paul. High dose multiple micronutrient supplementation improves villous morphology in

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Boruah et al. environmental enteropathy without HIV enteropathy: results from a double-blind randomised placebo controlled trial in Zambian adults. BMC Gastroenterology 2014;14(15):1-10. 17. Brenchley JM, Douek DC. HIV infection and the gastrointestinal immuneSystem.MucosalImmunology2008; 1:1:23-30. 18. Maini M K, Gilson RJC, Chavda N, Gill S, Fakoya A, Ross EJ et al. Reference ranges and sources of variability of CD4

A-387 counts in HIV-seronegative women and men. Genitourin Med 1996;72:27-31. 19. Anukam KC, OsazuwaEO, Osadolor HB, Bruce AW and Reid GR, Yogurt Containing Probiotic Lactobacillus rhamnosus GR-1and L. reuteri RC-14 Helps Resolve Moderate Diarrheaand Increases CD4 Count in HIV/AIDS Patients. J Clin Gastroenterol2008; 42:239-243.

*Corresponding author: Dr. Dibyajyoti Boruah, Scientist-‘D’ Department of Pathology, Armed Forces Medical College, Pune – 411040, Maharashtra, INDIA Phone: +919823553654, Fax : +91-20-26331776 Email: dibyajyotibh@yahoo.co.uk Date of Submission : 01.02.2017 Date of Acceptance : 17.04.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

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Original Article DOI: 10.21276/APALM.1417

Clinicopathological Analysis of Undifferentiated Malignant Neoplasms of The Sinonasal Tract Ashok kumar S1*, Srigayathri S2 and Geetha Devadas1 Department of pathology, Govt Stanley Medical college, Chennai, TN. India Sri Ramachandra Medical College And Research Institute, Chennai, TN, India 1

ABSTRACT Background: Sinonasal tumours are characterized by low incidence, non specific symptoms, and late presentation. Aggressive, nonâ&#x20AC;&#x201C; squamous cell epithelial and nonepithelial malignant neoplasms of varying histogenesis occurring in sinonasal region are grouped under the term undifferentiated malignant neoplasms. Frequently, these undifferentiated malignancies share clinical and light microscopic features, which make differentiation of one from the other virtually impossible without the use of adjunct immunohistochemical analysis . Methods: Undifferentiated malignant sinonasal tumors biopsied or surgically excised over a period of 5 years were studied. Result: We encountered 22 cases of undifferentiated malignant sinonasal tumors with an incidence of 0.05%, with a mean age of 41 to 50 years and the male to female ratio was 2.1:1. Epithelial tumors (18 cases) constituting 81.82 % predominated over non epithelial tumors (4 cases) constituting 18.18% with a ratio of 4.5:1. 16 tumors involved the nasal cavity (72.7%) and 6 involved paranasal sinuses (27.3%). The most common clinical presentation was mass in the nose 19 cases (86.36%). The most common Undifferentiated malignant tumor encountered was Sinonasal undifferentiated carcinoma-10 cases (45.45%). Conclusion: A variety of undifferentiated malignant neoplasms occur in the sinonasal tract with overlapping clinical and pathologic findings. In limited biopsy material, differentiation of these tumor types can be challenging and differentiating these tumors with the help of immunohistoochemistry has clinical importance because advances in therapeutic intervention may increase survival with good quality of life, and in some instances may achieve a cure. Keywords: Undifferentiated, Malignant, Sinonasal, Neoplasms, Immunohistochemistry

Introduction

The nasal cavity and paranasal sinuses including the maxillary, ethmoid, sphenoid and frontal sinuses are collectively referred to as the sinonasal tract. Although the nasal cavity and paranasal sinuses occupy a relatively small anatomic space, they are the site of origin of some of the more complex histologically diverse group of tumors of the entire human body. [1, 2] These include neoplasms derived from mucosal epithelium, seromucinous glands, soft tissues, bone, cartilage, neural/ neuroectodermal tissues, hematolymphoid cells and the odontogenic apparatus. Many of the tumours are similar to those found elsewhere in the body but a few such as olfactory neuroblastoma are unique to this site. [3]

Materials and Methods

The surgical specimens received in the Institute of Pathology, Madras Medical College, Chennai from the Upgraded Institute of Otorhinolaryngology, Government General Hospital, Chennai for the period of five years formed the material for this study. Small biopsy specimens

and excision biopsy specimens and resection specimens were included. Inadequate or unrepresentative biopsy material was excluded from the study. Informed written consent from the patient was obtained. Ethical committee clearance was obtained. The clinical features such as age and sex of the patient, site of lesion and type of surgery done were noted. The tissues were routinely processed and paraffin blocks were made and histological sections of 5 to 6 micrometer were taken in Leica microtome and routinely stained with hematoxylin and eosin stains. The microscopic analyses were done from all the available slides. These included the histological pattern, cellular features, pleomorphism, mitosis, necrosis, vascularity and secondary changes. Provisional histological differential diagnosis was made. Special stains periodic acid (PAS), reticulin and Masson Fontana were done wherever necessary. For undifferentiated malignant neoplasms , we employed the panel of immunohistochemical markers that include: Cytokeratin, Synaptophysin, Neuronspecific

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Enolase,CD 99,vimentin,CD 45,Desmin,S100,HMB 45. After interpretation final diagnosis was made. Microphotographs were taken.

vimentin, desmin, S100, CD99 and CD45. 10 cases showed diffuse, moderate staining for cytokeratin and no reactivity for other markers except for 2 cases which in addition to cytokeratin showed focal reactivity for NSE. All those 10 cases were finally diagnosed as Sinonasal undifferentiated carcinoma (SNUC).

Result

We encountered 22 cases of Undifferentiated malignant tumors with an incidence of 0.05%. Undifferentiated malignant tumors occurred in patients with a mean age of 41 to 50 years and the male to female ratio was 2.1:1. Among Undifferentiated malignant tumors, epithelial tumors predominated over non epithelial tumors.

5 cases showed diffuse moderate staining for cytokeratin, NSE, synaptophysin, and were finally categorized as Sinonasal neuroendocrine carcinoma (SNEC). SNUC was the predominant variant (66.67%) followed by SNEC (33.33%) as shown in table-1.We had 5 cases of small round cell tumor (SRCT) for which immunohistochemistry panel was employed.

The age range varied from second to ninth decade of life but the peak age of presentation was 5th decade followed by 7th decade as shown in table-1.Males showed a higher incidence than females with a male to female ratio of 2.1:1 as shown in table-1.

Among them we observed that 2 cases showed reactivity for NSE, synaptophysin, S100 and was finally diagnosed as esthesioneuroblastoma(ENB) constituting 40%.Among the other 3 cases ,2 cases were diagnosed as PNET which showed reactivity for CD 99 and vimentin. 1 case was diagnosed as SNEC and showed reactivity for cytokeratin, NSE and synaptophysin as shown in table-2.

Out of the 22 cases 16 tumors involved the nasal cavity (72.7%) and 6 involved paranasal sinuses (27.3%) as shown in table-1. Amongst paranasal sinuses, maxillary sinus was the commonest site in our study as shown in table-1.The most common tumor encountered was Sinonasal undifferentiated carcinoma(SNUC) 10 cases(45.45%) as shown in table-1. The most common clinical presentation in our study was mass in the nose which constituted 19 cases (86.36%) as shown in table-1.

We had 2 cases of malignant melanoma with a incidence of 1.87% .Both cases presented in the nasal cavities of males aged 40 and 54 years respectively. Histopathology showed features similar to other undifferentiated neoplasms of sinonasal tract showing malignant epithelioid and spindle cells with pleomorphic nuclei and eosinophilic nucleoli. Immunostaining revealed tumor cells which were positive for markers HMB45, S100 confirming the provisional diagnosis of melanoma.

In our study we had 15 cases of undifferentiated carcinoma for which we employed a panel of immunohistochemical markers that included cytokeratin, NSE, synaptophysin,

TABLE-1: Distribution of Undifferentiated Malignant Tumors of Nasal Cavity, Paranasal Sinuses According to The Incidence, Sex Ratio, Age and Site of Presentation Diagnosis

No.of.cases

M:F

Peak age(decade)

Nasal cavity

PNS

Undifferentiated malignant tumors 22 Melanoma

1.87

M only

4&6

3.44

Olfactory Neuroblastoma

1.87

1:1

2&5

1.72

2.04

SNUC

9.35

4:1

12.06

6.12

SNEC

5.61

1.5:1

3&5

8.62

2.04

PNET

1.87

M only

1.72

2.04

TABLE 2: Small Round Cell Tumors (SRCT) SRCT

Esthesioneuroblastoma

PNET

SNEC

Total

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Fig. 1: Neoplastic cells showing positivity for HMB 45 in Malignant melonoma (IHC400x)

Fig. 2: Neoplastic cells showing membranous positivity for CD99 in case of PNET (IHC400X).

Olfactory Neuroblastoma

Fig. 3: Neoplasm composed of small round cells with Homer-Wright rosettes in Olf. neuroblastoma (H&E400X).

Fig. 4: CT scan showing tumor in the left nasal cavity and ethmoid sinus with Intracranial extension.

Fig. 5: Neoplasm arranged in diffuse dense cellular sheets in PNET (H&E100x).

Fig. 6: Cellular neoplasm in solid sheets with focal rosette formation lacking neurofibrillary background (H&E 100X) SNEC

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Discussion

The present clinicopathological study of sinonasal tumors includes 22 undifferentiated tumors of nasal cavity and paranasal sinuses for five years. During the period of five years 44730 specimens were received at the general surgical pathology laboratory of our institute. Out of which 22 were undifferentiated sinonasal tumors with incidence of undifferentiated sinonasal tumors representing 0.05%. The rare nature of the tumors is almost a universal finding. [4, 5, 6, 7] Epithelial tumors (18cases) predominated over nonepithelial tumors (4 cases) constituted 81.82%.The ratio of epithelial to nonepithelial tumors was 4.5:1. Male to female ratio was 2.1:1 similar to other studies (Lilly-Tariah da, 1999; Mundy et al., 1985 ;). Age range was 10 to 70 years with an average of 41 to 50 years. The maximum number of cases were present in nasal cavity (72.7%) followed by paranasal sinuses (27.3%).Among the paranasal sinuses maxillary sinus(22.72%) was the commonest site of presentation. Mass in the nose (86.36%) was the most common clinical presentation in our study. Both sides (Right&Left) involved with equal frequency (50%). We encountered 10 cases of sinonasal undifferentiated carcinomas which showed an incidence of 9.35 %.Studies by other authors revealed the incidence of sinonasal undifferentiated carcinoma had a varied range of incidence from 1.7 to 17%.We noted a peak incidence in the 5 th decade in our study. Males dominated over females in our analysis. In a study done in Taiwan by Jeng YM and Sung MT, 36 cases of sinonasal undifferentiated carcinoma was reviewed .They found that median age of presentation was 53 years with a male female ratio of 2:1. The most common locations were nasal cavity and ethmoidal sinus. The commonest site in our study was nasal cavity. Histopathology showed either small or large tumor cells and was predominantly arranged in nests, ribbons, thick trabeculae or sheet like pattern and had coarse chromatin, prominent nucleoli and necrosis. [8, 9, 10] We had 2 cases of malignant melanoma with a incidence of 1.87.Both cases presented in the nasal cavities of males aged 40 and 54 years respectively similar to Zafer et al(2008). Histopathology showed features similar to other undifferentiated neoplasms of sinonasal tract showing malignant epithelioid and spindle cells with pleomorphic nuclei and eosinophilic nucleoli with evidence of melanin deposition.Masson Fontana stain showed positivity in the cytoplasm of tumor cells. Immunostaining revealed tumor cells which were positive for markers HMB45, S100 confirming the diagnosis of melanoma (Fig.1). [11, 12, 13]

A-391 study. For confirmation and further subcategorization a panel of immunohistochemical markers that include cytokeratin,NSE,synaptophysin, CD99,vimentin,CD45, desmin,S100 and HMB 45 were employed.Among the 5cases of SRCT, 2 cases showed positivity for NSE, synaptophysin, and S100 confirming the diagnosis of olfactory neuroblastoma.2 cases showed positivity for CD99 and vimentin and were negative for other markers thus confiming with PNET (Fig.2). [14]One case showed positivity for cytokeratin, NSE, synaptophysin and a final diagnosis of SNEC was made. The incidence of olfactory neuroblastoma in our study was 1.87%.Of our 2 cases of olfactory neuroblastoma,one presented in a 20 year old female involving the ethmoid sinus and the other in 48 year old male involving the nasal cavity. We found a bimodal peak in age similar to other studies. [15, 16] The tumor was disposed in lobules and nests and composed of small round cells with high N/C ratio, small uniform hyperchromatic nuclei with salt and pepper type of chromatin, Homer-Wright rosettes in a fibrillary background with a Hyams grade I (Fig.3). [15, 16, 17, 18] One of our cases had intracranial extension and cervical lymph node metastasis with a Kadish stage C(Fig.4). FNAC of cervical node revealed small round cells with occasional rosette formation confirming metastasis to cervical node. Our 2 cases of PNET presented in 15 and 17 year old males respectively with an incidence of 1.87%.One presented in the nasal cavity and the other in the maxilla. Histopathology showed diffuse densely cellular sheets of uniform small to medium sized round cells with scant cytoplasm and round nuclei with delicate chromatin (Fig.5). One small round cell tumor diagnosed as SNEC presented in the nasal cavity of a 48 year old female. Of the Undifferentiated neoplasms we noted that10 cases showed diffuse positivity for cytokeratin alone with focal positivity for NSE only in 2 cases with a final diagnosis of SNUC. 5 cases showed diffuse positivity for both cytokeratin and neuroendocrine markers namely NSE, synaptophysin confirming the diagnosis of SNEC.The incidence of SNEC in our study was 5.61%.There was a slight preponderance in males. Nasal cavity was the commonest site and peak age was found in 5th decade .[19] Histopathology showed sheets of large round cells having moderate amount of cytoplasm, fine chromatin with inconspicuous nucleoli and focal rosette formation without fibrillary background (Fig.6). [19, 20, 21]

Conclusion

We had 5cases of small round cell tumors (SRCT) and 15 cases of undifferentiated carcinomas in our

Tumours of the nasal cavity and paranasal sinuses are rare pathologies with extremely varied etiopathology, clinical behaviour, treatment and prognosis. The symptoms of the

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neoplastic processes are essentially similar to inflammatory pathology of the sinonasal tract with resultant delay of diagnosis. The clinical and radiological features of masses of nasal cavity and paranasal sinuses are overlapping and often only a provisional diagnosis is possible. Definite diagnosis requires histopathological examination as most of the lesions are inaccessible for fine needle aspiration or FNAC is not recommended because of fear of haemorrhage. The key in the diagnosis and treatment of sinonasal tumours remains a high index of suspicion and early diagnosis as late presentation and delay in early diagnosis are major constraints to favourable outcome of treatment. This study highlights the characteristics of the rare sinonasal undifferentiated malignancies encountered and emphasizes the importance of Immunohistochemistry(IHC) in distinguishing esthesioneuroblastoma (ENB) from non ENB which has a bearing on prognosis and therapeutic intervention.

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De Paula Araujo R, Gomes EF, de Menezes DB. Rare sinonasal tumors: Case series and literature review. Braz J Otorhinolaryngol. 2008; 74:307-14.

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Casiraghi O, Lefèvre M. Undifferentiated malignant round cell tumors of the sinonasal tract and nasopharynx. Ann Pathol. 2009 Sep; 29:296-312.

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10. Ejaz A, Wenig BM. Review Sinonasal undifferentiated carcinoma: clinical and pathologic features and a discussion on classification, cellular differentiation, and differential diagnosis. Adv Anat Pathol. 2005; 12:134-43. 11. López F, Rodrigo JP, Cardesa A. Update on primary head and neck mucosal melanoma. Head & neck. 2016; 38:147-155. 12. Clifton N, Harrison L, Bradley P J, Jones N S. Malignant melanoma of nasal cavity and paranasal sinuses: report of 24 patients and literature review. Journal of Laryngology and Otology.2011; 125:479–485. 13. Thompson LDR, Wieneke JA, Miettinen M. Sinonasal Tract and Nasopharyngeal Melanomas.A Clinicopathologic Study of 115 Cases With a ProposedStaging System. The American Journal of Surgical Pathology. 2003;27: 594–611. 14. Kawabata M, Yoshifuku K, Sagara Y, Kurono Y.Ewing’s sarcoma/primitive neuroectodermal tumour occurring in the maxillary sinus. Rhinology. 2008; 46:75-8. 15. Shah K, Perez-Ordóñez B.Neuroendocrine Neoplasms of the Sinonasal Tract: Neuroendocrine Carcinomas and Olfactory Neuroblastoma. Head Neck Pathol. 2016; 10:85-94. 16. Bragg, Taryn McFadden, Scianna, Joseph , Kassam, Amin, Emami, Bahman. Clinicopathological review: esthesioneuroblastoma. Neurosurgery. 2009; 64:764-770. 17. Theilgaard SA, Buchwald C, Ingeholm P, Larsen SK, Eriksen J: Esthesioneuroblastoma: a Danish demographic study of 40 patients registered between 1978 and 2000. Acta Otolaryngol. 2003; 123:433-9. 18. Dulguerov P,AllalAS, Calcaterra TC. Esthesioneuroblastoma: a metaanalysis and review. Lancet Oncol 2001; 2:683-90. 19. Menon S, Pai P, Sengar M, Aggarwal JP, Kane SV.Sinonasal malignancies with neuroendocrine differentiation: case series and review of literature. Indian J Pathol Microbiol. 2010; 53:28-34. 20. Rischin D, Coleman A. Sinonasal Malignancies of Neuroendocrine Origin. Hematol Oncol Clin N Am. 2008; 22:1297-316. 21. Babin E, Rouleau V, Vedrine PO, Toussaint B, de Raucourt D.Small cell neuroendocrine carcinoma of the nasal cavity and paranasal sinuses. J Laryngol Otol. 2006; 120:289-97.

*Corresponding author: Ashok kumar S, Assistant professor, Department of pathology,Govt Stanley Medical college, Chennai, TN- 600001,India, Phone: +91 044 2528 1351 Email: ashoknithelan@gmail.com Date of Submission : 16.03.2017 Date of Acceptance : 29.04.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Original Article DOI: 10.21276/APALM.1421

An Analysis of Quality Control in Pap Cytology in A Tertiary Care Centre By Using ASC to SIL Ratio Hemalatha. J*, Deepak Kumar B, Srinivasa Murthy V and Vani BR Dept of Pathology, ESIC MC & PGIMSR, Rajajinagar, Bangalore, Karnataka India

ABSTRACT Introduction: Atypical Squamous Cells(ASC) is a common gynaecologic cytologic abnormality, comprising around 5% of Papanicolaou test results. It reflects a diagnosis of uncertainty and is used as an intra-laboratory & inter-laboratory comparison tool for quality control purposes. For this purpose, ASC/SIL ratio can be used as a quality control measure. Bethesda system suggests that ASC/SIL ratio for an individual or laboratory should be less than 2:1 or 3:1. Objectives: To assess the ratio of ASC/SIL categories and thereby evaluation of quality control in gynaecologic cytology smears. Methods: The present study being a retrospective study was conducted in the Dept. of Pathology ESIC Medical College& Postgraduate Institute of Medical Sciences and Research, (ESIC MC & PGIMSR), Rajajinagar, Bangalore. Archived cases of preceding 5 years from Jan 2012 to Nov 2016 were taken. The clinical details were retrieved from records. The study included conventional pap stained smears of ASCUS, ASC-H, LSIL, HSIL and SCC. A total of 436 cases were studied. ASC/SIL ratio was calculated. ASC component included ASC-US and ASC-H. SIL component included LSIL, HSIL and SCC. Results: Of the total 436 cases, 226 cases were ASCUS and ASC-H and 210 cases were LSIL, HSIL and SCC. ASC/SIL ratio was obtained by dividing the sum of all ASC cases by the sum of all SIL cases. The ratio obtained was 1.1:1 which is below the upper bench mark of 3:1. Conclusion: Monitoring the ASC/SIL ratio of a laboratory is a useful quality control measure. Bethesda system have suggested that the ratio should be less than 3 and others have suggested that lower ratios are desirable. As diagnosis of ASCUS conveys uncertainty, a low ratio decreases the uncertainty produced by laboratory and may reduce the percentage of women with negative biopsy results. Keywords: Atypical Squamous Cells, Squamous Intraepithelial Lesion, Quality Control

Introduction

The incidence and mortality from cervical cancer is decreasing as a result of successful screening by cervical cytology. The carcinoma of cervix is the commonest malignancy reported in developing countries. About 122,844 new cases are detected every year in India and 67477 die, out of 432.2 million female population aged 15years and older.[1]To promote optimal patient care, quality control measures for Pap smear are very essential. Quality control forms an integral part of any laboratory system. Quality control is defined as a system for verifying and maintaining a desired level of quality in an individual test or process.[2]The main objective of quality control is to eliminate the false negative test results. [3]For practical purposes, quality control can be divided into internal and external quality control. Internal quality control helps in improving the performance of single laboratory, where as external quality control helps in evaluation of the performance of different laboratories and thus can help in inter-laboratory comparability.[4]The various internal quality indicators that are used in pap cytology are atypical

squamous cells rate, correlation of ASC cases with results of biopsy, correlation of ASC-US cases with high risk HPV positivity rates and calculation of ASC/SIL ratio.[5,6]. Atypical Squamous Cells (ASC) constitutes around 5% of Papanicolaou test results and is considered as a common gynaecological cytologic abnormality.[7]The Bethesda System for reporting Pap Cervical Cytology defines ASC as cells having cytologic changes suggestive of Squamous Intraepithelial Lesion (SIL) but lack insufficient data either qualitative or quantitative, for a definite diagnosis of SIL.[8] It can be used as an intra-laboratory & interlaboratory comparison tool for quality control purposes as it reflects a diagnosis of uncertainty and to ensure that this interpretation is not overused.[7] As there is high prevalence of ASCUS interpretation, if incorrectly used affects the clinical management of patients and can result in over or under treatment of significant numbers of patients.[9]According to interim guidelines ASCUS diagnosis may be expected in about 5% of patients and greater frequency may constitute misuse of the term.[10]

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The ratio of ASC to SIL interpretations has been adopted as a preferred measure for the frequency of cervical dysplasia in different populations. The ASC/SIL ratio has been used as a surrogate marker for the level of certainty and specificity and can be calculated for the entire laboratory or for individual cytopathologists.[7] Bethesda authors have suggested ASC/SIL ratio of less than 3:1 for an individual or a laboratory which helps them to assess their ratio as well as the laboratory as a whole. [11,12] This study shows how ASC/SIL ratio depends on underlying SIL prevalence and also helps to analyse the laboratory performance in reporting pap smears. Objective of study is to assess the ratio of ASC/SIL categories and thereby evaluation of quality control in gynaecologic cytology smears.

Materials and Methods

The present study being a retrospective study was conducted in the Dept. of Pathology ESIC MC & PGIMSR, Rajajinagar, Bangalore. Archived cases of preceding 5 years from Jan 2012 to Nov 2016 were taken. The clinical details were retrieved from records. The study included conventional pap stained smears of ASC-US, ASC-H, LSIL, HSIL and SCC.A total of 436 cases were studied. ASC/SIL ratio was calculated. ASC component included ASC-US and ASC-H. SIL component included LSIL, HSIL and SCC. The cytologic criteria that was used for classification of the above cases was according to the Bethesda System for Reporting Cervical Cytology 2014.[13] The following is a brief description of the criteria explained with respect to various squamous lesions as described in The Bethesda System for Reporting Cervical Cytology 2014. ASC-US: Nuclei are approximately two and half to three times the area of the nucleus of a normal intermediate squamous cell with slightly increased ratio of nuclear to cytoplasmic area (N/C). Minimal nuclear hyperchromasia and irregularity in chromatin distribution or nuclear shape and atypical parakeratosis are characteristic. ASC-H: Cells usually occur singly or in small fragments of less than 10 cells. Cells are the size of metaplastic cells

with nuclei that are about 1½ to 2½ times larger than normal. Ratio of nuclear to cytoplasmic (N/C) area may approximate that of HSIL. LSIL: Nuclear enlargement more than three times the area of normal intermediate nuclei results in a slightly increased N/C ratio. Variable degrees of nuclear hyperchromasia are accompanied by variations in nuclear size, number and shape. Chromatin is uniformly distributed and coarsely granular. Nucleoli are absent or inconspicuous. Perinuclear cavitation (“koilocytosis”) consisting of a sharply delineated clear perinuclear zone and a peripheral rim of densely stained cytoplasm is a characteristic feature. Alternatively, the cytoplasm may appear dense and orangeophilic (keratinized). HSIL: Degree of nuclear enlargement is more variable than that seen in LSIL. Cytoplasmic area is decreased, leading to a marked increase in N/C ratio. Contour of nuclear membrane is irregular and frequently shows prominent indentations. Nucleoli are absent. Cytoplasm is immature, lacy and delicate. Squamous Cell Carcinoma (SCC): Few cells are seen as singles and less commonly in aggregates with marked variation in cellular size and shape with caudate and spindle cells that contain dense orangeophilic cytoplasm. Nuclei vary markedly in size, nuclear membranes may be irregular in configuration, and numerous dense opaque nuclei are often present. Associated keratotic changes (“hyperkeratosis” or “pleomorphic parakeratosis”) may be seen.

Results

Of the total 436 cases, 226 cases were ASC component which included ASCUS and ASC-H (Table 1)and 210 cases were SIL component which included LSIL, HSIL and SCC (Table 2). ASC/SIL ratio was obtained by dividing the sum of all ASC cases by the sum of all SIL cases. The ratio obtained was 1.1:1 which is below the upper bench mark of 3:1.

Discussion

The objective of quality control is to reduce the rate of false negative test results. There are various quality assurance

Table 1: Number of cases in ASC component PARAMETER

NUMBER OF CASES

ASC-US

100

ASC-H

126

ASC COMPONENT

226

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Table 2: Number of cases in SIL component PARAMETER

NUMBER OF CASES

LSIL

HSIL

SCC

SIL COMPONENT

210

ASC/SIL RATIO: 226/210= 1.1:1

Table 3: Comparison of ASC/SIL ratios in various studies. STUDY

ASC/SIL RATIO

Davey D D et al

[14]

Renshaw A A et al [7]

2.5

Nascimento A F et al [8]

1.9

Davey D D et al

1.3

[15]

Renshaw A A et al [12]

3.2

Present study

1.1

monitors which may be utilized to evaluate the laboratory’s utilization of ASC which are as follows:[5] 1. Correlation of ASC-US cases with high-risk HPV positivity rates; should be in the range of 40–60%, or in essence that ASC-US is a 50–50 proposition between SIL (usually LSIL) and cellular changes unrelated to HPV; 2. Correlation of ASC cases colposcopically directed biopsy;

with

results

3. Review of ASC cases by a second cytopathologist. 4. Calculation of ASC/SIL ratio 5. Atypical squamous cell rate; refers to ASCUS and ASCH categories divided by number of all gynaecologic cytology cases.[6] However, calculation of ASC/SIL ratio is a simplest measure for assessing the quality of reports by pathologists and as well as laboratory. In our study a total of 436 cases were studied retrospectively, out of which 226 cases belonged to ASC component and 210 cases belonged to SIL component. We obtained a ratio of 1.1:1 which is below the upper bench mark of 3:1. Studies done by various authors with reference to ASC/ SIL ratio as a quality control indicator have been tabulated below (Table 3).

control measure as it helps in analysing their performance. The frequency of ASCUS interpretation in a general population should not be greater than 5% to avoid the misuse of the term. As ASC/SIL ratio are less dependent on patient population, they increase with more number of high risk patients in a laboratory.[16] Although ASC/SIL ratio is a measure of a cytopathologist’s uncertainty, is certainly not a measure of cytopathologists diagnostic accuracy.[8]Overall performance evaluation stake not just ASC/SIL ratio but also other parameters such as biopsy and HPV correlation data.[8]As monitoring the ASC/SIL ratio is a useful quality control measure, allows individual CPs to assess their ratio as well as the laboratory as a whole and against the 3:1 benchmark. Bethesda system suggests that the ratio should be less than 3 and the lower ratios are more desirable as low ratio decreases the uncertainty produced by laboratory and may help to reduce the percentage of women with negative biopsy results.[12] A study conducted by Renshawet al. in his study concludes as follows: “A laboratory depends on its cytotechnologists to maintain adequate sensitivity and on its cytopathologists to maintain adequate specificity”.[16]

Conclusion

A study conducted by Juskevicius et al.[8]showed that by providing ASC/SIL ratio of individual cytopathologists (CPs) in a confidential communication was a useful quality

ASC/SIL ratio serves as a good surrogate marker for screening the sensitivity of cytopathologists. Confidential feedback of ASC/SIL ratio of individual cytopathologists helps in diminution of their ratio if it exceeds the upper bench mark. For cytopathologistswhose ratio correlates with the established bench mark, acts as a positive reinforcement and helps to maintain a stable ratio.

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Our study showed concordance results Davey D D et al.[9] who obtained a ratio of 1.3.

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References 1.

Sreedevi A, Javed R, Dinesh A. Epidemiology of cervical cancer with special focus on India. Int J Women’s Health. 2015;7:405-414.

10. Davey DD, Naryshkin S, Nielsen ML, Kline TS. Atypical squamous cells of undetermined significance: interlaboratory comparison and quality assurance monitors. DiagnCytopathol. 1994;11:390–96.

Krieger PA, McGoogan E, Voojis GP, Amma NS, CochandPriollet B et al. Quality Assurance / Control issues. ActaCytologica. 1998;42:133-140.

11. Juskevicius R, Zou KH, Cibas ES. An analysis of factors that influence the ASCUS/SIL ratio of pathologists.Am J ClinPathol. 2001;116:331-35.

Tobias AHG, Amarlal RG, Diniz EM, Carneiro CM.Quality Indicators of Cervical Cytopathology Tests in the Public Service in Minas Gerais, Brazil. Rev Bras GinecObst.2016;38:65–70.

Azara CZS, Manrique EJC, Alves de Souza NL, Rodrigues AR, Nascimento Tavares SBD. Quality Control of Cervical Cytopathology: Interlaboratory Variability. Acta Cytologica.2013;57:585–590.

Bibbo M, Wilbur DC ; Comprehensive Cytopathology. 3 rd ed. Saunders: Elsevier; 2008.

Auger M. Selection of Monitoring Parameters for Gynecologic Cytology—Beacons of Light for Quality Assurance. Cancer Cytopathology 2014; DOI: 10.1002/ cncy.21349

Renshaw AA, Deschênes M, Auger M. ASC/SIL Ratio for Cytotechnologists: A Surrogate Marker of Screening Sensitivity. Am J ClinPathol. 2009;131:776-81.

Nascimento AF, Cibas ES. The ASC/SIL ratio for Cytopathologists as a Quality Control Measure: A Followup Study. Am J ClinPathol. 2007;128:653-56.

Ko V, Nanji S, Tambouret R H, Wilbur D C. Testing for HPV as an Objective Measure for Quality Assurance in Gynecologic Cytology Positive Rates in Equivocal and Abnormal Spec imens and Comparison With the ASCUS to SIL Ratio. Cancer Cytopathology 2007;111(2):67-73.

12. Renshaw AA, Genest DR, Cibas ES. Should Atypical Squamous Cells of Undetermined Significance (ASCUS) Be Subcategorized?: Accuracy Analysis of Papanicolaou Smears Using Receiver Operating Characteristic Curves and Implications for the ASCUS/Squamous Intraepithelial Lesion Ratio. Am J ClinPathol. 2001;116:692-95. 13. Nayar R, Wilbur DC. The Bethesda System for Reporting Cervical Cytology. Definitions, Criteria, and Explanatory Notes. 3 rd ed. New York: Springer; 2015. 14. Davey DD, Woodhouse S, Styer P, et al. Atypical epithelial cells and specimen adequacy: current laboratory practices of participants in the College of American Pathologists Interlaboratory Comparison Program in Cervicovaginal Cytology. Arch Pathol Lab Med. 2000;124:203-11. 15. Davey DD, Nielsen ML, Naryshkin S, et al. Atypical squamous cells of undetermined significance: current laboratory practices of participants in the College of American Pathologists Interlaboratory Comparison Program in Cervicovaginal Cytology. Arch Pathol Lab Med. 1996;120:440-44. 16. Catteau X,Simon P, Noel J C. Evaluation of the Oncogenic Human Papillomavirus DNA Test with Liquid-Based Cytology in Primary Cervical Cancer Screening and the Importance of the ASC/SIL Ratio: A Belgian Study. ISRN Obstetrics and Gynecology 2014; http://dx.doi. org/10.1155/2014/536495.

*Corresponding author: Dr. Hemalatha. J, Department of Pathology, ESIC Medical College & Postgraduate Institute of Medical Sciences and Research, Rajaji Nagar, Bangalore, Karnataka (India) - 560010 Phone: +91 9740614607 Email: drhemalathaj@gmail.com Date of Submission : 21.03.2017 Date of Acceptance : 18.05.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Original Article DOI: 10.21276/APALM.1427

Correlation of Histopathological Study of Breast Lesions with Cytology and Mammography as a Measure of Internal Quality and Diagnostic Accuracy Anusha Mulka, Vaishali Dhananjay Kotasthane*, Rajendra S Dhaka and Dhananjay Shrikant Kotasthane Department of Pathology, Mahatma Gandhi Medical college and Research Institute, Pillaiyarkuppam, Pondicherry, India

ABSTRACT Background: Breast cancer is the one of the leading causes death in women. Triple test approach has been widely accepted in asymptomatic women for the preoperative diagnosis of the breast lesions. Triple test includes clinical examination, mammography and Fine Needle Aspiration Cytology (FNAC) to differentiate between neoplastic and non-neoplastic lesions, histopathological confirmation being the gold standard for neoplasticlesions and best management plan for the patient. The aim was to study the histopathological spectrum of breast lesions and correlate the cytological and radiological findings with histopathological examination and to determine sensitivity, specificity and diagnostic accuracy of FNACand mammography in the diagnosis of the breast lesions. Methods: The study comprised of 552 casesofhistopathologically diagnosed breast lesions over the period of twelve years in a rural tertiary health care centre catering rural population. Results: Of the 552 cases examinedhistopathologically, 375 cases (67.9%) were benign lesions, 177 cases (32.1%) were malignant. Fibroadenoma was the commonest benign lesion, whereas infiltrating ductal carcinoma was the commonest malignant tumour. Incidence of phyllodes tumour was significantly higher compared to other studies. Overall sensitivity, specificity and diagnostic accuracy of FNAC and mammography were 95.9%, 98%, 96.6% and 84.7%, 78.5%,81.1% respectively. Conclusion: Histopathology plays an important role in diagnostic and therapeutic management of neoplastic breast lesionswith preoperative Triple test being an important supportive investigation in rapid diagnosis withfairly good sensitivity,specificity and diagnostic accuracyin neoplastic lesions. Significantly higher incidence of phyllodes tumor needs further investigation for causative mechanism. Keywords: Triple Test, Histopathology, Cytology, Mammography, Breast lesions, Fibroadenoma, Infiltrating Ductal Carcinoma

Introduction

Breast lesions are a heterogeneous group of disorders ranging from inflammatory lesions to invasive cancers. [1] Diseases of the breast are showing a rising trend worldwide.[2] Carcinoma of breast is the most common malignant tumour, causing death in women with more than 1,000,000 cases worldwide annually.[3,4] In India, breast cancer is the second common malignancy after cervical cancer and it is detected in 20 per 1,00,000 women.[5] About 5-55% of all females suffer from breast diseases in their lifetime. Benign lesions of the breast are usually seen in the reproductive age, these are thought to be hormone induced and there is drastic fall in incidence, after menopause due to absence of ovarian stimulation. [6,7] Benign Breast Diseases are more common than malignant ones.[8] Benign breast lesions deserve attention because of their high incidence, their impact on women’s life and due to cancerous potential of some histological types.

Clinically, palpable lump is the commonest presentation followed by nipple discharge and pain along with other symptoms.[10]Many countries performing breast cancer screening programs aimed at detecting early disease in asymptomatic women.[11] “Triple diagnostic technique” which includes Clinical examination, Mammography and FNAC, which determines the diagnosis and assess the need for open biopsy, was suggested by Kreuzer and Boquoi and Hermansen et al.[12,13] Excision biopsy was practiced in the past, but presently, imaging and needle biopsy makes it possible to reduce surgical excision of benign breast lesions to a minimum.[14] Main aim of imaging is early detection of lesions and reducing mortality. The main purpose of fine needle aspiration cytology (FNAC) of breast lump is to confirm cancer pre-operatively and to avoid radical surgery in specific benign conditions.[15] [9]

However, the aspiration cytology is not a substitute for conventional surgical histopathology as a definitive

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diagnosis is not always possible by cytology, it also has some limitations but categorization of disease and differential diagnosis can be provided in the majority of cases.[16] With this in mind, an attempt was made to evaluate the breast lesions in context with histopathological correlation of cytological diagnosis to understand the lesion more clearly and also one of the objective tool used for Internal Quality Control. Similar correlation was done with mammography. The aim of the study was to describe different histopathological lesions of breast in context with age, sex and mode of presentation and to compare these findings with cytological and mammographic findings to calculate sensitivity, specificity and diagnostic accuracy as a quality control measure. The lesions were classified according to recent World Health Organization (WHO) classification tofind out the most common benign and malignant lesions of breast.

Materials and Methods

This was an analytical study, which was conducted at the Department of Pathology, Mahatma Gandhi Medical College and Research Institute (MGMCRI), Pondicherry which is a rural tertiary care hospital catering rural population of Pondicherry and near-by Cuddalore district of Tamilnadu. The Institutional Human Ethics Committee approved this study.The breast biopsies received through mastectomy or lumpectomy from January 2005 to January 2016 in the Department of Pathology, Mahatma Gandhi Medical College and Research Institute, Pondicherry were included in study. The specimens were analysed with reference to clinical features, gross and light microscopic findings with final histopathological diagnosis. Five hundred and fifty-two breast specimens were received during this period. Histopathology of the tumors was studied along with clinical variables namely age, sex. The tumors were classified according to WHO classification. These findings were analyzed and compared with findings of cytology and mammography wherever available.

Results

This study included 553 specimens of breast lesions in the Department of Pathology, MGMCRI, Pondicherry. During the study period, the occurrence of breast lesions was maximum in third decade, accounting for 153 (27.7%) cases. Benign lesions were more common in this age group. There were 89 cases (16.1%) in the fifth decade, in which most of the lesions were malignant lesions. In the present study, female predilection was seen. Among the 553 cases studied, 527 cases were females and 26 cases were males. In the present study,most common clinical presentation was breast lump accounting for 90% followed by nipple discharge and pain.

In histopathological examination of 553 cases, 67.5% (375) cases accounted for benign lesions. In these benign lesions, 72.3% were benign neoplasm, majority were Fibroadenomas. Distribution of Benign lesions is shown in Table 1. On Histopathological Examination,32.1%(177) cases were diagnosed as malignant lesions.Out of these 177 malignant lesions, 154 showed invasive ductal carcinomas and two cases were diagnosed as invasive lobular carcinoma.Distribution of remaining malignant lesions was as shown in Table 2. Cytological Correlation of Breast Lesions: Out of 553 cases, cytological evaluation was available for 537 cases. Table 3 shows correlation of Cytology and Histology in these 537 cases.[Fig 1 & 2].Fifteen cytology cases accounted for false negative and four cases for false positive. The sensitivity, specificity, accuracy, positive and negative predictive value of FNAC was found to be 95.9%, 98%, 96.6%,99.1% and 91.3% respectively. Radiological correlation of Breast lesions: In present study, out of 553cases, 143 patients with palpable breast lump underwent mammography and subsequent pathological examination.[Fig 3 & 4] Mammography showed more malignant lesions than benign because mammography was done after 35 years of age. Comparison ofMammographic diagnosis and Pathological diagnosis was as shown in Table 4. Table 5 shows Radio-Histopathological correlation. I n present study, sensitivity, specificity, accuracy, positive and negative predictive value of mammography was 84.7%, 78.5%, 81.1%,73.5% and 88% respectively.

Discussion

Breast lesions are one of the most common lesions encountered in clinical practice. In the present study, the frequency of benign lesions was higher than malignant lesions, as is seen in other studies.[17-22] The present study showed that majority of non-neoplastic cases were in the third decade followed by second decade.Malignant lesions were common after 5th decade. Astudy conducted by other authors also showed similar findings.[19,23,24] Among the benign lesions, fibroadenoma (64.30%) was the commonest benign breast lesions and fibrocystic disease (13.9%) was the second common benign breast lesion with peak occurrence in 3rd decade of life,as seen in other studies. [18,19] In the present study, infiltrating ductal carcinoma was the most common type of invasive carcinoma. Our findings correlated with studies conducted by other authors.[18,25,26,27] In our study, 15 cases reported on cytology turned out to be false negativity.Out of these,10 cases reported as fibrocystic disease of breast on cytology showed features

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Table 1: Frequency of individual benign breast lesions histopathologically(n=375). Histopathological diagnosis No. Of cases 1. Inflammmatory Lesions[n=24 (6.4%)] 09 • Breast abscess

• • •

Chronic mastitis Granulomatous mastitis Duct ectasia

Percentage 2.4%

1.9%

1.6%

0.5%

2. Benign proliferative disorders [n=55(14.7%)]

•

Fibrocystic change

13.6%

• •

Sclerosingadenosis Atypical epithelial hyperplasia

0.5%

238

63.5%

0.3%

6.1%

0.8%

0.5%

04 25 375

1.1% 6.7% 100%

3. Benign neoplasm[n=271 (72.3%)]

• • • • • •

Fibroadenoma Lactating adenoma Phylloidetumour Benign Intra ductal papilloma Lipoma Tubular adenoma

• •

Gynecomastia Total

4. Miscellaneous[n=25(6.6%)]

Table2: Frequency of individual malignant breast lesions histopathologically Histopathological diagnosis No. Of cases Invasive duct carcinoma 154 Invasive lobular carcinoma 02 Ductal carcinoma insitu 12 Medullary carcinoma 02 Papillary carcinoma breast 01 Mucinous carcinoma 02 Malignant phylloides 03 Metastatic carcinoma 01 Total 177

Percentage 87% 1.12% 6.8% 1.12% 0.56% 1.12% 1.69% 0.56% 100%

Table3:Cytological and histopathological correlation in general of all breast lesions Cytology diagnosis (n=537 cases)

HISTOPATHOLOGICAL DIAGNOASIS(n=537 cases)

Benign

Carcinoma

Suspicious of malignancy

Unsatisfactory

Benign lesions (363)

354[TN]

3[FP]

1[FP]

Malignant (174)

15[FN]

145[TP]

14[TP]

369

148

Total(537)

TN=true negative,TP=true positive,FN=false negative,FP=false positive

Table 4: Radiological correlation of breast lesions BIRADS n=143(%) BIRADS -I 0 BIRADS-II-27 (19%)

Histopathological diagnosis acute suppurative lesion ductal carcimona-in-situ Fibroadenoma fibrocystic disease

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BIRADS n=143(%) BIRADS-III-32(22%) BIRADS-IV- 25(17%) BIRADS-V- 36 (25%) BIRADS-VIâ&#x20AC;&#x201C;23 (16%)

Histopathological diagnosis Benign Malignant Benign Malignant Benign Malignant Malignant

Total No. Of cases(143) 25 07 15 11 03 33 23

Table 5: Radio â&#x20AC;&#x201C;histopathological correlation Mammographic findings(n=143) Benign [BIRADS I,II,III] (59) Malignant[BIRADS IV,V,VI] (84)

Histopathological diagnosis Benign Malignant 50 09 18 66

Fig. 1: Fibroadenoma showing predominantly intracanalicular pattern.(H&E 10x) Inset: cytology showing monolayered sheets of monomorphic cells with bare nuclei in the background (H&E 10x)

Fig. 2: Benign Phyllodestumor showing leaf-like proliferation of stromal cells(H&E 10x). Inset: Cytology showing spindle shaped stromal cells.(H&E 4x).

Fig. 3 :Fibrocystic change showing epitheliosis, cystically dilated glands. (H&E 10x). Inset: Mammography showing low density round calcification in multiple lobules.

Fig. 4: Infiltrating ductal carcinoma NOS type.(H & E 10x) Inset:mammography showing irregular radio-opaque lesion with speculated margins.

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Mulka J et al. of low grade DCIS on histology. Five cases reported as benign proliferative breast disease with atypia on cytology showed features of Infiltrating Ductal Carcinoma in 3 cases and lobular carcinoma in 2 cases on histology. This was due to inappropriate sampling and non targeted FNAC. This is one the common pitfalls and diagnostic dilemma of cytology, due to non-availability of architecturalfeatures and low grade cytological atypia on cytology smears.USG guided targeted FNAC can be helpful in diagnosis of missed out lesion. Careful evaluation of cytological features and adhering to criteria of adequacy of smear could be helpful to overcome these pitfalls.Also, reporting on smears with low cellularity should be avoided by repeating FNAC so that adequacy criteria are satisfied.In our study, four cases accounted for false positive cases.This can be avoided by correlating with mammographic and clinical features and advising biopsy in suspicious cases.The overall accuracy of cytology in diagnosis of breast lesions has been reported to be 95% to 100% in various studies in literature.The sensitivity, specificity and diagnostic accuracy of FNAC in other studies was comparable with our study.[28,29]FNAC is the mainstay in the diagnosis of breast lesions, but it cannot always be relied upon in isolation.FNAC should be used with the idea of ‘complimenting’, not competing with routine Histopathological examination. In the present study, pitfalls were also found in mammography. Fifteen cases reported as BIRADS IV on mammography, turned out to be benign on histopathology and three cases reported as BIRADS V, turned out to be malignant on histopathology.In the present study, sensitivity and specificity of mammography was 84.7% and 78.5% respectively. These results were close to Taori et al’s findings, where as studies conducted by Warren and Baker found higher diagnostic accuracy of 85 % and 89% respectively in mammography.[30,31,32] This indicates that histopathological evaluation remains the golden Standard. The current study provides Hospital based epidemiological data on Correlation of Histopathological diagnosis with Cytological and Radiological diagnosis for a period of 12 years.

Conclusion

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Goehring C, Morabia A .Epidemiology of Benign Breast Disease, with Special Attention to Histologic Types. Epidemiol Rev.1997; 19(2):310-22.

10. Dixon J M, Mansel RE.ABC of breast diseases. Symptoms assessment and guidelines for referral. BMJ. 1994; 309(6956):722-26. 11. Sankaranarayanan R, Ramadas K, Thara S, Muwonge R, Prabhakar J, Augustine P et al. Clinical breast examination: preliminary results from a randomized controlled trial in India. J of Natl Cancer Inst.2011; 103:1476-80. 12. Kreuzer G, Boquoi E. Aspiration biopsy cytology, mammography and clinical exploration: a modern set up in diagnosis of tumors of the breast. Acta Cytol. Jul-Aug1976; 20(4):319-23. 13. Hermansen C, Skovgaard Poulsen H, Jensen J, Langfeldt B, Steenoskov V, Frederiksen P, Myhre Jensen O. Palpable breast tumors: “triple diagnosis” and operative strategy: Results of a prospective study. Acta Chir Scand.1984;150(8): 625-28. 14. Ahmed HG, Ali AS, Almobarak AO. Frequency of breast cancer among Sudanese patients with breast palpable lumps. Indian J Cancer 2010; 47:2326. 15. Hand U, Mohan H, Bharadwaj S, Punia RPS. Fine needle aspiration as a diagnostic tool in breast lesions.I J S 2000; 62: 125-27.

The present study highlights importance of histopathological correlation to find the true nature of the lesion. Histopathological study acts as an internal quality measure for Cytological and Mammographic diagnosis and is the gold standard for diagnosis of neoplastic lesions.

16. Hari S, Srivastava A, Thulkar S. Scope of Breast Imaging in Developing Countries.NJR. 2013;vol 3(2):11-30.

References

18. Malik R, Bharadwaj VK. Breast lesions in young females - a 20 year study for significance of earlyrecognition. Indian J Pathol Microbiol. 2003; 46:559-66.

Guray M, Sahin A. Benign Breast Diseases: Classification, Diagnosis and Management. Oncologist. 2006; 11(5): 435-49.

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17. Amr SS, Rahman A, Sadi M, Ilahi F, Sheikh SS. The Spectrum of Breast Diseases in Saudi Arab Females: A 26 yr Pathological Survey at Dhahran Health Center. Ann Saudi Med. 1995; 15(2):125-32.

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19. Kulkarni S, Vora I M, Ghorpade K G, Shrivastava S. Histopathological spectrum of breast lesions with reference to uncommon cases. Obst Gynecol India. 2009; Vol.59(5):444-52. 20. Rasheed A, Sharma S, Rasool M, Bashir S, Hafiz A, Bashir N. A three year study of breast lesions in women aged 1570 years in a tertiary care hospital. Sch J App Med Sci. 2014;2(IB):166-68. 21. Iyer SP, Gore MA. Epidemiology of Benign Breast Diseases in Females of Child Bearing age group.(internet). http:// www.bhj.org.in/journal/2000_4201_jan00/original_141.htm 22. Mayun AA, Pindija VH, Babayo UD. Pattern of histopathological diagnosis of breast lesion in Gombe, Nigeria. Nigerian J Med. 2008; 17(2):159-62. 23. Bagale P, Dravid NV, Bagale S,Ahire N. Clinico-pathological study of benign breast diseases. Int J Health Sci Res. 2013; 3(2):47-54.

26. Mudholkar VG, Mashal SN, Kawade SB. Histopathological Study of Neoplastic Lesions of Breast.Indian Medical Gazette. 2012;145(9):353-64. 27. Bane AL, Beck JC, Bleiweiss I, Buys SS, Catalano E, Daly MB et al. BRCA2 Mutation- associated Breast Cancers exhibit a distinguishing phenotype based on morphology and molecular profiles fromtissue microarrays. Am J Surg Pathol. 2007;31:121-28. 28. Pant I, Singh PK, Singh SN, Agarwal A, Singh NB. Cytomorphologic study of palpable breast lesions and histopathologic correlation. Journal of Cytology. 2003; 20 (3): 129-32. 29. Vala MT, Goswami A, Suri SK. Comparative study of cytological and histopathological finding in breast lesion. IOSR Journal of Dental and Medical Sciences (IOSRJDMS). 2014;13(7): 5-7.

24. Ibrahim IM, Iliyasu Y, Mohammed AZ. Histopathological review of breast tumors in kano, Northern Nigeria. SubSaharan Afr J Med. 2015; 2: 47-51.

30. Taori K, Dhakate S, Rathod J, Hatgaonkar A, Disawal A,Wavare P. Evaluation of Breast Masses Using Mammography and Sonography as First Line Investigations. Open Journal of Medical Imaging.2013;3: 40-49.

25. ur-rehman A. Histopathological Analysis of Consecutive 161 cases of Breast Lumps. Pak J Med Health Sci. 2013; 7(3): 624-25.

32. Baker LH. Breast Cancer Detection Demon- stration Project: Five-year summary report. CA. 1982;32:194-225.

31. Warren SL. Roentgenologic study of breast. AJR 1930; 4:113.

*Corresponding author: Dr. Vaishali Dhananjay Kotasthane, Department of Pathology, Mahatma Gandhi Medical college and research Institute, Pillaiyarkuppam, Pondicherry- 607402,India Phone: +91 8124054684 Email: vaishalikotasthane@gmail.com, Date of Submission : 22.03.2017 Date of Acceptance : 25.04.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Original Article DOI: 10.21276/APALM.1432

Cytological Evaluation of Two Methods of Effusion Cell Block Preparations Deepa Siddappa Masur1 and Shilpa Somashekhar Biradar2* Department of Pathology, S. Nijalingappa Medical College and HSK hospital and research centre. Bagalkot,India Department of pathology, Akash Institute of Medical Sciences and research center, Prasannahalli road. Devanahalli, Bangalore, India 1

ABSTRACT Background: Cell Block (CB) procedures have now become an established part of cytological diagnostics because of its pivotal role in diagnosis and ancillary studies. Hence the present study was undertaken to emphasize the role of CB technique over Conventional Smear (CS) in serous effusions and to compare the Plasma-Thrombin (PT) block to Formalin Method block (FM) in assessment of morphological preservation and cellularity. Aim: To obtain simple , cost effective and ideal CB preparation where in maximal number of cells are displayed within a small area Methods: The sample was divided into three Parts(A,B,C). After centrifugation of all three parts of sample at 3000rpm for 15min – Part A sediment was used to prepare two CS for Papanicolaou (PAP) and May Grunwald Giemsa(MGG) stains. Part B sediment was subjected for 1hr and 24hr fixation in 1:1 solution of 5ml ethylalcohol and 10% formalin. To the Part C sediment 2drops of finger prick plain blood was added, mixed well and allowed to clot. The sediment of Part B and the clot of Part C were then processed for paraffin embedding. Result: 110 fresh effusion samples were evaluated for cellularity , retention of architectural patterns and volume of background. FM block’s were inconclusive in 12 cases due to low cellularity. PT block’s were all evaluable with best preservation of architecture and pale background. Conclusion: The CB technique revealed better architectural patterns and increased the sensitivity of cytodiagnosis. PT block’s had sufficient to abundant cellularity with evenly distributed cells in small area. PT preparation is simple and cost effective. Keywords: Serous Effusions, Conventional Smear, Cellblock, Plasma Thrombin Method, Formalin Cell Block.

Introduction

Bahrenberg in 1895 introduced CB technique for routine processing of fluids and Mandelbaum in 1917 devised a technique for the preparation of CB.[1] Ceelen in 1957 demonstrated bright red granules in the cytoplasm of mesothelial cells by CB’s.[2] De Girolami developed a technique to cover the sediment with few drops of plasma and few drops of thrombin in order to entrap cells within the artificial clot. The clot was then fixed in 15% formalin processed in paraffin and stained like tissue specimen. [3] Histochemical stains and various special stains such as PAS, Diastase, Ziehl-Neelsen and Gomori-Methenamine Silver nitrate can easily be performed on the sections prepared from CB.[4] The customary methods of investigation of serous effusions are cytologic examination of the fluid by both CS and CB sections, biopsy of the pleura, peritoneum, pericardium, and supplemented by immunocytochemistry, biochemical, bacteriologic or cytogenetic investigations.[5] Various studies conducted in the past and in recent years strongly advice to process the remainder of the specimen as a CB.[6]

Ideally, cellular components revealed by CB’s are expected to exhibit the following features: resemblance to corresponding cells in alcohol-fixed PAP stained smears, adequate preservation of nuclear-cytoplasmic details, easy recoverability, and suitability for ancillary studies such as immunostaining.[7] An ideal method for preparation would be simple, reproducible, and readily adaptable in a routine setting.[8] CB study is valuable in cytopathology because it provides histopathologic correlation and additional material for immunohistochemical studies.[9] Although the CB method is not new, handling of the specimens in a conventional manner results in considerable loss of material.[6] Instead of separating the cells by preparing smears, the principle of blood clotting can be applied to the sediment obtained. In such a way, the intercellular relationships and morphologic features of tissues in human organs could be maintained.[3] In today’s era of personalized medicine with an increasing array of molecular tests being applied to cytological specimens, there is a need for a standardized protocol for CB optimization to enhance cellularity. This study serves

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as a baseline to launch further investigation of the pros and cons of different CB preparation techniques. For this reason, in this study an attempt was made to prepare and analyze both CS and CB from the same specimen. To assess the feasibility of both CB preparations and emphasize the role of PT and FM CB’s in assessment of morphological preservation and cellularity which would be useful for immunohistochemistry and other special stains if required.

Materials and Methods

This is a one year prospective study performed in Department of Pathology of Shri. Nijalingappa. Medical. College and HSK hospital Bagalkot, with approval from the institutional ethics committee. Written informed consent was also obtained while sample collection. Serous effusions from the body cavities comprising of pleural, peritoneal and pericardial fluids were included. All fluids other than pleural, peritoneal, pericardial fluids were excluded. Fresh serous fluid samples received were first submitted for naked eye examination for physical characteristics and divided into three parts. Part A sample was centrifuged at 3000 rpm for 15 min and two CS were prepared for PAP and MGG stains. Part B sample was processed for FM CB. The sample was added to 1:1 solution of 5 ml of ethanol and 10% formalin and fixed for 1 hr. The mixture was agitated often for uniform fixation of the material. After fixation, the specimen was centrifuged at 3000rpm for 5min. The supernatant was decanted and the sediment completely drained off by inverting tube over Whatman filter paper. To this sediment 1:1 mixture of ethanol and 10% eosin tinted formalin was added and kept for fixation for 24hrs. Then after discarding the supernatant fixative, the pellet formed was removed with a pointed spatula and placed on top of the lens paper inside the tissue cassette and processed for paraffin embedding. Part C sample was processed for PT CB (Fig 1). The fluid was centrifuged at 3000 rpm for 5min. The supernatant was decanted and the excess fluid was removed by inverting over Whatman filter paper. To this sediment 2 drops of finger prick plain blood was added and mixed well and allowed to clot for 30 seconds. Then the clot was dislodged from the test tube and fixed in 1:1 mixture of ethanol and 10% eosin tinted formalin for 1hr. Further the clot was transferred to the lens paper in the tissue cassette and then processed for paraffin embedding. Instead of plasma and commercial available thrombin separately we have used 2 drops of finger prick blood to make the method cost effective.

Interpretation of Conventional Smears and Cellblocks. After evaluating the available clinical data, the CS and CB sections were than objectively analyzed for cellularity, architectural arrangement and background. The point scoring system (Table 1) as described by Mair et.al [10]was used to group the cases into following three categories difficult for evaluation, b) sufficient for evaluation, and c) superior .

Result

A total of 110 body cavity fluid samples were studied, of which 54 were pleural fluids, 51 were peritoneal fluids and 5 were pericardial fluids. 77 fluid samples were from male patients and 33 were from female patients. Male : Female ratio was 2.3:1. The maximum number of samples were from patients in the age group of 41 - 50 years. ( Table 2) Cellularity in Cell Blocks ( PT & FM) and Conventional Smears The cellularity in both the CB’s preparation was sufficient to abundant for evaluation (Table 3). FM CB’s did not contain cellular material in 12 cases and were inconclusive for evaluation (Fig 4). But the same samples had low cellularity and not easy for evaluation in CS (Fig 2) but evaluable in PT block ( Fig 3). So not a single case was inconclusive or difficult for evaluation by PT method. Volume of the Obscuring Background : As per the criteria described in Table 1 the volume of the obscuring background was considered to be large when the diagnosis was greatly compromised, moderate when diagnosis was possible and minimal when the background was clear. The CS were difficult for evaluation due to large and obscuring background of hemorrhage and degenerating cells ( Fig 5). In FM CB’s, the background was clear in all the samples but had artifactually crowded cells which was scattered in various parts of the slides (Fig 7). The PT CB’s (Fig 6) revealed uniform distribution of the cells with clear nuclear and cytoplasmic preservation in pale background. Preservation of Architectural Patterns. The CB’s concentrated material in smaller fields with more frequent appearance of organoid pattern and cells in the same focal plane. The serial sections of even minute amount of cellular material from various types of sample showed high cellularity with better morphologic preservation (Table 4). Architectural pattern preservation was excellent in all the PT blocks(Fig 3 &6) and only sufficient for evaluation in half of FM blocks and CS. Malignant Effusions Diagnosed by Cellblock Method. Even though the primary aim of the present study was not to detect the diagnostic accuracy for malingnancy, additional 15.5% malignancies were diagnosed by cellblock

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preparations. Total number of 27 body fluid samples were diagnosed as malignant effusions by CB’s and diagnosis of metastatic malignant effusion was rendered. Primary was identified in 12 cases and was unknown in 15 cases. 3 cases expired before diagnosis and 3 cases discontinued

the treatment. The remaining 4 unknown cases were cases of alcoholic liver disease and cirrhosis and primary was diagnosed by immunohistochemistry from the liver. In another 2 cases primary was not known, as patients lost for follow up study.

Table 1: Criteria for the assessment of the quality of CS and CB preparations. Criterion

Quantitative description

Amount of cellular material

Retention of appropriate architectural pattern

Volume of obscuring background

Point score

Low (5-10 nucleated cells /hpf)

Sufficient (10-100 nucleated cells/hpf)

Abundant ( >100 nucleated cells /hpf)

Minimal or Absent (evaluation not possible)

Sufficient for evaluation

Abundant

Large amount, evaluation greatly compromised

Moderate amount, evaluation possible

Clear

Table 2: Showing Number of patients in each Age Group Age group in yrs

No. of males

No. of females

Total No.

Percentage (%)

0 – 10

3.6

11 – 20

2.7

21 – 30

9.1

31 – 40

41 – 50

29.1

51 – 60

61 – 70

8.2

71 – 80

4.5

81 – 90

1.8

Table 3: Morphology of PT CB’s ( H&E stain) and CS ( PAP and MGG stain) Method

Cellularity

Preservation

Background

Sufficient/ abundant

low

Sufficient / abundant

Minimal/ absent

Large/ obscuring

Clear / pale

PT-CB

110

FM-CB

Table 4: Categories depending upon the scoring system Difficult for evaluation

Sufficient for evaluation

Superior

PT-CB

FM-CB

Method

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Table 5: Architectural Patterns in CS and CBâ&#x20AC;&#x2122;s (PT & FM ) Architectural pattern

Singly scattered

50.9

19.09

25.5

Cell balls

16.4

Cell clusters

19.09

34.5

Papillae

18.2

63.6

45.4

Glands

9.09

54.5

Sheets

11.8

55.5

Fig. 1: Flow chart for preparation of PT CBâ&#x20AC;&#x2122;s. ( The figure is constructed and derived from various textbooks and sources.).

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Fig. 2: Small cluster of suscpicious cells on CS (PAP stain 40X).

Fig. 3: Single acinar pattern in PT CB ( H&E stain 40X).

Fig. 4: Inconclusive FM CB when cellurity was less(H&E stain X40).

Fig. 5: Suscpicious Papillae on CS (PAP stain 40X).

Fig. 6: Uniform distribution of cells in small area in PT CB (H&E stain 40X)

Fig. 7: Shrunken scattered cells all over the fields in FM CB (H&E stain40X).

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Discussion

In the present study, we evaluated CS and two types of CB’s – PT block and FM block preparations for cellularity, volume of obscuring background and the cellular preservation of morphology. The CB’s concentrated the cellular material into a small area which was useful in screening the material in lesser time.[1,9,11,12,13] The cellularity, in present study denotes the number of cells per field and not the diagnostic tumour or metastatic cells. Similar criteria were used by Mair S et.al,[10] Thapar et.al,[12] and Nigro et.al.[14] These studies categorised the cellularity into Minimal or Absent - when the cells were not sufficient for evaluation, Adequate - when sufficient for evaluation, and Abundant - when increased number of cells were present. In our study we denote positive for cells and also the number of cells. The primary aim of the present study was not to detect the diagnostic accuracy of the preparations, but additional 15.5% malignancies were diagnosed by CB preparations. PT CB’s had better cellularity when compared to CS and FM CB. The agitation of plasma and thrombin present in blood facilitates the trapping of cells by the fibrin strands, which coalesce into a fibrinous clot. Thus all the cellular material present will be efficiently captured into a clot. The clot is also very easy to embed and will not be lost during processing. The eosin tint helps in easy location of the clot during processing. Similar type of blood clot principle was used by Yang et al [9], De.Girolami et.al,[3] Jing et.al,[7] Nigro et.al,[14] Karnauchow et.al,[15] Burt et.al,[16] Kulkarni et.al,[17] Mahzouni et.al,[18] Rowe et.al,[19] and Bhatia et.al.[20] PT block procedure is applied since 1964 and emphasizes the advantage of clustering the cells rather than isolated cells in the diagnosis of neoplastic or pathologic processes. The advantages of cellblocks are 1) Concentration of cellular material in one small area that can be evaluated at a glance with all cells lying in the same focal plane. PT method is successful in introducing such blocks. 2) Preservation of architectural pattern like cell balls, papillae and three dimensional clusters. 3) Intact cell membranes and crisp chromatin details 4) It bridges the gap between cytology and histology. The major disadvantage of the CB preparations is time, the delay in diagnosis when compared with CS. Another disadvantage is the loss of the cellular material and cytologic detail during processing.[21,22,23] PT preparation is cost effective, relatively easy and optimal for IHC.[24] They are most useful in CB preparation particularly in effusions with minimal cellularity.[10] Advantages of this method

include the assessment of an additional sample volume and thus reduction of sampling error, the possibility for unlimited storage and molecular testing similar to histological samples.[24] Additional studies with an in depth analysis to determine the appropriate method(s) is necessary. Determining and standardizing the most effective technique may alleviate the variability and provide consistency. As it is adapted to the introduction of immunohistochemistry and flow cytometry, cytology has to align itself with the multitude of molecular diagnostic tests.

Conclusion

PT block’s were highly cellular and demonstrated pale background with evenly distributed cells. The preparation is simple, cost effective and reproducible. Thus despite the fact that in majority of cases, the diagnosis can be made on the bases of either CS or CB alone, the study conclude that using the combined technique on the same specimen leads to a more accurate diagnosis. Based on these data, the PT method showed superior quality compared to CB’s prepared in a variety of other ways. Our study illustrates that there are a variety of ways to prepare CB’s and that subtle differences in technique can have an impact on important parameters and the overall quality of these specimens. Optimal CB preparation is important as the demand to do more with small specimens increases.

Reference 1.

Wojcik EM, Selvagi SM, Comparison of smears and cellblocks in the fine needle aspiration diagnosis of recurrent gynecological malignancies. ActaCytol 1991; 35(6): 773-776.

Ceelen GH: The cytologic diagnosis of ascitic fluid. ActaCytol 1964;8:175-183.

De. Girolami E. Applications of plasma thrombin cell block in diagnostic cytology. Part II. Digestive and Respiratory Tracts, Breast and Effusions. Annu Pathol 1997,12: 91-110.

Leung SW, Bedard YC. Methods in Pathology. Simple mini block technique for cytology. Mod pathol 1993;6(5):630632.

Shidham VB, Atkinson BF. Cytopathologic diagnosis of serous fluids. Elsevier WB Saunders, 2006; 1-55.

Koss LG: Diagnostic Cytology and Its Histopathologic Bases. Fifth edition. Philadelphia, Lippincott Williams and Wilkins. Pennysylvia. USA. 2006 Pg 919-1018

Jing X, Li QK, Bedrossian U, Michael CW. Morphologic and Immunohistochemical performances of effusion cell blocks prepared using 3 different methods. Am J Clin Pathol 2013;139:177-82.

Foot NC. Identification of types and primary sites of metastatic tumors from exfoliated cells in serous fluids. Am J Pathol 1954; 30(4): 661-677.

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Yang GC, Wan LS, Papellas J, Waisman J. Compact cell blocks, Use for body Fluid, Fine needle aspirations and Endometrial brush biopsies. Acta Cytol 1998; 42: 703-706.

10. Mair S, Dunbar F, Becker PJ, DuPlesis W. Fine needle cytology: Is aspiration suction necessary? A study of 100 masses in various sites. Acta cytol 1989;33:809-813. 11. Krogerus LA, Anderson LC, A simple method for the preparation of paraffin embedded cell blocks from fine needle aspirates, effusions and brushings. Acta Cytol 1998; 32(4): 585-587. 12. Thapar M, Mishra RK, Sharma A, Goyal V. Critical analysis of cell block versus smear examination in effusions. Journal of cytology 2009; 26(2):60-64. 13. Dekker A, Bupp PA. Cytology of serous effusions. An investigation into the usefulness of cellblocks versus smears. Am J Clin Pathol 1978; 70(6): 855-860. 14. Nigro K, Tynski Z, Wasman J, Abdul-Karim F, Wang N. Comparison of cell block preparation methods for nongynaecologic thinprep specimens. Diag Cytopathol 2007; 35(10): 640-643. 15. Karnachow PN, Bouin RE. ”Cell-block” technique for fine neddle aspiration biopsy. J Clin Pathol 1982;35:688. 16. Burt AD, Smillie D, Cowan MD, Adams FG: Fine neddle aspiration cytology: Experience with a cell block technique (letters). J Clin Pathol 1986; 39: 114-115. 17. Kulkarni Mb, Desai SB, Ajit D, Chinoy RF. Utility of the thromboplastin-plasma cell-block technique for fine needle

A-409 aspiration and serous effusions. Diag cytopathol 2009; 37(2):86-90. 18. Mahazouni P, Sharifani M. Direct smear vs cell Block (plasma-thrombin clot) method: diagnostic value in serosal cavities fluid cytology. Diag cytopathol 1999; 27 (2):77-80 19. Rowe LR, Marshall CJ, bentz JS. Cell block preparation as an adjunctive diagnostic technique in thinprep monolayer preparations: A case report. Diagn. Cytopathol 2001; 24: 142-144 20. Bhatia P, Dey P, Uppal R, Shifa R, Srinivasan R, Nijhawan R. Cell blocks from scraping of cytology smear – comparison with conventional cell block. Acta cytological 2007; 52(3): 329-333. 21. Crapanzano JP, Heymann JJ, Monaco S, Nassar A, Saqi A. The state of cell block variation and satisfaction in the era of molecular diagnostics and personalized medicine. CytoJournal 2014;11:7 22. Weihmann J, Weichert C, Petersen I, Gajda M. Evaluation of a cell block method in cytological diagnostics. Der Pathologe 2012;33:6 553-559. 23. Balassanian R, Ono JC, Wool GD, Olejnik-Nave J, Mah MM, Sweeney BJ, A superior technique for cell block preparation for fine needle aspiration. Mod Pathol 2013;26:83A. 24. Jain D, Mathur SR, Iyer V K. Cell blocks in cytopathology: a review of preparative methods, utility in diagnosis and role in ancillary studies. Cytopathology 2014; 25(6): 356-371.

*Corresponding author: Dr. Shilpa Somashekhar Biradar, Department of Pathology. Akash Institute of Medical Sciences and research center.Bangalore, India Phone: +91 9480716323 Email: shilpa.biradar@yahoo.co.in Date of Submission : 23.03.2017 Date of Acceptance : 25.04.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

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Original Article DOI: 10.21276/APALM.1455

Histomorphological Spectrum of Renal Lesion in An Autopsy Study Vaneet Kaur Sandhu*, Arun Puri and Navtej Singh Department of Pathology, Guru Gobind Singh Medical College and Hospital ,Faridkot , Punjab,INDIA

ABSTRACT Background: Autopsy provides normal as well as diseased human tissue for morphologic studies, for establishment of cell and organ culture as well for xenotransplantation. It provides the opportunity to discover new diseases, to evaluate toxic effects of drugs and therapies. The kidneys are often affected by chronic inflammatory lesions, neoplasms , toxic effects of various drugs and metabolic disorders. Material and Methods: This was a five year study from January 2011 to December 2015 in our department of pathology. The kidneys of medico legal autopsies performed during these years were subjected to our study. After excluding 30 cases of severely damaged tissue, 120 cases of well preserved renal medico legal autopsies were included in our study. The stained microscopic sections were examined by two histopathologists independently. Results: Seventy five of the 120 autopsies were males, while 45 were females. In 27 (22.5%) cases, the microscopic morphology was close to normal histology. Remaining 93(77.5%) cases had a nephropathological findings The percentage of non glomerular nephropathies (60.8%) was higher as compared to that of glomerular lesions (16%). 20 (16% ) cases exhibited glomerular alterations such focal global glomerular sclerosis, segmental glomerular sclerosis, nodular mesangial sclerosis, basement thickening and mesangial cellular proliferation. Tubular and interestium lesions were observed in 34.16% which included acute tubular necrosis, chronic pyelonephritis and tubercular pyelonephritis. Renal arteriosclerosis was observed in 25% cases. Renal cell carcinoma was incidentally detected in1.6% Conclusion: Our study provided satisfactory data in respect to morphological spectrum of various renal lesions in autopsy. Keywords: Autopsy, Renal diseases,

Introduction

The autopsy data continue to embellish the medical literature; it provides a unique opportunity for physicians to correlate their physical and laboratory findings with the pathologic changes of disease process. The autopsy pathologists uncover the changing patterns of disease. The autopsy aids in the education of students in medicine and other health related disciplines by providing teaching material for anatomy, histology and pathology. Further it plays an important role in establishment of diagnosis and whenever possible determines the possible cause of the death. Renal diseases are responsible for great deal of morbidity. Chronic kidney disease is now recognized as a major global public health problem and is an independent risk factor for cardiovascular disease.[1-2]CKD affects 10-15% of the adult population worldwide.[3-4]The increased prevalence of kidney diseases is a consequence of the accumulation of risk factors such as hypertension, diabetes, dyslipidaemia and obesity.[5]Pathologic examination of renal tissue in autopsy throws a light on renal histologic changes in the general population, might provide useful information

for preventing chronic renal diseases that tend to be asymptomatic and often go undiagnosed. The aim of the present study was to analyze varied spectrum of renal lesions detected on autopsy.

Material and Methods

This was a five year study from January 2011 to December 2015 in our department of pathology. The kidneys of medico legal autopsies performed during these years were subjected to our study. After excluding 30 cases of severely damaged tissue, 120 cases of well preserved renal medico legal autopsies were included in our study. The data pertaining to age, gender, and clinical findings were recorded from deceased post mortem papers. The thorough gross examination including weight, measurements, colours were recorded and then tissue was fixed in 10% neutral buffered formalin. The formalin fixed tissues were sampled, each sample included the cortico-medullary region then were further processed by automatic processor. The three micrometer thick sections were obtained from paraffin embedded tissue samples and were histochemically stained with haematoxylin and eosin. The special stains periodic acid Schiff reagent and silver methanamine were

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Sandhu et al. done as and when required. The microscopic sections were observed by two histopathologists indepentantly. The stenosis of renal arteries were graded on the basis of luminal narrowing of arteries and was grade from grade 0 ( normal ) to grade IV( complete obstruction) Grade 0: normal, Grade I: 1-25% narrowing of lumen, Grade II: 26-50% narrowing of lumen, Grade III: 51-75% narrowing of lumen, Grade IV: 76-100 narrowing of lumen.

Results

The age range of the autopsies was between 25 and 80 years. Seventy five of the 120 autopsies were males, while 45 were females. In 27(22.5%) cases, the microscopic morphology was close to normal histology. Remaining 93(77.5%) cases had a nephropathological finding at autopsy. Table 1 summarizes the various renal lesions in renal autopsies. The percentage of non glomerular nephropathies (60.8 %) was higher as compared to that of glomerular lesions (16%). Glomerular Findings: In 20 cases of renal autopsies glomerular alterations were observed of which 12 males and remaining 8 were were females. Ten cases (8.3%) exhibited focal global sclerosis. There were two cases (1.6%) of segmental glomerular sclerosis and three (2.5%) cases exhibited nodular glomerular sclerosis. Basement membrane thickening was observed in two (1.6%) cases. Three (2.5%) cases showed moderate degree of mesangial cellular proliferation.

A-411 Tubular and Interestium Findings:. There were tubular and interestium alterations in 41(34.16%) cases of which 29 were males and twelve were females. Acute tubular necrosis was seen in 27(22.5%) cases of renal autopsies fig1. The chronic pyelonephritis fig2 was noted in 8 (6.6%) cases out which 5 cases had diffuse interstitial fibrosis, tubular atrophy and diffuse global sclerosis. Remaining three cases had focal interstitial inflammation with focal tubular atrophy fig 3. In addition to chronic pyelonephritis three were accompanied by hydronephrosis and two cases had nephrocalcinosis. The tubercular pyelonephritis was observed in 6 (5% cases) of renal autopsies. The caseating epitheloid cell granulomas, langhan type of giant cells were noted in cases of tuberbular pyelonephritis fig 4 and fig 5. The Ziehl Neelson stain exhibited acid fast bacilli. Vascular Findings: The most frequent histological diagnosis was renal arteriolosclerosis observed in 30 (25%) of renal autopsies, of which 20 were males and 10 were females. Grade II renal arteriolosclerosis was observed in 18 (60%) cases followed by Grade I renal arteriolosclerosis in 6 (20%), Grade III in 4 (13.3%) and remaining had grade IV renal arteriolosclerosis 2(6.6%). The arterial nephrosclerosis was characterized by intimal fibrous thickening of arteries fig [6] The incidental renal masses in the study comprised of clear cell renal cell carcinoma in two renal autopsies, of which one was 65 year old male and other being 55 year old female.

Table 1: Distribution of various renal findings on autopsy study (120). Histopathological findings

Number

Gender M F

A.Glomerular lesions 1.Focal global glomerular sclerosis 2.Segmental glomerular sclerosis 3.Nodular glomerular sclerosis 4.Mesengium proliferation 5.Basement membrane thickening

20(16.6%) 10 (8.3%) 2 (1.6%) 3 (2.5%) 3(2.5%) 2 (1.6%)

12 8 73 11 21 12 11

B. Tubular and Interstitial findings 1.Acute tubular necrosis 2.Chronic pyleonephritis 3.Tubercular pyleonephritis

41(34.16%) 27(22.5%) 8(6.6%) 6(5%)

29 12 20 7 53 42

30 (25%) 18(60%) 6 (20%) 4(13.3%) 2(6.6%)

20 10 15 3 24 22 11

C. Vascular findings 1. Renal arteriosclerosis a.Grade II b.Grade III c.Grade I d.Grade IV D.Neoplasm 1.Renal cell carcinoma E.Normal histology Total

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2 (1.6%)

27(22.5%)

13 14

120

75 45

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Fig. 1: Sections shows a viable glomerulus with tubular epithelial cells exhibiting necrosis in case of Acute tubular necrosis H& E x 40.

Fig. 2: Gross appearance of a kidney from a case of chronic pyleonephritis shows dilation and blunting of pelvic calyces.

Fig. 3: Sections shows dense inflammatory infiltrate in the interestium with thyroidization of tubules in case of chronic pyleonephritis H& E x 40.

Fig. 4: Sections show epitheliod cell granulomas in the interestium in case of tuberbular pyleonephritis H& E x 10

Fig. 5: High power view of caseating epitheloid cell granulomas in case of tubercular pyleonephritis H & E x 40

Fig . 6: Sections show renal artery exhibiting arteriosclerosis with thickening of the wall and narrowing of the lumen H& E 10

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Discussion

Two (1.6%) cases of renal cell carcinoma (clear cell type) were observed during our study. Kozlowska Jolanta et al.[19] in their work observed renal tumors in 2.76% cases in post mortem examination. Shah VB et al.[20] and Sapna P et al.[21] in their respective autopsy studies revealed 5 cases and 4 cases of renal masses detected incidentally.

The distribution of renal lesions vary with geographic area, age, gender, environmental, nutritional and genetic factors.[6-7] In current analysis in 27 cases the microscopic findings were close to normal histology. This is in concordance with study conducted by Usta et al.[8] on 55 renal autopsy in which 23 cases exhibited almost normal histology. We observed nephropathological changes in 77.5% of renal autopsies .How ever slightly lower percentage of renal lesions were obtained by Monga et al.[9] and Martinez et al.[10] in their respective work on renal autopsies who found renal lesions in 68% and 59% cases respectively. The histopathologic findings in the present study revealed presence of non glomerular nephropathies in 73(60.8%) cases and glomerular lesions 20(16%) cases .A study conducted by Hailemariam S et al.[11] on 237 autopsies observed presence of glomerular or vascular pathology in 28% ,non glomerular lesion in 33% and 29% had combined lesions. We observed glomerular sclerosis in 20 (16.6%) cases of which 10 (8.3%) cases exhibited global focal glomerular sclerosis, two (1.6%) cases had segmental glomerular sclerosis, three (2.5%) cases had nodular sclerosis, three(2.5%) cases had moderate to severe degree of mesangial cellular proliferation and two (1.6%) case had basement membrane thickening. However Usta et al.[8] in their work observed focal global sclerosis in eleven cases, followed by 12 cases of mesangial cellular proliferation and one case of basement membrane thickening. In current analysis 30 (25%) cases exhibited renal arteriosclerosis .Mc Namara BJ et al.[12]in their work on 81 renal autopsies reported arteriolar nephrosclerosis in 34% cases . Nephrosclerosis at autopsy is associated with increasing age and is more frequent in blacks than whites. [13] Some observers suggest that renal arteriosclerosis is strongly linked with hypertension.[14-16] Among present work tubular and interstitium changes were observed in 41 (34.16%) cases of which27 (22.5%) cases had acute tubular necrosis .This might be attributed to death due to intake of toxic substance, drugs over dose and snake bite. Renal tuberculosis and chronic pyelonephritis were observed in 6 (5%) and 8 (6.6%) of cases respectively in our work. Tuberculosis has been described as a global emergency by WHO and in developing countries it still remains as a major cause of morbidity and mortality. [17] Renal tuberculosis develops in approximately 5 % of patients with active tuberculosis. Renal tuberculosis usually remains clinically silent and often detected incidentally in autopsy studies.[18] www.pacificejournals.com/apalm

Conclusion There is still no substitute for autopsy study which throws immense light on pathogenesis of disease, reveals hazardous effects of therapies and drugs administered and lastly often reveal cause of death. The present study on renal autopsies showed renal vascular and tubulointerstitial lesions outnumbered in comparison to glomerular lesions. We observed 1.5% cases of renal cell carcinoma. Our study provided satisfactory data in respect to morphological spectrum of various renal lesions in an autopsy study however it does not reflect the actual incidence of renal lesions in a population.

Referances 1.

Levey AS, Atkins R, Coresh J. Cohen EP, Collins AJ, Ec Kardt KU. Chronic kidney disease as a global public health problem:approaches and initiatives- a position statement from Kidney Disease Improving Global Outcomes. Kidney Int 2007;72:247-259.

Schiffrin EL, Lipman ML, Mamm JF. Chronic kidney disease:effects on the cardiovascular system. Circulation 2007;116:85-97.

Imai E, Horio M, Watanabe T, Iseki K, Yamagata K, Hara S. Prevalence of chronic kidney disease in the Japanese general population.Clin Exp Nephrol 2009;13:621-630.

Hall YN, Hsu CY, Iribarren C Darbiman J, Mc Culloch CE, Go Alan S. The conundrum of increased burden of end stage renal disease in Asians. Kidney Int 2005;68:2310-2316.

Nagata M, Ninomiya T, Doi Y, Yonemolo K, Kubo M, Hata J. Trends in the prevalence of chronic kidney disease and its risk factors in a general population:the Hisayama Study. Nephrol Dial Transplant 2010;25:2557-2564.

Kurnatowaska I, Jedrzejka D Malyska A, Danilewicz MW, Danilewicz M, Nowicki M. Trends in the incidence of biopsy proven glomerular dieases in the adult population in central Poland in the years1990-2010.Kidney Blood Press Res 2012;35:254-258.

Zaza G, Bernich P, Lupo A. Triveneto Register of Renal Biopsies (TVRRB).Incidence of primary glomerulonephritis in a large North Eastern Italian area:a 13 year renal biopsy study.Nephrol Dial Transplant 2013;28:367-372.

Usta U, Tastekin E, Isler E, Kutlu AK, Puyan FO. Histopathological and immune alterations in autopsied kidneys.Saudi Med J 2014;35:1331-38.

Monga G, Mazzucco G, Boldorini R, Cristina S, Giacalone A, Fortunato M et al. Renal changes in patients with acquired immunodeficiency syndrome.a postmortem study on an

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unselected population in northwestern Italy.Mod Pathol 1997;10(3):159-67. 10. Martinez PM, Penta JM, Arnalich F, Garcia M, Adridanto O, Gonzalez J et al. Spectrum of renal pathology in HIV infection:descriptive of 85 autopsies and clinic-pathologic correlation.Rev ClinEsp 1996;196(9):557-83. 11. Hailemariam S, Walder M, Burger HR, et al.Renal pathology and premortem clinical presentation of Caucasian patients with AIDS: An autopsy study from the era prior to antiretroviral therapy.Swiss Med Wkly2001;131:412-17. 12. Bridgette J. Mc Namara, Boucar Diouf, et al. Renal pathology, glomerular number and volume in a West African urban community. Nephrol Dial Transplant2008;23:2576-2585. 13. Marcantoni C, Fogo AB:A perspective on arterionephrosclerosis: from pathology to pathogenesis.J Nephrol .2007;20:518. 14. Tracy RE. Recent declines of hypertensive renovasculopathies in New Orelans blacks.Am J Hypertens 2000;13:966-972.

15. Tracy RE, Rios-Dalenz JL. Rarity of hypertensive stigmata in aging renocortical arteries of Bolivians.Virchows Archiv 1994;424:307-314. 16. Tracy RE, Mulvad G, Pederson HS. Blood pressure in people in Greenland assed by renovasculopathies of hypertension at autopsy. Am J Hypertens 1996;9:560-565. 17. Rastogi P, Kanchan T, Menezes RG. Sudden unexpected deaths due to tuberculosis.An autopsy based study.Journal of Forsensic Medicine and Toxicology.2011;28:81-5. 18. Eastwood JB, Corbishley CM, Grange JM. Tuberculosis and the kidney.J AMSoc Nephrol.2000;12:1307-1314. 19. Kozlowska J, Okon K. Renal tumors in postmortem material. Pol J Pathol.2008;59:21-25. 20. Shah VB, Deokar MS. Spectrum of incidental renal masses detected at autopsy.Bombay Hosp J.2009;51:432-36 21. Patel S, Rajalakshmi BR, Manjunath GV. Histopathologic findings in autopsies with emphasis on interesting and incidental findings-A Pathologists Perspective.J of Clinicla and Diagnostic Research2016;10:8-12.

*Corresponding author: Dr Vaneet Kaur Sandhu, Associate Professor, Department of Pathology, Guru Gobind Singh Medical College and Hospital Faridkot 151203 Punjab India Phone: +91 9815683434 Email: vaneetsandhu@gmail.com Date of Submission : 02.04.2017 Date of Acceptance : 25.05.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Original Article DOI: 10.21276/APALM.1226

Study of Lipid Profile in Patients of Polycystic Ovarian Syndrome Before and After Metformin Therapy Geetika Singh1, Nishat Afroz1, Noora Saeed1*, Sheelu Shafiq Siddiqi2, Aaliya Ehsan3 and Mohd Rafey3 Department of Pathology, JNMCH, AMU, Aligarh, U.P, India Rajiv Gandhi Centre for Diabetes and Endocrinology, JNMCH, AMU, Aligarh, U.P, India 3 Department of Pathology, JNMCH, AMU, Aligarh, U.P, India 1

ABSTRACT Background: Polycystic ovary syndrome (PCOS) is the most common metabolic and endocrine disorders affecting 5-10% of women in their reproductive age. Objective:To evaluate the effect of metformin on serum lipid profile in patients of polycystic ovary syndrome. Methods: An observational prospective study conducted at Rajiv Gandhi Centre for Diabetes and Endocrinology and Department of Pathology,JN Medical College AMU from December 2012 to November 2015. All PCOS women were randomized into two group; one group were received Metformin therapy for 6 months duration and the other group were kept on placebo and followed for the same period. All studied women were subjected to measurement of overnight fasting lipid profile which included total cholesterol,triglycerides,high density lipoprotein cholesterol and low density lipoprotein cholesterol. Results: For women who received Metformin, there was significant improvement in the biochemical profile with a significant p value of <0.001. Conclusion: Metformin have been shown to improve the lipid profiles leading to increase in the high density lipoprotein cholesterol, the main predictive of cardiovascular disease in the women with polycystic ovary syndrome. Keywords: Metformin, Polycystic Ovary Syndrome, Lipid Profile.

Introduction

Polycystic ovarian syndrome (PCOS) is the most common metabolic and endocrine disorders affecting 5-10% of women in their reproductive age[1] and is a leading cause of infertility. Itâ&#x20AC;&#x2122;s main features are lack of regular ovulation and excessive amount or effect of androgenic hormones. [2,3] While the causes of PCOS are still unknown, insulin resistance secondary to obesity is strongly correlated to this syndrome.[4]Obesity increases hyperandrogenism, hirsutism, and infertility and pregnancy complications both independently and by exacerbating PCOS. In general population, obesity and insulin resistance further increase type 2 diabetes (DM2) and cardiovascular disease (CVD). Likewise, in PCOS obesity worsens insulin resistanceand exacerbates reproductive and metabolic features.[5] Furthermore, women with PCOS have increased risk factors for DM2 and CVD,increased impaired glucose tolerance (IGT), DM2 and potentially increased CVD.[6] Metformin, a biguanide antihyperglycemic drug has been shown to have a beneficial effect on circulating lipid level by decreasing the plasma level of triglycerides and

total LDL cholesterol and increasing the level of HDL cholesterol independently of the improvement of glycemic control.[7] However there are only a few studies specifically concerning the effect of Metformin therapy on lipid profile in women with PCOS.[8]Moreover Metformin is offered as first line treatment to clomiphene resistant women with PCOS.[9]

Material and Methods

This observational study included 98 patients of Polycystic Ovarian Syndrome (PCOS) in the reproductive age group, attending the outpatient services of the Rajiv Gandhi Centre for Diabetes and Endocrinology and Department of Pathology, JN Medical College AMU. The cases were studied prospectively from December 2012 to November 2015. Informed consent was taken from each patient and the study design was approved by institution review committee. Metformin form one of the treatment approaches for PCOS and previous studies [10] suggest its therapeutic effect in control of the syndrome. Thus this study was performed in order to evaluate the therapeutic effect ofmetformin as treatment for PCOS.

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Lipid Profile in Patients of PCOS

Inclusion Criteria of the Study and Placebo Groups: Forty nine diagnosed cases of Polycystic Ovarain Syndrome were randomly selected who had already been tested for fasting blood sugar (FBS) serum levels of Luteinizing hormone (LH), Folllicle Stimulating Hormone (FSH), LH/ FSH, testosterone and dehydro-epiandrosterone sulphate (DHEAS) and selected for metformin treatment were included in the study group.

6 months. Both the groups were asked to follow same dietary and exercise regime during the study period. After completion of treatment , all the biochemical parameters i.e serum cholesterol, triglyceride, high density lipoproteincholesterol and low density lipoprotein-cholesterol were analyzed and compared with their pre-intervention values in study and placebo group respectively.

Similarly for placebo group age and weight matched 49 patients of PCOSwere included in the placebo group and were selected for placebo drugs

The mean age in the study and placebo group was 27.88 and 28.06 respectively and the age difference between the two groups was found to be insignificant (p-value=0.87), while the least common age group in both the groups were 36-40 years.(Table 1) (Figure 1)

Results

Exclusion Criteria; Women with prior history of glucose intolerance (including gestational diabetes or type 2 Diabetes mellitus, late onset congenital adrenal hyperplasia cushing’s syndrome, hypothyroidism, hyperthyroidism or patients taking medications to alter the hormonal profile were excluded both from study and placebo group.

Regarding the BMI, out of 98 patients a total of 37 (37.7%) patients were overweight with BMI >25kg/m2, while 25 (25.5%) patients were obese with BMI >30kg/m2. So a total of 62 cases (63.3%) were overweight and obese. (Table 2)

Material and Method

In patients of PCOS the most common complaint was infertility which was seen in 88.7%, followed by obesity which was noted in 63.2% of the patients. (Table 3)

After initial clinical assessment, all women who fulfilled the inclusion criteria were explained respective methodology of our study and an informed consent was obtained before the study. Pre-therapy serum lipid profile, thyroid function tests and serum prolactin levels were assessed besides complete history and detailed examination for each patient. The serum of patient was collected after an overnight fast of 10 -12 h, separated after centrifugation into two parts. The blood samples were centrifuged at 3000 rpm for 5 minutes, it was essential to ensure that the serum did not hemolyse. The clean serums were transferred to clean plastic tubes by micropipette. The tubeswere topped by plastic stoppers and stored at -200C in order to avoid variation, till the time of analysis. In all tests internal quality control tests was performed to detect the accuracy of the results.

Assessment of preintervention lipid profile in 49 PCOS patients of the study group revealed dyslipidemia in form of hypercholesterolemia in 43 (87.7%) cases, high LDL-C in 14 (28.5%) cases and hypertriglyceridemia in 37 (75.5%) patients. Mean serum HDL level was 37.90±5.452mg/dl. After 6 months of Metformin treatment the altered lipid profile in study group patients came down to normal level in 31 (63.3%) of patients and the decline in serum levels of total cholesterol, LDL-C and triglyceride was found to be highly significant (p<0.001), simultaneously at the same time there was also significant rise in serum HDL-C from 38.16±4.838 to 55.62±3.619, when compared to preintervention levels and to placebo group patients who had either insignificant or minimal alteration in their lipid profile after 6 months of followup (TABLE 4).

Out of the total number of 98 patients of PCOS reported, 49 PCOS patients were administered with Metformin at a dose of 500mg TDS for a period of 6 months while rest 49 PCOS females were prescribed a placebo drug for

Fasting plasma glucose showed improvement after metformin treatment but it was not significant (p-value=0.3)

Table 1: Age frequency of the PCOS patients in study and placebo group. Age in years

STUDY GROUP (%)

PLACEBO GROUP(%)

20-25

17 (34.7)

16 (32.7)

26-30

18 (36.7)

19 (38.7)

31-35

09 (18.4)

11 (22.4)

36-40

05 (10.2)

03 (6.2)

TOTAL

49 (100)

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Table 2: Body mass index frequency of the patients in study and placebo groups. BMI (kg/m2) Underweight <18.5 Normal weight (18.5-24.99) Overweight (25-29.99) Obese >30 Total

STUDY GROUP (%) 00 21 (42.8%) 16 (32.6) 12 (24.4) 49

PLACEBO GROUP (%) 00 15 (30.6) 21 (42.8) 13 (26.5) 49

Table 3: The clinical presentations of patients of PCOS. Symptoms Infertility Overweight/ obesity Irregular mensturation Hirsutism

No. Of patients (%) 87 (88.7) 62 (63.2) 38 (38.7) 21 (21.4)

TABLE 4: Lipid Profile Comparison IN The Study and Placebo Group Study group Variable (mg/dl) total chol LDL-C TG HDL-C

Placebo group

Pre-t/t

Post-t/t

p-value

Pre-t/t

Post-t/t

p-value

323.82±64.845 308.98±64.180 293.66±52.757 38.16±4.838

170.40±14.058 172.96±14.144 167.54±12.216 55.62±3.619

<0.001 <0.001 <0.001 <0.001

274.46±70.225 249.32±77.158 251.22±66.259 37.90±5.452

275.50±70.243 249.84±77.404 251.46±66.138 38.38±4.085

0.26 0.477 0.855 0.394

Fig. 1: Agewise distribution of PCOS patients.

Discussion

PCOS has been a subject of research and debate over past six decades. Insulin resistance accompanied by compensatory hyperinsulinemia is a common feature of PCOS and both obese and non-obese women with the syndrome are more insulin resistant and hyperinsulinemic than age and weight matched normal women.[11] Insulin resistance in muscles and adipose tissues increases plasma free fatty acid (FFA) and

insulin concentration, that stimulate synthesis and secretion of VLDL in the liver resulting in hypertriglyceridemia, which in turn enhances post-prandial accumulation of lipoproteins(LDL,VLDL) in plasma with lowering of HDL cholesterol.[12] Hyperinsulinemia plays a pathogenetic role in PCOS cases by increasing ovarian androgen production and decreasing the serum sex hormone binding globulin concentration.[13]

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Almost 70-80% patients of PCOS are obese. Similarly in our study, almost 64% cases were overweight and obese with BMI > 25. Obesity is associated with insulin resistance which is a well known fact by many studies, however patients of PCOS have much more insulin resistance than that of obese women in the general population.[14]

4. Futterweit W, Mechanick JI. Polycystic ovarian disease: etiology diagnosis, and treatment. ComprTher. 1988;14:12–20. 5.

Dahlgren E, Janson PO, Johansson S, et al. Polycystic ovary syndrome and risk for myocardial infarction. ActaObstetGynecolScand 1992;71:599–603.

The current study shows that a total of 43 out of 49 PCOS patients in study group had high total cholesterol (TC). The fall in TC was significant (p<0.001) when compared to placebo group after 6 months of Metformin treatment. Similarly LDL-C and triglyceride were also raised in 22 (44.8%) and 46(93.8%) of cases respectively.These two parameters also showed a significant fall after Metformin treatment (p<0.001),when compared to placebo group after Metformin treatment. HDL-C rose significantly from mean level of 38.16±4.838 to 55.6±3.619mg/dl after Metformin treatment (p<0.001) when compared to placebo group. The current study results of positive effect of Metformin treatment on lipid profiles in PCOS patients was also corresponding with the results obtained by Karimzadeh et al (2007) and Bratati et al. (2010).Other studies done by Moghetti et al and Velazquez et al showed only a negligible or no effect on serum lipids in women with PCOS.These discrepancies in the result among researches could be explained by difference in the studied population or by shorter duration of treatment.[15] It has been found by some studies done by Morin-papunen et al and Glueck et al that the beneficial effect of Metformin on serum lipid is related to the duration and the dosage of the treatment.

Hopkinson Z, Satter N, Fleming R et al. Polycystic ovarian syndrome: the metabolic syndrome comes to gynaecology, BMJ 1998;317:329-32.

Kiddy DS, Sharp PS, White DM, et al. Differences in clinical and endocrine features between obese and non-obese subjects with polycystic ovary syndrome: an analysis of 263 consecutive cases.ClinEndocrinol (Oxf) 1990; 32:213-20.

Shaw LJ, BaireyMerz CN, Azziz R, et al. Postmenopausal women with a history of irregular menses and elevated androgen measurements at high risk for worsening cardiovascular event-free survival: results from the National Institutes of Health-National Heart, Lung, and Blood Institute sponsored Women’s Ischemia Syndrome Evaluation. J ClinEndocrinolMetab 2008, 93:1276-1284.

Messaoudi S, Rongen GA, Riksen, “The cardioprotective effects of metformin”. Current Opinion in Lipidology 2011;22: 445–53.

Conclusion

Metformin- an insulin sensitizer has been shown to improve the serum lipid profiles leading to increase the HDL cholesterol, the main predictive of cardio vascular disease in the women with PCOS.

References

10. Dunaif A, Insulin resistance and polycystic ovary syndrome: mechanism and implication for pathogenesis. Endocrine Review 1997; 18: 774-800. 11. Bengtsson C, Bjorkelund C, Lapidusal, Lissnerl. Association of serum lipid concentration and obesity with mortality in women. Twenty years fellow up of participants in prospective population study in Gothenbrg, sweden. Br Med J 1993:1385-88. 12. Zawadski JK, Dunaif A . Diagnostic criteria for polycystic ovary syndrome. Towards a rational approach in: polycystic ovary syndrome. 1992:377-84. 13. Jacobs HS. Polycystic ovaries and polycystic ovary syndrome.GynecolEndocrinol 1987;1:113-31.

Ehrmann DA. Polycystic ovary syndrome. N Engl J Med 2005; 352: 1223-36.

Al-Inany H, Johnson N. Drugs for anovulatory infertility in polycystic ovary syndrome. BMJ 2006; 332:1461–62.

14. Fadhil AZ, Al-Dahhan F. The Effect of Metformin on Serum Lipids in Overweight and Obese Patients with Polycystic Ovary Syndrome.The Iraqi Postgraduate Medical Journal. 2014;13:3.

Michelmorek, Balen AH, Dunger DB, Vezelymp.Poycystic ovaries and associated clinical and biochemical features in young women.Clinendocrinol(Oxford).1999;51:779-86.

15. Lobo RA, Carmina E. The importance of diagnosing the polycystic ovary syndrome. Ann intern Med 2002;17:2495-99.

*Corresponding author: Noora Saeed, Senior resident, department of pathology, JNMCH, AMU, ALIGARH, U.P- 202002(India) Phone: +91 9997240844 Email: dr.noorasaeed@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 26.12.2016 Date of Acceptance : 11.06.2017 Date of Publication : 31.08.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Original Article DOI: 10.21276/APALM.1228

Estimation and Correlation of Different Hemoglobin Levels in HbE Hemoglobinopathies in Indian Population Using Capillary Electrophoresis Method Abhijit Kalita1* and Avinanda Mahanta2 2

1 Department of pathology, Pathcare Labs Pvt. Ltd., Hyderabad, India. Department of Biochemistry, Guwahati Medical College Hospital, Guwahati, India

ABSTRACT Background: Capillary electrophoresis (CE) estimates Hemoglobin E (HbE) in HbE hemoglobinopathies, which was previously not possible with other method due to combined elution of HbA2 with HbE . Associated hemoglobin abnormalities can be estimated with separation of HbA2 from HbE. Methods: The study is retrospective using CE to detect abnormal HbE and differentiate the HbE syndromes. Student t-test was used for statistical analysis. Result: 119 cases of HbE syndromes were identified and divided into HbE heterozygotes (71), HbE heterozygotes with borderline HbA2 (15), compound heterozygotes of HbE with Thalassemia (13 HbE with β-Thalassemia / 8 HbE with α-Thalassemia), compound heterozygotes of HbE with HbS (HbSE) (2) and HbE homozygotes (10). Mean HbA2 shows increasing pattern with increasing severity of HbE syndrome. However, compound heterozygote of HbE and β-Thalassemia (HbE-β-Thal) shows maximum mean level of HbA2 (5.46%). HbA2 of HbE heterozygote and HbE heterozygote with borderline HbA2 was not found to be significantly different, statistically. Fetal haemoglobin (HbF) of HbE homozygotes is found significantly higher than that of HbE heterozygotes, but significantly lower than that of HbE-Thalassemia. The HbE values of HbE with α-Thalassemia (HbE-α-Thal) and HbE-β-Thal were found to be below -3SD value (14.77%) and beyond +3SD value (37.77%) of mean of HbE of HbE heterozygote (26.27%), respectively. Conclusion: The study identifies range of different hemoglobin levels in HbE syndromes, with special reference to HbA2. Concurrent iron deficiency anaemia also needs to be kept in mind in dealing with a population where iron deficiency is very common. Keywords: Capillary Electrophoresis, Hemoglobin E heterozygote, Hemoglobin E

Introduction

clinical laboratories (11).

Hemoglobin E (HbE) was the fourth abnormal hemoglobin described (1). HbE is the second most prevalent hemoglobinopathy after Sickle cell hemoglobinopathy (HbS), showing the highest prevalence in South-East Asia (2) . In India, it is prevalent in the north-eastern states (2,3,4,5). Heterozygosity and homozygosity of HbE are clinically mild, whereas compound heterozygosity for HbE and HbS (HbSE) and compound heterozygosity for HbE and β-Thalassemia (HbE-β-Thal) are clinically severe (3,6,7,8,9). The co-inheritance of HbE with a host of other globin mutants results in a wide spectrum of hemoglobinopathies with varying degrees of severity (HbE disorders or HbE syndromes) (1,10).

Capillary electrophoresis (CE) has the ability to completely separate HbA2 from HbE (12,13,14). This enables to quantitate exact levels of HbE and HbA2 , assisting in identification of co-inherited hemoglobinopathies.

All the methods used previously were not able to distinguish between HbE and HbA2. While reverse phase HPLC has been reported to provide an estimate of HbA2 in the presence of HbE, this method is not routinely used in

The present study is a retrospective study conducted for a period of one year (June 2015 to May 2016). Capillary electrophoresis method was used for identification of abnormal hemoglobin variants. Patients with abnormal

The present study is conducted to identify levels of different hemoglobins in HbE syndromes and to find their correlation, if any. Most of the studies conducted previously on hemoglobinopathies in the Indian population have highlighted the increased prevalence of HbE and associated syndromes. This study attempts to define the range of abnormality in the HbE syndromes.

Materials and Methods :

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HbE peaks and without any treatment history (particularly of blood transfusion) were included in the study, to have an accurate estimation of the hemoglobin levels. Cases with abnormal hemoglobin E peaks were correlated with the Complete blood count (CBC) and peripheral blood smear (stained with Leishman’s stain) to conclude a final diagnosis. Cases were classified as HbE heterozygotes, HbE homozygotes and compound heterozygotes of HbE with other hemoglobinopathies (like sickle cell and Thalassemia). Another category, HbE with borderline HbA2, was classified in the present study, to find out if any significance exists. The mean values of HbA2 were compared statistically (using student t-test) amongst the HbE categories / syndromes, and also with control group of 30 normal individuals and 30 β-Thalassemia cases.

compound heterozygotes of HbE with Thalassemia (both α-Thalassemia and β-Thalassemia combined) was found to be significantly higher (p-value <0.05) than that of HbE heterozygotes, HbE heterozygotes with borderline HbA2 and HbE homozygous, but there is no significant difference (p-value >0.05) with that of β-Thalassemia control group. Compound heterozygotes of HbE with HbS was not considered for statisitical analysis due to small size. There is an increasing trend in the mean value of HbA2 from HbE heterozygote to HbE homozygous, as shown in Table 1. However, the mean of HbA2 in HbE-β-Thal exceeds that of HbE homozygotes.

Result

A total of 119 cases of HbE syndromes were identified during the study period, out of which 71 were HbE heterozygotes, 15 were HbE heterozygotes with borderline HbA2 , 21 were compound heterozygotes of HbE with Thalassemia (13 with β-Thalassemia and 8 with α-Thalassemia), 2 were compound heterozygotes of HbE with HbS and 10 were HbE homozygotes. The distribution of the cases into the different categories, along with levels of HbA2 are shown in Table 1. On statistical analysis, there was no significant difference (p-value >0.05) between the mean value of HbA2 in HbE heterozygotes, HbE heterozygotes with borderline HbA2 , and normal control group. The mean of HbA2 in

The mean value of HbE was found to be 26.27% in HbE heterozygotes, 31.08% in HbE heterozygotes with borderline HbA2 and 83.67% in HbE homozygotes. The compound heterozygotes of HbE with Thalassemia have been divided into two groups - HbE-β-Thal (Mean HbE level 51.97%) and HbE-α-Thal (Mean HbE level 16.65%). Although the sample size for HbSE was too small for calculation, however, the mean HbE in HbSE was calculated to be 9.75%. The fetal hemoglobin (HbF) level (Table 2) was found to be increased (than normal range in adults) in compound heterozygotes of HbE-α-Thal (Mean 19.71%), compound heterozygotes of HbE with β-Thalassemia (Mean 9.28%), compound heterozygotes of HbE with HbS (Mean 4.9%) and HbE homozygotes (Mean 5.41%). It was within normal range in HbE heterozygote (Mean 0.48%) and HbE heterozygote with borderline HbA2 (0.36%).

Table 1: HbA2 levels in different HbE syndromes. Sl no.

Type of HbE (with total number of cases)

HbA2 level(%)(Mean,±2SD)

HbE heterozygote (71)

2.98, ±0.8

HbE heterozygote with borderline HbA2 (15)

3.76, ±0.31

Compound heterozygote of HbE with β- Thalassemia (13)

5.46, ± 2.48

Compound heterozygote of HbE with α- Thalassemia (08)

3.10, ± 1.64

Compound heterozygote of HbE with HbS (2)

3.1, ±0.56

HbE homozygotes (10)

3.52, ±2.0

Table 2: Fetal haemoglobin (HbF) level in HbE syndromes Sl no.

Type of HbE (with total number of cases)

HbF level(%)(Mean,±2SD)

HbE heterozygote (71)

0.48,±1.68

HbE heterozygote with borderline HbA2 (15)

0.36, ± 1.28

Compound heterozygote of HbE with beta-Thalassemia (13)

Compound heterozygote of HbE with alfa-Thalassemia (8)

9.28, ± 7.84

Compound heterozygous of HbE and HbS (2)

4.9, ± 6.50

HbE homozygotes (10)

5.41,± 6.17

19.71,± 26.60

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Kalita et al.

Discussion

The CE method allows detection and separation of most of the common hemoglobin variants (15,16). The complete separation of HbA2 from HbE deserves note (17). The data obtained from 117 cases shows an increasing trend of the level of HbA2 in the HbE syndromes with increased severity of HbE. However, the level of HbA2 in HbE-β-Thal is significantly higher than all other categories, including HbE homozygous. This trend is similar to the study conducted by Mais DD et al (6), but the significant increased level of HbA2 seen in HbE heterozygotes compared with that of normal control group in the study of Mais DD et al (6) , is not seen in the present study. Morever, the category defined in this study “HbE with borderline HbA2” also does not have significant difference of HbA2 level from the normal control and HbE heterozygous categories. The study conducted by Keren DF et al (17) suggested that the levels of HbA2 are over – estimated by capillary electrophoresis method but the study also argues that in HbE syndromes, relative increase in HbA2 levels is expected, because HbE is a structural form of beta - Thalassemia. The range defined for HbA2 for HbE heterozygotes in the present study (Table 1) is lower than that described by Mais DD et al (6) (Mean ± 2Sd, 3.4% ± 0.4%) and Winichagoon P et al (18) (Mean ± 2SD, 3.5% ± 0.4%). However, the range of HbA2 defined for HbE heterozygous with borderline HbA2 is found to be in comparison with the mentioned studies. This can be attributed to concurrent iron deficiency anaemia, particularly in the Indian population, which lowers the levels of HbA2 , as evidenced by studies of Rao S et al (19) and Denic S et al (20). The fetal hemoglobin (HbF) of HbE homozygotes is found to be significantly higher than that of HbE heterozygotes, but significantly lower than that of HbE - Thal. The level of HbF inversely correlate well with proportion of HbA in the haemoglobin variant. In HbE homozygous, however, the HbE levels lowers the HbF levels, inspite of low HbA level (Mean HbA 7.4%). HbF is affected significantly by alfa / Beta - globin chain imbalances, as evidenced by study of Lin WF et al (21). The mean of HbE levels of HbE heterozygote in the present study was found to be 26.27% ± 7.6% (Mean ± 2SD) which is in comparison with the study of Charoenkwan P et al (22) (23.3% ± 6.2%) and Hafiza A et al (23) (24.28% ± 6.76%). The HbE values of HbE with α – Thalassemia were found to be below the 3SD value (14.77%) of the mean of HbE level of HbE heterozygote (26.27%), with few exceptions. However, in those cases where HbE values are within 3SD of mean of HbE, the low hemoglobin level and the www.pacificejournals.com/apalm

A-421 peripheral blood picture were suggestive of an associated Thalassemic abnormality. Again, the HbE levels of HbE-βThal were beyond the 3SD value (37.77%) of the mean of the HbE heterozygote. This is in correlation with the study of Mais DD et al (6). Only two cases of HbSE were encountered during the study period, the mean of sickle peak being 23.55% and HbE 9.75%, with mean HbF peak being 4.9%. The peripheral blood picture showed elongated cells, suggestive of the sickle abnormality. However, blood transfusion history was elucidated in these patients, so exact quantification of the different hemoglobin levels were not possible. Inclusion of more number of cases is required for further statistical analysis.

Conclusion

The present study identifies the range of different hemoglobin levels in HbE syndromes, particularly with reference to HbA2 level, which helps to detect associated abnormalities. The concurrent iron deficiency anaemia also needs to be kept in mind in dealing with a population where iron deficiency is very common. Genetic studies need to be performed, particularly in compound heterozygotes, to define the exact range of HbE levels with each gene deletion.

Acknowledgements

I want to thank my seniors and technical staff for their support. There is no conflict of interest declared.

Reference : 1.

Kaushansky A, Lichtman MA, Beutler E , Kipps TJ, Seligsohn U, Prchal JT. editors. Williams Haematology. 8th ed. New York : McGraw-Hill Companies; 2010

Aggarwal S, Saluja S, Bhasin S, Sharma M, Gupta DK, Gupta B, Mittal V. HbE variants – Retrospective analysis in a tertiary care centre. J Indian Acad Clin Med 2011; 12(4): 263-265

Tyagi S, Pati HP, Choudhary VP, Saxena R. Clinico heemotological profile of HbE syndrome in adults and children. Haematology 2004; 9: 57-60

Deka R, Gogoi BC, Hundrieser J, et al. Hemoglobinopathies in northeast India. Hemoglobi. 1987; 11 :531-538

Singh RM, Choudhury B, Singh TS. Hemoglobin E distribution in four endogamous populations of Manipur (India). Eurasian J Anthropol 2010: 1(2): 109-117

Mais DD, Gulbranson RD, Keren DF. The range of hemoglobin A2 in Hemoglobin E heterozygotes as determined by capillary electrophoresis. Am J Clin Pathol 2009; 132: 34-38

Vichinsky E. Hemoglobin E syndromes. Hematology Am Soc Hematol Educ Program 2007: 79-83

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Different Hemoglobin Levels in HbE Hemoglobinopathies

Premawardhena A, Fisher CA, Olivieri NF, et al. Hemoglobin E β Thalassaemia in Sri Lanka. Lancet 2005; 366: 1467-1470.

Mishra P, Pati HP, Chatterjee T, et al. Hb SE disease: a clinic – hematological profile. Ann Hematol 2005; 84: 667-670

17. Keren DF, Hedstrom D, Gulbranson R, Ou C, Bak R. Comparison of S ebia Capillarys Capillary Electrophoresis With the Primus High – Pressure Liquid Chromatography in the Evaluation of Hemoglobinopathies. Am J Clin Path 2008; 130: 824-831

10. Greer JP et al .editors. Wintrobe’s Clinical Hematology. 12th ed. Philadelphia: Lippincott Williams & Wilkins; 2009 11. Schroeder WA, Shelton JB, Shelton JR et al. The estimation of HbA2 in the presence of HbC or HbE by reverse phase high performance liquid chromatography. Hemoglobin 1986; 10: 253-257 12. Keren DF, Hedstrom D, Gulbranson R et al. Comparison of Sebia Capillarys electrophoresis with the Primus high - pressure liquid chromatography in the evaluation of hemoglobinopathies. Am J Clin Pathol 2008; 130: 824-831 13. Cotton F, Changying L, Fontaine B et al. Evaluation of a capillary electrophoresis method for routine determination of hemoglobins A2 and F. Clin Chem. 1999; 45: 237-243 14. Jenkins MA, Hendy J, Smith IL. Evaluation of hemoglobin A2 quantification assay and hemoglobin variant screening by capillary electrophoresis. J Capillary Electrophor 1997; 4: 137-143 15. Cotton F, Malaviolle X, Vertongen F, Gulbis B. Evaluation of an automated capillary electrophoresis system in the screening for hemoglobinopathies. Clin Lab 2009; 55 (5-6): 217-221 16. Cotton F, Wolff F, Gulbis B. Automated capillary electrophoresis in the screening for hemoglobinopathies. Methdods Mol Biol 2013; 984: 227-23

18. Winichagoon P, Svasti S, Munkongdee T, et al. Rapid diagnosis of thalassemias and other hemoglobinopathies by capillary electrophoresis. Transl Res. 2008; 152: 178-184 19. Rao S, Kar R, Gupta SK, Chopra A, Saxena R. Spectrum of hemoglobinopathies diagnosed by cation exchange HPLC & modulating effects of nutritional deficiency anaemias from north India. Indian J Med Res 2010; 132: 513-519 20. Denic S et al. Hemoglobin A2 Lowered by Iron Deficiency and α- Thalassemia: Should Screening Recommendation for β-Thalassemia Change ISRN Hematol 2013: 1-5 21. Lim WF, Muniandi L, George E, Sathar J, The LK, Lai MI. HbF in HbE / β -thalassemia: A clinical and laboratory correlation. Hematol 2015; 20 (6): 349-353 22. Charoenkwan P, Wanapirak C, Thanarattanakorn P, Sekararithi R, Sae - Tung R, Sittipreechacharn S, Sanguansermsri T. Hemoglobin E levels in double heterozygotes of hemoglobin E and SEA - type alpha - Thalassemia. Southeast Asian J Trop Med Public Health 2005; 36 (2): 467-70 23. Hafiza A, Malisa MY, Khirotdin RD, Azlin I, Azma Z, Thong MC et al. HbA2 levels in normal, beta - Thalassaemia and hemoglobin E carriers by capillary electrophoresis. Malays J Pathol. 2012; 34 (2): 161-4

*Corresponding author: Abhijit Kalita, Address: House no 66, Happyvilla, Ujanbazar, Guwahati, Assam, India. Pin: 781003 Phone: +91 8811039142 Email: abhighy1985@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 25.12.2016 Date of Acceptance : 01.05.2017 Date of Publication : 31.08.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Original Article DOI: 10.21276/APALM.1247

A Prospective & Multicentric Study of RBC Parameters in Patients of Sickle Cell Disorder Prateek Pradeep Umrikar* and Alpesh Prahladpuri Goswami Department of Pathology, Government Medical College, Bhavnagar, INDIA

ABSTRACT Background: Sickle cell disease (SCD) is an inherited blood disorder caused by abnormal haemoglobin. Present study was done to study the relationship of clinical presentation & haematological findings in case of symptomatic & asymptomatic sickle cell disorder patients. Methods: In the present study, the RBC parameters like Hb, MCV, MCH, MCHC, RDW, RBC COUNT & PCV were recorded & results of sickling test & Hb electrophoresis were used to confirm a case of sickle cell disorder. Result: Categorical Variables (age &sex ) were expressed in actual number & percentages. Continuous variables ( Hb, MCV, MCH, MCHC, RDW, RBC COUNT & PCV ) were presented as Mean. Continuous variables were compared between Sickle cell disease subjects, sickle cell trait subjects & normal subjects by one way Analysis of Variance (ANOVA) test.p value < 0.05 was considered to be statistically significant Conclusion: The most number of symptomatic patients of sickle cell disorder were found in the 2nd decade of age, followed by 3rd decade. There was a greater percentage of female subjects detected as sickle cell Disease (52.7%) & Sickle cell trait (62.8%) which points towards female preponderance of sickle cell disorder. Weakness & fatigue were the predominant symptoms of presentations among both sickle cell disease & sickle cell trait subjects. The peripheral smear examination in sickle cell disorder predominantly shows normocytic to microcytic with hypochromic picture. Keywords: Sickle cell Disorder, RBC Parameters, Hb Electrophoresis, Sickling Test

Introduction

Sickle cell disease (SCD) is an inherited blood disorder caused by abnormal haemoglobin. It limits the oxygenating role of haemoglobin, resulting in the damaging or the “sickling” of the red blood cells .There is high prevalence of sickle cell disease in socioeconomically backward groups in India. It is highly prevalent among schedule castes , schedule tribes & other backward classes(10 %).[1] It is a 2nd most common haemoglobinopathy next to thalassemia in India, general incidence of sickle cell disease is 1-44%. The average frequency of haemoglobin–S (HBS) is 4.3 % in India.[2][3] Sickle cell disease refers to a group of genetic disorders, characterised by presence of sickled haemoglobin (HBS), anemia, acute & chronic tissue injury secondary to blockage of blood flow by abnormally shaped Red cells. It is one of the variants of disorders of haemoglobin inherited from both the parents in an autosomal recessive pattern. In this disease, there is single nucleotide substitution in codon 6 of beta-globin chain of haemoglobin molecule. Out of the total 287 amino acids at the position 6th of beta chain in haemoglobin molecule, there is substitution of one amino acid. This change occurs because of the substitution of Triplet GAG (guanine, adenosine, and guanine) which

codes for Glutamic acid by triplet GTG (guanine, thymine, guanine) which codes for Valine. There is polymerisation of HBS molecules inside the red cells which is responsible for sickling of red cells. People with SCD have abnormal hemoglobin, called hemoglobin S or sickle hemoglobin, in their red blood cells. In all forms of SCD, at least one of the two abnormal genes causes a person’s body to make hemoglobin S. When a person has two hemoglobin S genes, Hemoglobin SS, the disease is called sickle cell anemia. This is the most common and often most severe kind of SCD.

Materials and Methods:

This hospital based cross sectional study was carried out in the Department of Pathology. Sample size of this study was 100 subjects which includes Sickle cell disease, Sickle cell trait diagnosed by hemoglobin electrophoresis inclusive of Normal subjects. These 100 patients were grouped according to test results into 36 symptomatic patients of Sickle cell Disease, symptoms being: severe anemia, weakness, recurrent jaundice, recurrent fever & bone pain etc, 35 symptomatic & asymptomatic patients of Sickle cell trait & 29 Normal subjects. With the informed consent of these subjects, a case record form was filled, which included all the detailed information like name,

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RBC Parameters in Sickle Cell Disorder

age, sex, registration number, caste, address, patients chief complaints, family history, complaints related to this disease, lab investigation, general examination etc. Then according to these clinical details, these were grouped into symptomatic and asymptomatic patients. Details of the normal controls (AA) were also recorded.

Variance (ANOVA) test. ‘p’ value < 0.05 was considered to be statistically significant. SPSS-22 trial version software was used for Data analysis. The above table demonstrates the age of symptomatic presentationin sickle cell disease patients is predominantly between 11 & 20 years in the present study (47.2%)

Collection of blood: Under all aseptic precautions, 2 ml of blood was drawn from antecubital vein by clean venepuncture from each patient and collected in an EDTA (anticoagulant) tube for determination of investigations like Sickling test, CBC (Complete Blood Cell count), Hemoglobin electrophoresis. p value <0.05 was considered to be statistically significant. SPSS-22 trial version software was used for data analysis.

The above table demonstrates Female preponderance in cases of both sickle cell disease (52.7%) & Sickle cell Trait(62.8%). In the present study , Weakness & fatigue has been observed to be the predominant presenting symptom amongst subjects of sickle cell disease (94.4%) & Sickle cell trait (77.1%) ,followed by recurrent jaundice (66.6%) in Sickle cell disease subjects & (2%) in sickle cell trait subjects as the second most predominant symptom

Result

Categorical Variables (age &sex) were expressed in actual number & percentages. Continuous variables (Hb , MCV,MCH,MCHC, RDW,RBC COUNT & PCV) were presented as Mean . Continuous variables were compared between Sickle cell disease subjects, sickle cell trait subjects & normal subjects by one way analysis of

The above table displays the mean values & ‘p’ values of all the RBC Parameters. The ‘p’ values of all RBC parameters in patients of Sickle cell Disease & Sickle cell Trait are < 0.001 which indicates that these values are statistically significant.

Table 1: Age distribution of symptomatic presentation in patients of sickle cell disease. Age of Presentation (Years)

No. Of Sickle cell disease Patients

below 10

7 (19.4%)

11-20

17(47.2%)

21-30

10(27.7%)

31-40

02(5.5%)

41-50

51-60

61-70

Table 2: Sex distribution of sickle cell disorder. Males

Females

Sickle Cell Disease

17(47.2%)

19(52.7%)

Sickle cell Trait

13(37.1%)

22(62.8%)

Total Table 3: Clinical presentation of sickle cell disorder.

Sickle cell disease

Sickle cell trait

symptoms Weakness & fatigue

34 (94.4%)

27(77.1%)

Recurrent fever & cough

15(41.6%)

02(5%)

Recurrent fever & bone pain

09(25%)

01(2%)

Breathlessness

18(50%)

07(20%)

Recurrent jaundice

26(72.2%)

01(2%)

Recurrent hospitalisation

24(66.6%)

01(2%)

h/o frequent blood transfusions

24(66.6%)

01(2%)

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Umrikar et al.

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Table 4: Mean values & ‘p’ values of all RBC Parameters in Sickle cell disease & Sickle Cell trait subjects. Mean Value

Parameters

Sickle cell Disease

Sickle cell Trait

‘P’ value

Hb (gm/dl)

7.43

7.97

<0.001

MCV (fl)

73.69

75.08

<0.001

MCH (pg)

23.62

23.52

<0.001

MCHC (gm/dl)

31.76

31.37

=0.005

RDW (%)

20.07

18.02

<0.001

RBC Count (million/ul)

3.23

3.45

<0.001

PCV (%)

24.50

29.82

<0.001

Fig.1

Fig.2

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RBC Parameters in Sickle Cell Disorder

Fig.3

Discussion

The present study demonstrates the haematological profile of sickle cell Disease patients & Sickle cell Trait patients. Total 100 subjects were taken in our study which included 36 cases of Sickle cell Disease, 35 cases of Sickle cell Trait &29 cases of Normal Control subjects. The most number of patients were found in the 2nd decade of age followed by 3rd decade. There were a greater percentage of female subjects detected as sickle cell Disease (52.7%) & Sickle cell trait (62.8%) in their respective groups which points towards female preponderance of sickle cell disorder. In the present study weakness & fatigue are the predominant symptoms of presentations among both sickle cell disease & sickle cell trait subjects. This finding is comparable with many other reference studies In our study concerned with haematological parameters of sickle cell disease & trait subjects ,showed an overall decrease in the level of haematological indices as similarly reported by study done by Kohchale et al (2015).[4] Our study shows decreased levels of RBC count , MCV , Hematocrit (PCV) which correlates with the study done by Kar et al (1986).[5] Balgir et al (2000)[3] in his study showed lower MCV levels (60-113 fl).Rao et al (2012) [6] showed lowering of above parameters in their respective studies, thus supporting our study. In our study, it is observed that the mean MCV levels were low in sickle cell disease as compared to the levels in Sickle cell trait. One more significant observation in our study results are that ,with the decrease of MCH , MCHC , there is increase in Red cell Distribution Width (RDW) in both the groups ,which correlates well with the similar

studies done by Kar et al (1986) ,[5] Balgir et al (2000).[3] Mean PCV level in our study in sickle cell disease is 24.52 which is comparable with mean PCV level of 20.0 in study conducted by Ifeanyi et al. [7] Sickle cell Disease is associated with anemia in majority of cases. The mean haemoglobin value in sickle cell disease subjects in present study is 7.43 g/dl. Study by Sameer et al [8]& Study by Lagos State University Teaching Hospital Ikeja, Nigeria [9] observed Haemoglobin levels (7.93 g/ dl), while Kohchale and Raja[4] et al observed lower levels of Mean haemoglobin levels (5.454g/dl) . Thus all the studies demonstrate that there is a net significant fall in Haemoglobin levels in Sickle cell disease cases. The mean MCV levels in present study & study done by Sameer et al[8]&Kohchale and Raja[4] et al are lower than normal range & indicate an overall normocytic with mild microcytic picture in sickle cell disease. This finding comes close to the fact that usually the anemia in sickle cell disease is normocytic. The study by Lagos State University Teaching Hospital Ikeja, Nigeria ,[9] supports this finding. The mean MCH & MCHC Values in all the studies including the present study show an overall slightly lower value which are on borderline in some of the above studies. This indicates hypochromic picture in sickle cell disease patients, which can be due to the chronic anemia in them &also can be due to concomitant iron deficiency. The RDW levels show persistent elevation in all the studies which is a consistent finding related with sickle cell disease which generally shows an increase in RDW levels which indicates an increased variation in the red cell sizes, which is expected to occur in case of sickle cell disease due to

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Umrikar et al. recurrent hemolysis & ineffective erythropoiesis, resulting in anisocytosis. Hematocrit & RBC Counts are consistently lower in all the above studies including our study with a higher reduction observed in study by Kohchale et al.[4] As with case of any anemia, reduction in PCV & RBC count are the most obvious findings & likewise also in sickle cell disease which presents with severe haemolytic anemia. Sickle cell trait patients are usually associated with normal levels of Haemoglobin or slight reduction which comes below the normal range. The present study shows slightly lower levels of Hb levels due to the fact that all the sickle cell trait subjects in our study have presented with symptoms related to crisis or some concomitant stressful event which has lead to the deoxygenating event leading to an occasional sickling which has reduced the mean Hb levels. The MCH & MCHC levels show slight reduction in all studies due to the overall reduced reservoir of haemoglobin in the RBCs in sickle cell trait subjects related to recurrent & occasional mild sickling episodes during the stressful events which reduce the overall haemoglobin concentration inside the RBCs. The consistent reduction in RDW levels in all the above studies is related with the variation in the cell size (anisocytosis) associated with sickle cell trait patients. Similarly RBC count & haematocrit show a slight reduction in sickle cell trait patients in all the studies & this reduction corresponds with the mild anemia seen in sickle cell trait which is less severe as compared to sickle cell trait patients.

Conclusion

In this study we have correlated the clinical presentations of sickle cell disorder with the haematological parameters. Even one more step ahead, our results can be helpful in encouraging the high risk families of sickle cell disorder to go for proper genetic counselling, in which patients or relatives at risk of an inherited disorder , are advised of the consequences & nature of the disorder, the probability of developing &transmitting the disease & the options open to them in management & family planning in order to prevent, avoid or ameliorate it. This includes the neonatal screening programs, which demonstrates the benefit to small babies regarding morbidity & also premarital marriage counselling. The purpose of the study is that the

A-427 clinics involved in supervision of sickle cell anemia patients would become more up to date & make use of results of this study in their practice ranges. This study would make experts involved in inspecting Sickle cell anemia patients to make use of these findings in their skill to manage this genetic menace in the region.

Acknowledgements

Dr. S. N. Shah, Dr. S. N. Baxi, Dr. P. H. Shah, Dr. G. K. Chauhan, Dr. Prateek Umrikar, Dr. Vikas Sinha, all the member of Institutional Review Board (Human Ethics Committee) and Department of Paediatrics

Reference 1.

Kate SL, Lingojwar DP. Epidemiology of Sickle Cell Disorder in the State of Maharashtra. Int J Hum Genet. 2002; 2(3):161-67.

Balgir RS, Sharma SK. Distribution of sickle cell hemoglobin in India. Indian J Hemat. 1988; 6:1-14.

Mohanty D, Mukherjee MB. Sickle cell disease in India. CurrOpinHematol. 2002; 9:117.

Kohchale SR, Raja IA. Hematological Profile of Sickle Cell Anemic Subjects from Gadchiroli District, Maharashtra, International J. of Life Sciences, 2015; Special Issue, A3,:153-156.

Kar BC, Devi S. Clinical profile of sickle cell disease in Orissa. Indian J Pediatr. 1997; 64:73-7.

Rao SS, Goyal JP, Raghunath SV, Shah VB. Hematological profile of sickle cell disease from South Gujrat, India. Hematol Rep. 2012;4(2):e8.

Ifeanyi1 OE, Nwakaego OB, Angela IO, Nwakaego CC. Haematological parameters among sickle cell anemia in steady state & haemoglobin AA individuals at Micheal Okpara, University of agriculture, Umudike, Abia state, Nigeria. Int.J.Curr. Microbiol. App. Sci , 2014; 3:1000-1005

Sameer MA, Kate R, Dagar V, et al. Diagnosis & haematological parameters of various haemoglobinopathies in paediatric age group by using Cation exchange HPLC : A hospital based cross sectional study, IOSR Journal of Dental and Medical Sciences 2016;15:81-86.

Akinbami A, Dosunmu A, Adediran A, Oshinaike O, Adebola P. Haematological values in homozygous sickle cell disease in steady state and haemoglobin phenotypes AA controls in Lagos, Nigeria. BMC Res Notes. 2012 1;5:396. doi: 10.1186/1756-0500-5-396

*Corresponding author: Dr. Prateek Umrikar, 3rd year Resident, Department of Pathology, Govt. Medical College, Bhavnagar, Gujarat-364001 India Phone: +91 9925243016 Email: prateekeuro21@gmail.com Date of Submission : 02.01.2017 Date of Acceptance : 31.05.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

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eISSN: 2349-6983; pISSN: 2394-6466

Original Article DOI: 10.21276/APALM.1254

Determination of An Optimum Cut-off Point for % fPSA/tPSA to Improve Detection of Prostate Cancer Vineeth* G Nair and M. H. Shariff Department of Pathology, Yenepoya Medical College, Karnataka, India

ABSTRACT Background: Total serum prostate specific antigen (tPSA) and free prostate specific antigen (fPSA) are known to be useful in the detection of prostate carcinoma (PCa). It has been reported that %fPSA/tPSA is more accurate when it comes to distinguishing PCa from non-malignant conditions such as BPH. The recommended cut-off value of %fPSA/tPSA in western countries is 20-25%. Through this study, we aim to determine an optimum cut-off value for %fPSA/tPSA in an Indian population. Methods: This study was performed at our institution between September 2015 and August 2016. The study population included 181 patients who had prostate enlargement and who then underwent PSA based prostate cancer screening with tPSA and %fPSA/tPSA and whose diagnosis was later confirmed by histopathology. An ROC curve analysis was performed to determine sensitivity, specificity and other performance characteristics. The optimum cut-off value of %fPSA/tPSA was determined from ROC curve using Youden’s index. Result: Malignant histology was seen in 17 (9.4%) cases. ROC curve analysis of %fPSA/tPSA revealed an AUC value of 0.777. The cutoff value of %fPSA/tPSA having the optimum balance between sensitivity and specificity was found to be 12.07% (Sensitivity: 70.6%, Specificity: 84.8%, Positive predictive value: 0.324, Negative predictive value: 0.965, Positive likelihood ratio: 4.631 and Negative likelihood ratio: 0.347). Conclusion: The cut-off value of %fPSA/tPSA obtained from our study (12.07%), which was conducted on a South Indian population, is different from the cut-off values seen in western countries and in many studies conducted in western populations. Keywords: Prostate Specific Antigen, Prostatic neoplasms, ROC Curve, Prostatic Hyperplasia

Introduction

Serum PSA levels are commonly used for early detection of prostate cancer (PCa).[1] Many conditions – including benign ones - can, however, cause an increase in the serum PSA levels.[2] A few studies also indicate that not all cases of PCa are associated with a high PSA level.[3] PSA exists in the plasma as a complex with serine protease inhibitors. These include α1-antichymotrypsin, α1-protease inhibitor, and α2-macroglobulin.[4] However, approximately 10-30% of the total PSA (tPSA) is not bound to serum proteins.[5] This is termed free PSA (fPSA). [5] Studies have shown that a lower ratio/percentage of free PSA to total PSA (%fPSA/tPSA) is seen in PCa as compared to benign conditions.[5] It was then found that %fPSA/tPSA could be useful in detecting PCa in patients whose serum PSA values lie within the gray zone i.e. 4-10 ng/mL.[6] It has also been reported that %fPSA/tPSA is a more accurate when it comes to distinguishing PCa from non-malignant conditions such as BPH.[7] The recommended cut-off value of %fPSA/tPSA in western countries is 20-25%.[8][9] Through this study, we aim to

determine an optimum cut-off value for %fPSA/tPSA in an Indian population.

Materials and Methods:

This study was performed at our institution between September 2015 and August 2016. The study population included 181 patients who had prostate enlargement and who then underwent PSA based prostate cancer screening with tPSA and %fPSA/tPSA and whose diagnosis was later confirmed by histopathology. Serum tPSA levels were measured using an immunometric assay (Vitros 5600, Ortho-clinical diagnostics, Buckinghamshire, UK) with an analytical sensitivity of 0.036 ng/ML. Serum fPSA was analysed by immunometric assay (Vitros 5600, Ortho-clinical diagnostics, Buckinghamshire, UK) with an analytical sensitivity of 0.007ng/mL. %fPSA/tPSA values were automatically calculated by the VITROS 5600. Data analysis was performed in SPSS version 23. Continuous data was expressed as mean or as median. A confidence interval of 95% was employed if the distribution was not normal. Sensitivity, specificity, positive predictive value and negative predictive value was calculated via

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receiver operator curve analysis. Values of area under the curve were also calculated used the same. A p-value of less than the significance level alpha=0.05 was considered significant. Optimum cut-off value was calculated from the ROC using Youden’s index.

Result

The study population comprised of a total of 181 male patients with a mean age of 65.02 ± 8.62 years. The youngest was 45 years and the oldest 90 years of age. Prostate malignancy was detected in 17 cases (9.39%). Of these, no patient was below the age of 50 years and the mean age of patients diagnosed with prostate malignancy is 69.88 ± 8.61 years. The mean tPSA level for patients with prostate malignancy and benign histology were 93.82 ng/mL (95% CI: 30.46 – 157.19 ng/mL) and 5.26 ng/mL (95% CI: 4.10 – 6.42 ng/ mL) respectively (p value = 0.001). The mean fPSA level for patients with prostate malignancy and benign histology

were 18.31 ng/mL (95% CI: 0.67 – 35.94 ng/mL) and 0.84 ng/mL (95% CI: 0.68 – 0.99 ng/mL) respectively (p value < 0.001). The mean %fPSA/tPSA level for patients with prostate malignancy and benign histology were 13.41% (95% CI: 8.11%-18.71%) and 21.93% (95% CI: 20.1923.66) respectively (p value = 0.006). %fPSA/tPSA had an area under the curve (AUC) value of 0.77 (95% CI: 0.643-0.911) in detecting prostate malignancy indicating that, in our study, it did indeed serve as a good distinguishing marker. Fig.1 shows the ROC curve of %fPSA/tPSA ratio. According to Youden’s index applied to the ROC curve analysis, the %fPSA/tPSA value with the optimum balance between sensitivity and specificity was 12.07% (Sensitivity: 70.6%, Specificity: 84.8%, Positive predictive value: 0.324, Negative predictive value: 0.965, Positive likelihood ratio: 4.631 and Negative likelihood ratio: 0.347).

Table 1: Characteristics of Benign Histology Group and Malignant Histology Group. Age (yrs) Mean tPSA (ng/mL) Mean fPSA (ng/mL) Mean %fPSA/tPSA (%)

BENIGN HISTOLOGY (N=164) MALIGNANT HISTOLOGY (N=17) p VALUE (<0.05 is significant) 64.51 69.88 0.012 5.26 93.82 0.001 0.91 18.31 <0.001 21.93 13.41 0.006

Table 2: Sensitivity, Specificity, PPV(Positive predictive value), NPV(Negative Predictive Value), PLR(Positive Likelihood Ratio) and NLR(Negative Likelihood Ratio) of %fPSA/tPSA at different cut-off values and at cut-off value determined from the ROC curve by Youden’s index (12.07%). SENSITIVITY SPECIFICITY PPV NPV PLR NLR

5% 11.8 97.6 0.33 0.91 4.82 0.90

10% 52.9 92.1 0.45 0.95 7.89 0.50

15% 70.6 70.1 0.19 0.96 2.36 0.40

20% 82.4 46.3 0.14 0.96 1.53 0.38

25% 88.2 31.1 0.12 0.96 1.28 0.38

30% 94.1 18.9 0.11 0.97 1.16 0.31

12.07% 70.6 84.8 0.32 0.97 4.63 0.35

Table 3: Comparison between cut-off value of %fPSA/tPSA as determined by our study with other studies conducted in the past. STUDY Our Study Prcic et al (2016) Yilmaz et al (2015) Thakur et al (2014) Pormand et al (2012) Suzuki et al (2006) Chun et al (2006) Safarinejad et al (2006) Partin et al (2001) Dalva et al (1999) Catalona et al (1998)

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%FPSA/TPSA CUTOFF VALUE 12% 16% 10% 12% 13% 10% 25-31% 18% 15% 15% 25%

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Fig. 1: ROC curve of %fPSA/tPSA.

Discussion

PSA was first detected in serum in 1980 and since then it has become essential to the management of prostate cancer (PCa).[10] PSA in the human body is required for liquefaction of semen and it is secreted into the seminal plasma.[11] The release of significant quantities of PSA into the main bloodstream is rare in a healthy individual and as such happens only when there is destruction of the basement membrane of the prostate epithelium.[12] This occurs not only in PCa but also in benign conditions. [2] Hence, increased serum PSA levels are not prostate specific.[2] However, a strong correlation between serum PSA and prostate cancer has been proven.[13][14] The advent of immunoassays made it possible to measure fPSA in various forms. This made it possible to calculate %fPSA. %fPSA was first utilised by Stenman et al and Christensson et al.[4][15] It has been demonstrated to be more efficient in distinguishing or differentiating patients with benign prostate histology from those with malignant histology than serum tPSA levels alone.[16] It has been proven that patients with increased serum tPSA concentrations have a higher probability for PCa. It has also been proven that these same patients tend to have a lower %fPSA than patients with benign prostatic disease.[8] In our study as well, we found that there was a statistically significant lowering of %fPSA values in cases of malignant disease as compared to benign prostate disease. It has also been studied and demonstrated that %fPSA or f/tPSA helps improve the discrimination between PCa and benign conditions especially in cases where serum tPSA is between 4 ng/mL and 10 ng/mL. This helps reduce

unnecessary prostate biopsies by helping identify the cases where the need for a biopsy is clear and present.[17][18][19] An optimum cut-off value for %fPSA, as with any screening test, is essential as it can lead to better and more accurate detection of PCa. Many studies have been conducted amongst various populations to determine cut-off values for better distinguishing PCa from benign lesions. Our study shows that a cut-off value of 12% gives the optimum balance between sensitivity and specificity (Sensitivity: 70.6%, Specificity: 84.8%). This 12% cut-off value gives an excellent negative predictive value of 0.965 (96.5%) which means that a patient with a %fPSA value of more than 12% has a 96.5% probability of not having PCa. A study conducted by Safarinejad et al concluded that a f/tPSA cut-off value of 0.18 (%fPSA = 18%) is optimum having a sensitivity of 94.5%.[20] Partin et al suggested that a cut-off value of 15% would detect all advanced tumours while avoiding 80% of unnecessary biopsies, especially in men whose serum tPSA value lie in the â&#x20AC;&#x153;grayâ&#x20AC;? zone (4 to 10 ng/mL).[21] Catalona et al suggested that a cut-off value of 24% would help detect 90% of PCa and avoid appox 18% of benign disease in patients with a serum tPSA value of 2.6 to 4 ng/mL.[7] In a later update by the same authors, a variety of cut-off values were examined and they concluded that a cut-off value of 25% was optimal. [22] Chun et al suggested using median age-specific cut-off values for %fPSA which ranged from 25-31%, and below which the risk of prostate cancer was high.[23] Suzuki et al. reported a 26% decrease in the number of unnecessary biopsies and a sensitivity of 90% when a cut-off value of 10% was applied.[24] Prcic et al found the best combination

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of sensitivity and specificity (sensitivity = 72.3% and specificity = 50.4%) was at a cut-off of 16%.[25] Yilmaz et al suggested a cut-off of 10% whereas Pourmand et al arrived at 13%.[26][27] The study conducted by Dalva et al came to a cut-off value of 15%.[28] A study conducted on an Indian population (sample size – 101 patients) by Thakur et al determined a cut-off value of 12%.[29]

Conclusion

In conclusion, the current study shows that the optimum cut-off value for %fPSA/tPSA which gives the best balance between sensitivity and specificity is 12.07%. This value is seen to be different from the cut-off values determined by most other studies, but this may be explained by the differing populations that were the subject of these studies, most of which were conducted in Western countries. The study by Thakur et al which was done on an Indian population yielded a cut-off value almost identical to ours. As it stands, further study with larger sample sizes is warranted for confirmation of our findings in the Indian context.

Abbreviations and Symbols:

PCa – Prostate Cancer tPSA – Total serum prostate specific antigen fPSA – Free serum prostate specific antigen %fPSA/tPSA – Serum free-to-total prostate specific antigen ratio/percentage ROC – Receiver Operating Characteristic AUC – Area Under the Curve PPV – Positive predictive value NPV – Negative Predictive Value PLR – Positive Likelihood Ratio NLR – Negative Likelihood Ratio

differentiation of prostate cancer from benign prostate hyperplasia in Iranian population. Urol J. 2010;7(2):99-104. 6.

Luderer AA, Chen YT, Soriano TF, Kram WJ, Carlson G, Cuny C, et al. Measurement of the proportion of free to total prostate specific antigen improves diagnostic performance of prostate specific antigen in the diagnostic gray zone of total prostate specific antigen. Urology 1995;46:187e94.

Catalona WJ, Smith DS, Wolfert RL, Wang TJ, Rittenhouse HG, Ratliff TL, et al. Evaluation of percentage of free serum prostate-specific antigen to improve specificity of prostate cancer screening. J Am Med Assoc 1995; 274:1214e20.

Catalona WJ, Partin Aw, Slawin KM, et al. Use of the percentage of free prostate-specific antigen to enhance differentiation of prostate cancer from benign prostatic disease: a prospective multicenter clinical trial. Jama. 1998;279:1542-7.

Ito K, Yamamoto T, Ohi M, Kurokawa K, Suzuki K, Yamanaka H. Free/total PSA ratio is a powerful predictor of future prostate cancer morbidity in men with initial PSA levels of 4.1 to 10.0 ng/mL. Urology. 2003;61:760-4.

10. Polascik TJ, Oesterling JE, Partin AW. Prostate specific antigen: a decade of discovery—what we have learned and where we are going. J. Urol. 1999;162: 293–306. 11. Lilja H, Oldbring J, Rannevik G, Laurell CB. Seminal vesicle-secreted proteins and their reactions during gelation and liquefaction of human semen, J. Clin. Invest. 1987;80:281–285. 12. Stenman UH, Prostate-specific antigen, clinical use and staging: an overview. Br. J. Urol. 1997:79(Suppl.1):53–60. 13. Aus G, Damber JE, Khatami A, Lilja H, Stranne J, Hugosson J. Individualized screening interval for prostate cancer based on prostate-specific antigen level: results of a prospective, randomized, population-based study. Arch. Intern. Med. 2005;165:1857–1861.

Reference 1.

Polascik TJ, Oesterling JE, Partin AW. Prostate specific antigen: a decade of discovery e what we have learned and where we are going. J Urol 1999;162:293e306.

14. Thompson IM, Pauler DK, Goodman PJ, Tangen CM, Lucia MS, Parnes HL et al. Prevalence of prostate cancer among men with a prostate-specific antigen level b or =4.0 ng per milliliter, N. Engl. J. Med. 2004;350:2239–2246.

Dalva I, Akan H, Yildiz O, Telli C, Bingol N. The clinical value of the ratio of free prostate specific antigen to total prostate specific antigen. Int Urol Nephrol. 1999;31:675-80.

15. Christensson A, Bjork T, Nilsson O, et al. Serum prostate specific antigen complexed to alpha 1-antichymotrypsin as an indicator of prostate cancer. J Urol. 1993;150:100-5.

Carter HB. Prostate cancers in men with low PSA levels-must we find them? N Engl J Med. 2004;350:2292

Stenman UH, Leinonen J, Alfthan H, Rannikko S, Tuhkanen K, Alfthan O. A complex between prostate specific antigen and alpha 1-antichymotrypsin is the major form of prostatespecific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res. 1991;51:222-6.

Amirrasouli H, Kazerouni F, Sanadizade M, Sanadizade J, Kamalian N, Jalali M et al. Accurate cut-off point for free to total prostate-specific antigen ratio used to improve

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16. Catalona wJ, Smith DS, Ratliff TL, Basler Jw. Detection of organ-confined prostate cancer is increased through prostatespecific antigen-based screening. Jama. 1993;270:948-54. 17. Catalona WJ, Partin AW, Slawin KM, Brawer MK, Flanigan RC, Patel A, et al. Use of percentage of free prostate specific antigen to enhance differentiation of prostate cancer from benign prostatic disease: a prospective multicenter trial. J Am Med Assoc 1998;279:1542e7. 18. van Cangh PJ, De Nayer P, De Vischer L, Sauvage P, Tombal B, Lorge F, et al. Free to total prostate-specific antigen (PSA) ratio improves the discrimination between prostate

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cancer and benign prostatic hyperplasia (BPH) in the diagnostic gray zone of 1.8 to 10 ng/mL total PSA. Urology 1996;48:67e70. 19. Catalona WJ, Partin AW, Slawin KM, Naughton CK, Brawer MK, Flanigan RC, et al. Percentage of free PSA in black versus white men for detection and staging of prostate cancer: a prospective multicenter clinical trial. Urology 2000;55:372e6. 20. Safarinejad MR. Population-based screening for prostate cancer by measuring free and total serum prostate-specific antigen in Iran. Ann Oncol. 2006 Jul;17(7):1166-71. 21. Partin AW, Mangold LA, Lamm DM, Walsh PC, Epstein JI, Pearson JD. Contemporary update of prostate cancer staging nomograms (Partin Tables) for the new millenium. Urology 2001;58:843e8. 22. Catalona WJ, Partin AW, Slawin KM, Brawer MK, Flanigan RC, Patel A, et al. Use of percentage of free prostate specific antigen to enhance differentiation of prostate cancer from benign prostatic disease: a prospective multicenter trial. J Am Med Assoc 1998;279:1542e7. 23. Chun FK, Perrotte P, Briganti A, Benayoun S, Lebeau T, Ramirez A, et al. Prostate specific-antigen distribution in asymptomatic Canadian men with no clinical evidence of prostate cancer. BJUI 2006;98:50e3.

24. Suzuki H, Komiya A, Kamiya N, Takashi I, Koji K, Junichiro M, et al. Development of a nomogram to predict probability of positive initial prostate biopsy among Japanese patients. Urology 2006;67:131e5. 25. Prcic A, Begic E, Hiros M. Actual Contribution of Free to Total PSA Ratio in Prostate Diseases Differentiation. Med Arch. 2016;70(4):288-292. 26. Yilmaz H, Ciftci S, Yavuz U, Ustuner M, Saribacak A, Dillioglugil O. Percentage of free prostate-specific antigen (PSA) is a useful method in deciding to perform prostate biopsy with higher core numbers in patients with low PSA cut-off values. Kaohsiung J Med Sci. 2015;31(6):315-9 27. Pourmand G, Ramezani R, Sabahgoulian B, Nadali F, Mehrsai AR, Nikoobakht,MR et al. Preventing Unnecessary Invasive Cancer-Diagnostic Tests: Changing the Cut-off Points.Iran J Public Health. 2012; 41(2): 47–52. 28. Dalva I, Akan H, Yildiz O, Telli C, Bingol N. The clinical value of the ratio of free prostate specific antigen to total prostate specific antigen. Int Urol Nephrol. 1999;31:675-8. 29. Thakur V, Talwar M, Singh P. Low free to total PSA ratio is not a good discriminator of chronic prostatitis and prostate cancer: An Indian experience. Indian Journal of Cancer. 2014;51(3):335.

*Corresponding author: Dr. Vineeth G Nair, Room 412, “A” Block, Gardyenia PG Hostel, Yenepoya University Campus, Deralakatte, Mangalore, Karnataka – 575018, India Phone: +91 9972917978 Email: dr.vgn86@gmail.com Date of Submission : 03.01.2017 Date of Acceptance : 20.04.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Original Article DOI: 10.21276/APALM.1272

Study of Congenital Malformations in Fetal and Early Neonatal Autopsies Pradnya Pandurang Kale*, Sujata R. Kanetkar, Dhirajkumar B. Shukla, Atul Bhanudas Hulwan Pramod Borade and Nikita Vinod Vohra Department of Pathology, Krishna Institute of Medical Sciences, Karad, Satara, Maharashtra, India

ABSTRACT Background: Antenatal care in India is rising due to improvement in awareness; still few congenital malformations can be missed in routine check-ups. One lost baby due to birth defects raises questions like; did the malformation led to death, what was the exact nature of malformation, will it recur in next pregnancy and are there any preventive measures? Few congenital malformations can be diagnosed prenatally with ultrasonography techniques, various maternal serum assays, confirmation relies on actual examination of the fetus or neonate. These techniques cannot identify large proportion of congenital malformations for which perinatal autopsy remains the gold standard investigation. The result of a perinatal autopsy may have broad impact, in that obstetrical, fetal, maternal, paternal, and familial conditions may be uncovered. Methods: The descriptive analytical study was carried out in Department of Pathology of a tertiary care hospital from June 2014 to May 2016 for detection of congenital malformations in fetal and early neonatal autopsies in 5 years. Conclusion: Despite advances in imaging such as antenatal ultrasonography and serology, perinatal autopsy is superior and continues to play an important role in diagnosing congenital malformations. The findings of autopsy are not only of theoretical importance but also of practical significance to clinicians in the form of estimating the risk of recurrence and in genetic counseling. Keywords: Congenital Malformations, Perinatal Autopsy, malformation Syndromes.

Introduction

Congenital means â&#x20AC;&#x153;Present since birthâ&#x20AC;?. Congenital malformation is a physical, metabolic or anatomic defect which is apparent before birth, at birth or detected during the first year of life. Congenital malformations are a major cause of fetal and neonatal deaths as well as disability cases worldwide. The varying pattern and prevalence of congenital malformations over time or geographical location are known. It reflects differing methods of detection and recording. There are also true differences in frequency due to the complex interaction of known and unknown genetic and environmental factors including socio-cultural, racial, and ethnic variables. For quite some time, congenital malformations have been reported to be a major cause of mortality and morbidity in children in the developed countries.[1-6] In the recent years, congenital disorders are becoming to be public health issue in developing countries, due to an epidemiological transition, which involves significant decline in infant mortality rates due to reduction of infections and malnutrition and relative increase of morbidity and mortality due to congenital malformations. [7-14] Antenatal investigations, such as ultrasonography

(USG), maternal serum enzyme hormone assays cannot determine significant number of congenital malformations, for which autopsies are must.[15] Fetal and early neonatal autopsy in cases of congenital malformations not only confirms but also provides additional information and is helpful in counseling the parents regarding prevention of similar congenital malformations in future pregnancies. The variety and complexity of congenital anomalies found in perinatal and fetal autopsies are endless, and the Pathologist must be prepared to spend the necessary time demonstrating these anomalies. The majority of the malformations found in this perinatal population are lethal. It is essential to have basic epidemiological information on congenital malformation for planning health care services. This study is therefore undertaken to ascertain various congenital malformations and to classify those according to organ system involved and measure the utility of autopsy in final diagnosis.

Materials and Method

This descriptive analytical study was carried out in Department of Pathology of a tertiary care hospital from June 2014 to May 2016 for detection of congenital malformations in fetal and early neonatal autopsies.

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Consent for the autopsy was taken from the parents/ relatives on a proforma prepared according to guidelines provided by the Institutional Ethics Committee. Perinatal autopsies done in Department of Pathology between the period of June 2014 to May 2016 were studied and analysed. Also, perinatal autopsy specimens from June 2011 to May 2014 preserved in the department with medical records were studied and included in the study. Thus, data included in the present study is of 5 years – June 2011 to May 2016. Inclusion criteria • Autopsies of fetuses with gestational age above 20 weeks of intra uterine life and birth weight greater than 350 gms. •

Autopsies of neonates within 7 days of post natal life.

Exclusion criteria: • Autopsies of fetuses of gestational age <20 weeks, •

Autopsies of neonates above 7 days

•

Autolysed dead fetuses or neonates

Relevant clinical data (maternal history and antenatal investigations) was collected from the case sheets. During autopsy procedure, photographs were taken for unusual gross findings. Post mortem radiographs of abnormal features were taken whenever required. For each fetus or neonate, morphometric examination was done. Autopsies were performed by the standard technique adopted by EDITH. L. POTTER.[16] Sections from each organ were submitted for histopathological examination. Following routine tissue processing, paraffin embedding, section cutting, staining was performed by routine Haematoxylin and Eosin stains. Clinical data and the pathological findings were recorded in proforma. Autopsy findings were compared with antenatal ultrasound findings. Microphotographs were taken in cases with congenital malformations diagnosed on histopathological study (microscopy). Malformations were classified into organ systems according to World Health Organization (WHO) recommendations, International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10) Version for 2010, Chapter XVII (Q00-Q99) Congenital Malformations, Deformations and Chromosomal abnormalities.[17]

Results

During this period, total 223 perinatal autopsies were done and 57 autopsies showed various congenital malformations

comprising 25.56% of perinatal autopsies.These 57 perinatal autopsies with congenital malformations were further analysed. Out of total 57 cases 54 were fetal and 3 were early neonatal autopsies. There was slight male preponderance (52.63%) observed in the present study. Ambiguous genitalia was observed in 3 fetuses. Maximum fetuses (63.15%) were from gestational age group of 20-24 weeks. Among all the fetuses with congenital malformations, 71.92% were having low birth weight (ranging from 350-1000 grams) for the gestational age. Maximum number of fetuses 39 (68.42%) were born to mothers in age group of 20-24 years and 50.87% mothers were primipara. One of the three neonates was a case of Down’s syndrome and born to a mother of age 36 years with bad obstetrics history. Bad obstetric history was noted in 14 mothers, out of those 2 showed high titre for TORCH complex. History of consanguinity was present in 4 mothers (7.02%) out of all congenitally malformed cases.(Table No.1) There were significant additional findings observed in 31 autopsies when compared with antenatal ultrasonographic findings. Antenatal ultrasonographic diagnosis was totally changed in 3 cases after final autopsy diagnosis. (Table No. 2) In 13 perinatal autopsies, multiple organ systems were involved by congenital malformations suggesting insult during common embryological period of development. (Table No. 3) A case of acardiacacephalic fetus was diagnosed on morphological examination (Fig. No. 1) In the present study, the commonest system affected by congenital malformations was musculoskeletal system (19.30%).Varieties of malformations involving musculoskeletal system were Omphalocele, diaphragmatic hernia and Congenital Talipes Equinovarus. Central nervous system was involved in 9 cases of congenital malformations. Neural tube defects were the commonest subgroup. Rare case of Holoprocencephaly was also diagnosed. ( Fig. No. 2) Various other major systems affected by congenital malformations were cardiovascular system (12.28%); renal system (7.02%), respiratory system (3.51%). Polycystic kidneys were noted in a case grossly, and microscopically confirmed. (Fig. No. 3) Various rare syndromes were identified in the study viz. Pentalogy of Cantrell (Fig. No.4), Sirenomelia (Fig. No.5), Limb Body Wall Complex; one case of each. Down’s syndrome and Edward’s syndrome were 2 chromosomal abnormalities diagnosed morphologically in the study, confirmed on cytogenetic studies.

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Table 1: Maternal Factors seen in malformed fetuses& neonates. Character

Number (out of 57)

Percentage%

<20

20-24

68.42

25-29

24.56

>30

7.02

Primipara

50.87

Multipara

47.37

Grand multipara

1.76

Maternal Age in years

Parity

Consanguinity Yes

7.02

92.98

Yes

24.56

75.44

H/o Previous Abortion

Table 2: Correlation of Antenatal USG findings with Autopsy findings. Correlation of USG findings and autopsy findings

Cases

Percentage (%)

USG findings confirmed on autopsy

54 27 27

94.74 47.37 47.37

Changes in Diagnosis

5.26

Total

100

â&#x20AC;˘ â&#x20AC;˘

No change in diagnosis Additional findings noted in the autopsy

Table 3: System wise distribution of Various Congenital Malformations in the Study System affected in congenital malformations

Number of cases

Percentage (%)

Central Nervous system Spina Bifida Spina Bifida with single umbilical artery Spina Bifida, anencephaly, Craniofacial Rashischisis Spina Bifida, Hydrocephalus Anencephaly CraniospinalRashischisis Holoprocencephaly Meningomyelocele, Hydrocephalus

09 02 01 01 01 01 01 01 01

15.79

Musculoskeletal system Diaphragmatic hernia Omphalocele CTEV CTEV, Rocker Bottom foot Radial Ray anomaly Limb deformity Hypoplastic Nasal Bone Arthropogyrosis

12 03 01 02 01 02 01 01 01

21.05

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System affected in congenital malformations

Number of cases

Percentage (%)

Cardiovascular system Atrial Septal Defect alone Atrial Septal Defect, Ventricular Septal defect Atrial Septal Defect, Ventriculomegaly Ventricular Septal Defect alone Ventricular Septal Defect, Transposition of great vessels Abnormal aorto-pulmonary communication Hypoplastic heart syndrome

07 01 01 01 01 01 01 01

12.28

Urinary system Renal Agenesis Polycystic kidneys Polycystic kidneys Dysplastic kidneys

04 01 01 01 01

7.02

Respiratory system Congenital Cystic AdenomatoidMalformation Bilateral Hypoplastic lungs

02 01 01

3.51

Eye, Ear, Face & Neck system Bilateral Cataract

1.75

Lymphatic system Cystic Hygroma

1.75

Multiple systems Spina Bifida- Lumbar level, Low set ears, B/L CTEV Persistent cloaca, Bladder Obstruction syndrome, Limb deformity Right renal Agenesis, Persistent Cloaca, Bladder Obstruction syndrome, B/L CTEV Ambiguous genitalia, Omphalocele, Imperforate Anus, Kyphosis, B/L lower limb deformity, Vertebral deformity Spina Bifida with Scoliosis, Occiputo-cervical Cystic Hygroma Corpus Callosal Agenesis, Spina Bifida, Diaphragmatic hernia, B/L hypoplastic lungs, Dysmorphic facial features Spina bifida occulta, B/L cataract Right Testis agenesis, Undescended left testis, Limb deformity, Dysmorphic face B/L hypoplastic lungs, Fallot’s tetralogy, Diaphragmatic hernia Acephalicacardiacfetus B/L Hypoplastic lungs, B/L Hypoplastic kidneys, Potter’ s facies Hydrocephalus, Imperforate anus Cleft Lip, Cleft Palate, Bifid tongue

22.81

Syndromes identified Down’s syndrome Edward’s syndrome VACTERAL- H syndrome Limb body wall complex Sirenomelia Pentalogy of Cantrell Tethered cord syndrome Potter’s syndrome

08 01 01 01 01 01 01 01 01

14.04

Total

100

01 01 01 01 01 01 01 01 01 01 01 01 01

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Fig.1: Photograph showing acardicacephalicfetus.

Fig. 2: Cut open cranial vault showing Holoprocencephaly

Fig. 3: Photographs showing in situ examination of fetus with enlarged kidneys, cut section of Polycystic kidneys and photomicrograph showing multiple dilated cysts in kidney of fetus having grossly polycystic kidneys (H & E,100 x).

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Fig. 4 : Photograph and Postmortem Radiogram showing malformed fetus with thoraco-abdominal wall defect (Omphalocele), ectopiacordis (arrow), multiple limb and facial deformities, spinal defects in a case of Pentalogy of Cantrell.

Fig. 5 : Photograph and Postmortem Radiogram showing Kyphoscoliosis, Single Femur, Absent Tibia, Fibula in a case of Sirenomelia.

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Discussion The awareness regarding fetal and neonatal (perinatal) mortality and its preventable causes is increasing. It has led to increase in finding the preventable causes of death in perinatal period. Few of the malformations are preventable by acquiring few simple measures such as intake of folic acid during the child bearing age. As congenital malformations are known to recur, physicians are interested in finding the chances of recurrence. The presence of congenital malformation in baby has profound emotional effect on mother. In additional to that loosing such baby gives not only blame but also physical and mental burden to mother. During the most sensitive period of embryogenesis i.e. the 3rd - 8th weeks of gestation, along with genetic factors other factors like environmental, teratogenic and infectious agents play important role for the causation of malformations. Autopsy plays role in identification and confirmation of malformations. Out of 223 perinatal autopsies performed 57 (25.56%) had congenital malformations, similar incidence was noted by Mohan et al 2004 (38.7%). [18]However, Kapoor et al 2013 (69%) and Puri et al 2009 (63%) noted higher incidence in their study. Fetal Factors: On comparing the sex ratio, the congenital malformations were slightly more in males (52.63%) with male to female ratio being 1.25:1. Pattanaik T et al have found the incidence to be more in males (56%) with ratio of 1.37:1.[19]But many studies have reported almost same incidence in males and females.[20] In the present study, there were 3 cases (5.26%) where sex of the fetus was ambiguous. Similar finding was noted by study done by Hakverdi S et al (6.25%).[21] Gestational age of most of the fetuses with congenital malformation ranged from 20-24 weeks (63.15%). This could be attributed to therapeutic termination done in this period after detecting malformation on ultrasonography. The association of low birth weight for gestational age and malformations has been well documented.[20,22] Similar to other studies; fetuses with congenital malformations had the weight in the range of 350-1000 grams (low for gestational age) in the present study.[20,22,23]

similar with study done by Subhashini et al in 2015(41.8%) [24] and Kapoor et al (60%).[18] Bad Obstetric history, with one or more than one abortions; was present in 14 mothers (24.56%). Similar results were noted by Subhashini et al with 33.4%.[24] Among the malformed fetuses or neonates,50.87% were born to primigravida. Similar finding was seen by Kapoor K et al(50%),[18] (Subhashini et al (40.1%),[24] and Parmar A et al (42%)[20] Consanguinity has been described as an important factor contributing to increased congenital malformations. In this study, the consanguinity was noted in 7.02% cases, this is comparable to study done by Pattaanaik T et al, 2016 (6%).[19,25,26] Antenatal sonography is developed few decades back; however it continues to lag behind a complete fetal autopsy in accurately diagnosing the cause of fetal death. [27] Only few studies have shown comparison of antenatal ultrasonographic findings & autopsy findings. In the present study, autopsy diagnosis confirmed ultrasonographic findings in 54 cases (94.74%), among these in 27 cases (47.37%) it provided additional information, whereas primary diagnosis given by ultrasonography was changed in 3 cases (5.26%). The findings in the present study are similar to those of Sankar and Phadake et al. [23,28] The most common system involved was musculoskeletal followed by central nervous system. Similar finding was noted by Potekar et al, 2013,[22]Tomatir et al 2009,[8] and Andola US et al 2012.[23] Various combinations of systems involved in malformations were noted. This could be attributed to same embryological period of development of different systems.In this study the 13/57 (22.80%) cases showed multiple malformations occurring in single fetus or neonate. Variety of syndromes have been identified in the study like Downâ&#x20AC;&#x2122;s syndrome, Potterâ&#x20AC;&#x2122;s syndrome etc. Few rare syndromes were encountered like Limb body wall complex /Body stalk anomaly refers to a rare complicated polymalformative fetal malformation syndrome of uncertain etiology. Antenatal detection of such malformations is important, in planning the management, and achieving better outcomes, and to reduce recurrence.

Conclusion

Maternal Factors: Maternal age is an important parameter in the birth of congenitally malformed fetuses. [18] Maximum number of fetuses 39 (68.42%) were born to mothers in age group of 20-24 years. This finding was

Perinatal autopsy is useful in detecting and confirming congenital malformations in fetuses and neonates. Identification and classification of the visceral congenital malformations can be achieved with good autopsy

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technique. Bad obstetrics history, low birth weight for gestational age and prematurity, male sex are the most frequently observed factors associated with development of congenital malformations. Despite advances in imaging such as antenatal ultrasonography and serology, perinatal autopsy is superior and continues to play an important role in diagnosing congenital malformations. The findings of autopsy are not only of theoretical importance but also of practical significance to clinicians in the form of estimating the risk of recurrence and in genetic counseling.

References 1.

McCandless SE, Brunger JW, Cassidy SB. The burden of genetic disease on inpatient care in a children’s hospital. The American Journal of Human Genetics2004; 74(1):121–127. MacDorman MF, Atkinson JO. Infant mortality statistics from the 1997 period linked birth/infant death data set. National Vital Statistics Reports, 1999; 47(23):1–23.

Rosano A, Botto LD, Botting B, Mastroiacovo P. Infant mortality and congenital anomalies from1950 to 1994: an international perspective. Journal of Epidemiology & Community Health2000; 54(9): 660–666.

EUROCAT Working Group, Surveillance of congenital anomalies in Europe 1980–1999, EUROCAT Report 8,Universty of Ulster, Belfast, UK, 2002.

Fida NM, al-Aama J, Nichols W, Alqahtani M. A prospective study of congenital malformations among live born neonates at a University Hospital in Western Saudi Arabia. Saudi Medical Journal 2007; 28(9): 1367–1373.

Bower C, Callaghan A, Quick J. Report of the Birth Defects Registry of Western Australia, Tech. Rep. no. 15, King Edward Memorial Hospital, Women and Newborn Health Service, 2010.

Abdi-Rad I, Khoshkalam M, Farrokh-Islamlou H R. The prevalence at birth of overt congenital anomalies in Urmia, Northwestern Iran. Archives of Iranian Medicine 2008; 11(2):148–151. Tomatir AG ,Demirhan H, Sorkun HC, K¨oksal A, Ozerdem F, ilengir NC. Major congenital anomalies: a five-year retrospective regional study in Turkey. Genetics and Molecular Research 2009; 8(1);19–27. Taksande A, Vilhekar K, Chaturvedi P, Jain M. Congenital malformations at birth in Central India: a rural medical college hospital based data. Indian Journal of Human Genetics 2010; 16(3)159–163.

10. Agha MM, Williams JI, Marrett L, To T, Dodds L. Determinants of survival in children with congenital abnormalities: a long-term population-based cohort

study. Birth Defects Research A: Clinical and Molecular Teratology2006; 76(1): 46–54. 11. Taboo ZA. A prevalence and risk factors for congenital anomalies in Mosul City. Iraqui Postgraduate Medical Journal 2012; 22(2):140–146. 12. Shamim A, Chohan N, Sobia Q. Pattern of congenital malformations and their neonatal outcome. Journal of Surgery Pakistan.2010;15: 34–37. 13. al-Mendalawi MD. Pattern of neonatal and postneonatal deaths over a decade (1995–2004) at a military hospital in Saudi Arabia. Saudi Medical Journal2008; 29(10)1518–1521. 14. Penchaszadeh VB. Delivery of genetic services in developing countries. In: M. J. Khoury, W. Burke, and E. Thompson eds. Genetics and Public Health in the 21st Century. New York, USA: Oxford University Press2000: 301-327. 15. Long G, Spring A. A comparative study of routine versus selective fetal anomaly ultrasound scanning. American College of obstetricians and Gynecologists 1998; 5: 6-10. 16. Potter EL. Pathology of the fetus and infants. 1st ed, New York: Mosby Co;1997. 17. World Health Organisation. The ICD-10 version 2010, Classification of Congenital malformations, deformations and Chromosomal abnormalities. Geneva, 1994 (http:// apps.who.int/classifications/icd10/browse/2010/en#/XVII) accessed on 08/05/2016. 18. Burton JL, Underwood JCE. Necropsy practice after the ‘Organ retention Scandal’. Requests, performance, and tissue retention. J. ClinPathol 2003; 56: 537-541. 19. Pattanaik T, Samal S, Jena T. Study of Congenital Anomalies in a Tertiary Care Hospital. Indian Journal of Neonatal Medicine and Research 2016; 4(3):1-4. 20. Parmar A, Rathod SP, Patel SV, Patel SM. A Study of Congenital Anomalies In Newborn. NJIRM 2010;1(1): 13-17. 21. Hakverdi S, Guzelmansur I, Gungoren A, Toprak S, Yaldiz M, Hakverdi A U. Evaluation of Fetal Autopsy Findings in the Hatay Region: 274 Cases. Turk PatolojiDerg 2012;28(2):154-161 22. Potekar RM, JavalgiPA , Yelikar B R. Autopsy study to determine fetal anomaly: a retrospective Cohort study. Int J Pharm Bio Sci 2014; 5 (3): 64 – 69. 23. Andola US, Anita AM, Ahuja M, Andola SK. Congenital malformations in perinatal autopsies- A study of 100 cases. JCDR 2012;6(10):1726-1730. 24. Subhashini R, Uma N, Neeraja. Incidence and Evaluation of Congenital Malformations in Victoria Govt. Hospital Visakhapatnam, Andhra Pradesh. JMSCR 2015; 3(2):4022-4036. 25. Madi SA, Al-Naggar RL, Al-Awadi SA, Bastaki LA. Profile of major congenital malformations in neonates in Al-Jahra region of Kuwait. East Mediterr Health J 2005; 11:700–06.

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26. Ordonez MP, Nazer J, Aguila A, Cifuentes L. Congenital malformations and chronic diseases of the mother. Latin American collaborative study of congenital malformations 1971-1999. Rev Med Chil 2003; 131:404â&#x20AC;&#x201C;11.

27. Nayak SR, Garg N. Determination of antepartum fetal death. J ObstetGynecol India 2010; 60: 494-97. 28. Sankar VH, Phadke SR. Clinical utility of fetal autopsy and comÂŹparison with prenatal ultrasound findings. Journal of Perinatology2006; 26: 224-29.

*Corresponding author: Dr Pradnya Pandurang Kale, 286/C, SadashivPeth, Pune 411030, Maharashtra, India. Phone: +91 9890281018 Email: pradnyaj.patho@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 09.01.2017 Date of Acceptance : 06.06.2017 Date of Publication : 31.08.2017

eISSN: 2349-6983; pISSN: 2394-6466

Original Article DOI: 10.21276/APALM.1277

Cytological Study of Pleural Effusions and its Utility in Clinical Approach Pravin Gojiya*, Alpeshpuri Goswami and Shaila Shah Department of Pathology, Government Medical College & Sir Takhtasinhji Hospital, Bhavnagar, India

ABSTRACT Background: Cytological analyses of pleural effusions play an important role in the diagnosis of various lesions. Most importantly it gives a significant contribution in diagnosis and management. Methods: A retrospective study for one year duration from January 2015 to December 2015 was conducted in the Cytology section, Department of Pathology, Govt. Medical College & Sir- T hospital Bhavnagar. Pleural fluids which were come for cytological analysis from all the departments are included in the study, then physical examination and microscopy done by staining with Gimsa, Papanicolaou stain and Hematoxylin and Eosin Stains for evaluation. Result: 295 cases of pleural effusion evaluated in which 29% are female and 71% are male and 12% of effusions are Bilateral, 32% are left sided, 57% right sided pleural effusion, 95% non Malignant lesions and only in 5% of cases were reported as malignant and further non-malignant lesions comprises of pleural effusions with reactive mesothelial cells 11% while remaining fluids 40% are of lymphocytic predominant effusions and 13% are of acute inflammatory conditions with abundant polymorphs, remaining 36% of pleural effusions were scant-cellular and have no suggestive role by cytological evaluation. Conclusion: This study showed that meticulous evaluation of the pleural fluids for their cytological properties will help the clinicians in clinching the diagnosis as well as early approach in management of these cases. Keywords: Pleural Fluid Cytology, Benign Effusion, Reactive Mesothelial Cells, Malignant Effusions

Introduction

Pleural fluid cytology is a simple and a minimally invasive technique as the preliminary step for the diagnostic evaluation of pleural effusions, assisting the clinician in establishing the differential diagnosis. It may lead to final diagnosis and provide useful information for treatment. The diagnostic yield of the cytological analysis may be attributable to the cell population present in the sediment that is representative of a much larger surface area than the pleural biopsy [1, 2]. It is very useful in the very first initial work up for the management of the case of pleural effusion. It may also provide crucial clues for the identification of both non-malignant pleural and transudative effusions. Sensitivity will be higher if the clinical, radiological and laboratory results are collaborated. This study was carried out to determine the diagnostic utility of pleural fluid cytology.

Materials and Methods

The study was conducted in the Department of PathologyCytology section of Govt. Medical College and Sir T Hospital, Bhavnagar (Gujarat). Itâ&#x20AC;&#x2122;s a retrospective study of cytology impression of pleural fluids for one year duration from January 2015 to December 2015. Pleural fluids

which received for cytological analysis from the various departments of our institute with the proper complete history and clinical brief are included in the study. These fluid samples were examined for physical properties like the volume, color, appearance, turbidity, presence of clot coagulum. Fluids samples are then centrifuged at 2000 rpm for five minutes. Smears made from the sediment part of centrifuged fluid and stained with Giemsa, Hematoxylineosin and Papanicolaou stains. Smears were examined for the differential cell count and reported descriptively, final impression given as malignant or non-malignant pleural effusion. Malignant pleural effusions were further classified according to its morphometrics. Study results then analyzed with the help of tables and charts and discussed for its incidence rate.

Result

In our study during the year 2015 total 295 cases of pleural fluids were examined for cytology. Out of 295 pleural fluids specimens 84 samples were of females and remaining 211 were of males, so here male is to female ratio of pleural fluid specimens was 2.5:1CHART-1. The pulmonary department had sent pleural fluid examination mostly among the all other departments. Pleural effusions

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occurrence was more common in the age group 60 to 70 years in both males and females sexes although it occurs in all age group from 9 years to 90 years of age CHART-2. Out of 295 cases 164 cases were of right sided, 96 cases were left sided pleural effusions and remaining 35 cases were of bilateral pleural effusion. About 95% of pleural fluids were non-malignant effusions given in pleural fluid cytology, here out of 295 cases 280, comprises 36(13%) of acute

inflammations with neutrophilic preponderance(fig.1), 112(40%) with lymphocytic(fig.2), 30(11%) effusions have reactive mesothelial cells(fig.4) and remaining 102(36%) cases have no further suggestive outcome in pleural fluid cytology. Malignant pleural effusions were 15(5%) out of which, 6(40%) metastatic adenocarcinoma (fig.3) and remaining pleural effusions have non-specific malignant cells effusions TABLE-1.

Chart 1: Sex Distribution of Pleural effusion cases

Chart 2: Age distribution in both sexes Vs frequencies for pleural effusions

Table 3: Diagnostic outcome of pleural fluid cytology examinations. Diagnostic impression Malignant metastatic adenocarcinoma (20%) Malignant pleural effusion (5%) Malignant effusion (40%) Lymphoma (40%) Acute inflammatory effusion(13%) Lymphocytic effusion (40%) Non-Malignant pleural effusion (95%) Reactive mesothelial effusion (11%) Non specific effusion(36%)

Number of cases 3 6 6 36 112 30 102

Fig. 1: Centrifuged smear of pleural fluid shows Plenty of Polymorphs-Acute inflammation [H&E, 40X].

Fig. 2: Centrifuged smear of pleural fluid shows plenty of lymphocytes - Chronicinflammation [H&E, 40X].

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Pleural Effusions Fluid Cytology

Fig. 3: Malignant cells in Pleural fluid (Adenocarcinoma). Cells arranged in 3D ball pattern, shows pleomorphism & high N:C ratio[H&E, 40X].

Fig. 4: Centrifuged smears of pleural fluid shows Reactive mesothelial cells with high N:C ratio, vacuolated Eosinophilic cytoplasm [H&E, 40x]

Discussion

Pleural effusions are caused by pulmonary or nonpulmonary diseases. Although the etiologic spectrum is wide, most effusions occur due to malignancy, heart failure, tuberculosis or bacterial infection.[1,7,8,9] Because the effusions develop as a manifestation of an underlying disease, it is difficult to determine the precise incidence. Cytological examination is often the initial diagnostic step for its etiologic identification. It represents the whole pleural surface due to the existence of exfoliated cells in the fluid. Even if the initial examination is negative, the cellular profile of the fluid leads the clinician in the correct diagnostic pathway, as the results of our study suggests. Consequently, this minimally invasive method appears to be the best preliminary step for the assessment of pleural effusions. In patients with a malignant pleural effusion, cytological examination is a fast, efficient and minimally invasive procedure to establish the diagnosis.

study suggests, the absence or the scarcity of mesothelial cells along with the presence of more than 50% small lymphocytes should be regarded as a strong evidence for tuberculosis[1]. The absence of mesothelial cells is attributed to the deposition of fibrin on the pleural surface, either sealing off the mesothelial cells, destroying them or both [14]. Although the mechanism of eosinophil accumulation in the pleural space is unknown, eosinophils play an important role in idiopathic, allergic diseases, or drug reactions. The presence of air in the pleural space may also cause eosinophilia. It is well known that tuberculous pleural effusions rarely contain more than 10% eosinophils[15,16,17,18]. The presence of numerous mesothelial cells or eosinophils was useful to exclude tuberculosis in the differential diagnosis of exudative pleural effusions. In the present study, pleural fluid analysis was not diagnostic in approximately 30% of the patients and thereby indicating its limits.

Almost all adenocarcinomas were diagnosed with cytology, but the yield was less with squamous cell carcinomas, Hodgkinâ&#x20AC;&#x2122;s disease and sarcomas [1]. This may be due to the fact that the adenocarcinoma cells are more easily identified cytological and large tissue biopsies are usually needed for the diagnosis of lymphoma and sarcomas. The effusion may develop secondary to other factors such as infection, pulmonary emboli or lymphatic blockade. Even after thoracoscopy, 10% of the pleural effusions may remain undiagnosed [12,13]. A tuberculous pleural effusion is frequently a diagnostic challenge for the pulmonary clinician. It is impossible to differentiate a tuberculous effusion from a malignant pleural effusion on clinical grounds alone and usually more invasive diagnostic interventions are needed. As the results of our

As the results of our study suggests, cytology of the pleural fluid is the most informative and definitive initial diagnostic step in pathologic states involving the pleura. This simple and minimally invasive technique may be considered as the best initial diagnostic tool in the hands of an experienced cytologist without any serious complications of thoracocentesis. Examination of the pleural fluid can narrow the differential diagnosis considerably. Cytology can be the key to direct diagnosis or can indicate the next step leading the clinician in the correct pathway for final diagnosis even when not diagnostic on its own, and thus precluding unnecessary invasive interventions. In most diseases related to pleural effusion, the pleural fluid analysis yields important diagnostic information and in certain cases it provides the final diagnosis[1].

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Conclusion

In developing countries like ours, where investigations and health facilities are inadequate and cost of treatment is often un-affordable, pleural fluid analysis and cytology should continue to be a first line investigation to screen out the suspiciously malignant pleural effusion cases, as it is a very convenient, cost-effective and safe investigation. Pleural fluid cytology may be helpful in diagnosing primary as well as metastatic pleural malignancies. However, along with the proper clinical history and other supportive radiological, biochemical investigations the diagnostic impression of the suspicious disease can be achieved satisfactorily for further clinical management.

Acknowledgements

We are very much thankful to pulmonologists of Sir Takhtasinhji hospital for their valuable support.

Reference 1.

at a tertiary care centre in North India. Cytopathol. 2007;18:28-32.2211 6. Geisinger KR, Stanley MW, Raab SS, Siverman JF, Abati A. Modern Cytopathology. Philadelphia Churchill Livingstone; 2004. 7.

Sahn SA. Pleural effusions of extravascular origin. Clin Chest Med. 2006;27(2):285-308.

Light RW. The undiagnosed pleural effusion. Clin Chest Med. 2006;27(2):309-319.

Heffner JE. Diagnosis and management of malignant pleural effusions. Respirology. 2008;13(1):5-20.

10. Bouros D, Pneumatikos I, Tzouvelekis A. Pleural involvement in systemic autoimmune disorders. Respiration. 2008;75(4):361-71. 11. Dekker A, Bupp PA. Cytology of serous effusions. An investigation into the usefulness of cell blocks versus smears. Am J ClinPathol. 1978;70:855-860. 12. Loddenkemper R, Boutin C. Thoracoscopy diagnostic and therapeutic indications. EurRespir J. 1993;6:1544-1555.

Tetikkurt C, YÄąlmaz Kara B, Tetikkurt S, YÄąlmaz N, Ilknur Yasar Rian Disci. The Value of Cytology in the Diagnosis of Pleural Effusions British Journal of Medicine & Medical Research 2014;4(11): 2203-11

13. Kalomenidis J. New advances in the investigation of pleural diseases. Pneumologie. 2003;16:247-251.

Kushwaha R, Shashikala P, Hiremath S, Basavaraj HG. Cells in pleural fluid and their value in differential diagnosis. J Cytol. 2008;25(4):138-143.

15. Epstein DM, Kline RM, Albelda SM, Miller WT. Tuberculous pleural effusions. Chest. 1987;91:106-109.

BedrossianW. Diagnostic problems in serous effusions. Diagn Cytopathol.1998;19(2): 131-137.

Pereira TC, Saad RS, Liu Y, Silverman JF. The diagnosis of malignancy in effusion cytology: A pattern recognition approach. Adv Anat Pathol. 2006;13:174-184.

Awasthi A, Gupta N, Srinivasan R, Nijhawan R, Rajwanshi A. Cytopathologic spectrum of unusual malignant effusions

14. Hurwitz S, Leiman G, Shapiro C. Mesothelial cells in pleural fluid TB or not TB. S Afr Med J. 1980;57:937-939.

16. Light RW. Establishing the diagnosis of tuberculous pleuritis. Arch Intern Med 1998;158:1967-1968. 17. Sahn SA. The value of pleural fluid analysis. Am J Med Sci. 2008;335(1):7-15. 18. Sakuraba M, Masuda K, Hebisawa A, Sagara Y, Komatsu H. Pleural effusion adenosine deaminase (ADA) level and occult tuberculous pleurisy. Ann Thorac Cardiovasc Surg. Oct 2009;15(5):294-6.

*Corresponding author: Dr. Pravin Gojiya, Room no. 27, PG Hostel-1, Sir-T hospital campus, kala nala, Bhavnagar-364001,India Phone: +91 0278-2510236 Email: pgojiya@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 10.01.2017 Date of Acceptance : 31.05.2017 Date of Publication : 31.08.2017

eISSN: 2349-6983; pISSN: 2394-6466

Original Article DOI: 10.21276/APALM.1303

HbA2 and Fetal haemoglobin in The Diagnosis of Thalassemia and Hemoglobinopathies Sandhya Venkatachala1* and Manjula Rajendran2 Dept. of Pathology, Apollo Hospitals Bangalore, India Dept. of Pathology, Madurai Medical college, Madurai, India 1

ABSTRACT Background: Hemoglobin (Hb) disorders which include hemoglobinopathies and Thalassemia affect 7% of the world population. Capillary electrophoresis is useful for screening and follow up of Hb disorders . Aim: To evaluate HbA2 and HbF (fetalhemoglobin) in the diagnosis of Thalassemia and hemoglobinopathies. Material and Methods: 100 consecutive Capillary Hemoglobin electrophoresis done as a part of screening programme for Hb disorders from Jan2016 to june 2016 was included in the study. Children <1 yr of age and individuals with recent blood transfusion were excluded . Hb, RBC count and MCV were recorded. Results: Of the 100 Hb electrophoresis performed, 57 normal and 43 abnormal patterns were seen. Among the abnormal Hb patterns, β thalassemia trait (βTT) was the most common constituting 58.2% followed by Sickle cell (HbS) trait(11.7%), HbE trait(9.3%) and HbE/ β Thalassemia (7.0%). The HbA2 levels in normal, βTT, Sickle cell trait , HbE trait and HbE/ β thalassemia were 2.12%(SD 0.5), 4.9%(SD 0.62), 3.28%(SD 0.43), 3.98%(SD 0.67) respectively . The difference in HbA2 were significant (p value <0.0001).The difference in HbA2 levels between HbE trait and HbE disease was also significant (p value 0.036).Based on the HbF levels Sickle cell hemoglobinopathy was further classified. Conclusion: The difference in HbA2 levels in normal subjects, Thalassemia and hemoglobinopathies are statistically significant. The percentage of HbF in sickle cell gives information about coexisting hemoglobin disorder. In HbEhemoglobinopathy, HbA2 along with HbF identifies a specific group HbE/ β Thalassemia which often needs clinical intervention. Keywords: Hemoglobin A2, Fetal Haemoglobin, Hemoglobinopathy, Thalassemia, Capillary Electrophoresis

Introduction

Hemoglobin (Hb) disorders are a frequent genetic disease affecting 7% of world population.[1] Hemoglobinopathies result from a structural defect in the globin chain where as Thalassemias are due to a quantitative defect in the globin chain production.They are detected in populations during programmes run for prevention of Hbdisorders or in patients with clinical suspicion or familial history of Hb disorder. Capillary electrophoresis has been used for precise and accurate first line screening and follow up Hb disorders.[2] DNA or protein analysis are recommended for a definitive diagnosis in difficult cases. HbA2 and HbF along with the specific abnormal band, point to the diagnosis of Hb disorder. Few studies have separately evaluated HbA2andHbF in Hb disorders.[3,4] Capillary electrophoresis(CE) and high performance liquid chromatography (HPLC) were used respectively for hemoglobin separation respectively in these studies. But both HbA2 and HbF have not been evaluated together in Hb disorders by CE. So the present study was undertaken

to evaluate HbA2 andHbF in the diagnosis of Thalassemia and hemoglobinopathies .

Material and Methods

100 consecutive cases of Hb electrophoresis done as a part of screening programme for hemoglobin disorders from Jan 2016 to June 2016 were included in the study. Children less than one year of age and individuals with a history of blood transfusion in the past three months were excluded in the study. Hemoglobin electrophoresis was done by Sebia Minicap Flex piercing capillary electrophoresis method.[2] K- EDTA anticoagulated blood was used. Hb, RBC count and MCV were recorded in each case. Reference range by Sebia CE-HbA 96.8% to 97.8% and HbA2- 2.2% to 3.2% .

Results

Of the 100 Hb electrophoresis performed 57 showed normal pattern (Fig 1) with HbA ranging between 97.1% and 99%( Mean 97.85,SD 0.52) and HbA2 between 1% and 3.6% (mean 2.12,SD 0.5). HbF of 0.5% was seen in one of the cases. Hb, RBC count and MCV ranged

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between 5gm% to 15.8gm% ,4.15to5.4x1012 /L and 70-118 femtolitres respectively. The lowest HbA2 observed was 1% which corresponded to Hb of5 gm% and MCV of 70 fl. Marginally elevated HbA2 of 3.6% was seen in the case with MCV of 118 and Hb of 10gm%. The 43 abnormal electrophoresis pattern included 26 cases of Thalassemia ,8 cases each of sickle cell hemoglobinopathy and HbE hemoglobinopathy, one case of HbD hemoglobinopathy. Furthur classification and distribution of cases are shown in table 1. Thalassemia out numbered hemoglobinopathies. The most common abnormal Hb pattern was β thalassemia trait (βTT) (Fig2A ). An elevated HbA2 level between 3.8% and 6.2% (4.9, SD 0.62) was observed. HbF was seen in 13 of the 25 cases and varied between 0.3%to 6%.The mean RBC count and MCV were 5.59 x10 12(SD 0.84) and 64.2 (SD 4.08) (Table 2). A single case of α Thalassemia was seen with HbH band(28.3%) (Fig2B) .HbA of 69.6% , HbA2 of 2.1% were recorded. The Hb, RBC and MCV were 13gm/ dl, 4.8x1012 and 83 fl respectively. The details of eight cases of Sickle cell hemoglobinopathy are shown in table 3. The five cases of SCT showed HbS band (Fig3A ) in addition to HbA. Average HbS was 30.46 (SD7.2) and HbA2 was 3.28 (SD 0.43). HbF was seen in two cases amounting to 0.6% and 1.8%. The mean RBC count and MCV were 4.7x1012 (SD 0.51) and 81.88

fl (SD 1.1). One case each of Sickle cell disease(SCD) (Fig3B) , heterozygous HbS/ β thalassemia( Fig 4A) and heterozygous HbS/ Heriditary persistence of fetal hemoglobin (HPFH) (Fig 4B) were seen . HbF of 20.6%, 9.0% and 32.8% were seen in these cases respectively. In addition heterozygous HbS/HPFH showed elevated HbA2 levels of 8.4%. HbE hemoglobinopathy included eight cases (table 4). HbE trait was the most common .Charateristic HbE band was seen (Fig5A) in addition to HbA and HbA2. HbA, HbE and HbA2 were 79.95% (SD 19.6) , 22.9(SD 2.8) and 3.98 (SD 0.67). HbF of 0.6% was seen in one of the cases . The mean RBC count and MCV were 4.99x1012(SD 0.65) and 76.1fl (SD 1.33).A single case of HbE disease was present (Fig5B ).Three cases showed higher HbF of 11%(SD 1.05) and HbA2 of 4.33(SD 0.15) along with HbE levels of 84.66 (SD 0.97). HbA was nil. They constituted the double heterozygous HbE/β° thalassemia (Fig 5C) HbD hemoglobinopathy was comprised of a single case of HbD trait as shown in Fig6 with HbD of30.8%, HbA of 66.1% and HbA2 of 3.1% . The Hb, RBC and MCV were 10gm/dl, 4.99x1012 and 66.7fl respectively. MCV in the ascending order in HbS/ β thalassemia, HbE/ β thalassemia, HbE disease, βTT, HbD, SCD, HbE trait, SCT and α Thalassemia were 52.3fl,62.5fl (SD 1.5),63.4 fl, 64.2 fl (SD 4.08), 66.7fl , 70fl , 76.1fl (SD 1.33), 81.8 fl (SD 1.1) and 83fl respectively.

Table 1: Shows the distribution of Abnormal hemoglobins Abnormal hemoglobin electrophoresis patterns

No. of cases (n=43)

Percentage

Thalassemia : α Thalassemia (HbH) β Thalassemia trait

1 25

2.3 58.2

Sickle cell hemoglobinopathy : Sickle cell disease Sickle cell trait Double heterozygous Sickle cell/ β thalassemia Compound heterozygous HbS/HPFH

1 5 1 1

2.3 11.7 2.3 2.3

HbE hemoglobinopathy HbE disease HbE trait HbE/ β Thalassemia

1 4 3

2.3 9.3 7.0

HbD hemoglobinopathy

2.3

Table 2: Shows electrophoresis pattern, Hemoglobin, RBC count and MCV in Beta Thalassemia Trait. HbA% 94.3 94.6 88.7 95.1 3.4

HbA2% 5.7 5.4 4.8 4.9 4.7

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HbF% -----6.5 ---1.9

Hb(gm/dl) 10.4 11.9 8.6 12.2 10.1

RBC(x1012/L) 5.2 6.16 3.68 6.68 5.22

MCV(fl) 64.3 62.5 75.5 59.3 62.7

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HbA% 96.4 95.5 95.1 94.7 94.6 96.2 93.1 94.7 95.8 92.9 95.1 94.2 94.5 95 94.6 93.5 94.8 94.3 94.7 95.5

HbA2% 3.8 4.5 4.5 5.3 5.1 3.8 6.2 4.8 3.8 4.5 4.9 5.5 5.1 5 5.4 5.7 5.2 5.2 5 4

HbF% ------0.9 ---0.3 ---0.7 0.5 0.5 2.6 ----0.3 0.4 ------0.8 ---0.5 ----0.5

Hb(gm/dl) 8.4 8.7 11 9.8 12.5 8.9 10.3 9.6 11.6 13.1 12.4 12.9 11.5 8.9 10.4 11 12.5 9 10.5 10.8

RBC(x1012/L) 5.3 4.5 6.4 4.83 6.5 4.1 5.89 5.28 4.85 6.18 6.6 6.61 5.4 4.64 5.21 6.3 6.6 6 5.8 5.9

MCV(fl) 64.3 60.5 59.5 60.6 62.9 69.7 58 60.3 77 68.8 59.2 63.6 69.6 64 66 63 64 68 63 62.2

Table 3 : Shows electrophoresis pattern, Hemoglobin, RBC count and MCV inSickle cell Hemoglobinopathy : Sickle cell hemoglobinopathy (8 cases ) Sickle cell disease(1)

Sickle cell trait (5)

HbA%

HbA2%

HbS%

HbF%

Hb(gm/dl)

RBC(x1012/L)

MCV(fl)

----

2.8

76.6

20.6

8.3

3.81

60.6

3.7

35.7

---

11.1

4.3

80.4

57.7

2.7

39.6

----

4.8

82.6

74.9

3.2

21.3

0.6

11.5

5.5

69.1

3.1

27.8

----

4.7

81.2

66.6

3.7

27.9

1.8

10.5

4.2

83.2

Double heterozygous Sickle cell/ βThalassemia (1)

5.7

8.4

76.9

9.0

5.2

3.19

52.3

Compound heterozygous HbS/ HPFH ( 1)

---

1.3

65.9

32.8

10.2

3.27

91.2

Table 4: Shows electrophoresis pattern, Hemoglobin, RBC count and MCV in HbE hemoglobinopathy : HbE hemoglobinopathy (8) HbE disease (1)

HbE trait (4)

HbE/ β Thalassemia (3 )

HbA

HbA2

HbE

HbF

RBC

MCV

---

5.2

94.8

---

8.5

4.2

63.4

71.1

24.9

---

11.7

4.8

75.4

72.3

3.6

24.1

---

14.3

5.9

72.7

3.4

23.9

---

12.1

4.36

75.7

4.9

18.8

0.6

11.9

4.9

---

4.5

84.9

10.6

4.7

---

4.2

83.6

12.2

9.2

4.8

---

4.3

85.5

10.2

9.7

5.2

62.5

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Fig. 1: Shows normal pattern of hemoglobin electrophoresis.

Fig. 2: A Shows elevated HbA2 in beta thalassemia trait. B Shows HbH band in alpha thalassemia.Â

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Fig. 3: A Shows HbS in addition to HbA in sickle cell trait. B Shows HbS with HbF in the absence of HbA in Sickle cell disease.

Fig. 4: A Shows elevated HbA2, HbF and HbS in heterozygous HbS- beta thalassemia. B Shows elevated HbF in the presence of HbS in heterozygous HbS-HPFH.

Fig. 5: A Shows HbE in addition to HbA in HbE trait. HbA2 is increased . B Shows HbEonly withelevated HbA2 in HbE disease .C Shows elevated HbF and HbA2 in addition to HbE in HetrozygousHbE-beta thalassemia.

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Fig. 6: Shows HbD in addition to HbA and HbA2 in HbD trait.

Discussion

No screening programme is 100% specific and sensitive for the diagnosis of Hb disorders. A combination of haematological, biochemical and molecular analysis including Hb separation on CE, globin chain separation on reverse phase chromatography, sequencing of globin genes, detection of globin gene deletions by PCR and measurement of δ:β globin chain ratios using spectrometry would provide a complete work up for a Hb disorder.[5] However Assessment of HbA2 and HbF along with specific bands play a vital role in the diagnosis of Hb disorders. CE separates the Hb bands with sufficient clarity compared to the other methods of electrophoresis.[2,6] Mean HbA2 levels of 2.12% (SD 0.5) seen in the 57 normal Hb electrophoresisis below the mean HbA2 seen in βTT (4.9%) , HbE hemoglobinopathy( HbE trait -3.98%,HbE/β thal-4.33%) and SCT(3.28%). This difference was statistically significant ( p<0.0001). A low HbA2 of 1% was seen in a case of Iron deficiency anemia in the present study . Mosca and others have reported similar findings and have attributed the decreased HbA2 levels in Iron deficiency to the inhibition of δ globin synthesis by low iron levels and to the preferential binding of β to α chain rather than δ chains. One of the cases with MCV of 118fl showed HbA2 level of 3.6% and was a case of VitB12 deficiency. Megaloblastic anemia and hyperthyroidism elevate HbA2 levels .[7] A www.pacificejournals.com/apalm

0.5% HbF seen in one of our cases are in accordance with normal adult HbF levels of <1%.[4] βTT was the most common abnormal Hb followed by HbS trait, HbE trait and HbE/ β thalassemia. This is comparable with other studies.[8,9,10,11]HbS trait was the second most common Hb disorder in the present study while HbE trait occupied the second position in other studies across the Indian population.[9,10] However Balgir and others have found Sickle cell anemia more common in Orissa, Eastern India. HbA2 levels in βTT was 4.9% (SD 0.62) (range 3.8%to 6.2%) . A range of 3.5% to5.5% and 5.23%(SD 0.63) have been seen in otherstudies using capillary electrophoresis. [3,5] HbA2 levels upto 6.9% have been observed.[12] High HbA2 levels over 6.5% characterise a subgroup of β thalassemia caused by deletions that remove the regulatory elements in the promoter region of β gene. These are often accompanied by increase in HbF[5]An increase in HbF>1% in a healthy adult points to a genetic or acquired pathology. [4] HbF of 2% and 6.5% were seen in the present study in two cases of βTT . Polymorphisms of BCL11A gene has been associated with high levels of HbF in normal persons , β thalassemia and in sickle cell anemia.[4,13] Pernicious anemia, aplastic anemia, chronic renal failure and Diabetes mellitus are some of the acquired causes of elevated HbF.[4] HbA2 levels in sickle cell trait was( 3.28 % ,SD 0.43) significantly more than normal (2.12, SD 0.5) (p<0.0001) eISSN: 2349-6983; pISSN: 2394-6466

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Mean HbA2 levels 0.2%to 1% higher than normal were noted in sickle cell trait by Craver R and others.[12]The modest increase in HbA2 in SCT is due to the increased avidity of normal δ chains for α chains compared to HbS β chain.[14]No significant difference in HbA2 between normal subjects and SCT was recorded by Ajjack and others in their study of sickle cell hemoglobinopathy by CE who found a HbS of 39.3% (SD 13.8)in SCT.[15]This is comparable to HbS of 30.46% (SD 14.4) seen in the present study . Studies show mean HbF levels of 1.4% and 2.14% in SCT. [13,15] In the present study, fetal hemoglobin was identified in two of the five cases(mean 1.2%) Fetal Hb is the major modulator of hematological and clinical features in sickle cell disease . HbF levels declines at a lower rate in sickle cell disease compared to normal individuals stabilising at 5 yrs of age at 5%to 8%.[13] However some patients with sickle cell hemoglobinopathy have high levels of HbF. This includes 3 groups of sickle cell patients – sickle cell disease with Senegal/Saudi-Indian haplotype or BCL11A polymorphisms and compound heterozygous for HbS/HPFH. HbF in the former group varied between 11% to 20%, while HbF levels of 30% (SD 2) was associated with the latter group (HbS/HPFH). [16] Furthur in HbS/HPFH a pancellular distribution of HbF is a characteristic feature. In the present study two cases showed high HbF levels of 20.65 and 32.8% along with HbS in the absence of HbA and were designated as Sickle cell disease and compound heterozygous HbS/HPFH respectively. Elevated HbF along with elevated HbA2, HbS and HbA is double heterozygous HbS/β Thalassemia , a single case in the present study. HbE is the second common hemoglobinopathy after sickle cell hemoglobinopathy in South-east Asia which includesNorth eastern India, Thailand, Malaysia, Nepal, Bangladesh and Vietnam. [17] In the present study, HbE hemoglobinopathy accounted for 18.6% abnormal Hb similar to Sickle cell hemoglobinopathy. HbE trait was more frequent followed by HbE/β Thalassemia and HbE disease. HbA2 levels in HbE trait was significantly higher than normal subjects (p value <0.0001) and HbA2 levels in HbE disease was significantly higher than in trait (p value =0.035) .These findings are in corroboration with those of Mais DD who found higher HbA2 in HbE trait compared to control group and higher HbA2 in HbE homozygotes than in heterozygotes.[6] β chain of HbE is synthesized at a reduced rate compared to HbA. Hence δ chain combines with α chain increasing HbA2 –Thalassemic effect. Three cases of HbE hemoglobinopathy with elevated HbA2 (4.33%, SD 0.15) and HbF (11%, SD 1.0) in the absence of HbA were categorised as HbE/β thalassemia . Since there

was no HbA these cases probably represented HbE/β° thalassemia. Praising and others have similarly concluded that a combination of HbA2 levels > 6% and HbF varying from 5%to 15% would differentiate HbE/β thalassemia from HbE disease.[18] Identifying HbE/β Thalassemia is important since it is clinically associated with a more severe disease compared to HbE trait and HbE disease.[17] α Thalassemia and HbD were the less common Hb disorder in the present study similar to the other studies by Vani and others who reported 1.6% and 0.7% of these cases respectively.[8] The predominant feature in HbH disease is the presence of HbH ranging between 0.8% to 40% with normal or slightly reduced HbA2.[19]In the present study, HbH of 28.3% and HbA2 of 2.1% were observed. HbD common in Punjab also known as HbDLos Angels can be inherited in heterozygous state with HbA as in the present study and in the rarest form of homozygous state- HbDD. Association with HbS and Thalassemia also occur.[20].MCV was lower in HbE/ β Thalassemia compared to β Thalassemia, HbE trait and SCT in the ascending order . A similar pattern has been described by Vani and others.[8] The other Hb disorders were single cases inadequate for comparison.

Conclusion

Thus the quantification of HbF differentiates the three groups of sickle cell hemoglobinopathy patients, those of Senegal/Saudi Indian haplotype, BCL11A polymorphisms Vs heterozygous HbS/HPFH . Furthur the presence of elevated HbA2 and HbF in HbE hemoglobinopathy identifies HbE/β Thalassemia , an entity needing clinical intervention. Assesment of HbA 2 and HbF in conjunction with specific abnormal Hb band and HbA thus plays a pivotal role in the diagnosis of hemoglobinopathy and Thalassemia. Quantification of HbA2 and HbF thus throws light on the pattern of inheritance – homozygous/ heterozygous / double heterozygous which is confirmed by molecular analysis.

References 1.

Wajcman H, Moradkhani K. Abnormal hemoglobins: detection and characterisation. Indian J Med Res 2011;134:538-46.

Oyaert M, Van Laer C, Claerhout H, Vermeersch P, Desmet K, Pauwels S et al. Evaluation of SebiaMinicap Flex Piercing capillary electrophoresis for hemoglobinopathy testing. International Journal of Laboratory hematology 2015;37:420-5.

Alauddin H, Yusoff MM, Khirotdin A, Azlin I, Ning YZ, Ishak LM et al.HbA2 levels in normal , β thalassemia and haemoglobin E carriers by capillary electrophoresis. Malaysian J Pathol 2012;34:161-4.

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Mosca A, Paleari R, Leone D, Ivaldi G. The relevance of haemoglobin F measurement in the diagnosis of thalassemias and related hemoglobinopathies. Clinical Biochemistry 2009;42:1797-1801. Youssef E. Thalassemias: detection, characterisation and laboratory interpretation. The Biomedical Scientist 2012;34:363-8. Mais DD, Ronald D, Gulbranson MT, Keren DF. The range of Hemoglobin A2 in Haemoglobin E heterozygotes as determined by Capillary electrophoresis. Am J ClinPathol 2009;132:34-8.

Mosca A, Paleari R, Ivaldi G. The role of haemoglobin A2 testing in the diagnosis of thalassemias and related hemoglobinopathies. J ClinPathol 2009;62:13-17.

Chandrashekar V, Soni M. Hemoglobin disorders in South India. Hematology 2011.doi:10.5402/2011/748939.

Sachdev R, Dam AR, Tyagi G. Detection of Hb variants and hemoglobinopathies in Indian Population using HPLC: report of 2600 cases. IJPM 2010;53:57-62.

10. Rao S, Kar R, Gupta SK, Chopra A, Saxena R. Spectrum of hemoglobinopathies diagnosed by cation exchange-HPLC and modulating effects of nutritional anemias from north India. Indian Journal of Medical Research 2010;132:513-19. 11. Balgir RS. Spectrum of hemoglobinopathies in the State of Orissa, India: a ten years cohort study. Journal of Association of Physicians of India 2005;53:1021-26. 12. Craver RD, Abermanis JG, Warrier RP, Ode DL, Hempe JM. Hemoglobin A2 levels in healthy persons, Sickle cell

A-453 disease, Sickle cell trait and β thalassemia by capillary isoelectric focusing. Hematopathology 1996;107:88-91. 13. Akinsheye I, Asultan A, Solovieff N, Duyen N, Baldwin CT, Sebastiiani P. FetalHemoglobin in sickle cell anemia. Blood 2011;118:19-27. 14. Shokrani M, Terrell F, Turner EA. Chromatographic measurements of haemoglobin A2 in blood samples that contain sickle haemoglobin. Ann Clin Lab Sci 2000;30:191-4. 15. Ajjack EA, Awood HA, Abdalla SE. Hemoglobin patterns in patients with sickle cell hemoglobinopathies. International Journal of Hematological Disorders 2014;1:8-11. 16. Duyen An, Aygun B, Akinsheye I, Hankin JS, Bhan I, Luo HY et al. FetalHemoglobin levels and haematological characteristics of compound heterozygotes for haemoglobin S and deletional hereditary persistence of fetal haemoglobin 2011;156:259-64. 17. Bachir D, Galacteros F. OrphanetEncyclopedia 2004.

Hemoglobin

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18. Prasing W, Pornprasert S. Measurement of HbA2 by Capillary electrophoresis for diagnosing β thalassemia/HbE disease in patients with low HbF. Lab Medicine Summer 2014;45:226-30. 19. Harteveld C, Higgs DR. α–thalassemia. Orphanet journal of rare diseases 2010;5:13-34. 20. Torres LD, Okumura JV, Humberto De Silva DG, BoniniDomingos CR. Hemoglobin D-Punjab: Origin, distribution and laboratory diagnosis. Brazilian Journal of Hematology and Hemotherapy 2015;37:120-6.

*Corresponding author: Dr. Sandhya.V, D3 -508, L&T Southcity apartment, JP Nagar 7th phase, Arekere MICO layout, Bangalore-560076,India Phone: +91 9591627364, 080-26554334 Email: sandhyavenkatachala@yahoo.co.in Date of Submission : 01.02.2017 Date of Acceptance : 29.05.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

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Original Article DOI: 10.21276/APALM.1462

Clinical and Histopathological Correlation in Hansenâ&#x20AC;&#x2122;s Disease Muruganantham Arunagirinathan, Vennila Muniswamy* and J. Sivaraman Dept of Pathology, Govt. Vellore Medical College & Hospital, Tamil Nadu, India

ABSTRACT Background: The present study was carried out to correlate clinical diagnosis of new leprosy cases with that of histopathological diagnosis of skin biopsies stained with Haematoxylin and Eosin followed by Fite Faraco stain. Method: Skin biopsies of 70 newly diagnosed leprosy patients were fixed in 10% formalin and routine histopathological examination done followed by special stain (Fite Faraco) to evaluate the bacterial index. Results: From this study it was observed that, the commonest age group affected by leprosy was 31 to 50 years, males are twice more commonly affected than females (M:F = 1.9 : 1) and the most commonest clinically diagnosed spectrum was Tuberculoid leprosy (TT). The commonest histopathologically diagnosed spectrum was Indeterminate leprosy(IL). It was observed that there was complete agreement between clinical diagnosis and histopathological diagnosis in 62.85% cases and disagreement was observed in 37.15% cases. Conclusion: In case of confirmed discrepancy, the more advanced findings (ie. towards the lepromatous pole) should be given greater weightage and the case is to be classified and treated accordingly. Keywords: Clinical Diagnosis, Histopathological Diagnosis, Leprosy, Fite Faraco Stain, Agreement, Disagreement

Introduction

Leprosy (Hansenâ&#x20AC;&#x2122;s disease), is a chronic infectious disease that primarily affects the skin & the peripheral nerves. Leprosy is one of the oldest diseases of mankind[1]. Even though tremendous progress has been made in the field of leprosy, it still continues to be a global health problem. Despite an extensive global drug programme for leprosy, implemented by the WHO, leprosy is endemic in many countries with approximately 211,000 new cases reported every year. The overall prevalence of leprosy in India has declined from 5.27/10000 in the year 2000 to 0.66/10000 in the year 2016, but still it continues to be a sizable public health problem[2]. India represents approximately 60% of the global burden[3]. Leprosy expresses itself in different clinicopathological forms depending on the immune status of the host. Diagnosis of leprosy is based on different clinical parameters which involves detailed examination of skin lesions and peripheral nerves. Demonstration of acid- fast bacilli in slit skin smears by modified Ziehl- Neelsenâ&#x20AC;&#x2122;s staining also aids in the diagnosis of leprosy. A reliable diagnosis hinges around a good histopathological work up and demonstration of bacilli in histopathological sections.

Fite - Faraco staining procedure has proved most valuable in demonstrating lepra bacilli in tissue sections. Ridley and Jopling were the first to suggest a subclassification of leprosy based on immunological aspects, as five types; Tuberculoid (TT), Borderline Tuberculoid (BT), Mid borderline (BB), Borderline Lepromatous (BL) and Lepromatous Leprosy (LL)[4]. Later, they correlated clinical and bacteriological findings in each group with respective immunological and histological findings.

Materials and Methods

The study was conducted on the skin biopsies of patients newly registered at the Department of Dermatology, Venerology & Leprosy and subsequently reported to the Histopathology section of the Department of Pathology, Government Vellore Medical College & Hospital, Vellore, over a period of 2 years from January 2015 to Dec 2016. Newly diagnosed leprosy patients aged between 15 to 65 years with hypopigmented patches with loss of sensation were included in the study. Patients younger than 15 years and older than 65 years, old treated cases and those who where on multi drug therapy(MDT) were excluded from the study. After informed consent, enrolled patients were subjected to general and dermatological examination regarding

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the morphology, number, size, site, colour, anaesthesia, margins, surface, satellite lesion and central clearing of skin lesions as well as the involvement of peripheral nerves and cutaneous nerves was done. Reactions and deformities were also noted.

Clinical diagnosis of leprosy cases (as provided by Department of Dermatology, Venerology & Leprosy) using Ridley & Jopling scale was correlated with the results of histopathological examination of their respective biopsies.

Skin biopsy was done from the margin of the skin lesion and fixed in 10% formalin. The specimen was processed routinely in the histopathology lab and sections were stained with Haematoxylin and Eosin.

During the study period of 2 years from January 2015 to Dec 2016, 70 skin biopsy specimens of clinically diagnosed new untreated cases of leprosy were studied, which included 46 males and 24 females (M : F = 1.9 : 1) aged between 15 to 65 years.

Haematoxylin and Eosin stained sections of the skin biopsies of all the cases of leprosy included in the study were examined for : a) Epidermal atrophy, epithelioid granulomas, number and distribution of lymphocytes, histiocytes & foam cells. b) Infiltration of nerves, blood vessels and adnexa. c) Grenz zone. Sections stained with Fite- Faraco stain were examined for lepra bacilli. The following Fite Faraco staining procedure was carried out : 4 micron thick sections were kept in the incubator for 10 to 20 minutes to dewax the sections. The slides were kept in a mixture of xylene and oil (liquid paraffin) in the ratio of (3:2) for 40 minutes. The slides were drained, blotted, dried and rinsed in water for 1 minute, followed by this the sections were covered with carbol fuschin for 40 minutes. The slides were rinsed in water, decolourised with 5% sulphuric acid for 5 minutes. The slides were washed in water, counter stained with haematoxylin for 3 minutes. Followed by this, slides were washed in water, air dried and mounted with DPX. The stained slides were observed under microscope, mycobacterium appears as pink to red rods in a blue background. Based on Ridleyâ&#x20AC;&#x2122;s logarithimic scale, bacterial index (BI) was done. Histopathological findings were graded into tuberculoid (TT), borderline tuberculoid (BT), mid borderline (BB), borderline lepromatous(BL) and lepromatous leprosy (LL) according to Ridley & Jopling scale. Sections showing scattered non specific lympho-histiocytic infiltration with cellular reaction within the dermal nerve or presence of bacilli in arrector pilorum muscle / dermal nerve were classified as indeterminate leprosy and also included in the study for the purpose of analysis. Table 1: Histological Types of Leprosy HISTOLOGICAL TYPE TT BT BB BL LL HISTOID IL

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Results

Histopathological examination of the skin biopsies of 70 patients revealed that, the maximum histopathological cases were seen in IL (Indeterminate) type followed by TT and LL (with its variant Histoid type). Maximum number of cases were seen in the polar spectrum ie). TT & LL (including its variant Histoid) and indeterminate type. Based on the Ridley & Jopling logarthmic scale, bacteriological index (BI) was studied on Fite Faraco stained slides. BI observed was 0 (zero) in case of TT and 5+/ 6+ in cases of LL and its variant Histoid leprosy. The distribution of 70 cases on clinical leprosy spectrum based on Ridley & Jopling scale revealed maximum cases in polar spectrum: TT - 28 cases (40 %), LL and its variant Histoid - 18 cases (14 + 4) (25.71 %), borderline (BT + BB + BL) - 20 cases (28.57 %) and the least in IL - 4 cases (5.72%). Complete agreement of clinical and histological diagnosis was seen in Histoid Hansenâ&#x20AC;&#x2122;s (variant of LL) (100%) and IL (100%) followed by LL (85.71%) & TT (57.14%). Least agreement of clinical and histopathological diagnosis was observed in borderline spectrum (BT, BB, BL). Most number of cases which had disparity between clinical and histopathological diagnosis showed histological features of IL (20 cases) due to the absence of granuloma. In the present study the histopathological characteristics were consistent with the clinical diagnosis in 44 cases out of 70 cases. Complete agreement between clinical diagnosis and histopathology was observed in 62.85 % and disagreement was seen in 37.15% cases. NUMBER OF CASES 16 4 4 6 12 4 24

PERCENTAGE 22.85% 5.72% 5.72% 8.57% 17.14% 5.72% 34.28%

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Table 2: Correlation of Hpe Diagnosis and Bacteriological Index ( Fite Faraco Stain). HPE DIAGNOSIS BACTERIOLOGICAL INDEX ( BI ) TT 0 BT 1+ BB 2+ / 3+ BL 3+ / 4+ LL 5+ HISTOID 5+ / 6+ IL 0 / 1+ Table 3: Clinico- Histopathological Correlation CLINICAL DIAGNOSIS TT (28) BT (8) BB (4) BL (8) LL (14) HISTOID(4) IL(4)

TT 16 -

BT 2 2 -

HISTOPATHOLOGICAL DIAGNOSIS BB BL LL HISTOID 2 2 4 2 12 4 -

Table.4. Complete Agreement - Clinical Diagnosis & Histopathology CLINICAL CASES TYPES NUMBER TT 28 BT 8 BB 4 BL 8 LL 14 HISTOID 4 IL 4 TOTAL 70

Fig.1: Borderline lepromatous leprosy. Multiple bilateral more or less symmetrical copper coloured shiny anaesthetic patches.

IL 12 4 2 2 4

PARITY PERCENTAGE 57.14% 25% 50% 50% 85.71% 100% 100%

COMPLETE PARITY ( HPE ) NUMBER PERCENTAGE 16 57.14% 2 25% 2 50% 4 50% 12 85.71% 4 100% 4 100% 44 62.85%

Fig. 2: Borderline lepromatous leprosy : Atrophic epidermis, clear subepidermal grenz zone, collection of macrophages and lymphocytes.

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Fig. 3: Borderline lepromatous leprosy. Fite- Faraco stain. Bacterial Index ( BI ) = 4+.

Fig. 4: Clinico â&#x20AC;&#x201C; Histopathological correlation of various types of leprosy. Y- axis denotes the number of patients.

Fig. 5: Percentage of complete agreement of histopathological diagnosis with clinical diagnosis and percentage of disagreement.

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Discussion

A chronic disease like leprosy needs appropriate classification because of its varied manifestations. The most commonly accepted classification by research workers is that of Ridley & Jopling4 which is primarily based on immunity but has been correlated with clinical, histopathological and bacteriological findings. Despite having such an accurate classification, there are diversities between the clinical and histopathological features. Clinical spectrum of leprosy in the present study revealed maximum cases in polar spectrum - TT (40%), followed by LL and its variant Histoid (25.71%), borderline (ie) BT, BB, BL (28.57%) and IL (5.72%). Similar predominance of cases in polar spectrum was observed by Kalyani mitra et al5. In the present study the histopathological characteristics were consistent with the clinical diagnosis in 44 cases out of 70 cases (62.85%), similar to the observation made by Nadia et al6. Lepromatous cases (including its variant Histoid) seem to present the least problem in classification. Similar highest percentage of agreement between clinical and histopathological diagnosis of lepromatous leprosy cases was also observed by Shenoi & Sidappa7, Pandey & Tailor8, Bhatia et al9, Kalla et al10 and Shanker Naryan et al11 in their respective studies. Least agreement was seen in cases of borderline spectrum (BT, BB, BL) in this study, which is in concordance with observations recorded by Shenoi & Siddappa7, Nadkarni & Rege12, Bhatia et al9 and Singhi et al13. Maximum discordance (37.15%) between clinical and histopathological diagnosis was observed in borderline spectrum cases (BT, BB, BL) of the present study and the same was also noted by Singhi et al13 and Sandeep et al14. Borderline spectrum particularly midborderline leprosy is immunologically the least stable and a variety of clinical lesions of different morphology may be found in the same patient. It is therefore necessary to relate the histological features with the clinical characteristics presented by the particular morphological lesion subjected to biopsy. If this is done carefully, it may be possible to achieve a better clinical correlation with the histological changes. Tuberculoid and borderline tuberculoid leprosy often overlap clinically, histologically and immunologically but differ only in the degree and the same is true for borderline lepromatous and lepromatous leprosy. In the present, study, 24 cases (34.28%) were diagnosed as indeterminate leprosy histologically as against 4 cases (5.72%) clinically. Nadkarni & Rege12 and Kalyani

mitra et al5 had also diagnosed a sizeable proportion of the cases as indeterminate leprosy histopathologically, who were clinically classified as cases of TT, BT, BB or BL leprosy. Indeterminate leprosy is one which cannot be classified within the Ridley & Jopling spectrum due to lack of distinguishing features and this happens more often histologically (due to the failure to find a granuloma) than clinically. In the present study, the high percentage of indeterminate leprosy noted histologically in clinical cases of TT, BT & BB groups could have been due to immunological differences in host responses. The disparity between clinical and histological observation was anticipated because the parameters used for the histopathological classification are well-defined, precise and also take into account the immunologic response of the tissue, while the clinical classification gives recognition only to the gross appearances of the lesions, which is due to the underlying pathological changes. Moreover, a sizable proportion of leprosy cases (BT, BB, BL) are in a continuously changing immunological spectrum and the histological classification gives a better indication for any recent shift of a case position in the spectrum. In some early cases, clinical signs and symptoms may precede the presently known characteristic tissue changes, or vice versa9. If a biopsy is taken at an early stage, there is likely to be discordance between the clinical and histopathologic observation. As disparity depends upon the lesion biopsied at the time of study, biopsy from the lesion which is morphologically suggestive of clinical diagnosis, serial biopsies from the same lesion or from paired lesions should be studied for better clinico pathological correlation. From the present study it was observed that :

•

The commonest age group affected by leprosy was 31 - 50 years.

•

Males are twice more commonly affected by leprosy than females. (M : F = 1.9 :1).

•

The most commonest clinically diagnosed spectrum was tuberculoid leprosy (TT).

•

The commonest histopathologically diagnosed spectrum was IL, followed by TT & LL with its variant histoid leprosy.

•

It was observed that there was complete agreement between clinical diagnosis and histopathological diagnosis in 62.85% cases.

•

Disagreement between histopathological diagnosis and clinical diagnosis was observed in 37.15 % cases.

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•

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If there is discrepancy between clinical and histopathological classification, both the findings should be reviewed by independent experienced observers.

WHO – Fact sheet on Leprosy : Status of the disease in 2015.

Ridley DS, Jopling WH. Classification of leprosy accroding to immunity: a five- group ystem. Int J Lepr 1966; 34: 255.

In case of confirmed discrepancy, the more advanced findings (ie. towards the lepromatous pole) should be given greater weightage and the case is to be classified and treated accordingly.

Mitra K, Biswas S et al. Correlation between clinical and histopathological criteria for the classification of leprosy. Indian J Dermatol 2001; 46 (3) :135- 137.

Nadia S, Rashmi J, Sohaib A, Rawat SDS, Selvi Thamarai N, Meena H. Clinicopathological correlation of Leprosy : A 4 year’s retrospective study from a tertiary referral centre in North India. Int J Med Res Health Sci. 2015;4(2):350-354.

Shenoi SD, Siddappa K. Correlation of clinical and histopathologic features in untreated macular lesions of leprosy - a study of 100 cases. Ind J Lepr 1988; 60 : 202 - 06.

Pandey AN, Tailor HJ. Clinicohistopathological correlation of leprosy. Ind J Dermatol Venereol Lep 2008; 74 : 74- 76.

Bhatia AS, Katoch K et al. Clinical and histopathological correlation in the classification of leprosy. Ind J Lepr 1993 ; 61 : 433- 438.

In all cases of leprosy, in order to type the exact spectrum of disease for appropriate treatment, skin biopsy of the lesions with histopathological examination followed by special stain (Fite -Faraco) is recommended.

Conclusion

In clinical practice a case of leprosy is to be classified as per clinical criteria. Skin biopsy has to be taken from the most active site of the lesion. This will help in the confirmation of diagnosis and classification. If there is discrepancy between clinical and histopathological classification both the findings should be reviewed by independent experienced observers. In case of confirmed discrepancy the more advanced findings (ie. towards the lepromatous pole) should be given greater weightage and the case is to be classified and treated accordingly. This will prevent inadequate treatment of a particular case.

10. Kalla G, Salodkar A, Kachhawwa D. Clinical and histopathological correlation in leprosy. Ind J Lepr 2000 ; 68 : 184 -185. 11. Shanker Narayan NP, Ramu G et al. Correlation of clinical, histological and immunological features across the leprosy spectrum. Ind J Lepr 2001 ; 73 :329- 42. 12. Nadkarni NS, Rege VL. Significance of histopathological classification in leprosy. Ind J Lepr 1999 ; 7 : 325- 32.

References

13. Singhi MK, Kachhawa D, Ghiya BC. A retrospective study of clinic- histological correlation in leprosy. Ind J Pathol Microbiol 2003 ; 46 : 47- 48.

14. Sandeep M, Shamanur Murugesh. A Study of ClinicPathological Concordance in Leprosy Patients in the PostElimination Era. International Journal of Science and Research. 2016;5(2):1304-1306.

Jopling WH, McDougall. Definition, Epidemiology and World Distribution Hand book of Leprosy, Fifth edition. CBS Publishers and Distributors 1996: 1. National Leprosy Eradication Programme – Annual report for the year 2015 -2016.

*Corresponding author: Dr Vennila Muniswamy, Dept. of Pathology, Government Vellore Medical College and Hospital, Adukamparai, Vellore, Tamil Nadu 632011, India. Phone: +91 8939958993

Financial or other Competing Interests: None.

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Date of Submission : 06.04.2017 Date of Acceptance : 26.04.2017 Date of Publication : 31.08.2017

eISSN: 2349-6983; pISSN: 2394-6466

Original Article DOI: 10.21276/APALM.1463

Fine Needle Aspiration Cytology of Cervical Lymphadenopathy: Is There Anything Different At South Coastal Region of India? Priya R, Dhananjay Kotasthane*, Vaishali Dhananjay Kotasthane and Koteeswaran G Department of Pathology, Mahatma Gandhi Medical college and Research Institute, pillaiyarkuppam, Pondicherry (India)

ABSTRACT Background: Cervical lymphadenopathy is one of the commonest clinical presentations of the patients in all age groups. FNAC is simple, inexpensive and minimally invasive procedure that can be used as an outpatient procedure for diagnosing lymphadenopathy cases. Aims and Objective: To assess the various causes of cervical lymphadenopathy through FNAC and to see the distribution of various lesions with respect to age and gender and to correlate with histopathological findings. Materials and Methods: The present Analytical cross-sectional study was conducted in the Department of Pathology, Mahatma Gandhi Medical College and Hospital, Pondicherry from January 2010 to July 2016.FNAC was done by standard procedure. All the slides were reviewed and diagnosis was rendered. Cytology findings were compared with histopathological diagnosis where-ever excision biopsy was done. Results: A total of 411 patients were included in the present study. Overall, female preponderance was seen. In benign non-neoplastic lesions, peak occurrence was seen in the 3rd decade, whereas it was 7th decade in metastatic lesions. Benign lymphadenopathies were diagnosed in 75.1% of cases, majority being chronic reactive lymphadenitis(34.6%) followed by granulomatous lymphadenitis(30%). The proportion of granulomatous lymphadenitis was more in our study as compared to other studies(p<0.0001).In neoplastic lesions, majority (88%)were metastatic deposits. In metastatic lesions, higher proportion of metastatic thyroid carcinoma (13.9%) was seen which was statistically significant (p<0.0001) as compared to other studies. Unpaired t test was used to prove statistical significance. Cyto-histopathology correlation was done in 54 cases and FNAC showed sensitivity of 97%,specificity 95%and diagnostic accuracy of 96%. Conclusion: FNAC is safe, simple, rapid, minimally invasive and inexpensive procedure to diagnosis cervical lymphadenopathy with high sensitivity, specificity and diagnostic accuracy. Higher frequency of granulomatous lesions was seen in non-neoplastic cases. In neoplastic lesions, metastatic thyroid carcinoma showed higher frequency in our study conducted at iodine sufficient south coastal region of India. Keywords: Fine Needle Aspiration Cytology, Lymph Node, Histopathology

Introduction

Cervical lymphadenopathy is one of the commonest clinical presentations of all age groups attending the outpatient department.[1]The various etiologies for cervical lymphadenopathy are broadly categorized as microbial infections & their breakdown products, malignancies, iatrogenic conditions, autoimmune disorders.[2] The general approach to cervical lymphadenopathy includes: detailed clinical evaluation, fine needle aspiration cytology and open biopsy.[3] FNAC has become an important diagnostic tool for initial and rapid diagnosis and management of patients having lymphadenopathy, owing to its simplicity, reliability, early availability of results and it, being a minimally invasive procedure. It is a primary method for diagnosing whether lymphadenopathy is due to reactive, infective or metastatic causes, thereby avoiding unnecessary excisional biopsy and aiding in rapid onset of therapy.[3,4]

The cytomorphological features obtained through FNAC correlates well with histological appearance. Thus, FNAC is considered as an ideal method for diagnosing the nature of lesions.[5,6] The diagnosis of metastatic tumor to the lymph node by cytomorphological pattern is highly reliable and it is a sole indicative procedure for evaluating primary tumor in case of the occult primary.[7,8,9] In case of non-neoplastic lesions, the varied morphological patterns can mimic the other reactive conditions and it can lead to multiple differential diagnosis.[10,11,12] In case of primary lymphoid malignancies, FNAC is mainly used to assess the tumor staging and to recognize the residual and recurrence of lymphoid malignancy. The aim of the study was to describe different cytological lesions of cervical lymphnode in context with age, sex and mode of presentation and to compare these findings with histopathological diagnosis to calculate sensitivity, specificity and diagnostic accuracy.

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

Priya R et al.

Materials and Methods

The present Analytical study on 411 patients with cervical lymphadenopathy was done at the Department of Pathology, Mahatma Gandhi Medical College and Research Institute over a period of 6.5 years from January 2010 to July 2016 after obtaining clearance from institutional Human ethics committee. Aspirations from All cervical lymph nodes were included in the study i.e submental, submandibular, (level Ia, Ib) and posterior triangle including supraclavicular lymphnodes (level V). The relevant clinical details were noted from the accompanying requisition forms and/or from the patient case records. Clinical data including age, gender, clinical diagnosis, site and duration, consistency and any organ involvement like hepatosplenomegaly were noted. The cytology slides of all cases were examined to determine the cyto-morphological features. Cytology findings were compared with histopathological diagnosis wherever excision biopsy was done. The efficacy of FNAC was tested by using statistical test like Sensitivity, Specificity, Diagnostic accuracy, Positive predictive value and Negative predictive value after cyto-histopathology correlation. Unpaired t test was applied to compare our findings with other studies and p value <0.05 was considered as significant.

Results

Total 411 patients of all age groups with cervical lymphadenopathy were included in this study and the peak occurrence was observed in 3rd decade of life accounting for 23.6%(97 cases).We observed that non-neoplastic lesions were common in the 3rd decade with female preponderance, in contrast with metastatic lesions which were more common in the 7th decade with male preponderance. In the present study, out of 411 cases, majority of the patients presented with left sided cervical lymphadenopathy with frequency of 55.7%and right sided involvement in 40.4%. Bilateral involvement was seen in 3.9%cases. Out of total 411 cases, non-neoplastic lesions were more common accounting for 75.1%, followed by neoplastic 22% and non-diagnostic 2.9%.In non-neoplastic lesions, chronic reactive lymphadenitis was found to be the most common pathological lesion(34.6%) followed by granulomatous lesion(30%)[Fig1]. Distribution of remaining non-neoplastic cases was shown in Table 1.Ninetycytology cases showed neoplastic lesions and were further grouped into metastatic, Hodgkin’s and nonHodgkin’s lymphomas. Out of these, metastatic lesions were more common with an occurrence of 87.7%.The occurrence of metastatic Adenocarcinoma and Squamous cell carcinoma showed equal frequency in our study with www.pacificejournals.com/apalm

A-461 62 cases (39.2%) each. Metastasis occurred from primary carcinoma in breast, stomach, colon, rectum, and lung.[Fig 2&3]The occurrence of metastatic thyroid carcinoma in our study was 13.92%.[Fig 4] The youngest patient in our study was 5 year girl presented with nodular enlargement of thyroid with multiple group of cervical lymph node enlargement over a period of 6 months. In 3 cases, ultrasonography revealed multiple cystic areas admixed with solid areas which were diagnosed as metastasis from papillary carcinoma of thyroid, correlating with cytological diagnosis. In our study, metastatic malignant melanoma was reported in one case. Clinically, a 65 year old male patient presented with 4 x3 cm, raised, ulcerated wound present in the right plantar aspect of the foot for a duration of six months with multiple groups of lymph node involvement. On aspiration, highly cellular smear shows scattered and few clusters of polygonal shaped cells with eosinophilic cytoplasm with vesicular nuclei with prominent nucleoli. Both intracellular and extracellular melanin pigmentation is seen. Background shows Melanophage and melanin.[Fig 5] Cytologically, 5 cases were diagnosed as Non-Hodgkin’s lymphoma. The age group ranged from 32 to 60 years with male preponderance. Cytologically, 6 cases were diagnosed as Hodgkin’s lymphoma, majority of patients were in 2nd and 3rd decade with male preponderance. Clinically, all patients presented with cervical lymph node enlargement. Two patients presented with splenomegaly. Microscopically, 4 cases showed Classic Reed Sternberg cell and two cases showed mononuclear cells morphology. Correlation of Cytological Diagnosis with Histopathology: In this study period, 411 cases of cervical lymphnode were referred to Department of pathology for aspiration.Out of these, 12 smears were inadequate or non-diagnostic, hence exclude from further evaluation. Table 2 showed that for 90 cytologically diagnosed neoplastic lesions, histopathology was available in 33 cases. Similarly, for 309 non-neoplastic FNAC lesions, histopathology was available in 21 cases only as non-surgical treatment is usually preferred for non-neoplastic lesions. Thus, for 399 cytology cases, histopathology was available in 54 cases.[Table 2] From Table 2,metastatic (26 cases) and Hodgkin’s lymphoma (6 cases) showed 100% cyto-histopathology correlation, whereas in NHL, 3 out of 4 showed correlation with histopathological diagnosis of small cell lymphoma in two cases, one showed Diffuse Large cell lymphoma(DLCL)but remaining one case turned out to be granulomatous lymphadenitis accounting for false positivity. Similarly, in 21 non-neoplastic lesions, cytoeISSN: 2349-6983; pISSN: 2394-6466

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Cytology of Cervical Lymphadenopathy

histopathology correlation was seen in 20 cases and one case of granulomatous lesion showed features of NonHodgkinâ&#x20AC;&#x2122;s lymphoma on histology accounting for false negativity. Based on these results, sensitivity, specificity,

positive predictive value, negative predictive value and diagnostic accuracy of FNAC to diagnose cervical lymphnode lesions were calculated and was found to be 97%,95%,97%,95%,97% and 96% respectively.

Table 1: Age wise distribution of non -neoplastic lesions of cervical lymph Node. 01-10 11-20 21-30 31-40 41-50 51-60 61-70 71-80 TOTAL (n =309) Percentage % Chronic nonspecific reactive lymphadenitis

106

34.64

Granulomatous lymphadenitis Suppurative lymphadenitis Necrotizing lymphadenitis Tuberculous lymphadenitis Others(RDD) Percentage %

6 3 1 4 10

21 3 2 15 22.3

28 6 3 24 2 30

16 11 1 13 17.1

12 2 1 4 1 8.7

7 8 7.8

3 2 2.9

1 2 4.8

94 26 8 72 3 -

30.04 8.41 2.58 23.33 0.97 100%

Table 2: Cyto-Histo Correlation of Cervical Lymph Node Lesion(n=54).

FNAC DIAGNOSIS(N=399) Neoplastic (N=33)

HPE (N=54)

N eoplastic(N=33)

Metastasis (N=26)

HL (N=3)

3 -

Metastasis(N=79)

HL(N=6) NHL(N=5) N on-N eoplastic (N=309)

NHL (N=4) 3 1

Non -Neoplastic (N=21) -

1 20

Table 3: Comparison of Distribution of different pathological subtypes of metastasis with other studies. FNAC of metastatic LN Adenocarcinoma Squamous cell carcinoma Thyroid carcinoma

Ghartimagar D et al.26 67% 39.24% 2%

Rathod K et al25 2% 96% -

Pavithra et al20 9.37% 56% 4.68%

Present study 39.24% 39.24% 13.94%

Poorly differentiated carcinoma

25%

5.06%

Malignant melanoma

1.56%

1.26%

Fig. 1: Tuberculous lymphadenitis showing caseous necrosis and granuloma (H&E 40x).

Fig. 2: Aspirate shows dyscohesive clusters of cells with scant cytoplasm and pleomorphic nuclei with prominent nucleoli in Metastatic Infiltrating DuctalCarcinoma of breast (H& E Stain 40x).

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Fig. 3: Cytology smear adenocarcinoma (H&E 10x).

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showing

metastatic

Fig. 4: Aspirate shows papillary fragment containing cells with large nuclei havingintranuclear inclusion in metastatic papillary carcinoma thyroid (H & E 10x).

Fig. 5: Aspirate shows dispersed melanin laden pleomorphic cells in metastatic malignant melanoma(H &E 40x).

Discussion

Among the different groups of lymph nodes, cervical group is easy to access by FNAC and therefore it plays a major role in establishing rapid diagnosis. FNAC also plays important role in diagnosing the etiology of cervical lymph node enlargement due to early availability of results, better patient compliance and minimal trauma.[13] The diagnosis obtained through FNAC very well correlates with histopathological diagnosis after excision biopsy.[14] The gender distribution of cases in our study showed clear female preponderance with a male: female ratio of 1:1.7 in adulthood. Similar findings were noted by other authors. [15-18] However, paediatric and older age group showed male predominance in our study.

of our study showed correlation with the study conducted by Dukare et al, Pavithra et al and Kumar et al.[19,20,21]In our study, left sided cervical lymph node lesions were most commonly involved in both neoplastic and nonneoplastic lesions with an frequency of 55.7%, whereas right sided lesions accounted for 40.4%.Similar findings were noted by Attaullah et al.[17]In the current study, we observed that non-neoplastic lesions was the most common cause of cervical lymph node enlargement accounting for 74.5%. In other studies, similar findings were observed with frequency of non-neoplastic lesion ranging from 70 to 90%.[19,22,23]

In the present study, we observed that non- neoplastic lesions were common in the 3rd decade whereas neoplastic lesions were more common in 6th to 7th decade. Findings

Among non-neoplastic lesions, reactive lymphadenitis was the most common pattern with an occurrence of 34.6%, which showed mild preponderance over granulomatous lymphadenitis (30.04%).Other studies also showed majority cases belonging to reactive lymphadenitis ranging from 50% to 70%. Our study showed 63% cases of granulomatous

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lymphadenitis, which was higher than other studies, which reported these cases ranging from 25 to 30% [13,23,24,].This difference was found to be statistically significant using unpaired t test with p value <0.0001.In the present study, one cytologically diagnosed granulomatous lymphadenitis turned out to be Non-Hodgkin’s Lymphoma on histopathology accounting for false negativity probably due to non availability of architecture on cytology or sinus histiocytes aspirated on smear appeared as ill formed granulomas. In the neoplastic lesions, metastatic lesions accounted for 87.78%.Similar findings were noted by other authors. [19,20] Among metastatic lesions, both adenocarcinoma and squamous cell carcinoma has the occurrence rate of about 39.4% each, followed by thyroid carcinoma 13.9%. Most of the Indian studies observed that, metastatic lesion in cervical lymph node was mainly due to squamous cell carcinoma followed by adenocarcinoma.[20,25]But, in our study, adenocarcinoma showed the same frequency with that of squamous cell carcinoma(SCC). As SCC is common in head and neck region and we get less number of cases of this region in our hospital, metastatic adenocarcinoma in supraclavicular lymphnode showed equal proportion with SCC as compared to other studies. Ghartimagar et al also reported higher frequency of metastatic adenocarcinoma over squamous cell carcinoma in their study.[26][Table 3]. In our study, Metastatic thyroid carcinoma (predominantly papillary carcinoma) showed higher frequency of 13.9% as compared to other studies which showed frequency ranging from 2 to 5%. [20,26] [Table 3].Unpaired t test showed significant statistical difference with p value <0.0001.This could be due to higher iodinated salt intake because of our coastal region as documented in literature thatincreased iodine supplementation increases risk for thyroid malignancy, particularly Papillary thyroid carcinoma(PTC).[27]Similar findings were noted by Indian studies mentioning environmental conditions and dietary habits i.e diet rich in fish may be contributing factor for wide spread distribution of PTC occurring in coastal areas of Tamilnadu, Andhra Pradesh& Kerala which are iodine rich and the iodine content of soil is thought to modify the development of these cancers.[28]Previous studies conducted in our hospital also showed higher frequency of thyroid carcinomas predominantly papillary carcinomas which needs further molecular studies.[29] In neoplastic lesions, apart from metastasis, Hodgkin’s and Non-Hodgkin’s Lymphoma showed frequency of 6.6% and 5.5% respectively. Similar findings were observed by other authors.[21,23,30]In our study, one cytologically diagnosed Non-Hodgkin’s lymphoma turned out to be granulomatous lymphadenitis accounting for false positive diagnosis probably due to inadequate sampling or sampling predominantly of germinal centre centroblastic cells or large atypical cell appearance due to drying artifact

because of late fixation. Thus, cytology has limitations in diagnosing Lymphomas due to non-availability of complete architecture for complete assessment. Also, diagnostic accuracy is said to be lower for lymphomas compared to metastatic malignancy.[31] In our study, the overall diagnostic accuracy of FNAC in diagnosing cervical lymph node lesions was 97% with sensitivity 97%, specificity 95%, positive and negative predictive value to be 97% and 95% respectively which was comparable with other studies. In other studies, sensitivity ranged from 90% to 99% and specificity from 70 to 100%.[15,32,33]

Conclusion

Fine needle aspiration cytology offers a simple, safe, quick and minimally invasive technique which can be performed as an outpatient department procedure for rapid diagnosis of neoplastic and non-neoplastic lesions of cervical lymph nodes for better patient care management. Our study showed high sensitivity, specificity and diagnostic accuracy, thus making it reliable investigation in further management of cervical lymphnode lesions. Also, significant findings in our study were higher frequency of granulomatous lymphadenitis in non-neoplastic lesions and higher occurrence of metastatic thyroid carcinoma in this rural tertiary healthcare centre located at iodine sufficient eastern coastal region of South India.

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Hirachand S, Lakhey M, Akhter J, Thapa B. Evaluation of fine needle aspiration cytology of lymph nodes in Kathmandu Medical College, Teaching hospital. Kathmandu Univ Med J. 2009; 7(2):139–42.

Bazemore AW, Smucker DR. Lymphadenopathy and malignancy. American family physician.2002 Dec;66(11):2103-110.

Young JA. Fine needle aspiration cytopathology. J of Pathol. 1993 Jan;169(1):109-114.

Keith VE, Harsharan SK, Jerald GZ. Fine needle aspiration biopsy of lymph nodes in the modern era: reactive lymphadenopathies. Pathol Case Rev 2007; 12(1):27–35.

Howlett DC, Harper B, Quante M, Berresford A, Morley M, Grant J, Ramesar K, Barnes S. Diagnostic adequacy and accuracy of fine needle aspiration cytology in neck lump assessment: results from a regional cancer network over a one year period. J Lary & Otol. 2007 Jun 1; 121(06):571-79.

Kline TS. Handbook of Fine Needle Aspiration Biopsy Cytology. 2nded. New York: Churchill Livingstone; 1988.p.492.

Kollur SM, El Hag IB. Fine needle aspiration cytology of metastatic nasopharyngeal carcinoma in cervical lymph nodes: comparison with metastatic squamous cell carcinoma and Hodgkin’ and Non Hodgkin’ lymphoma. DiagCytopathol 2003;28:18-22.

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Miliauskas J. Diagnostic accuracy of FNAC of Lymph node lesions. In: Orell SR, Sterrett GF, editors. Fine needle aspiration cytology.5thed.New York: Churchill Livingstone;2012.p.85-86. Al-Mulhim AS, Al-Ghamdi AM, Al-Marzooq YM, Hashish HM, Mohammad HA, Ali AM, Gharib IA. The role of fine needle aspiration cytology and imprint cytology in cervical lymphadenopathy. Saudi Med J.2004;25(7):862–5. Haque MA, Talukder SI. Evaluation of fine needle aspiration cytology of lymph node in Mymensingh. Mymensingh Med J. 2003 Jan;12(1):33–35. Gupta AK, Nayar M, Chandra M. Reliability and Limitations of Fine Needle aspiration cytology of lymphadenopathies. An analysis of 1,261 cases. ActaCytol 1991; 35: 777-83. Ferrer R. Lymphadenopathy: differential diagnosis and evaluation. Am FamlPhysician 1998;58:1313-20. Khajuria R, Goswami KC, Singh K, Dubey VK. Pattern of Lymphadenopathy on Fine Needle Aspiration Cytology in Jammu. JK Sci J of Cytology. 2006; July-Sep;8 (3) : 157- 59. Steel BL, Schwartz MR, Ramzy I. Fine needle aspiration biopsy in the diagnosis of lymphadenopathy in 1103 patients. Role, limitations and analysis of diagnostic pitfalls. ActaCytol. 1995;39(1):76-81. Nesreen H. Hafez, Neveen S. Tahoun. Reliability of fine needle aspiration cytology (FNAC) as a diagnostic tool in cases of cervical lymphadenopathy. J Egyptian Nat Cancer Inst 2011;23: 105-114 Sharma P, Rana S, Gill MK, Singh P, Satarkar RN, Kalhan S. Spectrum of lymph node lesions on cytology in rural Haryana: a retrospective analysis. International J Res Med Sci. 2015 May;3(5):1125-30. Attaullah M, Shah W, Pervez SN, Khan S, Jehan S, Rahim S. cytomorphological pattern of superficial lymphadenopathy. Gomal Journal of Medical Sciences. 2014 Oct 1; 12(4):197-200. Nidhi P, Sapna P, Shalini M and Kumud G. FNAC in tuberculous lymphadenitis- Experience from a tertiary level referral centre. Indian Journal of tuberculosis. 2011 jul; 58(3): 102-107. Dukare SR, Jadhav DS, Gaikwad AL, Ranka SN, Kale PB, D’Costa G. Fine needle aspiration cytology of cervical lymphadenopathy - a study of 510 cases. Asian J Sci Technol. 2014 sep;5(9):537-40. Pavithra P, Geetha JP. Role of fine needle aspiration cytology in the evaluation of the spectrum of lymph node lesions. Int J Pharm Bio Sci. 2014;5(4):377-84.

A-465 21. Kumar H, Chandanwale SS, Gore CR, Buch AC, Satav VH, Pagaro PM. Role of fine needle aspiration cytology in assessment of cervical lymphadenopathy. Med J DY PatilUniv 2013;6:400-4. 22. Tanteo M, Garcia R.Clinical profile and histopathologic diagnoses of childhood peripheral lymphadenopathy: an MMC experience. Paed Infect Dis Soc. Phillipines. 2011;12(2):67-74. 23. Singh N, Singh A, Chauhan R, Singh P, Verma N. Fine needle aspiration cytology in evaluation of lymphadenopathy in pediatric age group: our experience at tertiary care centre. International Journal of Contemporary Medical Research. 2016;3(5):1347-51. 24. Sibanda EN, StanczukG. Lymph node pathology in Zimbabwe: a review of 2194 specimens. Quart J Med.1993;86:811-17. 25. Rathod KM, Shah SA. A Study of Metastatic Lesion of Lymph Node by Fine Needle Aspiration Cytology. Nat J Community Med.2012; 3(4):708-10. 26. Ghartimagar D, Ghosh A, Ranabhat S, Shrestha MK, Narasimhan R, Talwar OP., Utility of fine needle aspiration cytology in metastatic lymph nodes. Journal of Pathology of Nepal. 2011; 1:92-5. 27. Levi F, Vecchia CL, Randriamiharisoa A. Cancer mortality in Switzerland 1989. SozPreventimed. 1991; 36:112-126. 28. Sepuri M, Das B. Spectrum of Thyroid carcinoma in coastal Andhra Pradesh. Aretrospectivestudy. J. Evid Based Med. Healthcare.2016; 3(71): 3840-44. 29. Bharathidhasan I, Goneppanavar M, Dhaka RS. Changing trends in the incidence of thyroid lesions in coastal regions of south india. Int j Health Sci Res. 2015; 5(6):134-141. 30. Wilkinson AR, Mahore SD, Maimoon SA. FNAC in the diagnosis of lymph node malignancies: A simple and sensitive tool. Indian J Med PaediatrOncol 2012; 33:21-24. 31. Cardillo MR. Fine needle aspiration cytology of superficial lymph nodes. Diagnostic cytopathology. 1989;5(2):166-73. 32. Babu GS, Ramesh G, Kashyap B, Suneela S, Hiremath SS, Murgud S. Cytohistopathological evaluation of the cervical lymph nodes by fine needle aspiration cytology. Journal of Cranio-Maxillary Diseases.2014 ;3(2):101-5. 33. Paliwal U K,Nigam S K. Diagnostic accuracy of fine needle aspiration cytology in Cervical Lymph Nodes with Histopathological correlation. Journal of Evolution of Medical and Dental Sciences. 2013; (32): 5936-42.

*Corresponding author: Dr Dhananjay Shrikant Kotasthane, Mahatma Gandhi Medical college and Research Institute, pillaiyarkuppam, Pondicherry-607402 (India) Phone: +91 9092096244 Email: dskotasthane@gmail.com Date of Submission : 07.04.2017 Date of Acceptance : 29.04.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

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Original Article DOI: 10.21276/APALM.1486

Role of Mean Platelet Volume As An Adjunct in Evaluation of Acute Inflammation Nabila Afsar*, Idrees Akhtar, Afroze, Habeebunnisa Tahniath, Zakia Abid Department of Pathology, Deccan College of Medical Sciences, Kanchan Bagh, Hyderabad. India

ABSTRACT Background: Platelets are also known for their role in the pathophysiology of vascular inflammation. Activated platelets can promote vascular inflammation, causing endothelial inflammation and subsequent leucocyte extravasation. Increased MPV may be used as a marker of platelet activation. We aim to study the relationship of MPV with total WBC count in cases with neutrophilic leucocytosis and to assess if MPV may have any role as an inflammatory marker. Methods: A retrospective study was conducted including 97 cases of leucocytosis with neutrophilia. 95 age and sex matched healthy individuals were used as controls. MPV values and total WBC counts were obtained on automated cell counter Horiba pentra ES60 and manual differential count was performed. Results: MPV did not show any significant change in the study group compared to the control group. MPV was noted to be higher in cases with associated thrombocytopenia, while MPV was lower in cases with thrombocytosis. Conclusion: MPV has no direct role in the evaluation of acute inflammation. However the increased MPV noted in cases of thrombocytopenia with neutrophilic leucocytosis suggests that activation of platelets causes a rise in MPV due to shape change and may be implicated in impending sepsis and disseminated intravascular coagulation due to the production of thromboxane A2 by activated platelets, warranting the need for further workup and prospective studies in order to establish the role of MPV as a marker for impending sepsis and DIC in cases with neutrophilic leucocytosis. Keywords: MPV, Mean Platelet Volume, Acute Inflammation, Neutrophilic Leukocytosis, Platelet Activation

Introduction

Platelets are known for their role in the pathophysiology of vascular inflammation apart from their role in primary hemostasis and arterial thrombus formation. Activated platelets can promote vascular inflammation, causing endothelial inflammation and subsequent leucocyte extravasation via their stored cytokines and chemokines. [1] With the availability of platelet parameters in routine complete blood count reports by automated blood cell counters, these newer parameters may have an additional diagnostic and prognostic significance in various clinical conditions. Mean platelet volume is a potential marker of platelet reactivity and is calculated by dividing the plateletcrit by the number of platelets. MPV may be determined in the progenitor cell, the bone marrow megakaryocyte. It is presumed that young platelets, those released recently from bone marrow are larger and more dense, contain more of certain proteins (i.e. PF4), and exhibit some alterations in function compared to smaller platelets. Circulating platelets may, indeed diminish in size during a normal circulating lifespan by shedding some of their surface components.[2] The platelet volume is found to be associated with cytokines (thrombopoietin, interleukin-6

and interleukin-3) that regulate megakaryocyte ploidy and platelet number and result in the production of larger platelets.[2,3,4] Increased MPV indicates increased platelet diameter, which can be used as a marker of production rate and platelet activation. During activation, plateletsâ&#x20AC;&#x2122; shapes change from biconcave discs to spherical, and a pronounced pseudopod formation occurs that leads to MPV increase.[5] In this study, we aim to study the relationship of mean platelet volume with total white blood cell count in conditions resulting in neutrophilic leucocytosis and to assess if mean platelet volume may have any role as an inflammatory marker.

Materials and Methods

A retrospective study was conducted with retrieval of data from the data registers and electronic data base of the automated cell counter, during the months of June to July 2016 and included all inpatient and outpatient cases of leucocytosis with neutrophilia (WBC count above 11x103/cmm and neutrophils more than 75%). CBC values were obtained by Horiba Pentra ES60 following the standard operating procedure of sample collection in EDTA vacuotainers, stringent quality control maintenance

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and immediate processing of sample within 2 hours. The differential count was performed manually by counting 100 cells in a Leishman stained slide. 97 cases were included in the study, all showing neutrophilic leucocytosis . Complete blood counts of 95 healthy individuals with normal complete blood counts including 45 males and 50 females of age groups ranging from 18 to 70 years were used as controls. Inclusion Criteria: CBC of all cases with neutrophilic leucocytosis with WBC count >11x103/cmm and neutrophils more than 75%. Exclusion criteria: 1) Paediatric and Octogenarian patients; 2) Patients with very high WBC count above 50x103/cmm. 3) Patient with high WBC count without associated neutrophilia. 4) Patients with haematological malignancies and 5) Antenatal patients CBC reports of the 95 patients with neutrophilic leucocytosis were analysed and compared with the control group. Statistical analysis using mean, standard deviation, range was done using Microsoft excel and student ‘t’ test was done to obtain the p value to compare with the control group and establish the statistical significance.

Results

Ninety five cases of neutrophilic leucocytosis were analysed out of which 53(55.8%) were males and 42(52.6%) were females in the ratio of 1.2:1. 95 controls were also analysed

including 45(47.4%) males and 50(52.6%) females. (Table 1) The mean age of subjects was 48.8 years and ranged from 16 to 79 years. Mean age of controls is 36.1 years and ranged from 18 to 70 years. The mean WBC count noted in the cases with neutrophilic leucocytosis was 17.9 x 103/cmm and ranged from 11.4 to 38 x 103/cmm with a Standard Deviation of 5.77. Platelet count ranged from 21 to 682 x 103 /cmm with a mean of 271.4 x 103 /cmm and Standard deviation of 118.4. The mean platelet volume in the 95 subjects with neutrophilic leucocytosis ranged from 6.5-11.5 µm3, with an average of 8.28 and SD of 1.02, while the MPV was 8.23 with SD of 0.8 and ranged from 6.8-10.6. There was no statistically significant difference noted in the mean platelet volume noted in the 2 groups.(Table 2) The mean platelet volume was assessed in relation with the rising WBC count and showed no significant increase or decrease in the MPV with increasing WBC count . (Table 3) The mean platelet volume was assessed in relation with platelet counts and it was found that MPV was significantly higher in thrombocytopenic patients with neutrophilic leucocytosis, compared to those cases with normal platelet reference range (p=0.006). It was also noted that cases with thrombocytosis had a statistically significantly lower MPV when compared with cases with normal platelet reference range.(p=0.004) (Table 4)

Table 1: Patient Demographic Data. AGE 16-25 26-35 36-45 46-55 56-65 >66 Total

Male 5(5.3%) 5(5.3%) 8(8.4%) 12(12.6%) 11(11.6%) 12(12.6%) 53(55.8%)

CASES Female 9(9.5%) 8(8.4%) 2(2.1%) 8(8.4% 8(8.4%) 7(7.5%) 42(44.2%)

Table 2: Mean platelet volume in cases versus controls. MPV(µm3) Cases(n=95) Range 6.5 – 11.5 Mean 8.28 Standard deviation 1.02

Total 14(14.7%) 13(13.7%) 10(10.5% 20(21%) 19(20%) 19(20%) 95(100%)

Male 15(15.7%) 6(6.3%) 7(7.4%) 8(8.4%) 5(5.3%) 4(4.2%) 45(47.4%)

CONTROLS Female 22(23.2%) 9(9.5%) 7(7.4%) 3(3.2%) 8(8.4%) 1(1.1%) 50(52.6%)

Control(n=95) 6.8-10.6 8.23 0.80

Table 3: Mean platelet volume in relation with WBC in cases with neutrophilic leucocytosis. WBC count x103/cmm MPV(µm3) Range 11 to 15 (n=41) 8.1 6.5-10.5 16 to 20 (n=29) 8.3 6.7-11.5 21 to 25 (n=15) 8.6 7.3-10.4 26 to 30 (n=8) 8.6 6.8-9.3 31 to 40 (n=4) 8.4 7.2-9.2

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Total 37(38.9%) 15(15.8%) 14(14.7%) 11(11.6%) 13(13.7%) 5(5.3%) 95(100%) P value 0.74

SD 0.9 1.2 1.1 0.8 0.9

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MPV in Evaluation of Acute Inflammation

Table 4: Mean platelet volume in relation with platelet count in cases with neutrophilic leucocytosis. Platelet count MPV(µm3) Range 21-149(n=12) 9.56 8.2-11.5 150-459(n=76) 8.16 6.5-10.5 >460(n=7) 7.3 6.7-8.1

Discussion

The study was aimed to demonstrate if mean platelet volume could be used as an adjunct to evaluate patients with acute inflammation considering the fact that platelets play an important role in the pathophysiology of vascular inflammation. PLTs have not only hemostatic functions but also are considered as inflammatory anucleate cells because many studies demonstrated that PLT counts and PLT parameters are strongly associated with inflammatory and infectious condition. Platelet parameters can be easily obtained on a routine cell analyser report and could be used as a potential marker of platelet reactivity and activation. There is a well-known close relationship between leukocytes and platelets especially in inflamed endothelium . Leukocytes can roll on a template of adherent platelets, firmly adhere, and then transmigrate through the adherent platelets. [6] It is thought that PLTs are one of the first responding anucleate cells during the development of sepsis. Platelet activation readouts have been suggested as biomarkers for the development of septic complications and have been related to prognosis.[7] In the present study, we found that MPV did not show any statistically significant change with increasing WBC count, these findings were concordant with the study by Ozturk N et al who studied the changes in platelet parameters in leucocytosis and found that the MPV and PDW did not significantly differ in these patients.[8] Gurler M et al suggested that either increase or decrease in MPV in inflammatory conditions should be the result of the effects of inflammatory cytokines in bone marrow.[9] Activated platelets become larger in these conditions and cause an elevation in MPV values in hemogram tests. However, at an uncertain level, MPV tend to be decreased after utilization of these activated larger platelets in inflammatory processes thus, remaining smaller platelets lead a decrease in MPV in blood count tests. Z.A. Özturk et al found that in patients with active UC and CD, there was a statistically significant decrease in MPV, PDW levels and increase in PCT levels when compared to healthy controls. In remission phase of IBD while MPV levels were lower, PDW and PCT levels were higher than control group. There was statistically significant change in all platelet indices during diseases follow-up.[10] Zhe Fan et al showed that MPV was clearly lower in patients with acute gangrenous appendicitis. However, the MPV did not have a higher sensitivity compared with PDW.[11]

SD 1.0 0.87 0.47

Koç et al demonstrated that MPV level was significantly higher in Chronic recurrent sinusitis compared to control group and PLT level was borderline higher in CRS group compared to control group.[12] S.Bozkur et al showed that there was no statistically significant difference between patients with appendicitis (complicated or uncomplicated) and those having no appendicitis with respect to MPV level. However, the complicated appendicitis group had a lower MPV value compared to other groups.[13] Zareifar et al studied the change in platelet count and mean platelet volume during infectious and inflammatory diseases and demonstrated a higher level of platelet count and lower MPV in the patients with active disease compared to the recovered patients. These parameters were well correlated with the known disease activity markers.[14] An increase in MPV, a sign of larger PLT size, usually is indicative of compensated bone marrow PLT production following stress-induced platelet destruction, as septic shock develops , in fact, the MPV is inversely proportional to the degree of PLT maturity. [15] The present study demonstrated that MPV was significantly higher in thrombocytopenic patients with neutrophilic leucocytosis, compared to those cases with normal platelet reference range (p=0.006). This may be suggestive of impending sepsis. According to McMillan R, morphologically large platelets are seen on blood smears in patients with severe thrombocytopenia, thus suggesting that “hyperfunctional” platelets compensate for low numbers by their increased effectiveness. [16.17] This could explain the increase in MPV noted in our study. It was also noted in the present study that cases with thrombocytosis had a statistically significantly lower MPV when compared with cases with normal platelet reference range (p=0.004). Van der Lelie et al and Robbins et al also observed that patients with reactive thrombocytosis had considerably lower mean platelet volumes than those with normal subjects, which was similar to the observation in the present study.[18,19] While platelets can have a beneficial role in host response to an invading pathogen, during sepsis, platelet activation contributes to the development of complications such as DIC, multiple organ failure, acute lung injury (ALI) and acute kidney injury (AKI).[20] This observation highlights

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the need for a marker of platelet activation to facilitate early prevention and management of complications in patients with acute inflammation with impending sepsis.

Conclusion

In patients with neutrophilic leucocytosis suggesting acute inflammation due to varying etiology, MPV did not show any significant change compared to the control group of healthy individuals with normal WBC count. However, MPV was noted to be higher in those cases which had associated thrombocytopenia, while the MPV was lower in cases with thrombocytosis. The study infers that although MPV has no direct role in the evaluation of acute inflammation, the increased MPV noted in cases of thrombocytopenia with neutrophilic leucocytosis suggests that activation of platelets causes a rise in MPV due to shape change and may be implicated in impending sepsis and disseminated intravascular coagulation due to the production of thromboxane A2( a potent inducer of platelet activation) by activated platelets. This finding warrants the need for further workup and studies in order to establish the role of mean platelet volume as a marker for impending sepsis and DIC in cases with neutrophilic leucocytosis.

Acknowledgment

We would like to thank our technical staff who have of utmost help in the collection of data

References 1.

1. Projahn D, Koenen RR. Platelets. Key players in vascular inflammation. J Leukoc Biol. 2012;92(6):1167-1175.

2. Larsen SB, Grove EL, Hvas AM, Kristensen SD. Platelet turnover in stable coronary artery disease-influence of thrombopoietin and low-grade inflammation. PLoS One 2014;9:e85566.

Brown AS, Hong Y, de Belder A, Beacon H, Beeso J, Sherwood R, et al. Megakaryocyte ploidy and platelet changes in human diabetes and atherosclerosis. Arterioscler Thromb Vasc Biol 1997;17:802–7.

Osselaer JC, Jamart J, Scheiff JM. Platelet distribution width for differential diagnosis of thrombocytosis. Clin Chem 1997;43:1072–76.

Budak YU, Polat M, Huysal K. The use of platelet indices, plateletcrit, mean platelet volume and platelet distribution width in emergency non-traumatic abdominal surgery: a systematic review. Biochemia Medica. 2016;26(2):178-193.

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Smyth SS, McEver RP, Weyrich AS et al. 2009 Platelet Colloquium Participants: Platelet functions beyond hemostasis. J Thromb Haemost. 2009; 7(11) Gao Y, Li Y, Yu X et al. The impact of various platelet indices as prognostic markers of septic shock. PLoS One. 2014; 9(8) Ozturk N, Baygutalp NK, Bakan E, Altas GF, Polat H, Dorman E. Changes in platelet parameters in leukocytosis. The Pan African Medical Journal. 2016;24:185. Gurler, Mujgan; AKTAS, Gulali. A review of the association of mean platelet volume and red cell distribution width in inflammation. Int J Res Med Sci, 2016 4(1) 1-4. Özturk ZA, Dag MS, Kuyumcu ME, et al. Could platelet indices be new biomarkers for inflammatory bowel diseases? Eur Rev Med Pharmacol Sci; 2013;17:334-341 Fan Z, Pan J, Zhang Y, et al., “Mean Platelet Volume and Platelet Distribution Width as Markers in the Diagnosis of Acute Gangrenous Appendicitis,” Disease Markers, 2015,(2015),1-4. Koç S, Eyibilen A, Erdoğan AS. Mean Platelet Volume as an Inflammatory Marker in Chronic Sinusitis, Eur J Gen Med 2011;8(4):314-317 Bozkurt S, Köse A, Erdogan, et al. MPV and other inflammatory markers in diagnosing acute appendicitis. JPakMedAssoc; 2015;65(6): 637-641 Zareifar S., Farahmand Far MR, Golfeshan F, Cohan, N. Changes in Platelet Count and Mean Platelet Volume During Infectious and Inflammatory Disease and Their Correlation With ESR and CRP. J. Clin. Lab. Anal., 2014;28: 245–248. Van der Lelie J, Von dem Borne AK. Increased mean platelet volume in septicaemia. J Clin Pathol 1983;36: 693–696 McMillan R. Therapy for adults with refractory chronic immune thrombocytopenic purpura.Ann Intern Med 1997;126:307-314. Kaushansky K , Gerald J Roth. Megakaryocytes and platelets. In: John P Greer, John Foerster, John N Lukens, George M Rodgers, Frixos Paraskevas, Bertil Glader, editors. Wintrobe’s clinical Hematology. 11th edition. Philadelphia. Lippincott Williams & Wilkins; 2004: 632 Van der Lelie J, Von dem Borne AK. Platelet volume analysis for differential diagnosis of thrombocytosis. Journal of Clinical Pathology. 1986;39(2):129-133. Robbins G, Barnard DL. Thrombocytosis and microthrombocytosis: a clinical evaluation of 372 cases. Acta Haematol 1983;70:175-82. de Stoppelaar SF , van ‘t Veer C, van der Poll T. The role of platelets in sepsis. Thrombosis and Haemostasis 2014; 112: 627-842.

*Corresponding author: Dr. Nabila Afsar, Department of Pathology, Deccan college of Medical Sciences, DMRL X Road, Santoshnagar, Kanchan Bagh, Hyderabad, Telengana- 500058, India. Phone: +91 9848020386 Email: nabila_dr@yahoo.com Date of Submission : 19.04.2017 Date of Acceptance : 23.05.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

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Case Report DOI: 10.21276/APALM.1163

Benign Lymphoepithelial Cyst in An Adolescent Female Mimicking Lymphoma: A Diagnostic Dilemma in a Retrovirus Negative Patient

Somshankar Chowdhury, Sufian Zaheer*, Preeti Sharma and Ashish Kumar Mandal Department of Pathology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi- India

ABSTRACT Cystic lesions of the parotid gland are uncommon and may masqueradeneoplasms clinically. Benign lympho-epithelial cysts (BLEC) comprise the most common causes of the parotid gland swelling in human immunodeficiency virus (HIV)-positive patients. It is, however, less common amongst those not infected with HIV. We present a case of a12 years old female who presented with a swelling over left parotid region since one year.Local examination revealed a soft, cystic and non-tender swelling in the left parotid region measuring 5x4x3 cm. Serological investigations for viral markers were non-reactive.A differential diagnosis of granulomatous parotitis versus benign lymphoepithelial lesion was rendered on radiological investigations and superficial parotidectomy was planned. On histopathological examination, a cyst lined by cuboidal to low columnar lining was seen, underlying which, was dense lympho-plasmacytic infiltrate present in diffuse sheets. On immunohistochemistry, this lymphoid population was found to be reactive in nature (positive for kappa and lambda), thus excluding lymphoma. This case is being presented due to its rarity in HIV negative individuals and the histopathological diagnostic dilemma it presented with, mimicking a lymphoma. Keywords: Lymphoepithelial Cyst; Parotid; Lymphoma; HIV

Introduction

Cystic lesions within the parotid gland are uncommon, comprising approximately 5% of all salivary gland tumours, many of them representingcystic components of neoplasms. [1] Lymphoepithelial (LE) cysts are known to arise in the lateral cervical areas, referred to as branchial cleft cysts, or in the oral mucosa. [2] Albeit rare, the parotid gland has been recognized as a site for these unusual lesions.[3] The term â&#x20AC;&#x153;lymphoepithelial cystâ&#x20AC;? was denominated by Bernier and Bhaskar to lay emphasis on the non-embryonic derivation of this lesion.[4]These cysts are usually seen inhuman immunodeficiency virus (HIV) positive individuals and their rise in incidence has coincided with that of HIV infection, occasionally as the first manifestation of retroviral infection. [5, 6] However, they have also been seen in HIV negative individuals, although less frequently, thereby making its pathogenesis elusive with respect to HIV. [6, 7] Lesions of salivary glands with a prominent lymphoid component are a heterogeneous group of diseases that include benign as well as malignant lesions. These entities may, at times, pose difficulties in diagnosis. Lymphoepithelial sialadenitis, Warthinâ&#x20AC;&#x2122;s tumor, extranodal marginal zone B-cell lymphoma, chronic sclerosing sialadenitis and HIVassociated salivary gland disease including lymphoepithelial cysts are few such entities that might confound the clinician

as well as the pathologist. Our case highlights the diagnostic dilemma associated with lymphoepithelial cysts especially in retrovirus negative patients.

Case Report

A 12 years old female child presented in the out-patient department with complaints of a swelling over left parotid region. The swelling appeared one year ago and gradually increased in size to attain its present dimension of 5x4x3 cm. The overlying skin appeared unremarkable. There was no history of any pain while taking food. On examination, the swelling was soft, cystic and non-tender. No signs of facial nerve involvement was present. General physical examination and systemic examination was within normal limits. All the routine blood investigations including the haemogram, liver function tests and kidney function tests were within normal limits. Serological investigations for viral markers were nonreactive. Ultrasonography revealed bilateral bulky parotid glands (left >> right) with multiple intraparotid cystic lesions, the largest of which was 3.8x3.2 cm, within the left parotid. A differential diagnosis of granulomatous parotitis versus benign lymphoepithelial lesion was given. Magnetic resonance imaging (MRI) scan suggested the possibility of an impacted branchial cyst (Figure 1a, b). Fine needle aspiration yielded thin fluid. Smears were paucicellular with few inflammatory cells and macrophages against a fluid background. With a possibility

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Chowdhury et al. of a lymphoepithelial cyst, superficial parotidectomy was performed and sent for histopathological examination. The immediate post-operative period was uneventful. Gross examination revealed a grey brown encapsulated cystic structure, measuring 4.5x3.5x2 cm. On cut, a cyst was identified, filled with mucinous fluid. The wall thickness varied from 0.1 to 0.2 cm. Microscopic examination revealed a cyst lined by cuboidal to low columnar cells and surrounded by fibrocollagenous tissue. Beneath the lining were diffuse sheets of dense lymphoid infiltrate intermixed with plasma cells and macrophages (Figure 2a-d). At places, germinal centre formation was seen. No lymphoepithelial lesions were seen. Owing to the dense and diffuse lymphoid infiltrate, an immunohistochemical panel was applied to rule out the possibility of a lymphoma. On immunohistochemistry,the lymphoid cells were diffusely positive for leucocyte common antigen (LCA). Immunoreactivity for CD20 was observed within the germinal centres (Figure 3a) and for CD3 in the para-follicular areas (Figure 3b). Polyclonal expression of Kappa (Figure 3c) and lambda (Figure 3d) expression excluded the possibility of lymphoma.Based on the radiological, histo-morphological and immunohistochemical findings a final diagnosis of benign lympho-epithelial cyst of left parotid gland was rendered.On follow up, patient is currently healthy one year after excision.

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Discussion

Branchial cyst in the parotid gland was first reported in 1895 by Hildebrant. BLEC otherwise quite rare in general, is found most commonly in HIV-positive patients. Thus, a patient diagnosed with these cysts, should be tested for HIV, if the status is previously unknown. These cysts, when in HIV positive individuals, are bilateral in 80% cases and multiloculated in 90% cases. [8, 9] In our case, the cyst was unilateral and uniloculated, both features being in consistency with the HIV negative status of the patient. The age of patients in various case reports have ranged from 16 to 69 with the mean age of 44 years old. [10] Our patient was slightly younger than any of the reported cases, being 12 years of age. Histopathological examination is the gold standard for diagnosis of lymphoepithelial cysts. The dense lymphocytic component of these cysts often creates a diagnostic dilemma and a variety of differential diagnoses need to considered. Lymphoepithelial sialadenitis, Warthinâ&#x20AC;&#x2122;s tumour, extranodal marginal zone B-cell lymphoma and chronic sclerosing sialadenitis are some of the lesions that need to be distinguished from lymphoepithelial cysts. The most common lymphoma arising from salivary glands is extra-nodal marginal zone B cell lymphoma. Follicular and diffuse large B-cell lymphomas are the other common

Fig. 1: (a& b)MRI neck showed evidence of an altered signal intensity lesion measuring 3.2x2.7 cm in the left parotid suggestive of infected branchial cyst.

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Benign Lymphoepithelial Cyst in an Adolescent Female Mimicking Lymphoma

Fig. 2: (a& b) Photomicrograph showing cyst wall lined by cuboidal to low columnar cells underneath which is dense lymphoid infiltrate (H&E; x100) (c) Lymphoid infiltrate seen in diffuse sheets (H&E; x200) (d) Surrounding parotid gland acini showing presence of similar lymphoid infiltrate (H&E; x200).

Fig. 3: (a& b) Photomicrograph showing immunoreactivity for (a) CD 20 in the germinal centres (CD 20 immunostain x200) (b) CD 3 in the para-follicular regions (CD 3 immunostain x100) (c) Kappa in all lymphoid cells(Kappa immunostain x400) (d) Lambda in all lymphoid cells (Lambda immunostain x400).

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Chowdhury et al. ones. [11] Most of the marginal zone B-cell lymphomas (MZBCL) arise against a background pre-existing lymphoepithelial sialadenitis. A characteristic of diagnostic utility in such emerging MZBCLs is the presence of lymphoepithelial lesions. These are hyperplastic ductal epithelium, with the epithelial cells permeated by neoplastic lymphocytes. Surrounding these lesions, are proliferations of neoplastic marginal zone lymphoma cells. Admixed with the neoplastic lymphocytic proliferation is a nonneoplastic infiltrate comprising T-cells, B-cells, plasma cells, reactive germinal centers, and scattered centroblasts or immunoblasts. With such a picture, lymphoepithelial sialadenitis and lymphoepithelial cysts are the two principal considerations in the differential diagnosis. Our panel of immunohistochemistry showed polyclonal nature of the lymphoid cells, and a mixture of B cells in the germinal centres and T cells in the interfollicular areas. Lymphoepithelial sialadenitis is characterized by a benign lymphocytic infiltrate of salivary gland with parenchymal atrophy, ductal hyperplasia and lymphocytic epitheliotropism. Lymphoepithelial lesions are often prominent. It usually presents as a component of the autoimmune condition of Sjogren syndrome. Although T cells comprise the predominant population of lymphocytes, the lymphocytes infiltrating into the ductal epithelial cells are of B cell type. These patients have a much higher risk of developing lymphoma, and most, if not all, salivary extranodal MZBCLs are preceded by lymphoepithelial sialadenitis. Distinction from MZBCL is based on the number and distribution of marginal zone B cells. The origin of these cysts is controversial and four theories have been suggested till date. [12] The branchial apparatus theory and the cervical sinus theory, also known as classic theory, suggest that the cysts develop from the remnants of the branchial cleft. The Inclusion Theory, however, believes that the cysts arise from cystic changes in parotid gland epithelium that become entrapped in the upper cervical lymph nodes during embryonic life. [4] The pathogenesis of these cysts in HIV positive individuals is related to the “Ductal obstruction phenomena” where there is obstruction of parotid ducts along with a follicular hyperplasia in the periductal parotid lymph nodes, induced by the virus. To conclude, lymphoepithelial cysts are uncommonly encountered lesions of the parotid gland which are rarer in

C-101 children and retroviral negative patients. Furthermore, these cysts masquerade neoplasms of the salivary gland clinicoradiologically with histopathology being the gold standard for a decisive diagnosis. Our caseemphasises that this lesion should be kept in mind as a differential diagnosis while dealing with lymphoproliferative lesions of the parotid gland.

References 1.

Altman K and Bailey MW. Parotid cyst: a case report. Int J Oral Maxillofac Surg 1994;23:165-166.

Neville BW, Damm DD, Allen CM, Bouquot JE. Oral and maxillofacial pathology. 3rd ed. St Louis: Mosby;2008.

Terry JH, Loree TR, Thomas MD, Marti JR. Major salivary gland lymphoepithelial lesions and the acquired immunodeficiency syndrome. Am J Surg 1991;162:324-329.

Bernier JL, Bhaskar SN. Lymphoepithelial lesions of salivary glands. Histogenesis and classification based on 186 cases. Cancer 1958;11:1156-1179.

Ihrler S, Zietz C, Riederer A, Diebold J, Löhrs U. HIV-related parotid lymphoepithelial cysts. Immunohistochemistry and 3-D reconstruction of surgical and autopsy material with special reference to formal pathogenesis. Virchows Arch 1996;429:139-147.

Maiorano E, Favia G, Viale G. Lymphoepithelial cysts of salivary glands: an immunohistochemical study of HIV-related and HIV unrelated lesions. HUM PATHOL 1998;29:260-265.

Chetty R. HIV-associated lymphoepithelial cysts and lesions:morphological and immunohistochemical study of the lymphoid cells. Histopathology 1998;33:222-229.

Dave SP, Pernas FG, Roy S. The benign lymphoepithelial cyst and a classification system for lymphocytic parotid gland enlargement in the pediatric HIV population. Laryngoscope 2007;117(1):106-113.

Kooper DP, Leemans CR, Hulshof MC,et al. Management of benign lymphoepithelial lesions of the parotid gland in human immunodeficiency virus-positive patients. Eur Arch Otorhinolaryngol 1998;255(8):427-429.

10. Rahman S, Shaari R, Hassan R. Parotid lymphoepithelial cyst: A case report. Archives of Orofacial Sciences 2006;1:71-75. 11. Ahamed AS, Kannan VS, Velaven K,et al. Lymphoepithelial cyst of the submandibular gland. J Pharm Bioallied Sci 2014;6:185–187. 12. Camilleri AC, Lloyd RE. Lymphoepithelial cyst of the parotid gland. Br J Oral Maxillofac Surg 1990;28:329-332.

*Corresponding author: Sufian Zaheer, MD Pathology, Associate Professor, Department of Pathology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India Phone: +91 9650594818 Email: sufianzaheer@gmail.com Date of Submission : 01.11.2016 Date of Acceptance : 20.04.2017 Financial or other Competing Interests: None. Date of Publication : 25.08.2017

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Case Report DOI: 10.21276/APALM.1187

Cutaneous Rhinosporidiosis Mimicking Soft Tissue Tumour: A Case report with Review of Literature Pallavi Agrawal!, Richa Bhartiya1* and Rakesh Mehra2 Department of Pathology, Patna Medical College Hospital (PMCH), Ashok Rajpath, Patna(Bihar), India Department of Radiology, Patna Medical College & Hospital (PMCH), Ashok Rajpath, Patna(Bihar), India 1

ABSTRACT Objective: Rhinosporidiosis is a chronic granulomatous disease caused by Rhinosporidium seeberi. It is endemic in India & Sri Lanka. It usually involves the nasal cavity & rarely other sites including skin. Cutaneous rhinosporidiosis presents as papule & nodule and very rarely as soft tissue mass. Case Report: We report a case of cutaneous rhinosporidiosis in a 32-year old HIV seropositive male presenting as soft tissue mass on lateral aspect of left foot near ankle-joint. Conclusion: High index of suspicion is very important for an early diagnosis and management of these cases. Rarity of this lesion warrants its mention. Keywords: Rhinosporidium Seeberi, Subcutaneous, Nasal Cavity

Introduction

Rhinosporidiosis is a chronic granulomatous disease caused by Rhinosporidium seeberi.[1] It is endemic in India and Sri Lanka but has also been reported from United states, South America and Iran. In India, it is found more commonly in southern and central regions.[2]It usually involves nasal cavity but may rarely involve the lips, palate, uvula, maxillary antrum, epiglottis, larynx, trachea, bronchus, ear, scalp, vulva, vagina, penis, rectum and the skin.[3] Cutaneous rhinosporidiosis is a well known entity as most patients present with warty papule and nodule but cutaneous rhinosporidiosis mimicking clinically as soft tissue tumour is very rare. Only few such cases with tumoral presentations are reported in literature, however, their HIV status and geographic locale is not known.[4-9] We report a HIV seropositive case of cutaneous rhinosporidiosis in a 32-year-old male with brief review of literature regarding this rare presentation, and discuss the etiologic agent, pathogenesis and treatment of such cases.

Case Report

A 32-year-old male presented with a gradually progressive swelling over the lateral aspect of left foot near ankle joint for one year. He had not received any prior treatment for this swelling. Now the patient sought medical attention for recent onset of pain and difficulty in walking for one month duration. On examination, a firm, non-tender, irregular swelling measuring 7Ă&#x2014;5 cm was found on lateral aspect of

left ankle joint. The overlying skin was erythmatous, shiny with several prominent dilated veins. No ulceration was identified (Fig 1). No other verrucous lesion or cutaneous nodules could be identified. There was no regional lymphadenopathy. On anterior rhinoscopy no growth was identified in the bilateral nasal cavities. His serology for HIV infection by ELISA was positive. On radiological examination the underlying bone was free from the mass and the proximal tibia and fibula were unaffected. The patient was admitted with a differential diagnosis of squamous cell carcinoma vs soft tissue sarcoma. An excision biopsy of the mass was performed. Microscopic examination revealed numerous globular cysts of varying size. Thick walled sporangia could be identified containing numerous endospores. Also seen were trophocytes in various stages of development. The tissue between the cysts shows foreign body type of giant cell reaction and inflammatory infiltrate chiefly composed of plasma cells, lymphocytes, macrophages and few neutrophils (Fig 2). There were fresh and organizing thrombi in adjacent areas. The refractile sporangial wall and the cell walls of mature spores could be well highlighted on post-diastase Periodic Acid-Schiff stain (PAS). Trophocytes, immature spores and sporangia were weakly positive for PAS. The GrocottGomori methenamine silver method stained the sporangial wall uniformly black and outlined mature spores in the sporangia. Ziehl-Neelsen stain could not demonstrate any acid-fast bacilli. Based on morphology a diagnosis of

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Rhinosporidiosis was given. On further enquiry he gave a history of having undergone excision of nasal mass 20 years ago. Unfortunately, no histopathological records were available. Though Rhinosporidiosis responds well to chemotherapy but discomfort caused by massive lesion necessitates surgical excision. The patient was lost to follow up before complete surgical excision could be done. For the same reason other therapies could also be not given. Though the patient was seropositive for HIV, there was no other evidence of immunodeficiency, clinically or by other parameters.

Discussion

Rhinosporidiosis is a chronic granulomatous disease first described by Guillermo Seeber, in 1896 who considered

the sporangium of R. seeberi to be a sporozoan similar to coccidian. Though the agent was considered a fungus it was interpreted as a protozoan parasite, a cyanobacterium, and a carbohydrate waste product.[3] Although the taxonomy is controversial but recent biological and organismâ&#x20AC;&#x2122;s ribosomal DNA analysis suggest that R. seeberi is not a fungus but a protist belonging to Mesomycetozoa, which is located phylogenetically between the fungal and animal divergence.[10] Cutaneous rhinosporidiosis was initially described by Forsyth.[11] It usually affects males (70-90%) with a greater incidence in those aged between 20 and 40 years.[3] It is transmitted to humans by direct contact with spores through dust, through infected clothing or finger and bathing in stagnant water. Rhinosporidiosis frequently involves nasopharynx (70%) presenting

Table 1: Summary of cases of tumoral cutaneous rhinosporidiosis. Author

Year Age (yrs) Sex Clinical presentation

Clinical diagnosis

Histopathological examination

Aravindan et al[4]

1989

Left scapular mass

Chondrosarcoma

Rhinosporidiosis

Angunawela et al[5] 1999

Thigh and anterior chest wall mass with nasal polyps

Soft tissue sarcoma

Rhinosporidiosis

Date et al[6]

1995

Right thigh mass

Soft tissue sarcoma

Rhinosporidiosis

Houreih et al[7]

2006

Popliteal fossa mass

Soft tissue sarcoma

Rhinosporidiosis

Avadhani et al[8]

2008

Swelling right leg

Soft tissue sarcoma

Rhinosporidiosis

Prasad et al

2015

Right thigh mass

Soft tissue sacoma

Rhinosporidiosis

2015

Swelling left foot

Soft tissue sarcoma/ Squamous cell carcinoma

Rhinosporidiosis

Index case

[9]

Fig. 1: Soft tissue swelling on lateral aspect of left foot near ankle-joint.

Fig. 2: Photomicrograph of rhinosporidiosis showing sporangium containing many individual spores (HxE x200). Inset shows PAS stained spores at high power (x400).

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Rhinosporidiosis Mimicking Soft Tissue Tumor

as painless, polypoidal, pink or purple friable growth studded with minute white dots, which are sporangia containing the spores. Nasal obstruction and bleeding are most common symptoms. Cutaneous rhinosporidiosis may present as 1) satellite lesions, in which skin adjacent to the nasal rhinosporidiosis is involved secondarily 2) generalized cutaneous type with or without nasal involvement, due to hematogenous dissemination of the organism; and 3) primary cutaneous type associated with direct inoculation of organisms in the skin.[12] Most of the reported cases of cutaneous rhinosporidiosis presented with multiple warty lesions or subcutaneous nodules with or without nasal involvement but tumoral cutaneous rhinosporidiosis is very rare. Only few cases have been reported in literature so far (Table-1). Date et al proposed the term rhinosporidioma to such tumoral cutaneous rhinosporidiosis.[6] It is possible that the nasal mass removed 20 years back was rhinosporidiosis with seeding of the soft tissue at that time. Autoinoculation could be mode of spread. The infection was probably smoldering and gradually progressed to the current size.Microscopic examination shows mycotic elements consisting of sharply defined globular thick-walled cysts (sporangia), upto 0.5mm in diameter, which contain numerous rounded endospores, 6-7µ in diameter. Immature and collapsed sporangia are also present. Epithelioid granulomatous reaction may accompany the organisms along with fibroblasts and endothelial cells. The life cycle of the parasite is complicated. The mature forms of the organism, known as sporangia, contain multiple sporangiospores. The trophocytes, the immature forms of R. seeberi, are smaller and thinner than sporangia and do not contain endospores. Sporangiospores are released at maturity and thereafter develop into trophocytes. Wart like cutaneous lesion of rhinosporidiosis generally ulcerate. These ulcerated lesions have to be distinguished from squamous cell carcinoma, basal cell carcinoma, verruca vulgaris, tuberculosis verrucosa cutis, granuloma pyogenicum, veneral warts and donovanosis.[13] This disease must also be differentiated from coccidiomycosis which has a different clinical presentation and smaller fungal spherules (30-60µm). The immature centralnuclear form is not seen and the endospores have a simpler structure (2-5µm).[1] R. seeberi grows in cell culture but cannot be isolated in synthetic media. Some recent studies stressed on the need of fine needle aspiration cytology in these cases.[14] But histopathology remains the mainstay in providing definitive diagnosis of Rhinosporidiosis. Development of a cutaneous lesion

should be considered as indication of early dissemination and meticulous search should be made to exclude systemic involvement.[15]Surgical removal and electrodesiccation are the treatments of choice. Various drugs like dapsone, ketoconazole and ciprofloxacin have been suggested which may arrest the maturation of sporangia and accelerate degenerative changes in them. The organisms are then removed by an accelerated granulomatous response.[12]

Conclusion

The present case is reported to highlight the fact that cutaneous rhinosporidiosis apart from warty and nodular lesions can also present as large soft tissue masses clinically mimicking soft tissue tumour so a high degree of clinical suspicion is very important for early diagnosis and prompt management of such cases.

Reference 1.

Chung KG, Bennett JE. Medical mycology. London: Philadelphia,Lea & Febiger 1992:695-704

2. Grover S. Rhinosporidiosis. 1975;64:93-95

Ind

Med Assoc

3. Lupi O, Tyring SK McGinnis MR. Tropical dermatology: Fungal tropical diseases. J Am Acad Dermatol 2005;53:931-51 4.

Aravindan KP, Viswanathan MK, Jose L. Rhinosporidioma of bone. A case report. Indian J.Pathol. Microbiol. 1989;32:312-13

Angunawela P, Tissera AD, Dissanaike AS. Rhinisporidiosis presenting with two soft tissue tumors followed by dissemination. Pathology 1999;31:57-58

Date A, Ramakrishna B, Lee VN, Sundararaj GD. Tumoral rhinosporidiosis. Histopathology 1995;27:288-90

Houreih MA, Eyden B, Lucas SB, et al. Rhinosporidium seeberi: a case clinically mimicking soft tissue sarcoma. Histopathology 2006;49:208-210

8. Avadhani A, Pai K, Mohanty SP. Cutaneous rhinosporidiosis clinically masquerading as a soft tissue sarcoma- a rare occurrence. International J of Dermatol 2008;47:1153-54 9.

Prasad HLK, Rao C, Girisha BS, et al. Subcutaneous Rhinosporidiosis masquerading as soft tissue tumour: Diagnozed by Fine Needle Aspiration Cytology. Indian J Dermatol. 2015;60(2):215

10. Herr RA, Ajello L, Taylor JW, et al. Phylogenetic analysis of rhinosporidiosis seeberi’s 18S small-subunit ribosomal DNA groups this pathogen among members of the protoctistan Mesomycetozoa clade. J Clin Microbiol 1999;37:2750-54

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Agrawal et al. 11. Padmavathy L, Rao IL, Sevam SS, et al. Disseminated cutaneous rhinosporidiosis in a HIV seropositive patient. Indian J Dermatol Venerol Leprol 2001;67:332-333 12. Kumari R, Laxmisha C, Thappa DM. Disseminated cutaneous rhinosporidiosis. Dermatol Online J 2005;11:19 13. Kamal MM, Luley AS, Mundhada SG, Bobhate SK. Rhinosporidiosis. Diagnosis by scrape cytology. Acta cytol 1995;39:931-35

C-105 14. Mandol, Kumar P et al. Disseminated Cutaneous Rhinosporidiosis: a Tumour like lesion with Therapeutic challenge. Indian Journal of Pathology. 2014;9(4): 15. Gayathri P RS, Srinivasa Kannan SR, Parijatham BO, Ganapathy H, Subhashree AR. A rare case of disseminated cutaneous rhinosporidiosis. Journal of Clinical and Diagnostic Research 2015 Jan, Vol-9(1): EL01-EL02

*Corresponding author: Dr Richa Bhartiya, â&#x201E;&#x2026; Shri Vinay Kumar Shrivastava, Dy. CSTE, Bungalow No. 882, Railway Officersâ&#x20AC;&#x2122; Colony, Danapur (KHAGAUL), Patna (Bihar)- 801105, , Bihar, India Email: richabhartiya1972@gmail.com Date of Submission : 03.12.2016 Date of Acceptance : 14.04.2017 Financial or other Competing Interests: None. Date of Publication : 25.08.2017

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Case Report DOI: 10.21276/APALM.1231

Leiomyosarcoma with Unknown Primary Manika Khare*, Ashish Airun, Umesh babu sharma and S.K. Jain Dept. of Pathology, Jaipur National University, Institute Of Medical Sciences And Research Centre, Jaipur, India

ABSTRACT Leiomyosarcoma is a malignant tumor which does not arise from the leiomyoma. Leiomyosarcoma is an extremely rare form of cancer and accounts for 5–10% of soft tissue sarcomas, which are in themselves relatively rare. This case report shows unusual presentation of the multifocal leiomyosarcoma along with diagnostic dilemma. The prognosis and treatment modality are also discussed in the discussion. Keywords: Leiomyosarcoma, Multifocal, Poor Prognosis

Introduction

Leiomyosarcoma is a malignant tumor which do not arise from the leiomyoma.[1] Leiomyosarcoma is an extremely rare form of cancer and accounts for 5–10% of soft tissue sarcomas, which are in themselves relatively rare.[2] It is a resistant cancer, meaning generally not very responsive to chemotherapy or radiation. The best outcomes occur when it can be removed surgically with wide margins early, while small and still in situ.[3]

Case Report

A 32 years female presented with loose motions since 3-4 days, nausea since 1 month and bilateral foot oedema and right abdominal mass since 6 months. On general examination pallor was present along with bilateral pedal oedema and abdominal distension. Other system examination was within normal limits. Per abdomen examination showed a large palpable mass in the right hypochondriac region. There was the past history of hysterectomy 5 years back.on routine investigation patient was anemic with hemoglobin of 8.9 gm/dl. Other indices and biochemical investigations were within normal limits. USG abdomen was done which showed a large 90X 85 mm echogenic area along inferior aspect of liver along with a large solid cystic mass in the pelvis. Multiple peritoneal deposits of various sizes were also seen. Bilateral ovaries were not identified. Based on the above findings a possibility of ovarian carcinoma was given. Further CECT abdomen was done which showed a large complex solid cystic mass in bilateral adenexa. [Fig 1] Multiple variably sized solid and cystic deposits in peritoneum, mesentery, omentum and serosal deposits on surface of small and large bowel were seen. Large hepatic sub capsular deposits causing scalloping of liver and other adjacent structures were also noted. Hence final impression of carcinoma ovary (bilateral) with abdominal deposits was given.

Endocrinology showed a raised CA125 levels (205U/ml normal range – up to 35U/ml). FNAC of the lesion was done outside, and a diagnosis of benign spindle lesion was given. Laprotomy of the patient was done and specimen was sent to histopathology laboratory. We received multiple specimens including right lobe liver mass, appendix with mass, and multiple pelvic mass, omental pieces with mass and rectal sheath mass. On gross examination all the specimens were alike with outer nodular surface. Cut surface was grey white with many cystic areas. [Fig 2] Cysts were filled with necrotic material. Multiple sections were taken for the histopathological examination which showed the similar morphology. Tumor was well encapsulated with tumor cells predominantly arranged in sheets and at places in small interlacing fascicles. Tumor cells were predominantly spindle in shape with elongated hyper chromatic nuclei and ill-defined eosinophilic cytoplasm. At places few tumor cell were small with round nucleus and clear cytoplasm, indicating the epithelioid differentiation. Occasional mitosis was seen.[Fig3, 4] Areas of necrosis were also noted. Based on the above histomorphological findings two differntials were kept – extra intestinal stromal tumor and leiomyosarcoma. Further IHC panel was applied. Tumor showed cytoplasmic positivity for vimentin and smooth muscle actin and was negative for CD 117 and CD 34, excluding the possibility of extra intestinal stromal tumor.[Fig 5] Hence final diagnosis of leimyosarcoma of unknown primary was given.

Discussion

Leiomyosarcoma is the malignant tumor of mesenchymal origin. It originates from the smooth muscle component of the soft tissue. Most common primary site for leiomyosarcoma is the retroperitonium, which accounts 50% of all cases (in this case, there was no retroperitoneal mass on imaging)[4]. Soft-tissue leiomyosarcoma usually

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Fig. 1. Picture shows multifocal mass in the abdomen, largest seen in the abdomen.

Fig. 2. Picture shows multiple tumor mass of variable sizes, largest from the liver measuring 25X20 cms.

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Leiomyosarcoma with Unknown Primary

Fig. 3. Photomicrograph shows tumor cells predominantly arranged in sheets.

Fig. 4 photomicrograph shows epithelioid and spindle diffrentiation of the tumor cells.

Fig. 5 photomicrograph showing vimentin and smooth muscle actin positivity respectively

occurs in middle-aged or older persons, although it may develop in young adults and even in children. The cause of soft tissue leiomyosarcoma is unknown. The predominant occurrence of retroperitoneal and inferior vena cava leiomyosarcomas in women raises the question of hormonal influence, but this is unclear.[5]

Leiomyosarcoma of soft tissue typically forms a fleshy mass, with colours varying from grey to white to tan. A whorled character may be evident to some degree. Larger examples often display haemorrhage, necrosis, or cystic change. Leiomyosarcoma of somatic soft tissue has a number of histological subtypes including epithelioid

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leiomyosarcoma, myxoid leiomyosarcoma, inflammatory leiomyosarcoma, granular cell leiomyosarcoma and dedifferentiated leiomyosarcoma.

leiomyosarcoma range from 50% 3-year survival to 64% 5-year survival, making this tumor one of the more aggressive soft tissue sarcomas.[13]

Many case reports are published with multifocal leiomyosarcoma, most had primary in the GI tract. Roth and Carter et al and Friedie et al reported multicentric GI and extraGI leimyosarcomatosis in their case reports.[6,7] Friedie et al reported a polypoidal smooth muscle tumor in patients GI tract and extensive extra intestinal smooth muscle tumors in the patients liver, lungs, parathyroid gland, bone marrow of the vertebrae and visceral organs including diaphragm, which on histomorphology was diagnosed as leiomyosarcoma .[7] On histology the differentiation of leiomyosarcoma includes – GIST , fibrosarcoma and malignant schwannoma . All these differentials are best differentiated on the basis of IHC panels.[8] SMA and desmin are the specific markers for the diagnosis of the leiomyosarcomas.[9Raised CA125 in leiomyosarcoma is seen in uterine leiomyosarcoma.[10]Vellanki et al reported a case of pelvic mass with raised CA125, which on histology was diagnosed as uterine leiomyosarcoma.[11]

References

Local control of soft tissue sarcomas is usually achieved with surgical resection. Pre operative planning based upon radiographic and pathologic information is important to ensure adequate surgical margins. Achieving wide surgical margins is important in preventing local recurrence.[12] Radiation therapy is an important additional treatment for improving rates of local control when surgical margins are close, especially in high-grade sarcomas. Chemotherapy is employed for the treatment of the systemic diseases .These tumors display very aggressive biology. Neither size nor mitotic activity correlated with outcome, which may represent a reflection of the fact that most of these tumors are quite large on presentation. Local recurrences and metastases of soft tissue leiomyosarcoma usually become manifest within the first few years after diagnosis but may appear as much as 10 years later. For retroperitoneal leiomyosarcomas, the most common sites of metastases are the lungs and liver, whereas the lungs are the dominant location when the primary tumour is nonretroperitoneal. Metastases also occur with some frequency in skin, soft tissue, and bone. Overall reported survival for patients diagnosed with soft tissue

Horvai A. Bones, Joints and Soft Tissue Tumors. In: Kumar V, Abbas, AK, Aster JC, editors. Pathological Basis of Disease. 9 Edition. Philadelphia: Elsevier; 2015: 1179-226.

Weaver MJ, Abraham JA. Leiomyosarcoma of the Bone and Soft Tissue: A Review. ESUN.2007;4:2.

Basic info”. leiomyosarcoma

Golden T, Stout AP. Smooth muscle tumors of the gastrointestinal tract and retroperitoneal tissues. Surg Gynecol Obstet.1941;73:784.

Evans HL., Shipley J. Leimyosarcoma. In: Christopher D.M. Fletcher, K. Krishnan Unni, Fredrik Mertens, editors. World Health Organization Classification of Tumours, Pathology and Genetics of Tumours of Soft Tissue and Bone. Lyon: 2002. 131-34.

Fridie IL, Hong IS, Green WR. Multicentric gastrointestinal and extraintestinal leiomyosarcomatosis: a case report. J Natl Med Assoc.1992;84:7.

Roth JA, Carter H, Costabile DAn unusual multifocal leiomyosarcoma of the stomach: a light and electron microscopic study. Hum Pathol.1978;9:3.

Rodriguez FJ, Folpe AL, Giannini C, Perry A Pathology of peripheral nerve sheath tumors: diagnostic overview and update on selected diagnostic problems. Acta Neuropathol. 2012;123:3.

Schurch W, Skalli O, Seemayer TA, Gabbiani G. Intermediate filament proteins and actin isoforms as markers for soft tissue tumor differentiation and origin. I. Smooth muscle tumors. Am J Pathol. 1987;128.

www.leiomyosarcoma.info.

10. Juang CM, Yen MS, Horng HC, Twu NF, Yu HC, Hsu WL. Potential role of preoperative serum CA125 for the differential diagnosis between uterine leiomyoma and uterine leiomyosarcoma. Eur J Gynaecol Oncol. 2006;27:4:370. 11. Vellanki VS, Rao M, Sunkavalli CB, Chinamotu RN, Kaja S. A rare case of uterine leiomyosarcoma: a case report. J Med Case Rep. 2010;4:222. 12. Kandel R, Coakley N, Werier J, Engel J, Ghert M, Verma S. Surgical margins and handling of soft-tissue sarcoma in extremities: a clinical practice guideline. Curr Oncol. 2013;20:3. 13. Mankin, HJ, Casas-Ganem, J, Kim, JI, et al. Leiomyosarcoma of somatic soft tissues. Clin Orthop Relat Res. 2004;421.

*Corresponding author: Dr. manika khare, Jaipur National University, Institute Of Medical Sciences And Research Centre, Jaipur, India Email: drmanika09@gmail.com

Financial or other Competing Interests: None.

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LMS

Date of Submission : 27.12.2016 Date of Acceptance : 13.05.2017 Date of Publication : 25.08.2017

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Case Report DOI: 10.21276/APALM.1268

Touch Imprint Cytology Of Renal Tumors: Report Of Three Cases R.M.Potekar and Ambica C* Department of pathology, BLDEUâ&#x20AC;&#x2122;S Shri B. M. Patil Medical College, Hospital and Research centre, Vijayapur,Karnataka,India.

ABSTRACT Imprint is a touch preparation in which the cut surface of the tissue is touched gently on a glass slide which leaves behind its impression in the form of cells. Touch imprint cytology is an important diagnostic tool in surgical oncology. It is reliable and economical providing

excellent cellular details. The simplicity, speed and cost effectiveness along with the ability to maximize cell recovery even from a tiny tissue makes imprint cytology a valuable resource in diagnostic medicine. Renal tumors have distinct cytological features that facilitate diagnosis on touch imprint cytology. In the present study, correlation of imprint cytology with histopathological diagnosis was done in three cases of renal tumors. Keywords: Touch Imprint Cytology, Renal Cell Carcinoma, Wilms Tumor, Oncocytoma

Introduction

The outcome of any medical care is ultimately dependent on rapid and timely diagnosis for the effective management. [1] Touch imprint cytology (TIC) is on the rise in diagnostic cytology because of its main advantage to differentiate between benign and malignant lesions in a shorter time span.[1] Although, histopathological diagnosis is considered gold standard to establish definitive diagnosis, TIC can be considered a diagnostic approach especially during intraoperative period to arrive at a rapid diagnosis, thereby helping the surgeon in planning the management.[1,2,3] The diagnostic accuracy of TIC is comparable to frozen section with an added advantage of being economical and the access to be done in a low facility set-up.[2,4]Here, we present imprint cytology of renal cell carcinoma and Wilms tumor with histopathological correlation in three cases.

Case Reports

CASE 1A 45 year female presented with hematuria, burning micturition, pain and mass per abdomen since 8 days. On per abdominal examination, a right lumbar mass was noted measuring 10x8cm.After a thorough pre operative workup, a right nephroureterectomy with paracacval dissection was performed.Grossly, the specimen measured 17x15x5cm with attached ureter measuring 9cm. On cut surfaceâ&#x20AC;&#x201C; a solid fleshy growth was noted measuring 12x10cm with areas of hemorrhage and cystic change. Compressed renal tissue noted at foci. Imprint Cytology: Smears were highly cellular and cells were arranged in loose clusters and singly scattered. Individual cells were large round to polygonal having round to oval nuclei with prominent nucleoli and abundant finely granular eosinophilic to vacuolated cytoplasm

(Figure 1). Few spindle shaped cell clusters were also noted with elongated nuclei, inconspicuous nucleoli and loose chromatin. Cytoplasm was moderate in amount(Figure 1inset). Background showed foamy macrophages. Based on these findings,differential diagnosis of clear cell renal cell carcinoma and Chromophobe renal cell carcinoma (ChRCC) were given. Histopathology: Tumor tissue was predominantly arranged in diffuse sheets with focal trabeculae, papillary and glandular pattern. Individual cells were large round to polygonal having round vesicular nuclei, prominent nucleoli and abundant clear to granular cytoplasm, suggestive of clear cell RCC. Cystically dilated spaces lined by tumor cells and filled with RBCs and cyst macrophages were also noted (Figure 2). CASE 2: A 40 year male complained of pain abdomen since 8 days. On examination- a right lumbar mass was noted measuring 7x6cm. Ultrasonography and Computed Tomography suggested neoplastic lesion and cystic RCC respectively.Right nephrectomy was performed. Grossly, the kidney measured 14x9x6cm and was capsulated.Cut surface showed an encapsulated, solid, gray white mass in the lower pole which measured 8x7x5.5cm with large areas of hemorrhage and necrosis. Imprint Cytology: Smears were moderately cellular with many atypical cells arranged in monolayered sheets and singly scattered. These cells were round to polygonal with a round to oval eccentric nuclei. Few cells showed irregular nuclear membrane. Cytoplasm was abundant granular eosinophilic to flocculent with a distinct cell borders. Background showed neutrophilic inflammatory infiltrate.

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Potekar et al. Based on these findings, differential diagnosis of ChRCC and oncocytoma was given on imprint cytology(Figure 3). Histopathology: Tumor tissue was arranged in diffuse sheets. Individual cells were round to polygonal with small round, slightly irregular hyperchromatic nucleus, inconspicuous nucleoli and abundant amount of eosinophilic granular cytoplasm suggestive of ChRCC. Large areas of necrosis was also noted (Figure 4). Special staining by Haleâ&#x20AC;&#x2122;s colloidal iron showed positivity further confirming ChRCC (Figure 5). CASE 3: A 3 year girl complained of pain and distension of abdomen and hematuria since 3 months. Ultrasonography suggested Wilms tumor and a right radical nephroureterectomy was performed. Grossly, the specimen measured 13x10x8cm with lobulated appearance and congested surface. Cut section was gray white with areas of hemorrhage. Imprint Cytology: High cellular imprint smears showed predominantly blastemal component with cells arranged in small clusters and scattered singly. Individual cells were small, round with round to oval hyperchromatic nuclei, fine granular chromatin, inconspicuous nucleoli and scant, fragile cytoplasm. At foci few spindle shaped cells were also noted(Figure 6). Histopathology: Triphasic pattern of cells were noted comprising of blastemal, epithelial and mesenchymal stromal components. Blastemal cells were small, round to oval with dark staining nucleus and scant amount of cytoplasm. Epithelial component was predominantly

C-111 arranged in tubules and cysts comprised of cuboidal to columnar cells with round to elongated nucleus and moderate amount of eosinophilic cytoplasm. Mesenchymal component showed many spindle shaped cells.

Discussion

Renal tumors have distinctive cytological features that facilitate specific diagnosis on imprint smears. Renal cell carcinoma is the most common tumor of the kidney comprising about 80-90% of primary malignant renal neoplasm in adults.[5]The most common histological subtype of RCC is the clear cell or conventional type RCC accounting for 70-80%.[5]ChRCC is much less common (about 5%) as compared to clear cell RCC.[6,7]ChRCC is unique in having distinct morphological, histological, cytogentic and ultrastructural properties.[6,8] On imprint cytology, ChRCC show highly cellular smears with distinct cell borders and finely granular to transparent cytoplasm. The key feature which helps in diagnosis is the presence of distinct perinuclear halo and slight irregular to wrinkled nuclei. However, in the present cases (Case 1&2), perinuclear halo and wrinkled nuclei were not prominent, leading to confusion with other diagnosis. The main differential diagnoses of ChRCC on cytology include renal oncocytoma and clear cell RCC with granular cells. Differentiation of ChRCC from oncocytoma is not only challenging but also important in further management. Renal oncocytoma is a benign tumor and shows monotonous cell population on cytology. The cells have uniform nuclei, homogenous granular cytoplasm without reticulated clearing or vacuolization and without having well defined

Fig. 1: Microphotograph of imprint smear showing round to oval cells with abundant granular eosinophilic cytoplasm (H&E 20X). Inset showing spindle shaped cells (H&E 10X).

Fig. 2: Microphotograph showing round to polygonal cells with vesicular nuclei, prominent nucleoli and abundant clear to granular eosinophilc cytoplasm (H&E 200X)

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Imprint Cytology of Renal Tumors

Fig. 3: Microphotograph of imprint smear showing round to oval cells having eccentric nucleus and granular eosinophilic to flocculent cytoplasm in a necrotic background (Giemsa 200X).

Fig. 4: Microphotograph showing round to polygonal cells with round, irregular hyperchromatic nucleus, peinuclear halo and abundant amount of granular eosinophilic cytoplasm (H&E 400X).

Fig. 5: Microphotograph showing deep blue granules in the cytoplasm (Haleâ&#x20AC;&#x2122;s colloidal iron 200X).

Fig. 6: Microphotograph of imprint smear showing blastemal component and few spindle cells (Giemsa 200X)

cell borders as compared to ChRCC.[6-8] Special staining by Haleâ&#x20AC;&#x2122;s colloidal iron helps to distinguish the two by being strongly positive in ChRCC and negative in oncocytoma. [6] Immunohistochemistry by vimentin and cytokeratin helps in differentiation between ChRCC and oncytoma. Oncocytoma is positive for cytokeratin and negative for vimentin whereas ChRCC is positive for both the markers. [6,8] The prognosis of ChRCC falls between oncocytoma and clear cell RCC and usually is managed by simple or partial nephrectomy. Therefore, a correct cytological diagnosis aids in appropriate management. Distinguishing ChRCC from clear cell RCC with granular cells is often difficult. Clear cell RCC is associated with dirty, bubbly background containing foamy macrophages whereas ChRCC usually

has a clean background. Further, the cells in clear cell RCC have indistinctcell borders and friable cytoplasm. [6,8] Wilms tumor (Nephroblastoma) is the commonest renal neoplasm found in chidren accounting for 20% of all malignant tumors in this age group.[9]It arises from the metanephric blastema and primarily occurs in infants and about 50% of cases occur before the age of 3 years.[9,10] Cytomorphology shows triphasic pattern of cells comprising of blastemal, epithelial and mesenchymal stromal elements. Blastemal component is composed of small undifferentiated cells having scant cytoplasm.[9]The epithelial component show ill formed tubules, glomeruli like structures which resembles the appearance of metanephric tubules and

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Potekar et al. glomeruli respectively.[9]The mesenchymal spindle shaped cells have fibroblastic configuration.[9] Smears with predominance of blastemal cells could be mistaken for neuroblastoma, but the rosettes of neuroblastoma are pseudo rosettes which are multilayered containing central pink fibrillary material suggesting neuropil.[11]Further neuroblastoma is NSE positive and CK negative in contrast to Wilms tumor.[9]Other differential diagnoses for Wilms tumor include nonHodgkin lymphoma (NHL), rhabdomyosarcoma (RMS), Ewings sarcoma, desmoplastic small round cell tumor and other pediatric renal tumors like cystic nephroma, mesoblastic nephroma, rhabdoid tumor and clear cell sarcoma.[9,11]NHL presents as monomorphic population of tumor cells without forming clusters and showing LCA positivity. Lymphoglandular bodies may be seen in the background.[9,11]RMS show strap cells having cross striations. Further, the triphasic pattern and localization of the tumor to kidney favoursWilms tumor.[9,11] Chandrakar et al did a study to evaluate the relevance of TIC with histopathological correlation in 110 cases.Out of 110 cases, four cases were of the kidney lesion which included two RCC and two cases of Wilms tumor. Among the four lesions, three cases correlated with histopathological diagnosis and the remaining one case (Wilms tumor) was unsatisfactory on imprint smear.[1]Harnish B et al in their study on TIC comprising of 119 cases included a single case of transitional cell carcinoma of renal pelvis which was of non-diagnostic quality on imprint smear.[3]

Conclusion

TIC is a simple, rapid and inexpensive technique aiding in the early management. Renal tumors have distinct cytological features that facilitate diagnosis on touch imprint cytology. Although the major disadvantage of TIC is the failure to provide information on depth of invasion, early diagnosis helps in patients with high

C-113 clinical suspicion and where there is indication for preoperative chemotherapy and radiotherapy.The knowledge and experience of the cytopathologist, skillfulness of the technician and size of the tumor are important variables that influence the results of TIC.

References 1.

Chandrakar J, Srivastava S. Evaluation of the relevance of touch imprint cytology in the diagnosis of various neoplastic lesions. Int J Res Med Sci 2015;3:3046-50.

Kamatchi V, Babu AN, Sankari LS, Rajesh E. Imprint cytology. J Pharm BioallSci 2015;7:S207-8.

Harnish B, Nidhi V, Neena D. Usefulness of touch Imprint Cytology in Cancer diagnosis: A study of 119 cases. Int Res J Medical Sci 2014;2:19-25.

Ranjan A, Chandoke RK, Chauhan N, Kumari R. Study of Tumors by Imprint Cytology. Indian Journal of Clinical Practice 2013;24:472-7.

Ng CS, Wood GC, Silverman MP, Tannir MN, Tamboli P, Sandler MC. Renal Cell Carcinoma: Diagnosis, Staging, and Surveillance. AJR 2008;191:1220-32.

Mamatha K, Arakeri SU, Patil G. Chromophobe variant of renal cell carcinoma masquarding as renal oncocytoma on cytology. Int J Pharm Bio Sci 2015;6:(B) 59-63.

Granter SR, Renshaw AA. Fine needle aspiration of chromophobe renal cell carcinoma. American cancer society 1997;81:122-8.

Lee W. Imprint cytology of the chromophobe renal cell carcinoma: correlation with the histological and ultrastructural features. J Cytol 2011;28:77-80.

Dorothy LF, Pooja S. Giant adult Nephroblastoma â&#x20AC;&#x201C; A case report. International J of medical and applied sciences 2013;2:346-9.

10. Rajwanshi A. Cytology of soft tissue tumors: Malignant small round cell tumors. J cytol 2008;25:89-92. 11. Alam K, Prasad S, Maheshwari V, Aggarwal S, Chana RS. Diagnostic role of fine needle aspiration cytology in Wilms tumor. J Cytol 2007;24:134-36.

*Corresponding author: Dr Ambica C, Post Graduate, Department of pathology, BLDEUâ&#x20AC;&#x2122;s Shri B. M. Patil Medical College, Hospital and Research centre, Vijayapur-586103. Karnataka,India. Phone: +91 9986149480 Email: dr.ambica04@gmail.com Date of Submission : 08.01.2017 Date of Acceptance : 12.05.2017 Financial or other Competing Interests: None. Date of Publication : 25.08.2017

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Case Report DOI: 10.21276/APALM.1285

Inflammatory Myofibroblastic Tumour of Ovary Simulating Malignancy with Review of Literature Ruchi Sinha, Shuchismita, Iffat Jamal*, Shashikant Kumar, Nishi and Mamta Kumari Department Of Pathology , All India Institute Of Medical Sciences-Patna(India)

ABSTRACT Inflammatory myofibroblastic tumour (IMT) of ovary is an uncommon benign neoplasm that can mimic malignancy. It often occurs in children and young adults. Lung is the most common site of IMT. Precise etiology is not known but recent reports suggested the role of benign reactive process in its pathogenesis. A 32 year old female patient presented to the gynaecology out patient department with complaints of lower abdominal pain and increased frequency of micturition with burning since 15 days. On per abdominal examination, an irregular lump of 18 weeks size with restricted mobility was palpable in suprapubic region. Laboratoratory investigations revealed normocytic normochromic anaemia with neutophilic leucocytosis and raised ESR. Her serum CA 125 levels were slightly raised (44.30 u/ml). Imaging studies showed an infiltrating mass in the pelvis. The patient underwent total abdominal hysterectomy with bilateral salpingo oopherectomy and omentectomy. Histopathological examination of which revealed IMT of right ovary with extension to right parametrium and adjacent bladder. As IMT is a mimicker of malignancy due to its infiltrative growth. Histopathological examination is mandatory for its diagnosis and treatment. Misdiagnosis can lead to unnecessary extensive debulking surgeries and even chemotherapy. Rarity of this entity in the ovary has made this case worth reporting. Keywords: Inflammatory, Myofibroblastic, Tumour, Malignancy, Salpingo-Oopherecromy

Introduction

irregular lump of 18 weeks size with restricted mobility was palpable. Laboratory investigations revealed low haemoglobin (8.4 gm%), neutrophilic leukocytosis, raised ESR and slightly raised serum CA 125 level (44.30 U/ml). Culture and sensitivity test of urine did not show any growth. Per vaginal examination revealed a firm lump of restricted mobility measuring 10 x 10 cm which appeared as an irregular mass separate from uterus. The rectal mucosa was free on per rectal examination. Peritoneal tap aspirated 40 ml haemorrhagic fluid which on cytological analysis showed inflammatory smear comprising of neutrophils, lymphocytes with few clusters of benign mesothelial cells. No evidence of malignancy was found. Cervical smear was reported as inflammatory cervical smear with bacterial vaginosis. Ultrasonography of abdomen showed a solid cystic pelvic mass measuring 11 x 9.45 cm. The mass was present anterior to the uterus. Endometrial thickness was 8 mm.

Case Report

Contrast- enchanced computed tomography (CECT) of the whole abdomen was performed which revealed a large lobulated right ovarian mass (11 x 7.5 x 7.5 cm) involving and adhering to the right anterior uterine wall, extending anteriorly upto left uterine cornu and involving both sides of uterus (FIG â&#x20AC;&#x201C; 1). The mass was isodense to

Inflammatory myofibroblastic tumour (IMT), also known as plasma cell granuloma, plasma cell pseudo tumour, inflammatory myofibrohistiocytic proliferation and inflammatory pseudotumour is a rare benign lesion of mesenchymal origin. The designation of IMT was adopted by the World Health Organization (WHO) classification of tumours of the soft tissue and bone in the year 2013.[1] It primarily affects children and young adults.[1] Precise etiology of this lesion is unknown. The neoplasm is composed of proliferating myofibroblastic spindle cells admixed with chronic inflammatory cells such as lymphocytes, plasma cells and eosinophils. It can arise in any organ but most frequent reported sites are lung, mesentry and omentum.[1] Female genital tract is one of the rarest site for occurance of IMT with most cases reported in the uterus.[2] Only few cases of IMT in ovary have been reported.[3] A 32 year old female presented to the gynaecology out patient department with complaints of lower abdominal pain, burning micturition and increased frequency of micturition since 15 days. There was no history of fever or vomiting. On physical examination of abdomen, a firm

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the uterus with similar enhancement pattern. The mass was solid with cystic component. The cyst wall was 1 cm thick and contained pus/necrotic material. The left ovary showed a 4.5 x 3.2 x 2.1 cm heterogenous mass . The fat and omentum anterior to the right ovarian lump seems to be infiltrated. Few satellite nodules measuring upto 1 cm in size were seen. Surrounding pelvic viscera appeared to be free on CECT. Enlarged lymph nodes were seen in both iliac chains, largest measuring 20 x 12 cm in size. Few enlarged paraotic lymph nodes were present, the largest one measuring 1.5 x 0.9 cm. A clinical diagnosis of stage IV-A ovarian carcinoma was given and total abdominal hysterectomy with bilateral salpingo oopherectomy and omentectomy (suboptimal debulking) was planned. Intra operatively, a solid-cystic mass measuring 5 x 5 cm was seen in right ovary and filled with approximately 3040 ml of pus. The pus was aspirated and sent for culture and sensitivity. The ovarian mass was densely adherent and infiltrating into uterus, bladder, vagina, gut, omentum and extending upto the lateral pelvic wall. Due to the dense adhesions urinary bladder was perforated while removing the ovarian mass and which was subsequently repaired. Pus culture showed growth of Staphylococcus aureus. Cytology revealed degenerated as well as intact polymorphs admixed with few mononuclear cells comprising of plasma cells and macrophages.

The excised specimen was sent for histopathological examination. On gross examination, cut section of the mass revealed solid grey white areas with pale yellow patches and cystic spaces (Figure 2). Microscopic examination showed proliferating myofibroblasts (at places arranged in fascicles) along with diffuse infiltration of chronic inflammatory cells comprising of lymphocytes with focal lymphoid aggregates, plasma cells, histiocytes and occasional eosinophils (Figure 3). Focal aggregates of foamy macrophages were seen. Areas of coagulative necrosis as well as sclerosed and congested blood vessels were present. No pleomorphism, atypical mitosis and lymphovascular invasion was noted. The mass was seen to entrap the fat spaces and infiltrating into the muscularis propria of bladder (detrusor muscle). Left ovary was not involved microscopically. Diagnosis of IMT of right ovary with extension to right parametrium, anterior uterine wall, peritoneal fat and detrusor muscle of bladder was given. Immunohistochemistry showed diffuse positivity for vimentin(FIG 4), smooth muscle actin(Fig.4) was positive in the fibrous septa and blood vessels. ALK was negative. Post operative period was unremarkable.

Discussion

IMT is a rare benign mesenchymal neoplasm. Proper detailed description of its pathological identity was done in 1954 by Iverson and Umiker, though it was described first by Brunn.[4] It often presents in first and second decades of life with slight female predominance.[1] Lungs are most

Fig. 1: Computed tomography scan showing a solid cystic right ovarian mass involving and adhering to the right anterior uterine wall, extending anteriorly upto left uterine cornu and involving both sides of uterus.

Fig. 2: serial sectioning of the right ovary revealed solid grey white areas with pale yellow patches and cystic spaces.

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Fig. 3: photomicrograph showing proliferating myofibroblasts along with diffuse infiltrate of chronic inflammatory infiltrate entrapping the fat spaces(H&E – 10X4). Inset showing the same in higher magnification (H&E – 10X40).

Fig. 4: photomicrograph showing diffuse positivity for smooth muscle actin and vimentin respectively.

frequently involved site.[5] IMTs of female genital tract are rare with uterus being the most common organ.[2] Only few cases of ovarian IMT have been reported.[3] Exact etiology is unknown though, infection with inflammation has been proposed to be a causative factor. Recently the role of human herpes virus-8 DNA sequences and over expression of human interleukin 6 and cyclin D1 has been reported in seven cases.[6] Cases of IMT have been reported post treatment of Wilm’s tumour in oesophagus, stomach and liver.[7] Recent report suggests that it is a neoplastic process because of its aggressive behaviour, involvement of chromosome 2p23 and cytogenetic clonality.[8]

The symptoms of IMT vary according to the site of involvement. Patients with intra-abdominal tumors most commonly complain of abdominal pain or an increasing abdominal mass and occasionally with signs and symptoms of gastrointestinal obstruction. Pulmonary IMT presents with chest pain and dyspnoea.[9] Some patients have prominent systemic manifestations, including fever, night sweats, weight loss and malaise. Laboratory abnormalities are present in up to one – third of patients and include an elevated erythrocyte sedimentation rate, anemia, thrombocytosis, and polyclonal hypergammaglobulinemia, which often resolve when the lesion is excised.[10]Computed tomography (CT) scan and

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magnetic resonance imaging (MRI) studies reveal a solid mass with infiltrative growth.[11] Grossly, most lesions present as exophytic, nodular or polypoidal intraluminal lesions that may infiltrate deeply into visceral organs from which they arise and also into the adjacent structures. They range in size from 1.0 cm to upto 17 cm.[1] The consistency of lesion depends upon the relative amounts of fibrous and myxoid stroma present. Microscopically, IMT is composed of myofibroblasts and inflammatory cells.The spindled myofibroblasts and inflammatory cells of IMT are arranged in three basic histological patterns.[5] In first pattern, myofibroblasts are loosely arranged in an oedematous myxoid background with abundance of blood vessels and chronic inflammatory infiltrate comprising of plasma cells, lymphocytes and eosinophils. This pattern mimics granulation tissue, nodular fasciitis, or other reactive processes. A second pattern is characterised by fascicular and storiform arrangement of myofibroblasts with variable myxoid and collagenised areas and mature lymphoid and plasma cell infiltrate with germinal centres.. Ganglion like myofibroblast are often seen in these two patterns. This pattern resembles fibromatosis, fibrous histiocytoma, or smooth muscle neoplasm. The third pattern is paucicellular with sparse inflammatory infiltrate and dense collagenisation. It resembles a scar or desmoids-type fibromatosis. Highly atypical polygonal cells with vesicular nuclei, prominent nucleoli and variable mitoses are seen in rare IMTs which undergo histologic malignant transformation.[5,12] Invasion into the muscularis propria of the urinary bladder is a common finding and some even infiltrate into the perivascular adipose tissue. IMT needs to be differentiated from IgG4 related disease due to the prominent plasma cell infiltrate and fibrosis in both the lesions.[1]From an immunophenotypic standpoint, the spindle cells of IMT expresses diffuse cytoplasmic reactivity for vimentin.[1] ALK positivity is detectable in approximately 50% of IMTs.[1,14] Focal to diffuse reactivity for smooth muscle actin and muscle specific actin is also demonstrated in spindle cells cytoplasm.[1,14] One third of cases show immunoreactivity for cytokeratin. Sometimes it is mistaken as immunoglobulin G4 subclass (IgG4) related sclerosing disease, however IMT is negative for IgG4.[1] Surgery is the principal modality of treatment. Extrapulmonary IMTs have a recurrence rate of 25% and depends upon anatomical site, resectability and multinodularity.[1] Metastasis is also reported in <5% of cases.[1] Factors related to adverse prognosis are morphological atypia, ganglion like cells, expression of TP53 and aneuploidy.[12] www.pacificejournals.com/apalm

The differential diagnosis of IMT comprises of low grade myofibroblastic sarcoma, benign reactive or neoplastic mesenchymal lesions such as, solitary fibrous tumour, spindle cell carcinoma, nodular fasciitis and peripheral nerve sheath tumour.

Conclusion

The clinicians and pathologists should be familiar with this entity and diagnosis of IMT should be considered in a mass presenting with inflammatory reactions. As IMT is a benign mimicker of malignancy, histopathological examination is mandatory for its diagnosis and treatment. Misdiagnosis can lead to unnecessary morbidity and mortality related to extensive debulking surgeries and even chemotherapy.

Reference 1.

Fletcher CDM, Bridge JA, Hogendoorn PCW et al. Inflammatory myofibroblastic tumour. In: Fletcher CDM, Bridge JA, Hogendoorn PCW, Mertens F, editors. Lyon International Agency for Research on Cancer. World Health Organization Classification of Tumours of Soft Tissue and Bone. 4th edition;2013: 83-4.

Olgan, S, Saatli, B et al. Hysteroscopic excision of inflammatory myofibroblastic tumor of the uterus: as case report and brief review. Eur. J. Obstet. Gynecol Reprod. Biol.2011;157: 234–240.

Shintaku, M, Fukushima A. Inflammatory myofibroblastic tumor of the uterus with prominent myxoid change. Pathol. Int.2006; 56 :625–628.

Bharatnur S, Ramkumar V, K Natrajan. Inflammatory Pseudo-Tumor Of Ovary - A Case Report. The Internet Journal of Gynecology and Obstetric.2012;16:1.

Sahnoun L, Elezzi O, Maazoun K, Krichene I et al. Ovarian inflammatory myofibroblastic tumor in children. J Pediatr Adolesc Gynecol. 2007 ;20(6):365-6.

Wang TY, Chou JW, Shih YS et al. Inflammatory myofibroblastic tumor mimicking adrenal incidentaloma. Intern Med. 2011;50: 165–6.

Coffin CM, Humphrey PA, Dehner LP . Extrapulmonary inflammatory myofibroblastic tumor: a clinical and pathological survey. Semin Diagn Pathol .1998;15: 85-101.

Coffin CM, Watterson J, Priest JR, Dehner LP. Extrapulmonary inflammatory myofibroblastic tumor (inflammatory pseudotumor). A clinicopathologic and immunohistochemical study of 84 cases. Am J Surg Pathol.1995; 19: 859-872.

Gomez-Roman JJ, Vinyals GO,Velasco PS et al. Presence of human herpesvirus-8 DNA sequences and overexpression of human IL-6 and cyclin D1 in inflammatory myofibroblastic tumor (inflammatory pseudotumor). Lab Invest.2006; 80:1121-1126.

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10. Vujanic GM, Milovanovic D, Aleksandrovic S. Aggressive inflammatory pseudotumor of the abdomen 9 years after therapy for Wilms tumor. A complication, coincidence, or association? Cancer.1992; 70: 2362-2366. 11. Poh CF, Priddy RW, Dahlman DM. Intramandibular inflammatory myofibroblastic tumour: A true neoplasm or

reactive lesion? Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2005;100:460–6. 12. Volker HU, Scheich M, Holler S et al. Differential diagnosis of laryngeal spindle cell carcinoma and inflammatory myofibroblastic tumour: Report of two cases with similar morphology. Diagn Pathol. 2007;2:1–7.

*Corresponding author: DR.IFFAT JAMAL, Flat No-D/2,Phase-1,Sapna Apartment, Nayatola, Patna(Bihar)-800004 India Phone: +91 9835498843 Email: iffatjamal111@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 15.01.2017 Date of Acceptance : 03.04.2017 Date of Publication : 25.08.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Case Report DOI: 10.21276/APALM.1295

Synchronous Primary Ovarian Mucinous Carcinoma and Endometrioid Endometrial Carcinoma : A Rare Case Report Mohan Krishna Pasam1, Rajendiran S1*, Susruthan M1 and Usha Rani2 Department of pathology, Sri Ramachandra Medical college and Research Institute ,chennai(India) . Department of obstetrics and gynaecology . Sri Ramachandra Medical College and Reasearch Institute , Chennai(India) 1

ABSTRACT Introduction: Synchronous tumours of female genital tract are a rare . The most common organs involved are ovary and endometrium. Endometrioid carcinoma is the most common malignancy detected in majority of cases . Synchronous cancers with dissimilar histology are a very rare phenomenon . We report a case of 51 year old lady with synchronous primary mucinous carcinoma of ovary and primary endometrioid endometrial carcinoma . Case Report: a 51 year old lady presented with post menopausal bleeding and foul smelling vaginal discharge of 1 month duration . Her CA125 was elevated. Magnetic resonance imaging showed increased endometrial thickness and a12.5x10x9.3cm mass in the fundus of uterus suggestive of a leiomyoma along with and a 13.8x11.5x11cm mass in the left adnexa. Endometrial aspiration done showed Complex Hyperplasia with Atypia. Total abdominal hysterctomy and bilateral salphingoophorectomy along with lymphnode sampling was performed . The adnexal mass was pT1a pN0 cM0 grade 2 invasive mucinous carcinoma. The endometrial lesion was pT1b pN0 cM0 FIGO grade 2 endometrioid endometrial adenocarcinoma. Conclusion: Synchronous ovarian tumours of dissimilar histology is extremely rare and there are only a few reports of ovarian mucinous carcinoma with endometrioid endometrial carcinoma. Keywords: Synchronous, Ovarian Tumour, Endometrial Adenocarcinoma, Immunohistochemistry

Introduction

Simultaneously identified tumours in female genital tract represent any of the three following possibilities . Primary endometrial tumour with ovarian metastasis . Primary ovarian tumour with metastasis to the endometrium . Synchronous primary ovarian and endometrial carcinoma. Synchronous primary tumours of female genital tract account for only <2% of all gynaecological malignancies [1,2] . Ziano R et al reported that 5% cases of endometrial cancer have synchronous tumour in ovary and 10% cases of ovarian cancer show a synchronous tumour in the endometrium [3]. Scully et al proposed a clear pathological criteria for determining synchronous tumours which include histological dissimilarity, minimal invasion of endometrial tumour , presence of in situ carcinoma, absence of vascular space invasion of endometrial tumour , unilateral ovarian tumour involving parenchyma with no surface involvement, presence of ovarian endometriosis 4 We report a 51 year old woman presented in our centre with post menopausal bleeding diagnosed to have a rare synchronous association of primary ovarian mucinous carcinoma and endometrioid endometrial carcinoma

Case Report

A 51 year old lady came with post menopausal bleeding for 1 month and foul smelling vaginal discharge for 1

week. Her Body Mass index was 28kg/m2and is a known case of hypothyroid on treatment .She had undergone total knee replacement for osteoarthritis 3years back. Physical examination showed an abdominal mass corresponding to 18 to 20 weeks gestation. Magnetic resonance imaging was done which showed thickened endometrium of 57mm along with a heterogenous conglomerate mass in the fundus of size 12.5x10x9.3cm [fig.1]suggestive of a leiomyoma. A well circumscribed heterogenous and hyper intense adnexal mass of 13.8x11.5x11cm was also seen[Fig.1]. Serology showed an elevated CA125. Pre operative endometrial aspiration was done which showed Complex Hyperplasia with Atypia. A staging laparotomy was done and total abdominal hysterectomy with bilateral salphingoophorectomy was sent for frozen section. The adnexal mass weighed 2.5kg and measured 18x16x7cm with a smooth grey white nodular surface . The cut curface exuded yellowish mucoid material and showed 80% solid areas and 20% cystic areas with papillary projections [fig:2] . The uterus showed friable lesion in the lower uterine segment and body and was infiltrating >50% of the myometrium [fig:3].A grey white well circumscribed mass of size 13.5x8x7cm seen in myometrium. Histopathology of the sections from the adnexal mass showed mucinous carcinoma of endocervical type which was invading the

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ovarian stroma[Fig.4].while the endometrium showed endometrioid type endometrial adenocarcinoma with myometrial invasion[Fig.5] . Further pelvic lymph node sampling was done and a final diagnosis of pT1a pN0 cM0 grade 2 invasive mucinous carcinoma of left ovary and pT1b pN0 cM0 FIGO grade 2 endometrioid endometrial

adenocarcinoma was given. The grey myometrial mass showed features of a leiomyoma and opposite ovary showed endometriosis[Fig.6]. Immunohistochemistry done on both ovarian and endometrial tumours showed a positive nuclear staining of estrogen receptor in endometrial carcinoma[Fig. 7] and was negative in ovarian mucinous carcinoma .

Fig. 1: MRI showing an adnexal and uterine mass.

Fig. 2 : ovarian mass showing a multiloculated lesion with papillary architecture and mucin.

Fig. 3 : uterus with endometrial lesion and leiomyoma.

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Fig. 4: H&E , invasive mucinous carcinoma of ovary : 200x.

Fig. 5: H&E endometrium showing invasive endometrioid adenocarcinoma : 100x.

Fig. 6: H&E : opposite ovary with endometriosis 40x.

Fig. 7: immunohistochemistry positive for Estrogen receptor in endometrial carcinoma 400x

Discussion

75% were endometrioid carcinomas8. They reported 7 cases of ovarian mucinous carcinomas with endometrioid endometrial carcinomas in their study .Sylwia et al reported 10 cases of which 1 was ovarian mucinous carcinoma with endometrioid endometrial carcinoma[9] . Age of presentation in our case was 51 years which is correlating with previous studies[7].

Our case fulfilled all the pathological criteria for a synchronous malignancy . In a study done by Y Chiang et al done on 23 cases of synchronous ovarian and endometrial tumours 63% showed similar histology[5]. A 10 year study done by Young Kuei Lim et al on 75 patients with synchronous tumours showed 73.9% being endometrioid endometrial cancers and 26.1 % with tumours of different histological type[6] . Yuantaoliu et al reported 43 cases of synchronous tumours of which 30% showed dissimilar histology[7].There are only a few reports of synchronous endometrial carcinoma with an ovarian carcinoma of mucinous morphology. Catharina et al reported 53 cases of synchronous endometrial and ovarian tumours of which www.pacificejournals.com/apalm

Presenting symptom of synchronous tumours is that of individual primary tumours [7] . In our case it was post menopausalbleeding .Soliman PT et al reported that 30 to 40% of patients with synchronous tumours were obese (BMI> 30kg/m2)[10]. Though obesity is a known risk factor for endometrial carcinoma, there is no specific data to eISSN: 2349-6983; pISSN: 2394-6466

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support the association of obesity with synchronicity. Majority of patients with synchronous tumours show elevated CA125 levels .Yuantaoliu et al reported a mean CA125 value of 161.7IU/ml in their study[7] . Endometriosis of ovaries is seen in majority of cases with synchronous tumours [3]. Though our case had endometriosis in the opposite ovary its association with mucinous ovarian carcinoma could not be explained. Synchronous tumours clinically present at a lower stage at diagnosis when compared to the tumours of individual organs. Previous reports do suggest that majority of synchronous tumours present at stage I [4,7] . The treatment of synchronous endometrial and ovarian cancer depends on the pathological characteristics of each tumour[4, 11]. Surgery with an adjuvant chemotherapy remains the main stay of treatment . However reports show that stage I and II ovarian tumours which are grade 1 and endometrial tumours which are grade 1 with only superficial myometrial invasion do not require chemotherapy . In our case both endometrial and ovarian tumours were stage I. Long term prognosis of synchronous tumours was found to be relatively good . The prognosis often depends on the stage of each tumour rather than the histological type. Studies have shown that 5 year survival rates are excellent. Zaino R et al reported 86% 5 year survival and 80% 10 year survival [3]. Another report has shown a mean survival of 84%6. Yuantaoliu et al reported a mean survival of 109 months and rate of 93.5% in stage I and stageIItumours[7].Presentation at an early stage may be attributed to the overall good prognosis . Pathogenesis of synchronous ovarian and endometrial tumours is still unclear . Various theories like shared molecular receptors in different organs of female genital tract which respond to same carcinogen may hold good for tumours of similar histology . But This theory cannot explain the occurrence of tumours with dissimilar histology . Molecular study done by Soliman et al showed on 7 % of cases with criteria suggestive of a Lynch syndrome[10] . Wether the synchronicity of tumours of different histology is incidental or due to a common molecular carcinogenesis is yet to be determined .

There are very few reports of synchronous mucinous carcinoma ovary and endometroid endometrial carcinoma . This case is reported for its rarity , to emphasise the importance of thorough radiological evaluation of endometrium in all adnexal mass lesions and to emphasise the need of endometrial aspiration in suspicious cases . There is a dire need for extensive molecular analysis for determining the pathology of these synchronous tumours with dissimilar histology.

Acknowledgements

Dr Sandhya Sundaram , Head, Department of pathology , Sri Ramachandra Medical college , Chennai.

Reference 1.

Tong SY, Lee YS, Park JS, Bae SN, Lee JM, Namkoong SE. Clinical analysis of synchronous primary neoplasms of the female reproductive tract. European Journal of Obstetrics &Gynecology and Reproductive Biology. 2008 Jan 31;136(1):78-82.

Eisner RF, Nieberg RK, Berek JS. Synchronous primary neoplasms of the female reproductive tract. Gynecologic oncology. 1989 Jun 1;33(3):335-9.

Zaino R, Whitney C, Brady MF, DeGeest K, Burger RA, Buller RE. Simultaneously detected endometrial and ovarian carcinomas—a prospective clinicopathologic study of 74 cases: a gynecologic oncology group study. Gynecologic oncology. 2001 Nov 1;83(2):355-62.

Scully REYR, Clement PB: Tumors of the Ovary, Maldeveloped Gonads, Fallopian Tube, and Broad Ligament: Atlas of Tumor Pathology. Bethesda, MD, Armed Forces Institute of Pathology, 1998

CHIANG YC, CHEN CA, HUANG CY, HSIEH CY, CHENG WF. Synchronous primary cancers of the endometrium and ovary. International Journal of Gynecological Cancer. 2008 Jan 1;18(1):159-64.

Lim YK, Padma R, Foo L, Chia YN, Yam P, Chia J, KhooTan HS, Yap SP, Yeo R. Survival outcome of women with synchronous cancers of endometrium and ovary: a 10 year retrospective cohort study. Journal of gynecologic oncology. 2011 Dec 31;22(4):239-43.

Liu Y, Li J, Jin H, Lu Y, Lu X. Clinicopathological characteristics of patients with synchronous primary endometrial and ovarian cancers: A review of 43 cases. Oncology letters. 2013 Jan 1;5(1):267-70.

van Niekerk CC, Bulten J, Vooijs GP, Verbeek AL. The association between primary endometrioid carcinoma of the ovary and synchronous malignancy of the endometrium. Obstetrics and gynecology international. 2009 Dec 15;2010.

Szmich SD, Czernek U, Krakowska M, Frąckowiak M, Zięba A, Czyżykowski R, Kulejewska D, Potemski P.

Conclusion

Primary ovarian carcinoma with a synchronous primary tumour in the endometrium though a rare phenomenon is occasionally encountered in gynecological practice. Majority of synchronous tumours in the ovary and endometrium are of endometrioidtype. The occurrence of tumours with dissimilar histology is a rare event.

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Pasam et al. Synchronous primary ovarian and endometrial cancers: a series of cases and a review of literature. PrzMenopauzalny. 2014 Mar;13:64-9. 10. Soliman PT, Slomovitz BM, Broaddus RR, Sun CC, Oh JC, Eifel PJ, Gershenson DM, Lu KH. Synchronous primary cancers of the endometrium and ovary: a single institution

C-123 review of 84 cases. Gynecologic oncology. 2004 Aug 31;94(2):456-62. 11. Signorelli M, Fruscio R, Lissoni AA, Pirovano C, Perego P, Mangioni C. Synchronous early-stage endometrial and ovarian cancer. International Journal of Gynecology& Obstetrics. 2008 Jul 1;102(1):34-8.

*Corresponding author: Rajendiran S, Department of pathology, Sri Ramachandra Medical college and Research Institute , Chennai (India) Phone: +91 9480755187 Email: srajendiran@sriramachandra.edu.in, Date of Submission : 24.01.2017 Date of Acceptance : 24.05.2017 Financial or other Competing Interests: None. Date of Publication : 25.08.2017

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Case Report DOI: 10.21276/APALM.1453

Prerectal mucinous cystadenoma: A case report and considerations about its origin Jaime Zuloaga1, Javier Arias-Diaz2*, Maria Jesus Fernandez-Aceñero3, Cristina Diaz-del-Arco4 Julio Mayol1 and Antonio Torres1 Dept. of Surgery, Complutense University of Madrid, Instituto de Investigación Sanitaria Hospital Clínico San Carlos, Spain 2 Dept. of Surgery, Complutense University of Madrid, School of Medicine, Instituto de Investigación Sanitaria Hospital Clínico San Carlos, Spain 3 Dept. of Pahology, Complutense University of Madrid, Instituto de Investigación Sanitaria Hospital Clínico San Carlos, Spain 4 Dept. of Pathology, Instituto de Investigación Sanitaria Hospital Clínico San Carlos, Spain 1

ABSTRACT Mucinous cystadenoma is a generic denomination usually applied to cystic formations filled with mucinous content and showing signs of epithelial proliferation. They usually arise in ovaries, pancreas and appendix, but there have been occasional reports in many other locations. Most retroperitoneal and mesenteric mucinous cystadenomas have affected women, and a potential müllerian origin has been postulated, although for most of them their histogenesis remains uncertain. Some cases have shown malignant behavior. We herein describe a case of mucinous cystadenoma affecting a young man with an unusual prerectal location. An exhaustive study was performed as a basis for the discussion of possible origins for this tumor. This lesion showed premalignant proliferative changes, suggesting an early stage in a potential malignant transformation. Keywords: Mucinous Cystadenoma; Tailgut cyst; Cysts/pathology; Rectal Diseases/pathology; Rectal Diseases/surgery

Introduction

Mucinous cystadenoma usually arise in ovaries, pancreas and appendix, but they have been reported in many other locations.[1] Most retroperitoneal and mesenteric mucinous cystadenomas have affected women, and a potential müllerian origin has been postulated, although their histogenesis remains uncertain for most of them.[2][3] Some cases have shown malignant behavior.[1] We herein describe a case of mucinous cystadenoma affecting a young man with an unusual prerectal location. This lesion showed proliferative changes, suggesting an early stage in a potential malignant transformation.

Case Report

A 45-year-old male presented with a history of low rectal bleeding and a palpable rectal mass at 6 cm of the anal margin. At colonoscopy the mass had a rounded surface and a submucosal origin. Transrectal ultrasonography identified a 4x3.5 cm extraluminal tumor, which appeared encapsulated and independent of both the mucosa and muscular layers of the rectum.Pelvic MRI scan showed a lobulated cystic tumor, measuring 5 cm of diameter and located 7 cm above pectinate line, anteriorly to the rectum (Figure 1). The tumor was independent of the rectal mucosa and also was separated from the prostate and seminal vesicles by a fatty dissection plane. The tumor

capsule appeared thin and uniform, and no adenomegalies are found. RMI findings were thus concordant with the diagnosis of an epidermoid or duplication cyst.No additional pathological findings were observed in the abdominal US. Routine blood tests were normal, and serum tumor markers CA 15.3, CA19.9 and CEA were within normal range. Feminization traits were discarded, male and female sex hormone levels and those of sex hormone binding globulin were within normal limits. At laparotomy, a 5-cm mass was found anterior to the rectum. It was noted to be intimately involved with the muscular layers of the rectum wall but it did not appear to involve the prostatic fascia. A low anterior resection with stapled colorectal anastomosis and a loop ileostomy were performed. The patient showed uneventful recovery and he is alive and well eighteen months after discharge. The lesion was sent as a whole. It was found multilocular when cut, with thin delicate septa within its wall and filled with a sticky viscous material. Serial sectioning revealed no areas of macroscopic papillary growth or thickening in the cyst wall. The rectosigmoid colon was normal. Microscopic analysis of hematoxylin-eosin sections of the cyst wall revealed that the mass was growing within the adventitia and the muscular coat of the rectum with no mucosal involvement. The cyst loculi were filled

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Zuloaga et al. with amorphous bluish material (mucin) and sparse inflammatory cells. The outer cyst wall corresponded to fibrous paucicellular tissue with no muscular layer or adventitial tissue and the muscular coat of the rectum was compressed by the tumor growth. The inner wall was partially denuded of epithelium, but in some areas it showed a well-preserved layer of columnar tall cells (Figure 2a). These cells were relatively uniform and showed mucin production, mainly in the apical cytoplasm. The nuclei were all basally located and showed neither atypia nor mitosis (Figure 2b). Some foci of pseudostratification with micropapillary formations were noted (Figure 2c), but the basal membrane was preserved. No other mucin lakes or epithelial inclusions were noted in other areas of the rectal specimen sent for examination after thorough sampling. Immunohistochemistry performed on the cyst wall revealed intense positivity for pancytokeratins (low

C-125 and high molecular weight), cytokeratin 20 (marker of hindgut origin; Figure 3a), and carcinoembryonic antigen (CEA). Markers suggesting mesothelial (calretinin and mesothelin), mĂźllerian (cytokeratin 7), prostatic (p63, chromogranin A, vimentin and prostate specific antigen) or intestinal (CDX-2) origin, were all negative. Estrogen and progesterone receptors were also negative. Proliferative index with Ki67 (Figure 3b) was very low and restricted to the basal layer of cells in the foci of pseudostratification, therefore suggesting a benign nature of the lesion. The cyst did not communicate with the rectal lumen at any point, so we could not confirm diverticular disease as the origin of the lesion. Besides, the cyst wall did not show a muscle coat and it only had the epithelial inner lining and a peripheral narrow band of connective tissue, what seems to discard a duplication cyst of the rectal wall. Final diagnosis was mucinous cystadenoma.

Fig. 1: Pelvic MRI scan showing a lobulated cystic tumor, located anteriorly to the rectum and independent of the rectal mucosa. A fatty dissection plane separates the cyst from the prostate and seminal vesicles. A sagittal plane (a) and an axial one (b) are shown.

Fig. 2: A. Low-power view of the cyst wall showing the relation to the bowel mucosa (lower left). Note the cyst wall is only composed of fibrous tissue and compresses the muscular coat. B. Medium power view of the cyst wall showing a partially detached monolayer of epithelial cylindrical cells with apical mucin. C. Medium power view of the areas with pseudostratification of nuclei and papillary ingrowths into the cyst lumen (A, H-E x 40; B and C: H-E, x 200).

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Fig. 3: A. Immunohistochemical staining for cytokeratin 20 confirms hindgut origin of the epithelial lining. B. Immunohistochemical staining for Ki67 confirms low mitotic activity, limited to basal layers. (A. Immunohistochemistry for CK20, x 200; B. Immunohistochemistry for Ki67, x200).

Discussion

The pathologic diagnosis was eventually based on pure morphological grounds. We had a multilocular prerectal cyst lined by mucin secreting epithelium with micropapillary projections and pseudostratification, but without any sign of stromal invasion suggestive of malignant transformation. The histopathological image was in all aspects similar to that seen in appendicular or ovarian mucinous cystadenoma with some features of the so-called borderline lesions in this group.[4] A thorough literature review of this issue revealed rare reported cases of mucinous cystadenoma originating in the retroperitoneum or the mesentery. Nevertheless, very few cases were located in the perirectal anterior area.[5] These reports affected mainly women and discussion regarding origin suggested most a m端llerian origin or a mesothelial one, based on the positivity of two cases for calretinin.[6] We consider that the mesothelial origin has been reasonably excluded in the present case, for the epithelial lining was negative for calretinin and mesothelin. The m端llerian remnant in male is the prostatic utricle. In our patient, there was a lack of continuity between the cystic mass and the prostate. In addition, the plasma levels of sex hormones were normal and immunohistochemical markers indicative of a m端llerian origin were absent. The simple cyst in the seminal vesicle is a unilocular cystic lesion located at lateral to the midline in the retrovesical area. In our case, the cyst was also independent of the seminal vesicles, and did not contain sperm fragments, which seems to be a characteristic finding in these cysts.[7] Another potential source for this lesion could be a teratoma with complete regression of all non-ectodermal

components, but even in this case it would be expected to have some mixture of epithelial types, which was not found in our patient. A cyst arising from remnants of the cloacogenic membrane could be another possibility. However, the cytokeratin 20 positivity makes it however very unlikely. The absence of any history of prior colorectal biopsy or procedure makes also unlikely for it to arise in a misplaced or ectopic colorectal mucosa. We consider that the most plausible explanation for our lesion would be a premalignant epithelial proliferation in a tailgut cyst with an unusual location.[5] Nevertheless, the prerectal location cast some uncertainty over this statement.[8] Tailgut cysts usually arise in the presacral space and are related to a lack of involution of the tailgut, which usually regresses completely between the 7-8th week of development. It is known that most of these cysts are lined by mucin-secreting epithelium of intestinal type and there are reports of aggressive mucinous adenocarcinomas arising in long-standing cysts of this type.[9] We can guess that the neoplastic growth of the mucin secreting epithelium lining the cyst wall has led to a papillary mucinous cystadenoma, similar to the ones originating in m端llerian structures. It might be suggested that this cystoadenomatous pattern can be a premalignant phase of the mucinous adenocarcinomas known to arise in these embryologic remnants. Given that tailgut is a dorsal growth in the developing embryo, it is puzzling that remnants of such a clearly retrocloacal structure should be present in prerectal location. In fact, the possible tailgut origin of prerectal cysts is opposed by some.[8]

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Zuloaga et al. Nevertheless, there have been a few reports of cysts affecting the anterior wall of the rectum that have been eventually labeled to be of tailgut origin.[5][10] In those reports, cysts arising in the prostate utricle or the seminal vesicle, and also duplication enteric cysts, have been considered in the differential diagnosis. The former possibilities have been excluded in the present case with immunohistochemical studies, and the latter can also be excluded based on the lack of a neatly developed muscular and adventitial coat around the cyst wall. One peculiar aspect of our case is that there was only one kind of epithelium. Tailgut cysts usually show different types of epithelial linings, including squamous and cuboidal ones. Besides, it is fairly frequent in these lesions to have intense inflammatory changes with erosion of the epithelium and substitution by macrophages or lymphocytes.[8] This inflammatory reaction was absent in our case. However, the reactivity for cytokeratin 20 supports this embryologic origin in tailgut structures.

Conclusion

We report a case of mucinous cystadenoma, which we consider related to a congenital tailgut cyst of unusual location and discuss on the histogenesis of this lesion based on immunohistochemical data.

Reference 1.

Tapper EB, Shrewsberry AB, Oprea G, Majmudar B. A unique benign mucinous cystadenoma of the retroperitoneum: a

C-127 case report and review of the literature.Arch Gynecol Obstet. 2010;281:167-169. 2.

Subramony C, Habibpour S, Hashimoto LA. Retroperitoneal mucinous cystadenoma.Arch Pathol Lab Med. 2001;125:691-694.

Isse K, Harada K, Suzuki Y, et al.Retroperitoneal mucinous cystadenoma: report of two cases and review of the literature. Pathol Int. 2004;54:132-138.

Brown J, Frumovitz M. Mucinous tumors of the ovary: current thoughts on diagnosis and management.Curr Oncol Rep. 2014;16:389.

Jang SH, Jang KS, Song YS, et al. Unusual prerectal location of a tailgut cyst: a case report. World J Gastroenterol. 2006;12:5081-5083.

Demirel D, Gun I, Kucukodaci Z,et al. Primary retroperitoneal mucinous cystadenoma with a sarcomalike mural nodule: an immunohistochemical study with histogenetic considerations and literature review.Int J Gynecol Pathol. 2013;32:15-25.

Aumuller G, Riva A. Morphology and functions of the human seminal vesicle. Andrologia. 1992;24:183-196.

Hjermstad BM, Helwig EB. Tailgut cysts. Report of 53 cases.Am J Clin Pathol. 1988;89:139-147.

Mathis KL, Dozois EJ, Grewal MS,et al. Malignant risk and surgical outcomes of presacral tailgut cysts. Br JSurg. 2010;97:575-579.

10. Au E, Anderson O, Morgan B, et al. Tailgut cysts: report of two cases.Int J Colorectal Dis. 2009;24:345-350.

*Corresponding author: Javier Arias-Diaz, MD PhD, Dpto. de CirugĂa, Facultad de Medicina, Universidad Complutense de Madrid, Ciudad Universitaria, Madrid, Spain Phone: +34 607715705 Email: javardi@gmail.com Date of Submission : 01.04.2017 Date of Acceptance : 23.05.2017 Financial or other Competing Interests: None. Date of Publication : 25.08.2017

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Case Report DOI: 10.21276/APALM.1456

Corynebacterium amycolatum Causing Breast Abscess: An Infecting Diphtheroid with A Difference Hena Butta1*, Feroz Pasha2, Reetika Dawar1, Vikas Kashyap3, Leena Mendiratta1, Upasana Bora1 and Raman Sardana1 Department of Microbiology, Indraprastha Apollo Hospitals, New Delhi, India Department of Surgical Oncology, Indraprastha Apollo Hospitals, New Delhi, India Department of Pathology, Indraprastha Apollo Hospitals, New Delhi, India

ABSTRACT Corynebacterium amycolatum has been rarely reported from cases of breast abscess/mastitis. We describe a case of Corynebacterium amycolatum causing breast abscess with fistula formation. The identification of the organism was done by MALDI- TOF (Matrix assisted Laser Desorption Ionization Time of Flight) Vitek MS (Mass spectrometry) [Biomerieux, France] and Vitek-2 (Biomerieux, France). The clinical significance of the organism was ascertained in view of presence of many polymorphonuclear cells along with Gram positive bacilli on Gram stain examination. The patient was successfully managed with surgical treatment followed by antimicrobial therapy. Keywords: Corynebacterium amycolatum, Breast Abscess, Diphtheroid

Introduction

Non diphtheriae Corynebacterium species (Diphtheroids) constitute the normal flora of skin and upper respiratory tract and are commonly isolated as colonizer or contaminant but have also been isolated as opportunistic pathogens. The commonly isolated species in clinical microbiology laboratory are C. jeikeium, C. glucuronolyticum, C. aurimucosum, C. amycolatum, C. striatum, C. pseudodiphtheriticum, C. urealyticum, and C. tuberculostearicum.[1]The identification of these organisms to species level is difficult in a routine clinical Microbiology laboratory and thus they may be regarded as contaminants even if clinically significant. Although Corynebacterium species have been found to be isolated from patients with inflammatory breast disease particularly from cases of Granulomatous lobular mastitis but the reports of Corynebacterium amycolatum causing breast infections are scarce or under-reported.[2,3]Here, we report a case of breast abscess due to non diphtheriae Corynebacterium which was identified as C. amycolatum by MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight) Vitek MS (Mass Spectrometry) [Biomerieux, France] and Vitek-2 (Biomerieux, France).

Case Report

A 35 years old female, resident of Bangladesh, presented with purulent discharge from left nipple and hardness of breast. Patient had a history of lactational mastitis followed by left breast abscess for which she was operated there one year back. The patient was non-diabetic and otherwise

immunocompetent with no past history of any other illness or long term medication. On local examination, there was purulent discharge from periareolar area with hard lump in central and left upper outer quadrant. The systemic examination was within normal limits. Total leucocyte count was 15,500/mm3 with 73% neutrophils and 22% lymphocytes. Excision of fistulous tract and chronic inflamed tissue was done under general anaesthesia and pus and tissue specimens were sent for microbiological and histopathological examination respectively. The histopathologic examination was suggestive of duct ectasia with dense acute on chronic mastitis, and foci of suppurative inflammation. It showed several large neutrophilic micro abscesses and collections of foamy histiocytes and foreign body type multinucleated histiocytic giant cells within the background inflammatory infiltrate. Occasional small loosely formed epithelioid cell granulomas with Langhanâ&#x20AC;&#x2122;s type giant cells and devoid of central necrosis were also seen immediately around the ducts. Associated pathology like epithelial hyperplasia, ductal carcinoma in situ (DCIS), malignancy or tuberculosis was not seen. On Gram stain examination of pus, Gram positive bacilli and plenty of polymorphonuclear cells (PMNs) were seen (Figure-1). AFB (Acid fast bacilli) stain and fungal smear (KOH) examination were negative. Aerobic bacterial culture showed growth of dry, white opaque colonies on Columbia sheep blood agar and these were identified as Corynebacterium amycolatum by both MALDI TOF Vitek MS and Vitek-2 ANC card (Biomerieux, France). The antimicrobial susceptibility testing was done by disc diffusion

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method on 5% sheep blood agar and interpretation was done using CLSI guidelines applicable to Staphylococcus aureus ATCC25923.The organism was found to be susceptible to Amoxycillin + clavulanic acid, tetracycline, ciprofloxacin, vancomycin and chloramphenicol. Patient was initially put on oral Cefuroxime 500mg BD which was changed to Amoxycillin + clavulanic acid 1000mg (875/125mg) BD and Doxycycline 200 mg OD for seven days following the culture report. Patient showed good response to treatment and recovered completely.

Indian study by Reddy et al, C. amycolatum was found to be the most commonly occurring Corynebacterium amongst clinically significant Non diphtheria Corynebacterium.[4] Ojaydin I et al reported a case of recurrent breast abscess by C. amycolatum.[7]Paviour S et al in their retrospective study found maximum isolation of C. kroppenstedtii [14] followed by C. amycolatum(Three) from breast tissue, pus or deep wound swabs of 24 women. In literature, breast tissue infections due to C. kroppenstedtii has been reported more frequently in comparison to C. amycolatum.[2,8] This may be because of the lipophilic nature of C. kroppenstedtii.C. accolens which is also lipophilic has also been reported from case of breast abscess associated with granulomatous mastitis.[2,9]It has been stated that the significance of Corynebacteria can be determined by the presence of Gram positive bacilli along with PMNs in the clinical sample.[10] The occurrence of Gram positive bacilli along with many PMNs and pure growth of Corynebacterium amycolatumin our case strongly signifies this organism to be the only causative pathogen for the infection. It is also interesting to note that female gender can also be a risk factor in infections due to C. amycolatum. Female gender has also been reported as a statistically significant species specific risk factor for C. amycolatum endocardial infections.[11]

Conclusion Fig. 1: Gram stain examination (1000X)- showing many pus cells and Gram positive bacilli lying in palisade.

Discussion

We have isolated clinically significant C. amycolatum from a case of breast abscess with fistula which was successfully treated with surgical drainage and excision of fistula tract followed by antimicrobial therapy. C. amycolatum is an aerobic or facultative anaerobic non spore forming, Gram positive bacilli and is amongst the few Corynebacterium species which lack mycolic acid in their cell wall.[4] It was first isolated from clinical specimens in 1988 and has been found to be distantly related to other Corynebacteria.[5] C. amycolatum has been isolated from significant human infections like surgical wound infections, pilonidal sinus, mastitis, native valve and nosocomial endocarditis and septic arthritis.[6]The case reports of breast infections due to Corynebacterium amycolatum are scarce. It may be because of the fact that Diphtheroids commonly exists as natural flora on skin and mucous membranes and their identification is difficult. So, these are not speciated further and are recognized as colonizer or contaminant. In an www.pacificejournals.com/apalm

We appraise Corynebacterium amycolatum as a significant cause of breast abscess even in immunocompetent patient. Direct microscopic examination of the clinical specimen and accurate identification of the isolate along with clinical details is very crucial for the correct diagnosis, appropriate antimicrobial treatment and thus optimum management of infections caused by these bacteria. MALDI TOF Vitek MS is a very useful tool for the rapid identification of these isolates.

References: 1.

Williams DY, Selepak ST, Gill VJ. Identification of clinical isolates of nondiphtherial Corynebacterium species and their antibiotic susceptibility patterns. Diagn Microbiol Infect Dis 1993;17:23â&#x20AC;&#x201C;8.

Paviour S, Musaad S, Roberts S, et al. Corynebacterium species isolated from patients with mastitis. Clinical Infectious Diseases 2002;35:1434-40.

Zhou F, Yu LX, Ma ZB et al. Granulomatous lobular mastitis. Chronic diseases and translational Medicine 2016;2:17-21.

Reddy BS, Chaudhury A, Kalawat U, Jayaprada R et al. Isolation, speciation, and antibiogram of clinically relevant

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non-diphtherial Corynebacteria (Diphtheroids). Indian J Med Microbiol 2012;30:52-7. 5.

Mohammadi NS, Mafakheri S, Abdali N et al. Identification and characterization of the channel-forming protein in the cell wall of Corynebacterium amycolatum. Biochemica et Biophysica Acta 2013;1828:2574-82. Winn WC, Allen SD, Janda WM, Koneman EW, et al. Koneman’ s Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. USA: Lippincott Williams and Wilkins;2006. Aerobic and facultative Gram- positive bacilli.p.765-876. Ozaydin I, Yildirim M, Sahin I et al. Recurrent Breast Abscess Caused by Corynebacteriumamycolatum: A Case

Report. Turk J Med Sci 2009;39:147-9. 8.

Tauch A, Fernandez-Natal I, Soriano F. A microbiological and clinical review on Corynebacterium kroppenstedtii. Int J Infect Dis 2016;48:33–9.

Ang LMN, Brown H. Corynebacterium accolens isolated from Breast Abscess: Possible association with Granulomatous mastitis. J Clin Microbiol 2007;45:1666–8.

10. Funke G, Von Graevenitz A, Clarridge JE 3rd, Bernard KA. Clinical Microbiology of coryneform bacteria. Clin Microbiol Rev 1997;10:125-59. 11. Belmares J, Detterline S, Pak JB and Parada JP. Corynebacterium endocarditis species specific risk factors and outcomes. BMC Infect Dis 2007;7:4.

*Corresponding author: Dr Hena Butta, Department of Microbology, Indraprastha Apollo Hospitals, New Delhi-110076 Phone: +91 8447233605 Email: henavasdeva@yahoo.com

Financial or other Competing Interests: None.

Date of Submission : 02.04.2017 Date of Acceptance : 17.05.2017 Date of Publication : 25.08.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 4, July-August, 2017

Letter to Editor DOI: 10.21276/APALM.1160

Lipoma Arborescens Preethy Murthy1, Puvitha R.Duraisami2* and Murthy M.1 Department of Pathology,Karpagam Faculty of Medical Sciences and Research, Coimbatore, Tamil Nadu. (India).. 2 Department of Pathology, Coimbatore Medical College Coimbatore, Tamil Nadu (India).

Dear Sir

Lipoma arborescens is a rare intra articular lesion consisting of sub synovial villous proliferation of mature fat cells, presents with monoarticular or polyarticular . [1] It is an uncommon cause of intra-articular masses, as slowly progressive, painless swelling of the joint, with intermittent joint pain, in most of the cases it can be very well predicted by MRI. [2] This report describes a case a 46-year-old woman who presented with an intermittent left knee joint pain and effusion for more than year duration. The complete rheumatological work up and the routine blood investigations were done; all were within normal limits. However, radiological examination of bilateral knees, suggests features of osteoarthritis .The ultrasonographic (USG) examination of bilateral knees was done which revealed hyperechoic, frond like projections of the synovium with effusion suggestive of lipoma arborescence. The patient was advised arthroscopic synovectomy of knee joint. Grossly, the received specimen show frond like villous /papillary projections of fibro fatty tissue. Microscopically, the hyperplastic synovium show many papillary projections which are lined by hyperplastic synovium, with sub synovial tissue replaced with mature adipocytes concludes the diagnosis of lipoma arborescens.(Fig.1).

Albert Hoffa, a German surgeon, made the first description of lipoma arborescens in the year 1904; the Latin term arbor means â&#x20AC;&#x2DC;tree-like appearanceâ&#x20AC;&#x2122;, describing the characteristic villous and frond-like morphology of this condition. Lipoma arborescence has been observed in patients aged between 9 and 68 years, equally in men and women. There are two etiological hypotheses, primary and secondary; primary idiopathic, rare commonly seen in younger group, whereas the more common secondary type associates with an underlying chronic irritation, usually seen in elderly patients. [2] The differential diagnosis includes, synovial lipoma, pigmented villonodular synovitis, synovial chondromatosis, rheumatoid arthritis, synovial haemangioma,and xanthomata. The main differential is synovial lipoma, macroscopically it appear as fatty polyp with adipocytes, where in Lipoma arborescences show hyperplastic synovium with subsynovial adipocytes. PVNS show many giant cells with haemosiderin pigments, Synovial chondromatosis show atypical chondrocytes in the synovium Rheumatoid arthritis show lymphoplasmacytic infiltrate with variable germinal centers, in contrast to Lipoma arborescences. [3] Lipoma arborescences is a rare intra articular tumor with benign indolent course, awareness of its clinical and imaging findings and possible differential diagnosis is essential for early diagnosis and treatment, as well as to avoid misinterpretation with other aggressive articular masses.

Reference

Fig. 1: show papillary projections, with sub synovial tissue replaced with mature adipocytes.

Al-Sharim MM. Intra-articular lipoma arborescens of the knee joint. Ann Saudi Med 2011; 31(2):194-196.

Sanamandra SK, Ong KO. Lipoma Arborescens. Singapure Med J. 2014 Jan; 55(1):5-11.

Kamaran MSF, Kavin K, Sharma V, Shivanand G. Bilateral lipoma arborescens with osteoarthritis knee: Case report and literature review of Literature. J Clin Orthop Trauma.2015 Jan; 6(2):131-136.

*Corresponding author: Puvitha R. Duraisami, Kappiniya Gounder Layout M.K.P.Colony,Ganapathy,Coimbatore. - 641 032. Tamil Nadu. India. Phone: +91 9843112163. Date of Submission : 13.11.2016 Email: drrdpuvitha@yahoo.com Date of Acceptance : 03.04.2017 Financial or other Competing Interests: None. Date of Publication : 31.08.2017

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Letter to Editor DOI: 10.21276/APALM.1222

Cytology of Adenomatoid Tumour of Epididymis Meghna Singh1* and Jitendra Chaudhary2 1 Pathology, Shankar Diagnostic Centre, Agra (India) Radiodiagnosis, Shankar Diagnostic Centre, Agra (India)

Dear Sir

Adenomatoid tumour is a benign tumour of male and female genital tract with epididymis being the most common site in males. Fine needle aspiration cytology (FNAC) has an important role to play in the diagnosis of this tumour and pathologists should be aware of its cytological features so as to differentiate it from other paratesticular lesions. An 18 year old male presented with a painless left epididymal swelling for 3 months. There was no history of trauma. Local examination revealed a firm, mobile, mildly tender swelling measuring 2x1.5 cm in the left epididymis. Bilateral testes were normal. Clinically a diagnosis of tubercular epididymitis was made. Routine hematological and biochemical parameters were within normal limits. Ultrasonography revealed a well defined hypoechoic lesion measuring 1.9x1.4 cm with increased flow on colour doppler. FNAC was performed using 23 gauge needle and a blood mixed whitish aspirate was yielded. Smears were stained with May Grunwald Giemsa (MGG). Cytologic examination showed cellular smears with cells arranged in flat sheets, cords and vague glandular structures. Cells were round to oval with eccentric nuclei, pale chromatin, distinct nucleoli and moderate to abundant amount of cytoplasm. Cytologic diagnosis suggestive of adenomatoid tumour was given and histopathology was advised. The patient underwent a conservative testis sparing surgery with the excision of epididymal swelling. Grossly, the swelling measured 1.8x1.3 cm. Consistency was firm and cut surface was grey white. Histopathologic sections revealed cords and tubules of cells with a prominent intervening fibrous stroma. Individual cells were flattened to cuboidal with vesicular nuclei, small nucleoli and abundant amount of eosinophilic cytoplasm. A final diagnosis of adenomatoid tumour was made. Adenomatoid tumours are the most common tumours of male paratesticular tissues (epididymis, tunica or spermatic cord). In females it is seen in uterus, fallopian tube, ovary and paraovarian tissues.[1] They present mostly as slow growing, small, firm, asymptomatic intrascrotal lump peaking in third to fifth decade of life.[1] The histogenesis has been argued for years, the proposed cells of origin being mesothelial, mesonephric, mullerian

and endothelial. However, immunohistochemical and ultrastuctural evidence supports a mesothelial origin. [2,3] Cytological features of adenomatoid tumour have been described very briefly in literature. There are only few case reports of adenomatoid tumour of epididymis being diagnosed on cytology.[1,4,5] Initial cytological description of adenomatoid tumour was described by Perez-GuillemroÂ et al.[1] Cytological differential diagnosis of this tumour include the reactive mesothelial hyperplasia, papillary cystadenoma, malignant mesothelioma, and adenocarcinoma.[4] Reactive mesothelial cells with hyperplasia can be seen in hydrocoele fluid. They may not have definite arrangement of cells as seen in adenomatoid tumors. Cytologically papillary cystadenoma comprises of papillary structures composed of benign isolated cells with cytoplasmic vacuoles in a mucoid background. Malignant mesothelioma shows cells lying diffusely with cells having dense cytoplasm with nuclear enlargement and multinucleation. Metastatic adenocarcinoma show cytological features of malignancy and cells are positive for mucicarmine. Spermatic granuloma, tuberculous and chronic epididymitis are clinical differential diagnosis and can be ruled out by microscopy.[4] Spermatic granulomas have spermiophages in a dirty background, whereas tuberculous epididymitis consists of epithelioid granulomas and Langhanâ&#x20AC;&#x2122;s type giant cells in a background of caseous necrosis which can be confirmed by Zeihl Neelsenâ&#x20AC;&#x2122;s stain and culture. Chronic epididymitis shows a chronic inflammatory cell infiltrate. On histopathology, this tumour shows solid to cystic tubules and cords of vacuolated cells. The cells lining the tubules vary from flattened to cuboidal, usually with a prominent intervening fibrous stroma. Cells have vesicular nuclei, small nucleoli and abundant amount of eosinophilic cytoplasm. Immunohistochemical studies show positivity for CK, EMA and calretinin but negativity for endothelial markers, supporting mesothelial derivation,[2] as do ultrastructural studies.[3] The presentation of adenomatoid tumour is unique and easily recognized by FNAC. The preoperative cytology may be used as an important diagnostic tool to evaluate the paratesticular masses. Pathologist should be aware of cytological features of this tumour so as to avoid aggressive surgical procedures.

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Fig. 1: (a) Cytologic smear from the swelling showing clusters of cells with round to oval eccentric nuclei and moderate to abundant amount of cytoplasm( MGG, X400), (b) Histologic section showing dilated tubular structures lined by flattened to cuboidal cells with intervening dense fibrous stroma( H&E, X100).

References 1.

Perez-Guillermo M, Thor A, Lowhagen T. Paratesticular Adenomatoid tumors: The cytologic presentation in fine needle aspiration biopsies. Acta Cytol 1989;33:6–10 Delahunt B, Eble JN, Nacey JN, Thornton A. Immunohistochemical evidence for mesothelial origin of paratesticular adenomatoid tumour. Histopathology 2001;38:479

Mackay B, Bennington JL, Skoglund RW. The adenomatoid tumour: fine structural evidence for a mesothelial origin. Cancer 1971;27:109-115

Rege JD, Amarapurkar AD, Phatak AM. Fine needle aspiration cytology of adenomatoid tumor. A case report. Acta Cytol 1999;43:495–7

Gupta S, Garg S, Agarwal R, Sen R. Aspiration cytology of adenomatoid tumor of epididymis: An important diagnostic tool. J Surg Case Rep 2012(4):11

*Corresponding author: Dr Meghna Singh, 10 Laxman Nagar, Near Arjun Nagar, Agra, Uttar Pradesh,( INDIA) PIN- 282001, Phone: +91 8126738984 Email: dr.singhmeghna@gmail.com

Financial or other Competing Interests: None.

www.pacificejournals.com/apalm

Date of Submission : 22.12.2016 Date of Acceptance : 10.04.2017 Date of Publication : 31.08.2017

eISSN: 2349-6983; pISSN: 2394-6466

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