APALM 4.3 (2017) by PaGe

eISSN: 2349-6983 pISSN: 2394-6466

Annals of Pathology and Laboratory Medicine May-June 2017; Vol. 4, Issue 3

Cover design: Dr Prashant

DOI : 10.21276/apalm

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Co-Editor-in-Chief Dr Prashant Goyal Dr Shelly Sehgal

Annals of Pathology and Laboratory Medicine Co-Editor in Chief

Dr Niti Singhal Abu Dhabi, United Arab Emirates Dr (Prof) Severino Rey Quiron Hospitals and Pontifical Catholic University, Ecuador Dr Rajeshwar Reddy Prof. & Head, Dept. of Microbiology, Gandaki Medical College, Pokhara, Nepal Dr Nasser Said-Al-Naief ODRP/ Anatomic Pathology, Loma Linda Medical Center, Loma Linda, CA, United States Dr Hoda A Hagrass Clinical Pathology dept, Faculty od Medicine Zagazig University, Sharkyia, Egypt Dr Kemal Turker UlutaĹ&#x; Kadirli State Hospital, Central Laboratory, Osmaniye, Turkey Dr Dennis P Oâ&#x20AC;&#x2122;Malley Pathologist, Clarient Pathology Services, Columbia, Aliso Viejo, CA, United States Dr Parthasarathi Pramanik Consultant Forensic Pathologist, Forensic Science Laboratory, Kingston, Jamaica Dr Arvind Rishi Asst. Prof., Dept of Pathology, Hofstra North Shore-LIJ School of Medicine, New York, United States Dr Ahmad Mohammad Ragab, Senior Consultant Pathologist, Kameda Hospital & Oncology Center - JAPAN - National Medical Institute, Egypt Dr Shamim Sheikh Dept. of Pathology, M.P. Shah Medical College, Jamnagar, Gujarat, India Dr Viral M Bhanvadia Asst. Prof. Dept. of Pathology, Shri M.P. Shah Medical College, Jamnagar, Gujarat, India Dr Navin K Sinha Director-Lab, Artemis Health Institute, Gurgaon, India Dr Soumyesh Ghosh Dept. of Pathology, SDN Hospital, Delhi, India Dr Deepti Mittal Pathologist, Haryana, India Dr Amit Agravat Asso. Prof. Dept. of Pathology, PDU Medical College, Rajkot, Gujarat, India

Dr Prashant Goyal Director-Laboratory, Accuprobe Healthcare and Diagnostics, Delhi, India Dr Shelly Sehgal Specialist Pathologist, Department of Pathology, SDN Hospital, Delhi, India

Associate Editor

Dr Asitava Mondal Clinical Cytologist and Oncopathologist, Kolkata, West Bengal, India Dr Roque G. Wiseman Pinto Professor and Head of Dept. of Pathology, Goa Medical College, Bambolim, Goa, India Dr Sompal Singh Specialist Pathologist, Dept. of Pathology, N D M C Medical College & Hindu Rao Hospital, Delhi, India Dr Ruchika Gupta Pathologist (Scientist-C), Institute of Cytology & Preventive Oncology (ICPO), Delhi, India Prof. Vatsala Mishra HOD, Dept. of Pathology Moti Lal Nehru Medical College, Allahabad, India Dr Anil Parwani Vice Chair, Anatomical Pathology; Director of Pathology Informatics and Digital Pathology The Ohio State University Wexner Medical Center, Columbus, Ohio, United States Dr. Mohammad Zillur Rahman HOD & Associate Professor, Department of Pathology, Chittagong Medical College, Chittagong, Bangladesh Dr Manu Noatay Head Operations, Niche Theranostics, New Delhi, India Dr Manjusha Biswas Consultant Histopathologist & Oncopathologist, Bangalore, India, India Dr Mudit Agarwal Director Lab Services, Nishtha Pathology Lab, New Delhi, India Dr A S Ramaswamy Specialist Pathologist, Lifeline Hospital, Salalah, Sultanate of Oman Dr Harsh Vardhan Singh Senior Biochemist, N D M C Medical College & Hindu Rao Hospital, Delhi, India

Editorial Board Members

Advisory Editors

Dr Sarah Iqbal Ch Faculty of Pathology King Edward Medical University, Lahore, Pakistan Dr Naila Atif Associate Prof., Histopathology, Central Park Medical College, Lahore, Pakistan Dr Rajan Chopra King Fahad Hospital, Hufof, Saudi Arabia

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Dr Awanindra Kumar Head, Blood Bank & Pathology, SDN Hospital, Delhi, India Prof. Kuldeep Singh Prof. of Pathology, Govt. Medical College, Jammu, India Dr Shriniwas Rushi Histopathologist, KFCH, Riyadh, Saudi Arabia

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Contents

Original Article Soluble Transferrin Receptor levels of Apparently Healthy Adults in Port Harcourt, Nigeria A224-A229 Chilota Chibuife Efobi, Benedict Nwogo, Igwebuike Victor Onyiaorah, Oseikhuemen Adebayo Ejele Histomorphological Analysis of Lesions In Nephrectomy Specimens: A 4 Years Study In A Rural Hospital In India- Our Experience. Kishor H Suryawanshi, Rajshri P Damle, N V Dravid, Ashish Patil Rawandale, Akshay Surana Comparison Of Diagnostic Accuracy Of BIRADS Score With Pathologic Findings In Breast Lumps Navya BN, Shalu Thomas, Rudresh Hiremath, Sathyavathi R Alva A Cytohistopathological Correlation of Thyroid Lesions with Critical Evaluation Of Discordant Cases: An Experience At A Tertiary Care Hospital Shakuntala Sunil Aramani, Gururajaprasad C Coagulation abnormalities in patients with cirrhosis in a tertiary hospital Sangli : A prospective study Sheetal M Sale, Vaibhav P Mane, Vishrabdha R Pawar, Dajiram G Mote, Sushant N Mohite, Vanisha Dhaka Evaluation of pattern of angiogenesis in various menstrual disorders in different district in Gujarat, India. Killol Nathubhai Desai, Vidya Kantilal Satapara Clinicopathological study of Sinonasal Masses Prashant Shivaji Mane, Shubhangi Vinayak Agale Thrombocytopenia and Altered Platelet indices as potential Marker in complicated malaria caused by Plasmodium vivax: cross sectional descriptive study Bhavani K, Venkatraman J, Roopa urs A.N, Dhananjay Kotasthane Diagnostic Accuracy and Pitfalls in Fine Needle Aspiration Cytology Of Salivary Gland Lesions. Crysle S Saldanha, Hilda Fernandes, Zeeshanali Fazalbhoy, Jayaprakash C S, Sumanth D, Reshma Kini Spectrum of Thyroid lesions in a tertiary care hospital using Bethesda System for Reporting Thyroid Cytopathology. Mallikarjun A Pattanashetti, Ranjit P Kangle, Hema B Bannur Spectrum of Lymph Node Lesions by Fine Needle Aspiration Cytology: A Retrospective Analysis Shilpa Somashekar Biradar, Deepa Siddappa Masur Role of platelet indices in differentiating hypoproductive and hyperdestructive thrombocytopenia Shaheena Parveen, Mourouguessine Vimal Cytological and Histopathological Correlation of Breast Lump: A 3 Year Study at Tertiary Care Center Sanjay C Chauhan, Ankur N Sarvaiya Variant Hemoglobin Spectrum By Cation Exchange High Performance Liquid Chromatography: A Study 0f 2035 Subjects Ankur N Sarvaiya, Sanjaykumar C Chauhan Histopathological Spectrum of Non-Neoplastic Uterine Cervical Lesions in a Tertiary Care Centre Priyadarshini D, Arathi C. A Use of Mean platelet Volume as an initial diagnostic marker in evaluation of dengue fever Nabila Afsar, Idrees Akhtar Afroze, Shahmeen Humaira, Zakia Abid Chitotriosidase Enzyme Activity and miRNA-146a expression and their value as potential Biomarkers of subclinical Atherosclerosis in type 2 Diabetes Mellitus Hala Mourad Demerdash, Radwa Mohamed ElSharaby, Yasser Mohamed AblelRaouf

III

A230-A235

A236-A242

A243-A248

A249-A253

A254-A260

A261-A267 A268-A273

A274-A278

A279-A283

A284-A287

A288-A291

A292-A296

A297-A302

A303-A309

A310-A313 A314-A320

Case Report

Letter to editor

A study of histological types of leprosy along with clinico-histopathological A321-A324 correlation in a Tertiary Centre from North Maharashtra region. Maya Suresh Vasaikar, Bharati M Patil, Rajesh Y Thakur Study of histomorphological characteristics and itâ&#x20AC;&#x2122;s correlation with clinical, A325-A331 biochemical, serological and immunohistochemical parameters in incidentally detected hepatitis B patients Rakesh Kumar Gupta, Puja Sakhuja, Shahajad Ali, Sidharth Srivastava, Barjesh Chand Sharma, Amarender Singh Puri Bacteriological analysis including antimicrobial susceptibility pattern of blood A332-A337 stream infections in tertiary care hospital Iqbal M Desai, Hardik Kamlesh Bhavsar, Sachin M Darji, Jitendrakumar S Parmar Water Clear Parathyroid Adenoma: A report of 2 cases C66-C68 Subhash Yadav, Kritika Singh, Pragati Sathe Diagnosis of Bony Metastasis of Renal Cell Carcioma at a Rare Site on Fine Needle C69-C72 Aspiration Cytology: A Rare Case Report Vijay Kumar Bodal, Mohanvir Kaur, Deepika Gupta, Vikram Jassal Hamartomatous nodule, sertoli cell adenoma in complete androgen insensitivity C73-C77 syndrome with Wolffian/MĂźllerian duct remnants: An unusual case report Prashant Vijay Kumavat, Chetan S Chaudhari, Anita Padmanabhan, Nitin M Gadgil, Sangita Margam, Ganesh R Kshirsagar Vimentin Positive Primary Thyroid Diffuse Large B-Cell Lymphoma: cytological C79-C82 diagnosis of two cases Swati Bhardwaj, Charanjeet Ahluwalia, Ashish Kumar Mandal Cytology of Extrapulmonary Tuberculosis of Masseter Muscle: A Rare Case Report C83-C85 Ramya Rathinam Sunderaj, G Barathi, D Prathiba Echinococosis lung: A Cyto-histopathogical study in two cases C86-C89 Rimi Pandey, Nivedita Yadav, Padam Kumari Agarwal, Suruchi Shukla, Mustafa Ali, Pooja Singh Postpartum TTP-HUS Syndrome: A Rare Autopsy Case Report in a Tertiary Care Hospital. C90-C93 Ganesh R Kshirsagar, Chetan Sudhakar Chaudhari, Prashant V Kumavat, Nikita Patel, Nitin M Gadgil Extra abdominal Umbilical Vein Varix Causing Stillbirth: a Case Report C94-C97 Haneen Adnan Al-Maghrabi, Luis Humberto Cruz Contreras, Sareni Chavez Martinez Epithelioid glioblastoma, a newly described rare variant of common tumor describing L10-L12 immunohistochemical expression pattern Rakesh Kumar Gupta, Neha Garg, Vineeta V Batra, Lalit Garg

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Original Article DOI: 10.21276/APALM.1238

Soluble Transferrin Receptor Levels of Apparently Healthy Adults in Port Harcourt, Nigeria Chilota Chibuife Efobi1*, Benedict Nwogo2, Igwebuike Victor Onyiaorah3 and Oseikhuemen Adebayo Ejele4 1

Department of Haematology, Chukwuemeka Odumegwu Ojukwu University, Akwa Campus, Awka, Anambra State, Nigeria. 2 Department of Haematology, University of Benin, Benin city, Edo State 3 Department of Histopathology, Nnamdi Azikiwe University, Nnewi Campus, Nnewi, Anambra State 4 Department of Haematology, University Of Port Harcourt, Port Harcourt, Rivers State.

ABSTRACT Background: soluble transferrin receptor is an early marker of tissue iron deficiency before onset of anaemia. sTfR is one of the diagnostic markers for iron deficiency anaemia. Iron deficiency anaemia is one of the most prevalent causes of anaemia in our environment. However, there is no established reference range for this diagnostic marker sTfR in our environment. Objectives: To determine the soluble transferrin receptor levels in apparently normal adults in Port Harcourt Nigeria, determine the reference value of sTfR in the study population. Methods: This is a descriptive cross sectional study conducted at the university of Port Harcourt Teaching Hospital. One hundred and fiftyparticipants who satisfied the inclusion criteria were enrolled for this study. Full blood count and sTfR concentration were assayed on anticoagulated blood samples using a 3-part auto analyzer (Sysmex –KX2IN®) and Human sTfR ELISA kit by BioVendor respectively. The results were analysed using SPSS version 21. P value <0.05 was considered significant. Results: The mean sTfR concentration of the study population was 0.89±0.46ug/mL with a range of 0.3- 3.05ug/ml. The mean value of sTfR for males and females were 0.92±0.49ug/ml and 0.82±0.37ug/ml and the mean sTfR levels did not differ significantly for age and sex. Conclusion: The reference value of sTfR concentration in healthy adults in Port Harcourt was established as 0.3-3.05ug/ml. This study found no statistically significant relationship of sTfR between the different sexes and ages. Keywords: sTfR, Healthy Adults, Screening Tool, Iron Deficiency

Introduction

Iron deficiency anaemia (IDA) is the most common anaemia in the world despite abundance of iron in the environment. The examination of bone marrow aspirate, stained with Prussian blue stain for iron, is still considered as the best method for evaluating iron status in patients with indeterminate laboratory findings. Its draw backs are that it is invasive, expensive, the results may be operator dependent and the aspirate obtained may not be adequate. It has also been shown to be uncomfortable and therefore not practical for routine use. [1,2] Due to the high global burden of iron deficiency anemia which has made it a public health issue, there is a need to devise a screening tool that can be used to detect iron deficiency before onset of anemia to aid early intervention and reduce the mortality and morbidity associated with IDA. This necessitates the need for a less invasive, specific and sensitive means of detecting tissue iron deficiency before onset of anemia. Soluble transferrin receptor (sTfR) assay is a feasible option.[3] Transferrin receptors are expressed on all dividing cells and its rate of synthesis is related to the cell’s iron

requirement.[4]There are are up to 10,000 – 100,000 sTFr molecules per cell in actively dividing cells especially in erythroid precursors in the bone marrow and placental syncythiotrophoblast. sTfR concentration increases up to two folds in iron deficiency, making it an important tool for detecting early tissue iron deficiency before onset of anaemia. The other assays for determining iron deficiency, like serum iron, ferritin, total iron binding capacity, etc, are affected by inflammatory changes.[5] sTfR is not affected by inflammationmaking it superior to these other assays. [6,7] Sweet et al reported that sTfR measurement could replace other assays for iron deficiency state especially in subclinical iron deficiency present in their study population which was undetected by these other assays.[8] There is no difference between sTfR concentrations in healthy males and females, [9,10]but the values are higher in children compared to adults.[11]However, Blacks have a higher sTfR concentration than non- blacks.[12] This difference may be related to the well-known but unexplained difference in haemoglobin concentrations in Blacks and Caucasians.Therefore, misdiagnosis of

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Efobi R et al. iron deficiency can result if Caucasian values are used for blacks. Some studies have also shown that sTfR is increased in blood donors,[13]during physical exercise,[14, 15]cigarette smoking, [8]and high altitude,[16] where reduced oxygen tension necessitates increased erythropoiesisbut is reduced by ascorbic acid administration and iron supplementation. [17, 18] A blood donor loses about 200- 250 mg of iron with every unit of 450mls of blood donated. Some blood donors, especially commercial donors might donate more than three times a year for financial reasons, putting more pressure on their iron stores. Several studies have provided reference ranges for soluble transferrin receptor concentration in the white population. There is paucity of studies on sTfR in our environment hence this study was aimed to determine reference value of sTfR in our environment. A study done in the United States of America showed that the reference range of sTfR receptor is 10.3 – 29.1nmol/l. [12] Another study done in Kansas showed a reference value of 2.8- 8.5mg/l,[19]while one done on Arabs in the Sultanate of Oman showed a reference value of 10.7- 38.7nmol/l,[20] while another study showed 2.9-8.3mg/l.[21] The reference interval of serum transferrin receptor varies between different assay systems, depending on the choice of calibrators.It was proposed that recombinant serum transferrin receptor (rsTfR) preparation 07/202, may be designated a WHO Reference Reagent with assigned values of 21.7 mg/L and 30.3 nmol/L (when reconstituted with 0.50 mL distilled or deionized water).[22]

Patients and Methods

Study Design and Source: This is a descriptive study done at the University of Port Harcourt Teaching Hospital (UPTH), Rivers State, Nigeria, from September 2013 to November 2013. Study Population: A total of 150 participants, healthy non- fasting, males and females, within the age range of 17- 60 years were selected by simple random sampling method. A research questionnaire was used to obtain basic information from participants including age, sex, tribe, occupation, level of education, history of chronic illness, nutritional history, history of smoking, menopausal status, blood donation history. Blood samples were collected afterwards for determination of full blood count and sTfR levels. www.pacificejournals.com/apalm

A-225 Three milliliters of venous blood was collected by venipuncture into ethylene diamine tetra-acetic acid (EDTA) bottles for each participant. Samples to be analyzed for full blood count were run immediately. Plasma samples were obtained after centrifugation of anticoagulated blood at 2500 revolutions per minute for 2 minutes. Plasma samples of subjects that met the FBC inclusion criteria and were then stored at a temperature of -70o C. The full blood count was performed using 3 part autoanalyser (Sysmex –KX2IN®). Soluble transferrin receptor concentration was determined by an enzyme immunoassay (EIA) monoclonal antibody technique using Human sTfR ELISA kit (BioVendor® Research and Diagnostic Products D-69120 Heidleberg Germany). LOT- E13-012. Inclusion criteria: PCV for healthy males =39-50%, (haemoglobin concentration= 13.0-16.5g/dL) and normal reference valve for PCV for healthy females = 33 - 40% (haemoglobin concentration=12.0-15.0g/dL).[23]The total white cell count was 2.5x 109/L-11x109/L. MCV, MCH and MCHC were 80.0-92.0fL, 24.6 – 30.2pg, 31.2 – 34.6g/ dL respectively. Platelet count was 90- 350x109/l. Subjects with values outside these ranges were excluded. Excluded were subjects with febrile illness within 4 weeks prior to the study, pregnant and presently menstruating females, persons on iron supplements 3 months prior to study, subjects with haematological parameters outside the ranges stated above. Ethical Considerations: Ethical approval was obtained from the Research Ethical Committee of University of Port Harcourt Teaching Hospital (UPTH). A signed consent form was obtained from each participant before recruitment. Statistical Analysis: Data was analyzed using SPSS 21.0. Descriptive statistics such as means and standard deviations were calculated for quantitative variables like haematological indices. Tables were generated according to objectives. Statistical tools such as students t test, analysis of variances (ANOVA), were used as appropriate to make statistical inferences about population parameters. Pearson’s correlation test was employed to determine the strength of association between haematological indices and sTfR levels. A p-value of < 0.05 was considered statistically significant.

Results

Demographics A total of 150 respondents participated in the study consisting of 107 (71.3%)males and 43(28.7%) females. The ages of the respondents were grouped as: eISSN: 2349-6983; pISSN: 2394-6466

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sTfR Levels of Healthy Adults in PH Nigeria

21-30 which had 42 respondents(28.0%), 31-40 had 75 respondents(50%), 41- 50 had 28 respondents(18.7), 51-60 had 5 respondents(3.3%).Thirty six out of the 43 females were premenopausal, while 7 were postmenopausal. Majority of the respondents (64%) came from the southsouth geopolitical region. Details of general characteristics of the study is shown in table 1. Haematological Parameters: The mean PCV of the study population was 44.15%±3.96. The mean PCV for males was significantly higher than that of females 45.79±3.06 vs 40.06%±2.84; p<0.001. The mean haemoglobin concentration of the study population was 14.18±1.38g/ dL. The mean haemoglobin for males and females were 14.75±1.03g/dL and 14.05±1.60g/dL respectively p value= <0.001. The mean MCV, MCH, MCHC of the study population were 93.11±3.44fl, 29.84±0.06pg and 32.09±0.87g/ dL respectively. No statistically significant difference was found in red cell indices of males and females. The difference in the total white cell and platelet counts of males and females were statistically significant. Details are shown in table 2. Soluble Transferrin Receptor Levels: The total mean value of soluble transferrin receptor in the study population was 0.89±0.46ug/ml, with a range of 0.3-3.05ug/ml. Males had a higher sTfR level than females 0.92±0.49ug/ml vs 0.82±0.37ug/ml. however, the difference in the mean is not statistically significant(p value = 0.204) as shown in table Table 1: Sociodemographic Variables Variables Age group(years)

Sex

Regions

Level of Education

Occupation

3. There was no significant difference of sTfR amongst different age groups. The mean sTfR levels of male and female participants for various age groups is shown in table 3. For those in the age range of 21-30 yrs, sTfR was 0.88±0.48ug/dl and 0.80±0.47ug/dl for males and females respectively and for those aged 51-60, 0.98±0.53ug/dl and 0.97±0.13ug/dl for males and females respectively. The difference in mean sTfR across the various age groups were not statistically significant. sTfR Levels and Menopausal Status: Most of the participants of this study were males 107 (71.3) with a mean sTfR of 0.92 ± 0.49ug/dl. The females comprised 28.7% of the participants. Majority of the females were premenopausal 36 (24.0), while 4.7% of them were postmenopausal. The mean sTfR levels of females was 0.82 ± 0.37ug/dl. The sTfR level did not differ significantly between pre and post -menopausal women in the study population as seen in table 4. Correlation Between sTfR and Haematological Parameters: There was a weak positive correlation between sTfR concentration and some haematological parameters in the study group. The r values for PCV, Hb, WBC, Platelet, and MCHC were: 0.033, 0.032, 0.093, 0.092, and 0.002 respectively but these were not statistically significant. Aweak negative correlation was observed between MCV and MCH with sTfR r= -0.042 and -0.035 respectively which were not statistically significant as shown in table 5.

21-30 31-40 41-50 51-60 Males Females Premenopausal Postmenopausal South South South East Others Primary Secondary Tertiary Civil servants Healthcare Professionals Students Others

Frequency (%) N = 150 42 (28.0) 75 (50.0) 28 (18.7) 5 (3.3) 107 (71.3) 43 (28.7) 36 (24.0) 7 (4.7) 96 (64.0) 47 (31.3) 7 (4.7) 4 (2.7) 13 (8.7) 133 (88.7) 31(20.7) 85(56.7) 19(12.7) 15(10.0)

Key: N indicates number of variables

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Efobi R et al.

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Table 2: Stfr and Haematological Parameters in the Study Population. Total Mean±SD N=150

Variables

Male Mean±SD N=107

Female Mean±SD N=43

P Value

sTfR (ug/ml)

0.89±0.46

0.92±0.49

0.82±0.37

1.275

0.204

PCV (%)

44.15±3.96

45.79±3.06

40.06±2.84

10.576

<0.001

Hb (g/dl)

14.18±1.38

14.75±1.03

12.75±1.06

10.725

<0.001

WBC(x109/l)

7.40±1.56

7.21±1.52

7.86±1.57

-2.334

0.021

241.77±45.76

233.31±43.16

262.81±45.74

-3.722

<0.001

MCV(femtolitres)

93.11±3.44

92.83±3.26

93.82±3.82

-1.608

0.110

MCH(picograms)

29.84±0.06

29.87±0.73

29.80±0.91

0.516

0.607

MCHC (g/dl)

32.09±0.87

32.22±0.87

31.79±0.80

2.794

0.006

PLATELET(x109/l)

Key: SD- standard deviation. N-number of variables. sTfR- soluble transferrin receptor. PCV- packed cell volume. Hb- haemoglobin. WBC- white blood cells. MCV- mean cell volume. MCH- mean cell haemoglobin. MCHC- mean cell haemoglobin concentration.

Table 3: Stfr Levels by Age Group and Sex of the Study Population. Age groups 21-30yrs 31-40yrs 41-50yrs 51-60yrs

Sex

FREQUENCY(%)

Mean±SD

Male

33(22.0)

0.88±0.48

Female

9(6.0)

0.80±0.47

Male

52(34.7)

0.94±0.52

Female

23(15.3)

0.79±0.33

Male

20(13.4)

0.95±0.43

Female

8(5.3)

0.86±0.45

Male

2(1.3)

0.98±0.53

Female

3(2.0)

0.97±0.13

P Value

0.428

0.671

1.272

0.207

0.478

0.636

0.028

0.979

p value

1.275

0.204

0.758

0.470

Key: N= number of participants, SD- standard deviation, sTfR unit =ug/ml

Table 4: Stfr Levels by Sex and Menopausal Status Sex

Frequency (%)

Mean ± SD (ug/ml)

Males

107 (71.3)

0.92 ± 0.49

Females

43 (28.7)

0.82 ± 0.37

Premenopausal

36 (24.0)

0.82 ± 0.39

Postmenopausal

7 (4.7)

0.86 ± 0.20

Table 5: Correlation Between Stfr and Haematological Parameters in the Study Participants

PCV (%)

HB (g/dl)

WBCx109/l

PLTx109/l

MCV(fl)

MCH(pg)

MCHC(g/dl)

STFR(ug/ml)

0.033

0.032

0.093

0.092

-0.042

-0.035

0.002

P value

0.691

0.701

0.259

0.263

0.608

0.671

0.984

Key: SD- standard deviation. sTfR- soluble transferrin receptor. PCV- packed cell volume. Hb- haemoglobin.WBC- white blood cell count. MCV- mean cell volume. MCH- mean cell haemoglobin. MCHC- mean cell haemoglobin concentration.

Discussion

Various authors have reported varying reference values for sTfR depending on the study population. This study on apparently healthy Nigerians found the reference range of sTfR to be 0.3-3.05ug/ml. The upper limit is higher than those reported in some non-African based studies. [12, 20,24] Van den Bosch G et al,[24]Allen J et al and KnoxMaculay et al reported[12,20]reference values of 0.582.50mg/l,0.76- 2.77mg/l, 0.5-2.7mg/l respectively.

Similarly the reference range of the study population is higher than that of the Biovendor ELISA kit used for the study, which was 0.378-1.513ug/ml. [25]The mean sTfR in our study was0.89 ± 0.46ug/mL. Kohgo et al,[9]Van den Bosch G et al[24]reported lower values of 0.253±0.08ug/ ml and0.58±0.26mg/l respectively. Allen and KnoxMacaulay obtained a marginally higher mean in their studies of 1.4±0.3mg/l and 1.5±0.5mg/l respectively. Similarly, Simek M and his colleagues in another related study found mean values higher than that reported in the

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sTfR Levels of Healthy Adults in PH Nigeria

index study which was 1.84Âą 0.8mg/l.[26]Flowers and his colleagues[10] reported the sTfR value from their study to be 4.21-7.05mg/l, while Erhardt and his friends reported their own values to be 2.9-8.3mg/l.[21] These values are many folds higher than what we reported in our study, suggesting that there are variations in sTfR levels from one population to another. These variations might be due to environmental, racial, dietary and genetic factors and also assay methods used. The sTfR levels did not differ with age.(F= 0.160, p= 0.923) Von Schmiesing et al[27]in a similar study which though found relatively higher ranges for each age group when compared to the index study, did not also observe any significant difference in the mean sTfR across the various age groups which is similar to our observations in this study. This was affirmed by Raya et al[28]and other authors. [10, 11, 19, 24, 29] This is probably because the study population, being all adults, have all attained adult levels of sTfR. Contrary to this, Jong Weon Choi et al [30] observed age related differences in sTfR levels. This difference might be due to the fact that the study participants used by Jong Weon Choi and his friends were not limited to adults alone, unlike our study and other studies.[10, 11, 19, 24] We observed that the mean sTfR levels for both sexes were not significantly different though males had a slightly higher mean sTfR. Kohgo et al and Allen et al[9, 12]in their respective study, even though the former reported lower values and the latter higher sTfR values for each sex, did not find any significant difference in their mean sTfR as also seen in other related studies.[31,32]. On the contrary Knox- macaulay and co suggested otherwise from their work, stating that there was a statistically significant difference of sTfR values in men and women. [20]Another study also noted a higher sTfR levels in females compared to males.[29]The reason for these differences cannot be readily explained, however, this might require confirmation using a larger study population. There was no significant difference in sTfR with pre and postmenopausal women (.P=0.470),suggesting that menstrual blood loss of iron does not significantly affect sTfR levels in normal adults. This observation was affirmed by Allen and his colleagues.[12]

Conclusion:

This study using immunological assay has established reference range forsTfR to be 0.3-3.05ug/ml in heathy adult population in Port Harcourt Nigeria.sTfR did not differ significantly across various age groups and between males and females. Menstrual status in healthy females did not significantly impact on the sTfR level.

Acknowledgements

We are grateful to our supervisor and teacher, Prof O.A Ejele for his tutelage, advice and input in the course of this study. We thank Prof C.A Nwauche for his advice and encouragement. We also acknowledge Dr (Mrs) E.E Igbigbi, our Head of Department for all her support. We thank Mr Ben Alime for his assistance in the technical aspects of this study. Finally, we thank our families for their sacrifice, patience and understanding.

References 1.

Koulaouzidis A, Said E, Cottier R, Saeed A. Soluble transferrin receptor and iron deficiency, a step beyond ferritin. A systematic review. J Gastrointestin Liver Dis. 2009; 18(3): 345-352

Krause JR, Stolc V. Serum ferritin and bone marrow iron stores. Correlation with absence of iron in biopsy specimens. Am J Clin Pathol. 1979; 72: 817-820.

Jayarane S SP. Serum soluble transferrin receptor in hypochromic microcytic anemia. Singapore Med J. 2006; 47(60): 3-7.

Speeckaert MM, Speeckaert R, Delanghe JR. Biological and clinical aspects of soluble transferrin receptor.Crit Rev Clin Lab Sci. 2010; 47(5-6):213-28

Ejaz H, Muhammad A, Masood A, Waqar A MB. Evaluation of serum transferrin receptor in diagnosing and differentiating iron deficiency anemia from anemia of chronic disorders. JPMA. 2005; 55(13): 5-8

Jain S, Narayan S, Chandra J, Sharma S, Malhan P. Evaluation of serum transferrin receptor and sTfR ferritin indices in diagnosing and differentiating iron deficiency anemia from anemia of chronic disease. Indian J of Paediatr. 2010; 77(2): 179-83.

Chua E, Clague JE, Sharma Ak, Horan MA, Lombard M. serum transferrin receptor assay in iron deficiency anemia and anemia of chronic disease in the elderly. G J Med. 1999; 92:587-94.

Sweet DG, Savage G, Tubman TR, Lappin TR, Halliday HL. Study of maternal influences on fetal iron status at term using cord blood transferrin receptors. Arch Dis Child Fetal Neonatal Ed.2001;84(1):F40-3.

Kohgo Y, Niitsu Y, Kondo H, Kato J, Tsushima N, Sasaki K, et al. Serum transferrin receptor as a new index of erythropoiesis. Blood. 1987; 70(6): 1955-8.

10. Flowers CH, Skikne BS, Covell AM, Cook JD. The clinical measurement of serum transferrin receptor. The J Lab Clin Med. 1989; 114(4): 368-77. 11. Virtanen MA, Viinikka LU, Virtanen MK, Svahn JC, Anttila RM, Krusius T et al. higher concentrations of serum transferrin in children than in adults. Am J Clin Nutr. 1999; 69:256-60.

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Efobi R et al. 12. Allen J, Backstrom KR, Cooper JA et al. Measurement of soluble transferrin receptor in serum of healthy adults. Clin Chem. 1998; 44(1): 35-9. 13. Szymczyk-Nuzka M, Wolowiec D. Iron stores in regular blood donors. Polskie Archiwum Medycyny Wewnetrznej. 2003; 110(6): 1415- 21.

A-229 recombinant soluble transferrin receptor preparation. Clin Chem Lab Med. 2010 Jun;48(6):815-20 23. Umeh SO, Emelugo BN. A Survey Of Normal Haematological Variables Among Adults In Nigeria As Seen In Anambra State. Trop J Med Res.2008; 12 (1): 29-31.

14. Schumacher YO, Schmid A, Konig D, Berg A. effects of exercise on soluble transferrin receptor and other variables of iron status. Br J Sports Med. 2002; 36(3): 195-9.

24. Van den Bossche .G, Van den Bossche. J, Wagner C, De Schouwer P, Hugo N, Van De Vyvere M. Determination of iron metabolism- related reference values in a healthy adult population. Clin Chem. 2001; 47(8):1465- 7

15. Malczewska-Lenczowska J, Stupnicki R, GabryĹ&#x203A; T. Effects of exercise on markers of iron status in serum of crosscountry skiers. Biol. Sport. 2010; 29(4): 241-7.

25. Test principles for soluble transferrin receptor by Biovendor. https://www.biovendor.com/file/5074/PDS_06_sTfR_ ENG.004.A.pdf date accessed online:21/7/2014.

16. Cook JD, Boy E, Flowers C, Daroca MDC. The influence of high-altitude living on body iron. Blood.2005; 106(4):1441-6

26. Simek M, Remkova A, Kratochvilova H. Serum transferrin receptor in diagnosis of iron deficiency. Bratisl Lek Listy. 2002; 103(12): 449-53.

17. Tarng D-C, Hung S-C, Huang T-P. Effect of intravenous ascorbic acid medication on serum transferrin receptor in hemodialysis patients. J Am Soc of Nephrol. 2004;15(9):2486-93. 18. Zhu Yi, Haas JD. Response of serum transferrin receptor to iron supplementation in iron-depleted non-anaemic women 1-3. Am J Clin Nutr. 1998; 67:271-5 19. Skikne BS, Flowers CH, Cook JD. Serum transferrin receptor. A quantitative measure of tissue iron deficiency. Blood. 1990; 75(9): 1870-6. 20. Knox-macaulay H, Gravell D, Elender F. Serum Transferrin Receptor Status of Healthy Adult Arabs. Ann Clin Lab Sci. 2007;37(1):57-62. 21. Erhardt JG, Estes JE, Pfeiffer CM, Biesalski HK, Craft NE. combined measurement of ferritin, soluble transferrin receptor, retinol binding protein ad C- reactive protein by an inexpensive, sensitive and simple sandwich enzyme- linked immunosorbent assay technique. J Nutr. 2004; 134(11): 3127-3132. 22. Thorpe SJ, Heath A, Sharp G, Cook J, Ellis R, Worwood M. A WHO reference reagent for the Serum Transferrin Receptor (sTfR): international collaborative study to evaluate a

27. Von Schmiesing A, Schmidt SA, Krater W et al. Determination of reference values for the soluble transferrin receptor(sTfR) on a cross sectional population sample. Clin Lab. 2009; 55(5-6): 193-9. 28. Raya G, Henny J, Steinmetz J, Herbeth B, Siest G. Soluble transferrin receptor(sTfR): Biological variations and reference limits. Clin Chem Lab Med. 2001; 39(11): 1162-8. 29. Tijanic I, Vucic M, Golubovic LM. The significance of soluble transferrin receptors I diagnosing iron deficiency anemia. Revista Romana de Medicina de Laborator. 2015;23: 275-83 30. Jong Weon Choi, Soo Hwan Pai, Moon Wham Im, Soon Ki Kim. Change in transferrin receptor concentration with age. Clin Chem: 1999; 45(9) 31. Vernet M, Doyen C. Assessment of iron status with a new fully automated assay for transferrin receptor in human serum. Clin Chem Lab Med: CCLM/FESCC. 2000; 38(5): 437-42. 32. HA Huebers, Y Beguin, P Pootrakul, D Einsparh, CA Finch. Intact transferrin receptors in human plasma and their relation to erythropoiesis. Blood. 1990; 75: 102-107

*Corresponding author: Dr Chilota Efobi, Department of Haematology, Chukwuemeka Odumegwu Ojukwu University, Akwa Campus, Awka, Anambra State, Nigeria. Phone: +91 7030765610 Email: chylowb@yahoo.com Date of Submission : 30.12.2016 Date of Acceptance : 06.03.2017 Financial or other Competing Interests: None. Date of Publication : 31.05.2017

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Original Article DOI: 10.21276/APALM.1111

Histomorphological Analysis of Lesions In Nephrectomy Specimens: A 4 Years Study In A Rural Hospital In India-Our Experience Kishor H. Suryawanshi1*, Rajshri P. Damle1, N.V.Dravid1, Ashish Patil Rawandale2 and Akshay Surana1 Department Of Pathology, A.C.P.M. Medical College, Dhule, Maharashtra. India. 2 Department of Urology, A.C.P.M. Medical College, Dhule, Maharashtra. India.

ABSTRACT Background: Kidney is involved in various pathological conditions ranging from non-neoplastic lesions to neoplastic diseases like renal cell carcinoma. Nephrectomy remains the treatment of choice for patient care in end stage renal diseases from various causes and neoplastic lesions. Objective: To study the histomorphological spectrum and frequency of various lesions in nephrectomy specimens. Methods: A observational retrospective research study included 33 nephrectomy specimens in the department of pathology over a period of 4 years (April 2012 to April 2016).Clinical details and radiological findings were noted from records and clinico-histomorphological correlation was done. Results: A total of 33 nephrectomy specimens were studied. Age distribution varied from 16 to 71 years with male to female ratio of 2:1.Benign lesions comprised of maximum cases (75.76%) while malignant lesion were present in 24.24% cases. Chronic nonspecific pyelonephritis was the predominant non-neoplastic finding followed by xanthogranulomatous pyelonephritis. Renal cell carcinoma was the most common malignant lesion in this study. Conclusion: Chronic nonspecific pyelonephritis leading to end stage renal disease is the commonest causative factor in our region ; may be due to consumption of hard water in rural areas. Keywords: Chronic Nonspecific Pyelonephritis, End Stage Renal Disease, Nephrectomy

Introduction:

Kidney plays a vital role in excretion of waste product, water and electrolyte metabolism along with acid base balance, maintenance of blood pressure by secretion of renin-angiotensin and regulation of erythropoiesis by erythropoietin. Renal diseases are responsible for great deal of morbidity than mortality. Nephrectomy in the form of partial or radical is now-a-days became a common procedure in surgical and urological practice. Simple nephrectomy is indicated in patients with traumatic injury, end stage renal diseases resulting from obstructive uropathy, calculus disease, renovascular hypertension, chronic urinary tract infections and renal dysplasia. An indication of nephrectomy depends on the type of lesion, extent of damage, general condition of patient and status of contralateral kidney.[1,2] Moreover gold standard treatment renal tumors is radical or partial nephrectomy.[3]

Material and methods

This study was a observational retrospective research study conducted in Department of Pathology from April 2012 to April 2016 and included 33 nephrectomies specimen from

patients presented to Surgery /Urology department. Patients presented with complaints of hematuria, dysuria, loin pain, anuria and pyuria to Surgery /Urology department. Detail clinical history, significant findings, radiological findings were noted from records. All nephrectomies specimen were studied with clinical features, radiological findings, gross and microscopic findings. Representative sections were processed by paraffin embedding, stained with Haematoxylin and eosin stain. Special stains and immunohistochemistry were done wherever necessary. A final histopathological diagnosis was made with clinical and radiological correlations. The research study has been approved by ethical committee.

Results

The present study included 33 cases of nephrectomies specimen from patients presented to Surgery and Urology department. Age group of patients ranged from 16 years to 71 years. Maximum no. of patients were in the age group of 31-40 years (24.24%) followed by 41-50 years (24.24%). [Table 2] Out of 33 patients 22(66.67%) were males and

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11(33.33%) were females. Male to female ratio was 2:1. [Table 1] A greater percentage of both benign lesions (51.52%) and malignant lesions (15.15%) were observed in males as compared to females. Histomorphological distribution of lesions [Table-3] showed that chronic nonspecific pyelonephritis (51.51%) [Fig.1a] was the commonest finding followed by renal cell carcinoma in 8 cases (24.24%) and xanthogranulomatous pyelonephritis in 6 cases (18.18%)[Fig.1b].Age wise distribution of lesions [Table-4] showed that maximum number of cases of chronic nonspecific pyelonephritis were observed in 21-40 yrs age group while renal cell carcinoma was predominant in the age group of 41-60 yrs. Benign lesions(75.76%) comprised

maximum number of cases while malignant lesions were observed in 24.24% cases. The most common clinical presentation was loin pain followed by dysuria, hematuria, pyuria, fever and abdominal lump in three cases. The commonest clinical diagnosis made was nonfunctioning kidney in 21 cases (63.63%) based on clinical and radiological findings. In 10 cases renal cell carcinoma was suspected based on radiological finding of renal mass. Out of 8 cases of RCC, 4 were clear cell type,2 were papillary type and 1 case each of chromophobe RCC and collecting duct RCC was reported.[Fig.2-a,b,c,d]. One case was reported as multicystic nephroma.[Fig.1c & 1d]

Table 1: Sex wise distribution of benign & malignant lesions [Cases=33] Lesions

Males

Females

Total

Benign

17 (51.52 %)

08 (24.24%)

75.76 %

Malignant

05 (15.15%)

03 (9.09%)

24.24 %

Total

22 (66.67%)

11 (33.33%)

100 %

Table 2: Age-wise distribution of nephrectomy specimens Age group( yrs)

No. of cases

0-10

0.0

11-20

12.12

21-30

9.09

31-40

24.24

41-50

24.24

51-60

18.18

61-70

9.09

> 70

3.03

Table 3: Gender wise distribution of histopathological lesions Lesion

No. of cases

Male

Female

Chronic nonspecific pyelonephritis

Xanthogranulomatous pyelonephritis

Renal cell carcinoma

Multicystic nephroma

Severe hydronephrosis with secondary atrophy of renal parenchyma

Table 4: Age wise distribution of histopathological lesions Lesions

0-20 yrs

21-40 yrs

41-60 yrs

61-70 yrs

Chronic nonspecific pyelonephritis

Xanthogranulomatous pyelonephritis

Renal cell carcinoma

Multicystic nephroma

Severe hydronephrosis with secondary atrophy of renal parenchyma

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Histomorphological Analysis of Nephrectomy Specimens

Table 5: Sex wise distribution of RCC. Types of RCC

No. of cases

Male

Female

Clear cell RCC

Papillary RCC

Chromophobe RCC

Collecting duct RCC

Table 6: Comparison of Incidence of benign & malignant lesions in various studies. Author study Ghalayaini(2002)

[12]

Rafique(2007)[5] Mahesh KU(2012)

[1]

Aiman et al (2013)

[7]

Present study(2015)

Benign lesions (%)

Malignant lesions (%)

70.44

29.5

76.6

23.4

54.54

45.45

77.2

22.8

75.76

24.24

Fig. 1: (a) Chronic non-specific pyelonephritis (H&E; x100) ;( b) Xanthogranulomatous pyelonephritis (H&E; x100) ;(c) Multicystic nephroma (H&E; x100) and (d) Multicystic nephroma (H&E; x400).

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Fig. 2: (a) RCC ; clear cell type (H&E; x100) ;( b) RCC ; papillary type (H&E; x400) ;(c) RCC ; chromophobe pattern (H&E; x400) and (d) RCC ; collecting duct type (H&E; x100).

Fig 3: (a) Gross photograph-External surface showed enlarged kidney with bosselated surface and varying size nodules. ( b)&(c) RCC- cut surface showed variegated appearance with areas of necrosis and hemorrhage.(d) Multicystic nephroma showed immunopositivity for Pax 8 .(d).

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Histomorphological Analysis of Nephrectomy Specimens

Discussion

Gustav Simon, in 1869 and 1870, performed planned nephrectomy for urinary fistula and partial nephrectomy for hydronephrosis respectively.[4] Kidney is involved in variety of pathological conditions ranging from nonneoplastic to neoplastic lesions. Indications of nephrectomy depend on the type of lesion, extent of damage, general condition of patient and status of contralateral kidney. The present study included 33 nephrectomies specimen. Age group of patients ranged from 16 to 71 years with male to female ratio of 2:1.Similar results of male preponderance were observed by Rafique et al[5] and El Malik et al[6]. Aiman et al [7] reported female preponderance in their study. The highest percentage of patient [24.24%] were from 3rd decade and 4th decade which was in concordance with Rafique et al[5]. Loin pain followed by dysuria, hematuria, fever were commonest symptoms of patients in this study. Lump in abdomen was present in 3 cases who were later on diagnosed as cases of renal cell carcinoma [RCC] histopathologically. Out of 25 benign lesions chronic nonspecific pyelonephritis [CPN] was the predominant finding observed in 17 cases; xanthogranulomatous pyelonephritis [XGPN] in 6 cases; severe hydronephrosis and multicystic nephroma in one case each. Maximum no. of cases of CPN and XGPN were seen in 21-40 years. 2 cases of XGPN and 4 cases of chronic pyelonephritis were associated with renal calculus disease. Huland H [8] in his series reported 161 patients of end stage renal disease with 42 patients (26 %) had chronic pyelonephritis with bacteriuria in the past. All benign lesions were observed predominantly in males as compared to females. In the present study it is observed that RCC comprised all cases of malignant lesions. All malignant lesions were confirmed histopathologically as renal cell carcinoma with subtypes- 4 cases of clear cell type; 2 cases of papillary type and one case of each chromophobe RCC and collecting duct RCC. Amongst 8 cases of RCC, 4 cases of clear cell RCC showed Fuhrmanâ&#x20AC;&#x2DC;s nuclear grade 2, 2 cases of papillary RCC showed low nuclear features. Similar experience finding of RCC as predominant malignant lesion were reported by Rafique et al[5] and Popat et al[9]. Out of 8 cases of RCC 5 were found in males and 3 were in females.[Table-5] 5 cases were found in the age group 41-60 years; 2 cases in 21-40 years and one case in 61-70 years. Grossly majority cases (4) of RCC were found in left kidney with upper pole involvement in 4 cases. Five cases

showed variegated appearance with areas of necrosis and hemorrhage. [Fig. 3b&3c] One case of cystic mass in kidney suspected as RCC clinically was diagnosed as multicystic nephroma histopathologically and confirmed by immunohistochemistry study. It showed immunopositivity for Pax 8[Fig. 3d] and immunonegativity for p63/CD30. Multicystic nephroma is a rare benign lesion of kidney with bimodal age distribution. Adult onset multicystic nephoma is more common in postmenopausal females. In our case patient was a elderly male. Two cases of XGPN in older age group were suspected clinically as well as on grossly as RCC were confirmed as XGPN on histomorphology. In the present study out of 6 cases of XGPN 4 were seen in males due to calculus disease and 2 in females were associated with recurrent urinary tract infection. In the present study predominant cause of nephrectomy was non neoplastic lesions [75.76%] of kidney while neoplastic lesions with RCC as predominant lesion included only 24.24%. Geographical variation is found in indications of nephrectomy from review of literature. Study from Norway [10] and Nigeria [11] reported 68% and 67% respectively as a rate of nephrectomy for malignant lesions. Renal tuberculosis as a cause of nephrectomy was reported in 2.4% and 7.6% cases by Beisland et al[10] and Rafique et al [5] respectively. Renal tuberculosis is common in developing countries as compared to developed countries and patients present with sterile pyuria. But in our study we could not find renal tuberculosis as a cause of nephrectomy. While analyzing cause of end stage renal disease or non-functioning kidney from patients clinical details, radiological and histopathological findings, it was observed that calculus renal disease was the predominant cause of ESRD in our region. This may be due to consumption of hard drinking water in large area in rural population in our region. So education and screening programs are needed in such population to reduce rate of nephrectomy due to nonneoplastic lesions especially calculus renal disease. In comparison to various studies in the literature our findings were similar to findings of Ghalyaini et al[12], Rafique et al and Aiman et al. Mahesh KU et al[1] reported neoplastic lesions(54.5 %) as the predominant cause of nephrectomy in his study. Ghalyaini et al[12] reported youngest patient of 7-year-old child with RCC in his series.

Conclusion

To conclude, non-neoplastic lesions of kidney comprised the most common lesions in nephrectomy specimen. RCC was the most common malignant lesion with clear cell RCC as the most common subtype.

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References 1.

Mahesh KU, Yelikar BR,Patil G, Karigoudar MH, Pande P,Patil SB. Spectrum of Histopathological lesions in Nephrectomy specimens – A two year study in a tertiary care hospital. Int J of Research in Pharmaceutical and Biomedical Sciences 2012;3(4):1787-90.

Divyashree BN, Venkatesh K, Madhusudhan HR, Hanumantha Raju BK. “Pathological Spectrum of NonNeoplastic Diseases in the Nephrectomy Specimens”. Journal of Evidence based Medicine and Healthcare 2014; 1(15): 1909-1920.

Algaba F, Trias I, Scarpelli M, Boccon-Gibod L, Kirkali Z, Poppel HV. Handling and pathology reporting of renal tumor specimens. Eur Urol 2004;45: 437-43

Harry W. Herr. A history of partial nephrectomy for renal tumours. J Urol. 2005; 173(3): 705-708.

Rafique M. Nephrectomy: Indications, complications and mortality in 154 consecutive patients. J Pak Med Assoc 2007;57:308-11.

A-235 6.

El Malik EM, Memon SR, Ibrahim AL, Al Gizawi A, Ghali AM. Nephrectomy in Adults: Asir Hospital Experience. Saudi J Kidney Dis Transpl 1997;8:423-7. 7. Aiman A, Singh K, Yasir M. Histopathological spectrum of lesions in nephrectomy specimens: A five-year experience in a tertiary care hospital. J Sci Soc 2013;40:148-54. 8. Huland H, Busch R. Chronic pyelonephritis as a cause of end stage renal disease. J Urol 1982;127:642-3. 9. Popat VC, Kumar MP, Udani D, Mundra MP, Vora DN, Porecha MM. A study on culprit factors ultimately demanding nephrectomy. Internet J Urol 2010;7. 10. Beisland C, Medby PC, Sander S, Beisland HO. Nephrectomy indications, complications and post-operative mortality in 646 consecutives patients. Eur Urol 2000;37:58-64. 11. Eke N, Echem RC. Nephrectomy at the University of Port Harcourt Teaching Hospital: a ten year experience. Afr J Med Sci 2003;32:173-77. 12. Ghalayini IF. Pathological spectrum of nephrectomies in a general hospital. Asian J Surg 2002;25:163-9.

*Corresponding author: Dr. Suryawanshi Kishor H. Department Of Pathology, A.C.P.M.Medical College, Dhule, Maharashtra. India. 424005 Phone: +91 9403424244 Email: ompathologylab@gmail.com Date of Submission : 13.10.2016 Date of Acceptance : 13.02.2017 Financial or other Competing Interests: None. Date of Publication : 04.06.2017

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Original Article DOI: 10.21276/APALM.1124

Comparison of Diagnostic Accuracy of BIRADS Score With Pathologic Findings in Breast Lumps Navya B N1*, Shalu Thomas1, Rudresh Hiremath2 and Sathyavathi R Alva1 Department of Pathology ,K V G Medical College and Hospital, Karnataka, India Department of Radiology, K V G Medical College and Hospital, Karnataka, India

1 2

ABSTRACT Background: Breast lump is one of the commonest complaints with which patients present in breast clinics. As approximately 10% of breast masses ultimately lead to a diagnosis of breast cancer, it is important for women with a breast lump to receive appropriate evaluation. A confident diagnosis can be made in 95% of the cases through a combination of clinical examination, imaging and fine needle aspiration cytology. Histopathology is the gold standard for diagnosis. Methods: This is a prospective study conducted in the Department of Pathology, K.V.G Medical College, Sullia on 50 patients who presented with clinically palpable breast lump. Patients were subjected to sonomammography and Breast Imaging Reporting and Data System (BIRADS) scoring followed by FNAC and histopathological examination. Sensitivity, specificity, accuracy, positive and negative predictive values of sonomammogram in relation to the BIRADS score and FNAC taking histopathology as the gold standard was calculated. Result: FNAC had significantly higher sensitivity, specificity, positive and negative predictive values and accuracy compared to sonographical diagnosis using BIRADS score. Conclusion: FNAC could be considered as the first method to evaluate breast lesions, recognized by means of imaging techniques. Keywords: BIRADS, Fine-needle Aspiration Cytology, Histopathology

Introduction

Breast masses are localized swellings that feel different from the surrounding breast tissue. It is a symptom/sign for a variety of conditions.[1] Breast lump is one of the commonest complaints with which patients present in breast clinics.[2] As approximately 10% of breast masses ultimately lead to a diagnosis of breast cancer, it is important for women with a breast lump to receive appropriate evaluation.[1] Breast cancer is increasingly common in women worldwide. It is one of the leading causes of death among female malignancies. The incidence varies from region to region, more in developed countries (>80 per 100,000 populations) than in developing countries (<40 per 100,000 populations).[3] It commonly affects women older than 40 years of age. However, younger women can also be affected. Its management requires a multi-dimensional approach and a collaboration with different specialists. An accurate evaluation can maximize cancer detection and minimize unnecessary testing and procedures. Early detection and treatment is a key to preventing breast cancer from spreading.[4] A confident diagnosis can be made in 95% of the cases through a combination of clinical examination, imaging (including mammogram and/or sonomammogram) and fine needle aspiration cytology (FNAC).[1] Mammography

is cost efficient and widely accepted technique to evaluate clinically suspected breast lesions and used for screening of breast cancer. High resolution sonography is a useful adjunct modality and helps characterizing a mammographically non-detected palpable abnormality, especially in dense breast.[5,6] Sonomammography is noninvasive, easily available, cheaper and accurate tool in diagnosing breast masses. It is very helpful in pre surgical assessment of tumor size of even 2mm. It is the method of choice for differentiating solid from the cystic lesions, for further characterizing mammographic findings and better appreciating palpable breast lesions.[1] The American College of Radiology (ACR) created the breast imaging reporting and data system (BIRADS), to achieve trick verbal uniformity so as to get clear, unambiguous and standard language, not only among radiologist but also the treating physicians and surgeons. BI-RADS had 0-6 assessment categories.[6]  Category 0: need additional imaging evaluation  Category 1: negative  Category 2: benign finding  Category 3: probably benign finding; short-interval follow-up suggested

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Navya B N et al.  Category 4: suggestive abnormality; biopsy should be considered  Category 5: highly suggestive of malignancy; appropriate action should be taken  Category 6: known biopsy-proved malignancy FNAC has a high diagnostic accuracy rate (98.9%) in the hands of experienced cytopathologists. FNAC is a reliable method to differentiate whether a suspicious breast mass is benign or malignant.[1] In addition to its high diagnostic accuracy FNAC offers advantages such as minimal invasiveness, minimal discomfort, cost effectiveness and rapidity of results. FNAC is therefore an extremely vital tool in the evaluation of palpable breast lumps especially in resource limited settings.[7] However, the aspiration cytology is not a substitute for conventional surgical histopathology as a definitive diagnosis is not always possible by cytology, but categorization of disease and differential diagnosis can be provided in the majority of cases.[8] In the present study we aim to compare the diagnostic accuracy of BIRADS score in detecting benign and malignant lesions with pathological findings (FNAC and Histopathology) which is used for the final diagnosis in patients presenting with breast lumps, thus helping in avoiding unnecessary surgical procedures.

Materials and Methods

This was a prospective study conducted in the Department of Pathology, K.V.G Medical College, Sullia on 50 patients who presented with clinically palpable breast lump. The term “palpable breast lump” meant an area of denser breast tissue felt different from the surrounding tissue. Samples for the study were selected according to the inclusion and exclusion criteria. Inclusion Criteria • Female patients of all ages with complaints of breast lump. • Patients who have underwent breast imaging (including BIRADS scoring) and pathologic examination (i.e., both FNAC and histopathology). Exclusion criteria • Patients with recurrent lumps, history of prior irradiation to the chest or breast and cystic breast lesions • Pregnant and lactating ladies • Male patients The clinicians obtained a full history, performed breast examination, and then the patients were sent for sonommamography to the radiology department. www.pacificejournals.com/apalm

A-237 Sonommamography was performed on all cases presenting with breast lump. The images obtained were analyzed and categorized using the BIRADS score as benign and malignant. For the next part of the study, all the patients were subjected to FNAC and later on followed up with histopathology (Excision biopsy/Mastectomy specimens) findings which was used as the gold standard to confirm the diagnosis on sonomammography according to BIRADS classification and FNAC. FNAC was performed using a 23 G needle attached to 10 mL disposable plastic syringes, smeared on standard microscope glass slides and stained with haematoxylin and eosin (H&E) and Leishman stain. The slides were reviewed under light microscopy. For histopathological examination the tissue was fixed in 10% formalin and sections were taken from representative areas. The samples were labeled and processed, which involves a series of steps lasting 12 -16 hours. The slides were then stained with routine haematoxylin and eosin and examined under the microscope for histopathological diagnosis. Statistical analysis were performed to compute the sensitivity, specificity, accuracy, positive and negative predictive values of sonomammogram in relation to the BIRADS score and FNAC taking histopathology as the gold standard.

Results

The study included 50 patients presenting with breast lump. The youngest patient was 16 years of age and the eldest was 70 years of age [Table -1]. Maximum number of cases were seen in 15-25 years age group followed by 26-35 years age group. Results of sonomammogram according to BIRADS score is given in Table -2.There were no patients in categories 0,1 and 6.There were 14 patients( 28%) in category 2 revealing benign findings ,16 patients (32%) in category 3 revealing probably benign finding , 10 in category 4 (20%) revealing suspicious abnormality and 10 patients in category 5 (20%) indicating a high suspicion of malignancy. Findings were considered benign if score was 2 or 3 and malignant if score was 4 or 5. Sonomammography diagnosed 60% cases as benign and 40% cases as malignant. Distribution of cases on FNAC and Histopathological examination are given in Tables- 3 and 4. On FNAC, 32 cases were diagnosed as benign with majority being Fibroadenomatoid hyperplasia and Fibroadenoma (12 cases each) and 18 cases were diagnosed as malignant. On histopathological follow up, 32 cases were diagnosed as benign with majority being Fibroadenoma (15 cases) eISSN: 2349-6983; pISSN: 2394-6466

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and 18 cases as malignant with majority being Infiltrating ductal carcinoma (10 cases) followed by Infiltrating Lobular carcinoma (3 cases).

hyperplasia and the other as Ductal carcinoma in situ. Both were later confirmed as Infiltrating ductal carcinoma on histopathological examination. These cases constituted the false negatives in our study.

Four cases were given a score of 4 on BIRADS i.e., suspicious of malignancy of which 3 was diagnosed as Acute suppurative mastitis on FNAC and later on confirmed on histopathological examination as Fibrocystic disease with mastitis. The remaining case was diagnosed as Fibroadenoma / Intraductal papilloma on FNAC and as Intraductal Papilloma on Excision. These cases thus constituted the false positives in our study. Two cases were given a score of 3 i.e., probably benign on imaging, of which one was diagnosed as Atypical ductal Table 1: Age Distribution of Cases Age Group 15-25 26-35 36-45 46-55 56-65 66-75 Total

Tables -5 and 6 (Figure 1- a, b, c and Figure 2- a, b, c) show the comparison of BIRADS scoring and FNAC with histopathologic findings respectively. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy was 88%, 87.5%, 80%, 93% and 88% respectively for BIRADS score and 100% for all the parameters on FNAC. Table- 7 shows the overall accuracy of sonomammography using BIRADS scoring and FNAC in the diagnosis of breast lesions. Number of Cases 17 13 9 3 5 3 50

Table 2: Distribution of benign and malignant lesions on Sonomammogram SONOMAMMOGRAPHIC DIAGNOSIS NUMBER OF CASES (BIRADS) BENIGN (BIRADS SCORE -2 and 3) 30 MALIGNANT (BIRADS SCORE -4 and 5) 20 TOTAL 50

Percentage 34% 26% 18% 6% 10% 6% 100% PERCENTAGE 60% 40% 100%

Table 3: Distribution of lesions on FNAC LESIONS

NUMBER OF CASES

BENIGN • Fibroadenoma • Fibroadenoma /Intraductal papilloma • Fibroadenomatoid hyperplasia • Fibroadenomatoid hyperplasia with mastitis • Breast abscess • Intraductal papilloma • Acute suppurative mastitis

12 1 12 2 1 1 3 32 (64%)

TOTAL MALIGNANT • Carcinoma breast • Carcinoma probably medullary type • Carcinoma –Infiltrating ductal /Invasive Papillary Carcinoma • DCIS • Malignant phyllodes • Apocrine carcinoma • Infiltrating ductal • Atypical ductal hyperplasia TOTAL

7 2 1 4 1 1 1 1 18 (36%)

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Table 4: Distribution of lesions on Histopathology (Excision/Mastectomy) LESIONS

NUMBER OF CASES

PERCENTAGE

BENIGN •

Fibroadenoma

•

Fibroadenomatoid hyperplasia

•

Fibroadenoma with cystic change

•

Fibroadenomatoid hyperplasia with cystic change

•

Fibrocystic disease with mastitis

•

Intraductal papilloma

TOTAL

64%

MALIGNANT •

Infiltrating ductal carcinoma

•

Infiltrating lobular carcinoma

•

Medullary carcinoma

•

Colloid carcinoma

•

Malignant phyllodes

TOTAL

36%

TOTAL

100%

Table 5: Comparison of BIRADS score with Histopathology SONOMAMMOGRAM (BIRADS score)

HISTOPATHOLOGY BENIGN MALIGNANT

TOTAL

BENIGN

28 (TN)

2 (FN)

MALIGNANT

4 (FP)

16 (TP)

TOTAL Table 6: Comparison of FNAC with histopathology

HISTOPATHOLOGY BENIGN MALIGNANT

FNAC

TOTAL

BENIGN

32 (TN)

0 (FN)

MALIGNANT

0 (FP)

18 (TP)

TOTAL

Table 7: Overall accuracy of FNAC and Sonomammography in breast lesions FNAC

SONOMAMMOGRAM (BIRADS score)

Sensitivity

100%

88%

Specificity

100%

87.5%

Positive predictive value

100%

80%

Negative predictive value

100%

93%

Accuracy

100%

88%

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Fig. 1: (a) Breast ultrasound image of a solid lesion-BIRADS Category 3, (b) FNAC of the same case showing cell rich smear of elongated, branching fragments of ductal epithelial cells (H&E,10x) (c) Excision biopsy showing compressed ducts having a linear branching pattern with slit-like lumens- Fibroadenoma( H&E, 10x).

Fig. 2: (a) BIRADS category 5 lesion-well defined horizontally ovoid lobulated predominantly hypoechoic mass. (b) FNAC showing large neoplastic cells having pleomorphic hyperchromatic nuclei (H&E,100x) (c) Histopathology showing neoplastic Invasive Ductal carcinoma in glandular pattern, sheets and clusters (H&E, 100x).

Discussion

Breast diseases are common in females. In developing countries like India, females are unaware of breast pathologies and are hesitant to reveal, hence they are detected usually in advanced stages. Various benign breast lesions like fibroadenomas, breast abscess, galactocele, duct ectasia, enlarged lymph nodes and different malignancies are common pathologies of female breast .[9]

cancer. Although mammography is recognized as the best method of screening for breast cancer, breast sonography is now well-established as a valuable imaging technique, and, while there has been some controversy regarding its utility in evaluating solid breast masses for the likelihood of malignancy, several studies have suggested that sonographic appearance can be useful in differentiating malignant from benign solid breast masses.[10]

Patients with palpable breast lesions commonly present for radiology evaluation. Mammography is a primary method of detection and diagnosis of breast disease with sensitivity of 85% - 95%. But the false negative findings in mammography in evaluation of palpable breast mass is high, estimated between 4% & 12%.[9] The BIRADS lexicon was ďŹ rst developed in 1993 for reporting mammography. Since its establishment, several studies have found that it can be helpful to physicians in predicting the likelihood of

A wide variation in the sensitivity of sonomammogram in the diagnosis of breast lesions ranging from 67% to 97% have been reported .[11] In a study conducted by Shrestha M K et al the sensitivity and specificity of sonomammography in differentiating benign from malignant lesions using the BIRADS score was 78.9 and 95% respectively .[1] Shumaila S M et al in their study have reported mammography to be positive in 66(90%) and sonomammography to be positive in 68 (93%) out of

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Navya B N et al. 73 cases .[12] Ultrasonography can be used to differentiate benign from malignant lesions with negative predictive value of 99.5%, specificity of 67.8% and over all accuracy of 72.9% in a study according to Stavros et al.[13] Emine D et al did a study on 546 breast lesions with histopathology analysis, they reported sensitivity and specificity for sonomammogram to be 72.6 and 88.5% .[14] In the present study we got comparable results with a sensitivity of 88%, specificity 87.5%,positive predictive value of 80%, negative predictive value of 93% and an accuracy of 88% in differentiating benign from malignant masses in sonomammography using the BIRADS lexicon system. FNAC of breast lumps is an accepted and established method for determining the nature of breast lumps with a high degree of accuracy. Application of Fine Needle Aspiration for the diagnosis of palpable breast masses was first introduced by Martin and Ellis in 1930 and since then, it has been established as an important tool in the evaluation of breast lesions. Most of the patients with breast lumps are in a state of anxiety. So, in reducing anxiety and unnecessary surgical procedures as well as in minimization of delay in the diagnosis, FNAC proves very fruitful. FNA procedure is a safe method with only a few reported complications.[15] In the present study, 18 and 32 cytologically diagnosed malignant and benign cases were confirmed as malignant and benign respectively on subsequent histopathological examinations. So, in our study, a 100% cytohistopathological correlation was observed for breast lesions. Zhang Qin et al in their study similarly reported a 97.1 and 97.3% sensitivity and specificity respectively for FNAC in diagnosing breast lesions .[16] Tiwari M has also observed the similar results in their studies with a sensitivity of 83.3% and specificity of 100%.[17] The sensitivity, specificity, accuracy, negative predictive value, and the positive predictive value of FNAC was 98%, 100%, 98%, 100%, and 97%, respectively in the study conducted by Bukhari et al.[18] Our study showed higher values than the range reported with 100% sensitivity, specificity, positive and negative predictive values and accuracy. FNAC is not only useful in diagnosis and further planning of treatment without need for biopsy, but also helpful in prognostication of the tumor factors such as nuclear grading, mitotic index, hormone receptor status and DNA contents.[19] Histopathological examination should be performed for uncertain diagnostic cases and when the evaluation of the invasiveness or histological type of breast lesion is mandatory.[20] The gold standard test used in our study was the histopathological report which is valid, reproducible and has been accepted as the gold standard www.pacificejournals.com/apalm

A-241 internationally. For a good study, the reference test against which the diagnostic test in evaluation is compared should be gold standard.[11]

Conclusion

Ultrasonography with BIRADS score is an imaging technique and FNAC a tissue diagnostic technique. Both these diagnostic tools should be considered complimentary. The results of our study showed FNAC of breast lumps to be a reliable method to diagnose breast lump with high accuracy compared to sonographic categorization using BIRADS score. Considering patientâ&#x20AC;&#x2122;s comfort, lack of requirement of anesthesia, rapid analysis and reporting and an absence of false positive results, FNAC could be considered an ideal initial diagnostic modality in breast lumps recognized by means of imaging techniques. Further advancement in the technique of both these procedures like FNAC under imaging guidance, addition of immunohistochemistry in cytology and addition of Doppler in USG may increase their accuracy. However all clinically malignant or suspicious masses should be subjected to histopathological examination which is the gold standard for tissue diagnosis.

Reference 1.

Shrestha MK, Ghartimagar D, Ghosh A, Shrestha E, Bolar P. Significance of quadruple assessment of breast lump â&#x20AC;&#x201C; A Hospital based study. Journal of Pathology of Nepal. 2014;4:630-4.

Lalchan S, Thapa M, Sharma P, Shrestha S, Subash K, Pathak M et al. Role of Mammography Combined with Ultrasonography in Evaluation of Breast Lump. American Journal of Public Health Research. 2015;3(5A):95-8.

Chairat R, Puttisri A, Pamarapa A, Samintharapanya S, Tawichasri C, Patumanod J. Are Both Ultrasonography and Mammography Necessary for Cancer Investigation of Breast Lumps in Resource-Limited Countries? ISRN Oncology. 2013;Article ID 257942, doi:10.1155/2013/257942.

Arsalan F, Subhan A, Rasul S, Jalali U, Yousuf M, Mehmood Z et al. Sensitivity and specificity of BI-RADS scoring system in carcinoma of breast. Journal of Surgery Pakistan. 2010;15(1):38-43. Phurailatpam J, Sakalecha A, Prasad CS, Kumar BN, Hegde P. Evaluation Of

Mammography, Sonomammography In Correlation With Fine Needle Aspiration Of Breast Lumps. Int J Biol Med Res. 2014;5(3):4370-6.

Quershi S A, Rehman K, Muhammad D, Khan M I, Jaffra H. Validity of Mammogram according to Bi-RADS Scoring In Relation with Histopathology among Females Presenting with Clinically Palpable Breast lump or Nipple discharge. Ann Pak Inst Med Sci. 2014;10(3):150-4.

Daramola AO, Odubanjo MO, Obiajulu FJ, Ikeri NZ. Correlation between Fine needle aspiration cytology and histology for palpable breast masses in a Nigerian tertiary

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health institution. International Journal of breast Cancer. 2015;Article ID 742573, doi:10.1155/2015/742573. 8.

Yalavarthi S, Tanikella R, Prabhala S, Tallam US. Histopathological and cytological correlation of tumors of breast. Medical Journal of Dr. D.Y. Patil University. 2014;7 (3):326-31. Taori K, Dhakate S, Rathod J, Hatgaonkar A, Disawal A, Wavare P et al. Evaluation of Breast Masses Using Mammography and Sonography as First Line Investigations. Open Journal of Medical Imaging. 2013;3:40-49.doi. org/10.4236/ojmi.2013.31006.

10. Heinig J, Witteler R, Schmitz R, Kiesel L, Steinhard J. Accuracy of classiﬁcation of breast ultrasound ﬁndings based on criteria used for BI-RADS. Ultrasound Obstet Gynecol. 2008;32:573–78. 11. TakhellambamYS, Lourembam SS, Sapam OS, Kshetrimayum RS, Thoujam BS, Khan T. Comparison of Ultrasonography and Fine Needle Aspiration Cytology in the Diagnosis of Malignant Breast Lesions. Journal of Clinical and Diagnostic Research. 2013;7(12):2847-50. 12. Shumaila SM, Tayyiba A, Safdar AM. Mammographic – Sonographic co-relation in the diagnosis of breast lump. Biomedica. 2008;24:147-51. 13. Stavros AT, Thickman D, Rapp CL, Dennis MA, Parker SH, Sisney GA. Solid Breast Nodules: Use of Ultrasonography to Distinguish between Benign and Malignant Lesions. Radiology. 1995;196(1):123-34.

14. Emine D, Suzana M, Halit Y, Arben K. Comparative accuracy of mammography and ultrasound in women with breast symptoms according to age and breast density. Bosn J Basic Med Sci. 2009;9:131-6. 15. Vala MT, Goswami A, Suri SK. Comparative study of cytological and histopathological finding in breast lesion. IOSR Journal of Dental and Medical Sciences. 2014;13(7):05-07. 16. Qin Z, NieShigui, Yuhua C, Limei Z. Fine Needle Aspiration Cytology of Breast Lesions: Analysis of 323 Cases. The Chinese-German Journal of Clinical Oncology. 2004;3(3):172-4. 17. Tiwari M. Role of FNAC in diagnosis of breast lumps. Kathmandu University Medical Journal. 2007;5:215-17. 18. Bukhari MH, Arshad M, Jamal S, Niazi S, Bashir S, Bakhshi IM. Use of fine-needle aspiration in the evaluation of breast lumps. Patholog Res Int. 2011;doi:10.4061/2011/689521 19. Velu ARK, Srinivasamurthy BC, Rani J. Cytological evaluation of benign breast lesions with histopathological correlation. Indian Journal of Pathology and Oncology. 2016;3(1):7-10. 20. Moschetta M, Telegrafo M, Carluccio DA, Jablonska JP, Rella L, Serio G et al. Comparison between fine needle aspiration cytology (FNAC) and core needle biopsy (CNB) in the diagnosis of breast lesions. G Chir. 2014;35(7/8):171-6.

*Corresponding author: Dr Navya B N, Associate Professor, Department of Pathology, K V G Medical College and Hospital, Kurunjibhag, Sullia, D.K District- 574327,Karnataka, India Phone: +91 9886687911 Email: navyabn@rediffmail.com Date of Submission : 25.10.2016 Date of Acceptance : 09.03.2017 Financial or other Competing Interests: None. Date of Publication : 04.06.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Original Article DOI: 10.21276/APALM.1154

A Cytohistopathological Correlation of Thyroid Lesions with Critical Evaluation Of Discordant Cases: An Experience At A Tertiary Care Hospital Shakuntala Sunil Aramani1* and Gururajaprasad. C2 1

Department of Pathology, Gadag Institute of Medical Sciences, Mallasamudra, Gadag-Karnataka, India. 2 Department of Pathology, JSS Medical College, Mysuru, Karnataka, India.

ABSTRACT Background: Fine Needle Aspiration Cytology (FNAC) is important for pre-operative diagnosis of benign and malignant thyroid lesions. It is usually the first line of investigation and a minimally invasive diagnostic procedure whose essential role is to diagnose and distinguish benign from malignant lesions. Thus the present study was undertaken to determine sensitivity, specificity and accuracy of FNAC in the diagnosis of thyroid lesions and to correlate cytological findings with histopathology. Methods: A prospective study was conducted in a tertiary care centre to study FNAC of thyroid lesions along with its histopathological correlation over a period of 2 years starting from June 2013 to June 2015 Result: 56(93%) patients were females and 4(7%) patients were males. Patients age ranged from 15 to 65 years .Commonest lesion encountered in thyroid gland was colloid goitre, while papillary carcinoma was the most frequent malignant lesion. Cyto-histopathological correlation of 60 cases was done. Sensitivity ,specificity and diagnostic accuracy of the study for malignant lesions was 96.36%,100%,and 96.66% respectively. Conclusion: FNAC is a safe, simple, highly accurate, economical method for evaluation of palpable thyroid lesions. There is almost perfect cyto-histopathological concordance and the results are consistent with those available in the literature. FNAC helps in avoiding unnecessary surgeries in patients diagnosed to have a benign pathology based on cytology. Thus FNAC serves as a useful screening test to triage thyroid lesions before surgery. Keywords: Thyroid, Histopathological Correlation, FNAC, Sensitivity, Specificity, Diagnostic accuracy

Introduction

Head and neck lesions are very common and they range from inflammatory lesions to malignancies. It is evident that early diagnosis of these lesions provide the best chance of successful treatment. Among head and neck lesions thyroid lesions are most common with a prevalence rate ranging from 4-7%.[1]The majority of clinically diagnosed thyroid lesions are non-neoplastic ;only 5-30%,are malignant and require surgical intervention. [2] FNAC is a minimally invasive procedure which is safe, simple, cost effective reliable and produces a quick result. [3] FNAC has allowed a dramatic decrease in unnecessary surgeries without thyroid nodular disease, enhancing the percentage of malignant operated nodules over 50%. [4] Therefore the main aim of this diagnostic procedure in thyroid lesions is to detect the thyroid neoplasms for surgical resection and to identify non neoplastic lesions that may be managed conservatively.

Thus, this study was undertaken to study the cytology of various palpable thyroid lesions and also confirmation of the diagnosis by histopathological study.

Materials and Methods

The present study was a prospective, cross-sectional study which was undertaken to study FNAC of thyroid lesions along with its histopathological correlation at department of pathology , JSS Medical college, Mysuru over a period of 2 years from June 2013 to June 2015 . All the patients were clinically examined in detail, and a careful palpation of the thyroid gland was done to judge precisely the location for aspiration. After brief explanation about the procedure to the patient multiple passes (2 -4) were done under aseptic precaution using a 24-25 gauge needle using a 10 ml syringe. Multiple smears were prepared from aspirate and those immediately fixed in 95%ethanol were stained using Haematoxylin and Eosin (H & E) and Papanicolaou(Pap) stains and air dried smears were stained with May Grunwald Giemsa (MGG).

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Whenever the aspirate yielded fluid, it was cytocentrifused and the smears were prepared from the sediment and stained by the stains described earlier and the residual solid mass was sampled. The cases were followed by postoperative specimen received from the department of Surgery, department of ENT. Specimens were collected in 10% formalin in fresh state and allowed to fix for 24 hours. Detailed gross examination was done and bits were given. Paraffin embedded H&E stained sections were obtained and studied under light microscopy. All the patients who presented with palpable thyroid lesions and who underwent FNAC with subsequent histopathological examination were included in the study and those patients in whom FNAC was either acellular or non diagnostic and who underwent FNAC without subsequent histopathological examination were excluded from the study. Statistical Package for Social Sciences(SPSS) software was used for statistical analysis. Sensitivity, specificity, and diagnostic accuracy(efficacy) were calculated. Cytological diagnosis was correlated with the histopathology and the efficacy of FNAC is estimated by using the methodology of Galen and Gambino. These statistical values are interdependent statistical concepts indicating the accuracy of results.

Results

The present study includes a total number of 60 cases who presented with thyroid lesions. Patients age ranged from 15 to 65years with predominance of female patients with F:M ratio being 14:1. Graph-1 shows distribution of thyroid lesions with regard to sex, whereas Table-1 shows age and sex wise distribution of thyroid lesions. Majority of the

patients are in the age group of 21-40 years followed by 41-60years. In the present study, the non neoplastic lesions were more common then neoplastic lesions Colloid goitre: Out of 60 cases, 47 cases were diagnosed as colloid goitre by cytology. Histopathological study confirmed the cytodiagnosis in 40 cases. In the remaining 7 cases, 5 cases turned out be follicular adenoma, 1 case as follicular carcinoma, and the other 1 case as papillary carcinoma. Follicular neoplasm: Out of 60 cases,7 cases were diagnosed as follicular neoplasm by cytology.5 cases were confirmed by histopathology and the rest 2 cases turned out to be colloid goitre. Gross picture of follicular adenoma is shown in the fig-1,whereas fig-2 and the fig-3 shows the cytology of follicular neoplasm and histopathological section of follicular adenoma respectively. Hashimoto,s thyroiditis: 1 case was diagnosed as hashimoto,s thyroiditis which was confirmed by histopathology. Papillary carcinoma: Out of 60 cases, 5 cases were diagnosed as papillary carcinoma and all the cases were confirmed by histopathology. Of the 60 cases , 55 cases were diagnosed as benign and only 5 cases turned out to be malignant by FNAC, where as 53 cases were diagnosed as benign and 7 cases turned out to be malignant by histopathology resulting in 2 false negative cases. The present study showed 96.36% sensitivity, 100% specificity and 96.66% diagnostic accuracy in detecting malignant tumors.

Graph 1: Distribution of thyroid lesion with regard to sex (n=60)

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Graph 2: The various histopathological diagnosis of the thyroid lesions.

Fig. 1: Gross picture of Follicular neoplasm.

Fig. 2:Follicular neoplasm. Smear shows repeating units of microfollicles[H&E X100].

Fig 3:Follicular adenoma. Section shows an encapsulated lesion with compression of surrounding normal thyroid follicles [H&EX100]

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Table 1:Age and sex wise distribution of the thyroid lesions. Age groups(years) Sex

<20

21-40

41-60

61-80

Male

Female

Table 2: Analysis of the cyto-histopathological correlation of thyroid lesions. Cytological diagnosis

Histopathological diagnosis

Total

Comment

False negative-2

False positive-0

Benign

Malignant

Benign

Malignant

0 53

Discussion

In the present study , 2 cases were erroneous and were false negative cases. One case was diagnosed as Colloid goitre with cystic change by FNAC and it was subsequently diagnosed as cystic papillary thyroid carcinoma by histopathology. The greatest risk of a false negative diagnosis is in relation to cystic neoplasms, mainly cystic papillary carcinoma. Over 40% of the cystic neoplasms may be missed by FNAC. Amatya et al. [10] and Fernandes H et al. [11]also found a similar misdiagnosis in their study.

In the present study there was a female predominance with F:M ratio of 14:1.It was similar to the studies done by Ranjan et al. [1]and Tilak et al. [8]. Age of the patients ranged from 15-65 years and majority of the patients were in the 21-40 years age group. It is in concordance with the study done by Kumar et al. [9] Out of the 60 cases, Cytologically 55 cases were diagnosed as non-malignant and 5 cases as malignant lesions. Of these 55 cases, 53 cases proved to be non-malignant by histopathology resulting in 2 false negative cases. All 5 cases diagnosed as malignant by cytology were proven to be malignant by histopathology and there was no false positive case in the present study, probably it could be because of small sample size.

The cause of false negative results were the poorly cellular sample in a cystic papillary carcinoma and the thick fibrous capsule. Gagneten stressed the importance of doing multiple aspirations in a thyroid swelling in order to obtain representative material from different areas since the thyroid can be affected by more than one disease process. [12]

Thyroid FNAC was initially started by Martin and Ellis in 1930. It is usually the first line of investigation and a minimally invasive diagnostic procedure whose essential role is to diagnose and distinguish benign from malignant lesions. A total of 60 cases of thyroid lesions who underwent FNAC with subsequent histopathological examination were included in the study and all the lesions were analysed with their history, FNAC findings and histopathology.

The diagnostic error was most commonly due to inadequate specimens and cystic lesions. One must be careful in committing a false negative diagnostic error in cystic lesions that contain macrophages and scanty material, since these features do not exclude malignancy. Repeat FNAC or thyroidectomy is advised for persistent nodules. [13,14] Cystic thyroid lesions pose diagnostic difficulties. Cystic change and/or hemorrhage in neoplasms is seen in upto 25% of primary papillary carcinomas, In 20% of follicular neoplasms and in 26% of follicular carcinomas. [8]

When Galen and Gambino method is applied, FNAC of the thyroid lesions had a sensitivity of 96.36% and specificity of 100%.The overall diagnostic accuracy in detecting malignant tumors was 96.66%. This study is comparable with the other studies as shown in the table 3.

Table 3: Comparison of results of present study with previous studies in identifying thyroid malignancies Studies

Year

Sensitivity

Specificity

Accuracy

Gupta et al.5

2010

86.6

Pinky pandey et al.6

2012

57.14

80.28

Parikh et al.

2012

71.43

100

90.24

2014

82.14

86.8

83.60

Gamit et al.

2015

92.85

98.48

97.5

Present study

2015

96.36

100

96.66

Ranjan et al.

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Aramani et al. Different studies by Heimann A and Gritsman A suggest that, multiple criteria must be observed before making a confident cytological diagnosis of papillary carcinoma. Logistic regression analysis of various criteria suggested that a combination of a intranuclear cytoplasmic inclusion, papillary structure with or without adherent blood vessels and dense metaplastic cytoplasm were the three most important variables. [15] Commonest lesion encountered in thyroid gland was colloid goitre and most of the cases correlated with the histopathological diagnosis. Of the 45 cases diagnosed as goitre, all were correctly verified except 5 cases which were diagnosed as follicular adenoma. In these cases, the lesions were cystic, colloid material was aspirated, and the smears showed features of nodular goitre. However, on sectioning, there was nodular mass which turned out to be a follicular adenoma. [10] The cytological appearances in colloid goitre form a continuum which merge with those of follicular adenoma, and in this grey area, cytological criteria alone cannot reliably distinguish between the two. [16]\ 2 cases of goitre were misdiagnosed as follicular neoplasm in FNAC, it is a known fact that the cytological criteria cannot reliably differentiate between the two because of the overlap. Smears from micro follicular areas in a nodular goitre may show a repetitive pattern of micro follicles or rosettes similar to the follicular neoplasm, and if only such a focus is aspirated and sampled, the differentiation from follicular neoplasm is impossible. [15] In the present study similar type of microfollicular areas were sampled and lead to the erroneous diagnosis of follicular neoplasm. Another false negative case was a follicular carcinoma which was misdiagnosed as colloid goiter by FNAC. The differentiation of thyroid adenoma from follicular carcinoma based on cytologic criteria done is difficult and challenging as the cytological appearences of both are very similar. Lowhagen advocated that a cytologic report should only state that a follicular neoplasm is present with no implications of its benign or malignant nature. Friedman advised histologic examination in such cases for final diagnosis. [17] Thus FNAC is an efficient diagnostic procedure for assessing the thyroid lesions. The method is simple, safe and economical. Another advantage is that it is a time saving procedure which can be reported within minutes and hours instead of days needed for histopathology studies. Nevertheless, the specificity of 100%, sensitivity of 96.36%, and an overall diagnostic accuracy of 96.66% are very encouraging. These results show a strong correlation of FNAC with the histopathology of the thyroid lesions. www.pacificejournals.com/apalm

A-247 FNAC should be regarded as an extremely valuable complement to the conventional surgical histopathology and is just indispensible.

Conclusion

FNAC serves as a rapid , convenient and an accurate outpatient method for diagnosis of palpable thyroid lesions than any other combination of clinical laboratory tests. There is almost perfect cytohistopathological concordance with a sensitivity of 96.36% and specificity of 100% as evaluated in this study. These results are consistent with those available in the literature. It also helps as a guide for appropriate therapeutic management to either locally excise a benign tumor or plan for a radical surgery in case of malignancies. Hence, we conclude that FNAC serves as a first line investigation for palpable thyroid lesions and acts as a useful screening test to triage thyroid lesions before surgery.

References 1.

Agrawal R, Saxena M, Kumar P. A Study of Fine Needle Aspiration Cytology of Thyroid Lesions with Histopathological Correlation. Indian Journal of Pathology and Oncology.2015;2(4):277-283

Bakhos R, Selvaggi SM, DeJong S, Gordon DL, Pitale SU, Herrmann M and Wojcik EM. Fine-needle aspiration of the thyroid: Rate and causes of cytohistopathologic discordance. Diagn. Cytopathol.2000;23(4):Â 233â&#x20AC;&#x201C;237

Gamit MJ, Talwelkar SR, Dhruva GA. Histocytological Correlation Study of Thyroid Gland Lesions.International Journal of Science and Research. Nov 2015;4(11):777-780.

4. Yassa L, Cibas ES, Benson CB, Frates MC, Doubilet PM, Gawande AA et al. Long-term assessment of a multidisciplinary approach to thyroid nodule diagnostic evaluation. Cancer Cytopathology. 2007 Dec 25;111(6):508-16. 5.

Gupta M, Gupta S, Gupta VB. Correlation of fine needle aspiration cytology with histopathology in the diagnosis of solitary thyroid nodule. Journal of thyroid research. 2010 Apr 18;2010.

Pandey P, Dixit A, Mahajan NC. Fine-needle aspiration of the thyroid: A cytohistologic correlation with critical evaluation of discordant cases. Thyroid Research and practice. 2012 May ;9(2):32-39

Parikh UR, Goswami HM, Shah AM, Mehta NP, Gonsai RN. Fine needle aspiration cytology (FNAC) study of thyroid lesions (study of 240 cases). GMJ. 2012;67(2):25-8.

Tilak V. Dhaded AV, Jam R. Fine needle aspiration cytology of head and neck masses. Indian J Pathol Microbiol. 2002 Jan; 45(1):23-9.

Kumar SK, Seetharamaiah T, Rampure D, Ramakrishna C, Devi RY. Thyroid nodule: Cytohistological correlation. Scholar J Appl Med Sci.2013;1(6):745-747

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10. Amatya BB, Joshi AR, Singh SK, Panth R, Basnet RB. A study of fine needle aspiration cytology of head and neck masses and their corroboration by histopathology. Post graduate medical journal of national academy of medical sciences. 2009;6(2).

in Chinese patients. Acta Cytologica.1986Dec;31(6): 699-704.

11. Fernandes H, Dâ&#x20AC;&#x2122;Souza CRS, Thejaswini BN. The role of fine needle aspiration cytology in palpable head and neck masses. Journal of clinical and diagnostic research. 2009 Oct; (3):1719-1725. 12. Gagneten CB, Roccataglinta G, Lowenstein A et al. The role of fine needle aspiration biopsy cytology in the evaluation of the clinically solitary thyroid nodule. Acta Cytologica. 1987;31:595-598 13. Hsu CH, Boey J, Diagnostic pitfalls in the fine needle aspiration of thyroid nodules. A study of 555 cases

14. Goellner JR, Gharib H, Grant CS, Johnson DA. Fine needle aspiration cytology of the thyroid,1980 to 1986.Acta Cytologica.1986Dec;31(5): 587-90 15. OreIl SR, Sterrett GF, Walters MN-I, Whitaker D. Manual and atlas of fine-needle aspiration cytology, The thyroid gland. Third edition : Churchill Livingstone, 2003: 110 -140. 16. Orell SR, Sterrett GF, Whitaker D et al: The thyroid gland. In Manual and atlas of fine- needle aspiration cytology, 2nd Edn: Churchill Livingstone,1992:102-104 17. Lowhagen T, Sprenger E. Cytologic presentation of thyroid tumors in aspiration biopsy smear. A review of 60 cases. Acta Cytologica.1974;18(3):192-197.

*Corresponding author: Dr Shakuntala S Aramani, Assistant Professor, Department of Pathology, Gadag Institute of Medical Sciences, Mallasamudra, Gadag-582103.Karnataka, India. Phone: +91 9663935998 Email: shakkuaramani@gmail.com Date of Submission : 11.12.2016 Date of Acceptance : 03.03.2017 Financial or other Competing Interests: None. Date of Publication : 04.06.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Original Article DOI: 10.21276/APALM.1170

Coagulation Abnormalities in Patients with Cirrhosis in A Tertiary Hospital Sangli : A prospective Study Sheetal Mahesh Sale1, Vaibhav Pandurang Mane1*, Vishrabdha Rahul Pawar1, Dajiram G .Mote2, Sushant Narayan Mohiteand1 and Vanisha Dhaka1 Department of Pathology , Bharati Vidyapeeth Deemed University Medical College And Hospital , Sangli. India Department of Surgery , Bharati Vidyapeeth Deemed University Medical College And Hospital , Sangli, India

ABSTRACT Background: Clotting is a multistep process comprised of sequence of events of platelet plug formation , clotting process , clotting process termination and clot removal.Synthesis of clotting factors and clearance of their activation products took place in liver. The magnitude of clinical features and coagulation abnormalities will vary depending on liver dysfunction .Therefore wide spectrum of abnormalities were seen in patients of liver cirrhosis. Aims and Objectives: To study the various coagulation abnormalities in patients of liver cirrhosis. Methodology: This 1 year prospective study was conducted in a tertiary hospital for the evaluation of the frequency of coagulation abnormalities in patients with cirrhosis of liver. 82 patients presenting with cirrhosis of liver were selected and were evaluated for coagulation profile. The data was collected via questionnaire form and analyzed by SPSS (Statistical Packages for Social Sciences) version. Patients blood was tested for coagulation abnormalities including prothrombin time (PT), activated partial thromboplastin time (aPTT) and platelet count. In this study design control group of normal people without cirrhosis was not considered. Results: In the present study, out of 82, fifty (60.9 %) were males and thirty two (39.1 ) % were females. According to Child’s Pughs classification, 37(45.12 % ) cirrhotic patients were in class A, 13 (15.85% ) in class B and 32 (39.02 % ) in class C. The PT was prolonged (mean + SD = 20.67 ± 4.12 sec) in 44 (53.65 % ) patients, while38 (46.34 %) patients had normal PT which was less than 14 seconds (mean + SD = 12.13 ± 1.01sec). Activated partial thromboplastin time was prolonged in47 (57.31 % ) patients, while 35 (42.68 % ) patients had normal APTT which was less than 40 seconds (mean + SD = 33.05 ± 3.06 sec). PT and APTT were significantly raised in cirrhotic patients. Approximately 39% CLD cases had decreased platelet count. Relative risk of GI bleeding with abnormal clotting tests in CLD cases were weakly positive for PT (RR = 1.02; 95% CI, 0.49-2.10), negative for aPTT (RR=0.83; 95% CI, 0.47-1.45), strongly positive for decreased platelet counts (RR = 1.96; 95% CI, 1.08-3.56) . Conclusion: Coagulation abnormalities are commonly seen in cirrhotic liver disease.Decreased platelet count and increased PT and APTT are commonly seen in chronic liver disease. These parameters can be used as prognostic markers. Keywords: Cirrhosis, Prothromobin, Clotting , Coagulation Abnormalities, Chronic Liver Disease,

Introduction

The liver plays a central role in the clotting process. Acute and chronic liver diseases are invariably associated with coagulation disorders due to multiple causes including: decreased synthesis of clotting and inhibitor factors, decreased clearance of activated factors, quantitative and qualitative platelet defects, hyperfibrinolysis, and accelerated intravascular coagulation. [1,2,3,4,5 ] Most coagulation factors are produced in liver, but the response of each factor to liver disease is variable due to differences in biologic half lives and acute phase reactions. [5,6] The PT is usually prolonged first, then APTT. Factor VII: shortest biologic half life, often affected earliest with largest decrease in plasma level. Factor VII also decreases

earliest with warfarin treatment. Factor VIII: may be normal or elevated due to acute phase reactants. Factors XI and XII:have long biologic half lives, and may be normal until liver disease is advanced. [7,8,] However the D-Dimer is usually normal which is helpful to differentiate liver disease from DIC.[9,10,] The bleeding tendency accounts for increased risk of morbidity and mortality in patients with liver disease undergoing diagnostic or therapeutic invasive procedures. [11] Coagulation disorders in liver disease are extensive, complex and expensive to treat. Many mechanisms can cause the coagulation changes, but many can be attributed to cytokine activation. Sepsis further impairs hemostasis in patients with liver cirrhosis bleeding from esophageal

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varices. Thrombotic events, even if rare in cirrhotic patients, can occur.[12,13 ] In liver disease the platelet count is either normal or decreased. The liver is a source of thrombopoietin which stimulates platelet production. Mild splenomegaly may be present in cirrhosis resulting in platelet pooling. Alcohol intake inhibits the production of platelets by megakaryocytes. Folate deficiency, which may accompany cirrhosis may also cause thrombocytopenia. [14] The prolonged PT is related to the severity of liver failure and is one of the parameter used in commonly used prognostic indices of chronic liver disease such as ChildPugh or Mayo End-Stage Liver Disease (MELD) scores.[6,7] The PT is considered as a simple, inexpensive, qualitative and accurate prognostic marker of liver impairment and also a predictor of bleeding.[1] The degree of PT impairment an expression of decreased liver synthesis predicts the severity of portal hypertension and the presence of esophageal varices.[6] PT is related both to bleeding risk and mortality. Patients with moderately or severely prolonged PT have 5 to 10 fold higher mortality rates than patients with normal PT.[8] The aPTT is also prolonged in advance chronic liver disease.[9,16,17] To study the alteration in coagulation profile in cirrhotic liver diseases which helps to evaluate the risk of bleeding in patients with liver disease and to study the association of coagulation abnormality with the extent of liver disease.

Materials and Methods

This one year prospective study was conducted at a tertiary hospital .Confirmed cases of cirrhosis (by clinical, biochemical, radiological and prior biopsy if done as described earlier) were selected, both sexes with age >14 years (to exclude pediatric age group). The exclusion criteria of the study were, patients of cirrhosis with a previous history of coagulation disorders and drug intake that causes changes in the coagulation parameters e.g. Oral

Contraceptive, aspirin, heparin, warfarin, etc, pregnant ladies and other liver disease patterns (non-cirrhotic liver disorders). All cirrhotic patients admitted t, that meets the inclusion and exclusion criteria were enrolled in this study. At first an adequate history was taken regarding hematemesis, melena, hemoptysis and hematuria, the presence of petechial hemorrhages or bruises. A focused clinical examination was done regarding the presence of petechial hemorrhages, bruises, the presence of ascites, the grade of hepatic encephalopathy (according to West Haven System) and per rectal examination was done to assess the presence of melena. The clinical examination was focus on bleeding tendencies and the clinical parameters of Child Pugh Class of severity of cirrhosis of liver. Secondly blood tests were done for platelet count, PT and APTT. All the data was collected on predesign proforma and statistical package for social sciences (SPSS) version 16 was used for data processing purpose. Frequencies and means ± Standard Deviation of data like age PT and APTT was calculated. All values considered significant when p value is 0.05. Blood samples from the patients were collected and following three coagulation tests were performed. 1) PT. 2) APTT 3) Platelet count. Staging of chronic liver diseases is done by modified Child-Pugh classification with a scoring system of 5- 15. The Child-Pugh score is calculated by adding the scores of the five factors and can range from 5-15. (table-I) Recently the model for end stage liver disease (MELD) score is used for assessing the need for liver transplantation. MELD score= 9.57xlog ecreatinine(mg/dl)+3.78x log e bilirubin(mg/dl)+11.20xlog e International normalized ratio(INR)+6.43 A comprehensive history including presenting complaints, past history, family history, drug history was taken by asking questions through a structured questionnaire. Thorough, physical examination was performed and

Table 1: Child – Pugh classification of cirrhosis Factors

Units

Sr.Bilirubin

Mg/dl

< 2.0

2.0 to 3.0

>3.0

Sr.Albumin

g/dl

>3.5

3.0 to 3.5

< 3.0

Seconds prolonged

4- 6

International normalized ratio (INR)

<1.7

1.7 - 2.3

>2.3

Ascites

None

Easily controlled

Poorly controlled

Hepatic encephalopathy

None

Minimal

Advanced

Prothrombin time

StageA-score5-6. Stage B-score7-9. Stage C-score10 or more. Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Mahesh Sale et al.

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clinically CLD suspected patients were investigated for the confirmation of chronic liver disease through Liver Function Test (LFT) and ultrasound abdomen. The criteria used for the diagnosis of CLD were based on: Presence of cardinal signs and symptoms including ascites, hepatomegaly, splenomegaly, jaundice, palmar erythema, clubbing, leukonychia, oedema, pubic hair loss, spider naevi, purpura, gynaecomastia, flapping tremors, drowsiness and coma. Ultrasonoghaphy revealing coarse hepatic texture, change in liver size, dilated portal vein and splenomegaly.

A, 13 (15.85% ) in class B and 32 (39.02 % ) in class C. The PT was prolonged (mean + SD = 20.67 ± 4.12 sec) in 44 (53.65 % ) patients, while38 (46.34 %) patients had normal PT which was less than 14 seconds (mean + SD = 12.13 ± 1.01sec). Activated partial thromboplastin time was prolonged in47 (57.31 % ) patients, while35 (42.68 % ) patients had normal APTT which was less than 40 seconds (mean + SD = 33.05 ± 3.06 sec). PT and APTT were significantly raised in cirrhotic patients.

This one year study was carried out in a tertiary hospital .82 patients presenting with cirrhosis of liver were selected for the analysis of coagulation profile.

Approximately 39% CLD cases had decreased platelet count. Relative risk of GI bleeding with abnormal clotting tests in CLD cases were weakly positive for PT (RR = 1.02; 95% CI, 0.49-2.10), negative for aPTT (RR=0.83; 95% CI, 0.47-1.45), strongly positive for decreased platelet counts (RR = 1.96; 95% CI, 1.08-3.56) .

In the present study, out of 82, fifty (60.9 %) were males and 32 (39.1 ) % were females. According to Child’s Pughs classification,37(45.12 % ) cirrhotic patients were in class

Bleeding was present in 21 out of 32 cases in which platelet count was altered and 10 out of 50 cases in which platelet count was normal

Results

Table 2: Cirrhotic patients in relation to prothrombin time. Prothrombin time (Normal range: 12-14 sec) Normal Prolonged -3 4 -5 +6 Total

Frequency

40 42 15 19 08 82

48.4 50.82 18.15 22.99 9.68 100

P-value: 0.001 (Significant)

Table 3: Cirrhotic patients in relation to activated partial thromboplastin time. Activated partial thromboplsatin time (Normal range: 30-40sec) Normal Prolonged Total

Frequency

39 43 82

47.19 52.03 100

No. of patients 50 21 11 82

Percentage 60.5 25.41 13.31 100

P-value = 0.001 (Significant)

Table 4: Cirrhotic patients in relation platelet count. Platelet count /cmm Normal 50000 to 1 lakh Less than 50,000 Total

Table 5 . Distribution of cases according to Child-Pugh class in cirrhosis Child – Pugh Class Class A Class B Class C Total

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Number of cases 37 13 32 82

Percentage of cases 45.12 % 15.85 % 39.02 % 100 .00

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Table 6. Comparison of percentage of cases with altered coagulation profile in cirrhosis with previous studies Studies

Percentage of cases with altered coagulation profile Altered PT

Altered APTT

Altered Platelet

Siddiqui et al

87.7%

71.3 %

36.8

Nwokediuko et al

36.6 %

22.6 %

42.7 %

79 %

42 %

11 %

Nieuwenhuizen et al Devrajani et al

50.85 %

51.7%

Present Study

50.82 %

52.03 %

39 %

Discussion

The clotting is a multistep process comprised of sequence of events of platelet plug formation , clotting process , clotting process termination and clot removal .Synthesis of clotting factors and clearance of their activation products took place in liver. The magnitude of clinical features and coagulation abnormalities will vary depending on liver dysfunction . Therefore wide spectrum of abnormalities will be seen in patients of liver cirrhosis This study revealed that males suffer slightly more with chronic liver disease than females (male/female ratio was 1.56:1), more so, in the age group between 50 -70 years Prolong PT has been associated with an increased risk of gastrointestinal bleeding in chronic liver disease.[17,19] This, study reported that majority of CLD patients had raised PT) and presented with GI bleeding .Although relative risk of GI bleeding with elevated PT was not statistically significant. Elevated aPTT was also not found to be associated with GI bleeding in this study. Possible explanation for this result may be because of their limitation, PT and aPTT only measure procoagulants factors, they do not measure anticoagulants factors (protein C and antithrombin) and the normal haemostasis is the strict balance between procoagulant and anticoagulant factors.[3] Other likely causes of prolonged PT are oral anticoagulation, vitamin K deficiency and for prolonged aPTT are heparin contamination and non-specific inhibitors (lupus coagulants).[5] Nevertheless, these parameters do give a good estimate of the synthetic function of the liver and may still be used as a prognostic marker.[6,7] Bleeding is a frequent and often severe complication of liver cirrhosis. The most frequent and potentially lifethreatening site is the gastrointestinal tract. Haemodynamic alterations secondary to portal hypertension are considered the main cause of GI bleeding in cirrhotics.[20,21] Nonhaematological factors may also contribute to the bleeding diathesis in chronic liver disease. These include portal hypertension, renal failure and bacterial infection.[22]

In liver disease the platelet count is either normal or decreased. The liver is a source of thrombopoietin which stimulates platelet production . Mild splenomegaly may be present in cirrhosis resulting in platelet pooling. Alcohol intake inhibits the production of platelets by megakaryocytes. Folate deficiency, which may accompany cirrhosis may also cause thrombocytopenia. The bleeding tendency accounts for increased risk of morbidity and mortality in patients with liver disease undergoing diagnostic or therapeutic invasive procedures. Coagulation disorders in liver disease are extensive, complex and expensive to treat. Many mechanisms can cause the coagulation changes, but many can be attributed to cytokine activation. Sepsis further impairs hemostasis in patients with liver cirrhosis bleeding from esophageal varices. Thrombotic events, even if rare in cirrhotic patients, occur mainly in the portal and mesenteric veins. Liver Disease is a complex disorder that impacts coagulation testing. It must be diagnosed cost effectively and treated appropriately.

Conclusion

The study is concluded with the fact that the coagulation profile was significantly altered in cases of cirrhosis.The alteration of coagulation profile was not significant in cases of hepatitis and other liver diseases.The risk of bleeding was more in the cases of cirrhosis with altered coagulation profile.Coagulation abnormalities were significantly associated with the extent of liver disease.By earlier identification of patients at risk of bleeding, early treatment can be started.

Acknowledgement

The authors are thankful to the Department of Medicine, Technicial Staff , BVDUMC & H, Sangli, for their help and cooperation

References 1.

Amitrano L, Guardascione MA, Brancaccio V, Balzano A. Coagulation disorders in liver disease. Semin Liver Dis 2002; 22: 83-96.

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Peck Radosavljevic M. Review article: coagulation disorders in chronic liver disease. Aliment Pharmacol Ther 2007; 26 Suppl 1: 21-8.

Rverter JC. Abnormal hemostasis tests and bleeding in chronic liver disease: are they related? Yes. J Thromb heamost 2006; 4: 717-20.

Tripodi A. Tests of coagulation in liver disease. Clin Liver Dis 2009; 13: 55-61.

Thachil J. Relevance of clotting tests in liver disease. Postgrad Med J 2008; 84: 177-81.

Pugh RN, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973; 60: 649-9.

Formen LM, Lucey MR. Predicting the prognosis of chronic liver disease: an evolution from child to MELD. Mayo endstage liver disease. Hepatology 2001; 33: 473-5.

Garrison RN, Cryer HM, Howard DA, Polk HC. Clarification of risk factor for abdominal operations in patients with hepatic cirrhosis. Ann Surg 1984; 199: 648-55.

Hedner U, Erhardtsen E. Hemostatic disorders in liver disease. In: Schiff ER, Sorrell MF, Maddrey WC, editors. Diseases of the liver. Philadelphia: Lippincott Williams and Wilkins; 2003; pp 625-35.

10. Lechner K, Niessner H, Thaler E. Coagulation abnormalities in liver disease. Semin Thromb Hemost 1977; 4: 40-56.

A-253 14. Clinical and Laboratory Standards Institute (CLSI). Collection, transport and processing of blood specimens for testing plasma-based coagulation assays and molecular hemostasis assays; approved guideline. 5th ed.H21-A5. Pennsylvania USA; 2008. 15. Clinical and Laboratory Standards Institute (CLSI). Procedure for the Determination of Fibrinogen in Plasma; Approved Guideline.2nd ed.H30-A2.Pennsylvania, USA; 2001. 16. Bukhtiari N, Hussain T, Iqbal M, Malik AM, Qureshi AH, Hussain A. Hepatits B and C single and co-infection in Chronic liver disease and their effect on the disease pattern. J Pak Med Assoc 2003; 53: 136-40. 17. Nidegger D, Ragot S, Berthelemy P, Masliah C, Pilette C, Martin T, et al. Cirrhosis and bleeding: the need for very earlymanagement. J Hepatol 2003; 39: 509-14. 18. Patch D, Armonis A, Sabin C, Christopoulou K, Greenslade L, McCormick A, et al. Single portal pressure measurement predicts survival in cirrhotic patients with recent bleeding. Gut 1999; 44: 264-9. 19. Lecleire S, Di Fiore F, Merle V, Herve S, Duhamel C, Rudelli A, et al. Acute upper gastrointestinal bleeding in patients with liver cirrhosis and in noncirrhotic patients: epidemiology and predictive factors of mortality in a prospective multicenter population-based study. J Clin Gastroenterol 2005; 39: 321-7.

11. Bashour FN, Teran JC, Mullen KD. Prevalence of peripheral blood cytopenias (hypersplenism) in patients with nonalcoholic chronic liver disease. Am J Gastroenterol 2000; 95: 2936-9.

20. Escorsell A, Bordas JM, Castaneda B, Liach J, Garcia-Pagan JC, Rodes J, et al. Predictive value of the variceal pressure response to continued pharmacological therapy in patients with cirrhosis and portal hypertension. Hepatology 2000; 31: 1061-7.

12. Schepis F, Camma C, Niceforo D, Magnano A, Pallio S, Cinquegrani M, et al. Which patients with cirrhotic should undergo endoscopic screening for esophageal varices detection? Hepatology 2001; 33: 333-8.

21. Dellâ&#x20AC;&#x2122;era A, Bosch J. Review article: the relevance of portal pressure and other risk factors in acute gastro-oesophageal variceal bleeding. Aliment Pharmacol Ther 2004; 20 Suppl 3: 8-15.

13. Zaman A, Hapke R, Flora K, Rosen HR, Benner K. Factors predicting the presence of esophageal or gastric varices in patients with advanced liver disease. Am J Gastroenterol 1999; 94: 3292-6.

22. Lisman T, Bongers T, Adelmeijer J, Janssen H, de Maat M, de Groot P, et al. Elevated levels of von Willebrand factor in cirrhosis support platelet adhesion despite reduced functional capacity. Hepatology 2006; 44: 53-61.

*Corresponding author: Dr. Vaibhav Pandurang Mane, Flat No. 1 Shri Ramshailya Apartment, Neminath nagar, Vishrambag , Sangli. India Phone: +91 9422041490 Email: vaishnavilab1060@gmail.com Date of Submission : 31.01.2017 Date of Acceptance : 17.02.2017 Financial or other Competing Interests: None. Date of Publication : 15.06.2017

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eISSN: 2349-6983; pISSN: 2394-6466

Original Article DOI: 10.21276/APALM.1180

Evaluation of Pattern of Angiogenesis in Various Menstrual Disorders in Different District in Gujarat, India Killol Nathubhai Desai1* and Vidya Satapara2 Department of Pathology, GMERS Medical College, Junagadh, Gujarat, India 2 Department of Anatomy, GMERS Medical College, Junagadh, Gujarat, India

ABSTRACT Backgound: Menstrual disturbance is one of the commonest gynecological problems. Majority of these women have dysfunctional uterine bleeding (DUB), the rest being associated with leiomyomas, carcinomas, adenomyosis, endometrial polyps, hyperplasia and oral contraceptive uses. Aims: To study of alteration in blood vessels morphology in various menstrual disorders have significant role in prognosis and in various anti-angiogenic therapies. Methods and Material: A Cross-sectional study done at Anand, Surendranagar and Junagadh district from October 2011 to October 2016. 1867 endometrial specimens were studied to document the changes in blood vessels in various phases of menstrual cycle, menstrual disturbances and in unexplained infertility. 761 cases were taken as control and 1106 cases as study group which included cases of dysfunctional uterine bleeding (DUB), endometrial polyps, fibroids, adenomyosis, infertility, atrophic endometrium, endometrial hyperplasia, pill endometrium and carcinomas. Using light microscopy, the vascular morphology was studied. Statistical analyses were performed using the IBM SPSS (Statistical package for the Social Sciences v15.0) and Microsoft Excel 2007 software. Results: The blood vessels were concentrated more in basal layer in the proliferative phase and in functional layer in the secretory phase. Cases of complex hyperplasia and pill endometrium had significantly higher vessel concentration. Congestion and dilatation of blood vessels were significantly higher in cases of DUB. Conclusions: The present study showed a positive correlation between endometrial angiogenesis and menstrual disorders. The alteration in blood vessel morphology has significant role in prognosis and in various anti-angiogenic therapies. Keywords: Angiogenesis, Menstrual Disorders, Dysfunctional Uterine Bleeding, Endometrium

Introduction

Menstrual disturbance is one of the commonest gynecological problems for which a curettage or hysterectomy specimen is received by the pathologists. Majority of these women have dysfunctional uterine bleeding (DUB) of unknown pathology, the rest being associated with leiomyomas, carcinomas, adenomyosis, endometrial polyps, hyperplasia and oral contraceptive uses.[1] Poor understanding of the mechanisms underlying spontaneous and induced menstrual disturbances has hindered the development of non-surgical therapies for these complaints. Another problem encountered is that of unexplained infertility. Although some ultrastructural studies have been carried out in the past, they cannot form a part of routine endometrial evaluation in the infertility and other gynecological disorders. Therefore, the present study employed the routinely used stains to evaluate the number and morphology of endometrial blood vessels

associated with benign gynecological conditions resulting from menstrual disturbances and infertility.

Material and Methods

This study was carried out in three different Medical Colleges from October 2011 to October 2016 like 1) Department of Pathology, Pramukhswami Medical College, Karamsad, Anand, Gujarat, India, 2) Department of Pathology, C.U. Shah Medical College, Surendranagar, Gujarat, India and 3) Department of Pathology, GMERS Medical College, Junagadh, Gujarat, India. (Table 1) A total of 1867 endometrial specimens obtained by hysterectomy and dilatation and curettage (D and C) were included in the study. 761 cases were taken as control and 1106 cases as study group ( Control taken from hysterectomy specimens which are operated for non-hormonal conditions, due to lesson of hysterectomy specimen for control in my time duration and further no necessity of same case and control group). In the control group, endometrial specimens were

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obtained from the hysterectomy specimens performed for non-hormonal indications like prolapse, endometrial polyps, fibroids and adenomyosis in young patients in the age group of 28-40 years who had a normal and regular menstrual cycle and from endometrial curettage performed along with cervical biopsies with no suspicion of hormonal endometrial pathology in young patients in the age group of 28-40 years. We are only used specimens which are received in our histopathology laboratory for diagnosis purpose and HREC of Pramukhswami Medical College, Karamsad, Anand, Gujarat, India give permission with waiver of consent. So consent of patient is not required in my study. The study group included cases of DUB and local uterine lesions like, endometrial polyps, fibroids and adenomyosis. Cases of infertility and atrophic endometrium were also studied. The pertinent patient details like age, hospital, menstrual history, obstetric history, chief complaints, history of hormonal use and the clinical diagnosis of the patient were also studied. Required sections were taken from the hysterectomy specimens. Specimens of D and C and suction-evacuation were examined for adequacy and those containing only blood were not included in the study. The tissues were fixed in 10% formalin and processed routinely. Sections of 3 micron thickness were stained with Hematoxylin and Eosin (H and E) stain. Wherever required van Gieson stain was done. The appropriate controls were used with each batch of van Gieson stain. Van Gieson stain used for staining of collagen and other connective tissue, so it is used in endometriosis, stromal endometrial fibrosis, periglandular and perivascular fibrosis. Sections stained by H and E stain were used for dating of the endometrium and evaluation of various characteristics of endometrial blood vessels with respect to number, congestion and degree of dilatation. The overall vascularity of the endometrium was judged by counting the average number of blood vessels seen in 10 high-power fields (HPFs) in basal layer and functional layer separately and compared with that of the controls. As the curettages contained endometrium of the functional layer, an average was calculated after observing 10 HPFs. The statistical test used for comparison was Student’s unpaired t-test. P value less than 0.05 was considered significant. Vascular congestion was noted for its presence or absence. The degree of dilatation was graded as mild and moderate. These values were compared with that of the controls. The statistical test used for comparison was Z test. P value less than 0.05 was considered significant.

menstrual cycle in the basal and functional layers (P < 0.05). The blood vessels are mainly concentrated in the basal layer in the proliferative phase and were concentrated more in the functional layer in the secretary phase [Figures 1 and 2]. However, there was no statistically significant difference between the average endometrial (basalis + functionalis) blood vessel concentration per HPF in various phases of menstrual cycle. The study group included 1106 endometrial specimens of DUB, of which 369 were hysterectomy and 737 were D and C specimens. Some of the specimens also associated with atrophic uterus (14%), leiomyoma (13%), infertility (4%), polyps (3%) and adenomyosis (3%). In DUB, according to the histopathology diagnosis, the hysterectomy and D and C cases were distributed as in Table 2. The mean of the blood vessels per HPF in early proliferative, late proliferative, early secretory, mid secretory and late secretory were calculated together and placed into a group endometrium (proliferative + secretory) and compared with the control as in Table 4. Other histopathology categories included cystic glandular hyperplasia (CGH), simple hyperplasia, complex hyperplasia and pill endometrium. There was no statistically significant difference in blood vessel density in the hysterectomy specimens for DUB and control group. In D and C specimens, there was no significant difference between the study and control mean of average blood vessels per HPF in the group endometrium (proliferative + secretory), simple hyperplasia and CGH. The average blood vessels per HPF were significantly higher in complex hyperplasia (4.01 ± 0.12), pill endometrium (6.42 ± 0.67) and endometrial carcinoma (4.37 ± 0.093) as compared to control (3.80 ± 0.42), P < 0.01, Table 4). The number of cases in the group endometrium (proliferative + secretory) showed congestion was 43.58% as compared to 11.2% cases in the control group which was significantly higher (P < 0.001). Cases of CGH and simple hyperplasia that showed congestion were significantly higher (55.3%), when compared to 11.2% control cases (P < 0.001). Seventy-five percent (75%) of cases of complex hyperplasia showed significant congestion (P < 0.001). Cases of pill endometrium also showed significant congestion (P < 0.001).

In 761 cases of the control group, average endometrial blood vessels per HPF in hysterectomy specimens are given in Table 3. There was statistically significant difference in endometrial blood vessel density in different phases of

The number of cases showing mild dilatation in the group endometrium (proliferative + secretory) was 23.4% and moderate dilatation was 6.4%. Both these figures were significantly higher as compared to control (P < 0.001). The number of CGH and simple hyperplasia cases showing mild dilatation was 30.5% and moderate dilatation was 9.5%. These values were also significantly higher as compared to

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control [Figure 4]. In complex hyperplasia, mild dilatation and moderate dilatation were significant as compared to control. The percentage of pill endometrium cases showing mild blood vessel dilatation was 57.5% which was also significantly higher than in control [Figure 3]. In cases of fibroids (13%), the mean of the blood vessels in the stratum basalis and stratum functionalis was not significantly different as compared to the controls. The endometrial blood vessel congestion was 13.2% as compared to control (12.7%) which was not significantly different (P > 0.05). No significant difference in dilatation was seen between the two groups. In the cases of adenomyosis (3%) were included in the study. All were hysterectomy specimens. No significant difference was observed in the mean of blood vessel concentration in adenomyosis as compared to control.

In the cases of endometrial polyp (3%) were studied. The mean blood vessel concentration in polyp was (5.12 ± 0.77) which was significantly higher (P < 0.05) as compared to control. The stalk of the endometrial polyp showed the presence of large, thick-walled muscular arteries with wellcollagenized adventitial coat brightly staining red by van Gieson stain. Prominent arterioles were also seen beneath the surface lining epithelium. In the cases of infertility (4%) were included in the study. All were D and C specimens. There was no significant difference between the mean blood vessel concentration of control and infertility cases. None of the cases showed blood vessel congestion or dilatation. In the cases of atrophic endometrium (14%) were studied. The mean of average blood vessel concentration was not significantly different when compared to control.

Table 1: District wise case and control District

Case

Control

Total

Anand

533

303

836

Surendranagar

206

201

407

Junagadh

367

257

324

Total

1106

761

1867

Table 2: Histopathology diagnosis-wise distribution of dysfunctional uterine bleeding group (Case) Histopathology diagnosis

Hysterectomy (n)

D&C (n)

Total

Early proliferative

126

178

Late proliferative

126

156

Early secretory

134

Mid secretory

113

146

Late secretory

Cystic glandular hyperplasia

Simple endometrial hyperplasia (Typical+Atypical)

194

Complex endometrial hyperplasia (Typical+Atypical)

Pill endometrium

Endometrial carcinoma

Total

369

737

1106

Table 3: Average endometrial blood vessels in control group Histopathology diagnosis

Average blood vessels/HPF* (Mean) Basalis

Functionalis

Basalis + Functionalis (Mean)

Early proliferative

3.85 ± 0.2261

2.60 ± 0.4200

3.23 ± 0.3231

Late proliferative

3.62 ± 0.3881

3.68 ± 0.2666

3.65 ± 0.3274

Early secretory

3.28 ± 0.2865

4.0 ± 0.3331

3.64 ± 0.3098

Mid secretory

3.21 ± 0.4412

3.60 ± 0.6832

3.41 ± 0.5622

Late secretory

2.62 ± 0.5212

3.70 ± 0.7000

3.16 ± 0.6106

Total Mean

3.31 ± 0.3726

3.51 ± 0.4805

3.41 ± 0.4266

*HPF- High Power Field

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Table 4: Comparison of average endometrial blood vessels per high power field in case of Dysfunctional uterine bleeding. Mean

Histopathology diagnosis

p-Value

DUB*

Control

Proliferative + secretory endometrium

3.84 ± 0.34

3.80 ± 0.42

>0.05 NS***

Simple endometrial hyperplasia (Typical+Atypical)

3.92 ± 0.42

3.80 ± 0.42

>0.05 NS***

Cystic glandular hyperplasia

3.90 ± 0.22

3.80 ± 0.42

>0.05 NS***

Complex endometrial hyperplasia (Typical+Atypical)

4.01 ± 0.12

3.80 ± 0.42

<0.01 HS***

Pill endometrium

6.42 ± 0.67

3.80 ± 0.42

<0.001 VHS***

Endometrial carcinoma

4.37 ± 0.09

3.80 ± 0.42

<0.01 HS***

*DUB - Dysfunctional uterine bleeding, **CGH - Cystic glandular hyperplasia, *** NS - Not significance, HS=High significance, VHS=Very high significance

Fig. 1: Microphotograph of Secretory endometrium (H&E stain 10x view).

Fig. 2: Microphotograph of Proliferative endometrium (H&E stain 10x view).

Fig. 3: Microphotograph of Hormonal induced changes of endometrium (Pill endometrium) (H&E stain 10x view)

Fig 4: Microphotograph of Simple glandular hyperplasia of endometrium (H&E stain 10x view).

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Discussion

Endometrial angiogenesis is characterized by proliferation of vascular endothelial cells during proliferative phase, coiling of arterial system in secretory phase and repair of vascular bed during menstruation. Excessive or insufficient vascular growth contributes to numerous benign disorders. In many angiogenesis related diseases, there is defective remodeling of vascular bed and changes in vascular fragility. [2] In the present study, the endometrial blood vessels were evaluated using light microscopy on sections stained by H and E stain. This method was chosen because in the routine set-up, it provides maximum information and utility and can be readily applied in the study of endometrial vasculature as observed by Upadhyay and Mishra[3] and Sahasrabudhe et al.[4] The method used for the counting of blood vessels was obtained from Hourihan et al.[5] and Sahasrabudhe et al.[4] The endometrium in the control group was dated as early proliferative, late proliferative, early secretory, mid secretory and late secretory. The average number of blood vessels per HPF for hysterectomy cases was 3.54 Âą 0.20, which was comparable to that observed by Sahasrabudhe et al.[4] but lower as compared to the study of Shaw et al.[6] The blood vessels were concentrated more in the basal layer in proliferative endometrium and were distributed more in the functional layer by mid to late secretory phase, which was in agreement with those of Fanger and Barker,[7] Sahasrabudhe et al.,[4] and Nayha et al.[8] This is because, the spiral arterioles are extremely sensitive to the changes in levels of ovarian hormones and changes occur in the vessel density as the cycle progresses. However, there was no significant change in the average density of blood vessels (basalis + functionalis) during the various phases of the menstrual cycle like in the study done by Shaw et al. [6] and Rees et al. [9] This is because other elements such as glandular and stromal cells also increase in size and number in proportion to vascular growth in maturing endometrium and the relative number of blood vessels remains constant. [6] In cases of DUB, the mean blood vessels per HPF in complex hyperplasia were significantly higher as compared to control (P < 0.001) as observed by Abulafia et al.[10] This is due to significant increase in endothelial cell proliferation in the endometria of women with menorrhagia.[11] Similar finding was noted by Nayha et al. and they indicated that this microvascular density is associated with malignant transformation later.[8] The difference in the blood vessel mean (6.21 Âą 0.27)of pill endometrium as compared to control (3.86 Âą 0.36) was significantly higher as claimed by

Hourihan et al.[5] and Hickey et al.[12] However, the blood vessels were thin walled and congested. This effect reflects a combined estrogen progesterone effect which typically produces a weakly secretory pattern characterized by under developed non-coiled glands set within a spindled, vaguely predeciduated stroma containing thin-walled vascular channels. The increased density of blood vessels combined with the fragile nature of these vessels may be responsible for the hormone-induced breakthrough bleeding. Therefore, except for pill endometrium and complex hyperplasia, no significant difference was found in the concentration of blood vessels in curettage and hysterectomy specimens of DUB cases as compared to control which was in agreement with those of Rees et al., [9] Hourihan et al., [5] Sahasrabudhe et al., [4] and Mints et al. [2] Therefore, it is possible that excessive bleeding in DUB may be related to other qualitative changes in the blood vessels. Congestion was significantly higher in cases of DUB in the group endometrium (proliferative + secretory), CGH, complex hyperplasia and pill endometrium when compared with the controls . Vascular dilatation was graded as mild and moderate. Mild dilatation was significantly increased in cases of group endometrium, CGH, complex hyperplasia and pill endometrium. Moderate dilatation was also increased in cases of group endometrium and CGH. Sippe noted the presence of dilated vascular sinuses in 77% of cases of endometrial hyperplasia.[13] In the present study, such sinuses were found in 59.53% of cases including CGH and complex hyperplasia and 25.8% of cases of group endometrium (proliferative + secretory). This lower incidence is because unless the curettage or hysterectomy is carried out during an actual bleeding episode, a certain number of cases will not show this vascular changes. [13] However, similar lower incidence of dilated vascular channels (25%) was observed by Sahasrabudhe et al.[4] Thus, it is possible that the various morphological changes in the endometrial vasculature in DUB and rupture of the dilated and congested vascular channels could be responsible for the abnormal uterine bleeding. In cases of fibroids, there was no significant difference in the mean of the blood vessels, congestion or dilatation when compared with the controls as seen in the study carried out by Sahasrabudhe et al. [4] This is in contrast to the findings of Farrer-Brown et al., where dilated channels were noted in all cases of leiomyomatous uteri, [14] which was an injection study that might have distended the normally collapsed vascular channels by the injection material.[4]

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The cases of adenomyosis showed reduced vascularity in the adenomyotic foci as compared to the controls which was in agreement with that of Beilby et al. [15] This is contradictory due to the fact that adenomyosis is associated with abnormal uterine hemorrhage in a significant number of cases. In cases of endometrial polyp, the average blood vessel concentration was significantly higher as compared to control. This was in agreement with that of Beilby et al.[15] and Sahasrabudhe et al.[4] The large thick-walled muscular arteries with well-collagenized adventitial coat highlighted by van Gieson stain, which are never seen normally in the endometrium, were present mainly in the stalk of the polyp. Prominent arterioles were seen even beneath the surface lining epithelium. These morphological features find diagnostic application especially when a fragmented polyp is curetted. In the cases of infertility showed no statistically significant difference in the mean of blood vessels, congestion, or dilatation when compared with the control group. Thus, the present study could not establish inadequate vascularization of the endometrium as one of the factors involved in unexplained infertility as observed by Sahasrabudhe et al.[4] Deficient blood flow to the endometrium as studied by Edi-Osagie et al. and Steer et al., might be the reason in such cases.[16,17] In atrophic endometrium, mean of average blood vessel concentration was slightly lower as compared to control, but not statistically significant that was also observed by Hickey et al.[18] The endometrial glands were lined by mitotically inactive bland epithelium in a similar spindled inactive stroma. In some cases, the glands were cystically dilated and lined by cuboidal to flattened epithelium. These effects are due to withdrawal of endogenous ovarian steroids on the endometrial blood vessels.

Conclusion

The endometrial blood vessels showed characteristic changes in various phases of menstrual cycle. They were concentrated more in basal layer in the proliferative phase and were distributed more in functional layer in the secretory phase. The average endometrial blood vessels/ HPF in complex hyperplasia and pill endometrium were significantly higher when compared with the controls. The endometrial blood vessel congestion was significantly high in the group endometrium (proliferative + secretory), complex hyperplasia and pill endometrium when compared to the controls. The endometrial blood vessel dilatation was significantly high in the group endometrium (proliferative + secretory), complex hyperplasia and pill endometrium when compared with the controls. www.pacificejournals.com/apalm

The present study showed a positive correlation between endometrial angiogenesis and menstrual disorders. In the present era of newer anti-angiogenic therapies, endometrial angiogenesis and alteration in vascular morphology definitely has prognostic significance and thus helps to improve the treatment modalities and patient care. Early diagnosis of menstrual disorders improve patient’s symptoms, signs and newer anti-angiogenic therapies very effective at early stage of menstrual disorders. They also decrease probability of operative treatment procedure.

References

1. Sherman ME, Mazur MT, Kurman RJ. Benign diseases of the endometrium. In: Kurman RJ. Blaustein’s pathology of the female genital tract, 5th ed. USA: Springer; 2002. 421-66. 2.

Mints M, Blomgren B, Falconer C, Fianu-Jonasson A, Plamblad J.Microvascular density, vascular endothelial growth factor A and its receptors in endometrial blood vessels in patients with menorrhagia. Fertil Steril 2005;84:692-700.

Upadhyay SN, Mishra J. Observations on histopathological changes in the uterus in dysfunctional uterine bleeding. J Obstet Gynaecol India 1963;13:531-41.

4. Sahasrabudhe NS, Dalal SR, Jadhav MV. Endometrial blood vessels in health and disease. Med J West India 2000;28:27-31. 5. Hourihan HM, Sheppard BL, Bonnar J. A morphometric study of the effect of oral norethisterone or levonoregestrel on endometrial blood vessels. Contraception 1986;34:603-12. 6.

Shaw ST, Macaulay LK, Hohman WR. Vessel density in endometrium of women with and without intrauterine contraceptive devices: a morphometric evaluation. Am J Obstet Gynecol 1979;135:202-6.

Fanger H, Barker BE. Capillaries and arterioles in normal endometrium. Obstet Gynaecol 1961;17:543-50.

Nayha V, Viitanen T, Stenback F. Altered extent, pattern and characteristics of microvascular density are indicators of Neoplastic progression in the endometrium. Int J Cancer 2005;115:975-80.

Rees MC, Dunhill MS anderson AB, Turnbull AC. Quantitative uterine histology during the menstrual cycle in relation to measured menstrual blood loss. Br J Obstet Gynaecol 1984;91:662-6.

10. Abulafia O, Triest WE, Sherer DM, Hansen CC, Ghezzi F. Angiogenesis in endometrial hyperplasia and stage I endometrial carcinoma. Obstet Gynecol 1995;86:479-85. 11. Abulafia O, Sherer DM. Angiogenesis of the endometrium. 1999;94:148-53. 12. Hickey M, Fraser IS. Clinical implications of disturbances of uterine vascular morphology and function. Baillière’s Clin Obstet Gynecol 2000;14:937-51.

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13. Sippe G. Endometrial hyperplasia and uterine bleeding. J Obstet Gynaecol 1962;69:1015-9. 14. Farrer-Brown G, Beilby JOW, Tarbit MH. The vascular patterns in myomatous uteri. J Obstet Gynaecol Br Commonw 1970;77:967-75. 15. Beilby JO, Farrer-Brown G, Tarbit MH. The microvasculature of common uterine abnormalities, other than fibroids. J Obstet Gynaecol Br Commonw 1971;78:361-8. 16. Edi-Osagie EC, Seif MW, Aplin JD, Jones CJ, Wilson G, Lieberman BA. Characterizing the endometrium in

unexplained and tubal factor infertility: a multiparametric investigation. Fertil Steril 2004:82:1379-89. 17. Steer CV, Tan SL, Mason BA, Campbell S. Midluteal-phase vaginal color doppler assessment of uterine artery impedance in a subfertile population. Fertil Steril 1994;61:53-8. 18. Hickey M, Lau TM, Russell P, Fraser IS, Rogers PA. Microvascular density in conditions of endometrial atrophy. Hum Reprod 1996;11:2009-13.

*Corresponding author: Dr. Killol Nathubhai Desai, A-301, Staff Quarters, GMERS Medical College, Paddock road, Junagadh, Gujarat, India. Pin: 362001, Phone: +91 9428050253 Email: drkilloldesai@gmail.com Date of Submission : 23.11.2016 Date of Acceptance : 12.02.2017 Financial or other Competing Interests: None. Date of Publication : 05.06.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Original Article DOI: 10.21276/APALM.1207

Clinicopathological Study of Sinonasal Masses Prashant Shivaji Mane* and Shubhangi Vinayak Agale Department of Pathology, Grant Government Medical College, Mumbai Maharashtra India.

ABSTRACT Background: Sinonasal mass is a common finding in the Otorhinolaryngology Department. These can be non-neoplastic or neoplastic. Nasal obstruction is the most common clinical presentation. Imaging studies are not always conclusive in these cases. So, the present study aimed at clinical presentation and histopathological classification of sinonasal masses. Methods: All the specimens received as sinonasal masses were included in the present study. The tissues were routinely processed for histopathological examination and were stained by Hematoxylin and Eosin stain. Special stains were used wherever required. Immunohistochemistry was carried on cases with diagnostic difficulties. Results: Non-neoplastic lesions outnumbered the neoplastic lesions. Among the neoplastic lesions, benign tumours were more common than malignant tumours. Non-neoplastic lesions and benign tumours were commonly seen in middle age group while malignant tumours were seen in adult patients. Males were predominantly affected in non-neoplastic lesions and benign tumours. Malignant tumours showed female dominance. Nasal obstruction was the most common complaint. Overall, inflammatory nasal polyps were most common lesions. Inverted papillomas were most common benign tumours. Sinonasal undifferentiated carcinomas accounted for majority of malignant tumours. Conclusion: Sino-nasal masses or polyps can be non-neoplastic or neoplastic lesions and histopathological examination remains the mainstay in differentiating these lesions. Keywords: Nasal Cavity, Paranasal Sinus, Polyp, Nasal Obstruction.

Introduction

The nasal cavity and paranasal sinuses are collectively referred as sinonasal tract. Sinonasal area is exposed to various infective agents, chemicals, antigens, mechanical and many other influences. These deleterious exposures lead to formation of tumour like and neoplastic conditions.[1]Most of these lesions in Otorhinolaryngology Department present as polypoid masses, making it difficult to distinguish non-neoplastic polyps from polypoid neoplasms clinically.[2] Inflammatory polyps are a common cause of nasal obstruction, with a prevalence of 4% in the general population.[3]Benign tumours are relatively common, but malignant neoplasms are rare. Malignant tumours account for 0.2% to 0.8% of total malignancies and only 3% of all malignant tumours of upper aerodigestive tract.[4]Nasal obstruction is the most common symptom. Other symptoms include nasal discharge, epistaxis and disturbances of smell.[5]Fine needle aspiration of paranasal sinus lesions is difficult due to closed architecture, and only one study has been documented in the literature.[6] Intraoperative cytology and frozen section examinations of lesions of nose and paranasal sinuses are useful, quick, and reliable diagnostic technique for rapid and early diagnosis in the operation theatre and can be used as an adjunct to histopathology for better management of patients.[7]

The presenting features, symptomatology and advanced imaging technique help to reach a presumptive diagnosis but histopathological examination remains the mainstay of final definitive diagnosis.[8,9] The present study was carried to study the age and sex distribution of sinonasal masses, their clinical presentation and to categorize them histopathologically.

Materials and Methods

The present study was three year retrospective and two year prospective, carried out in the Department of Pathology, Grant Govt. Medical College & Sir J. J. Group of Hospital, Mumbai. All the specimens received as sinonasal mass were included in the present study. Lesions of the nasopharyngeal region and lesions arising from the external nose were not included in the study. The tissues were routinely processed for histopathological examination and were stained by Hematoxylin and Eosin stain. Special stains were used wherever required. The clinical details and imaging studies were obtained from medical record section. Detailed microscopic study was done and then the final diagnosis was given. Typing of the neoplastic lesions was carried out following WHO classification. Immunohistochemistry was carried on cases with diagnostic difficulties.

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

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Approval for the study was given by the Institutional Ethical Committee.

Result

Out of 36,829 specimens received during 5 year study period, 135 specimens (0.36%) involved lesions of nose and paranasal sinuses. Repeat biopsies were received in 4 cases. In 5 cases opinion was not possible due to inadequate biopsy or necrotic tissue, so these cases were excluded from the study. Thus, final study included total 126 cases. Non-neoplastic lesions (83.33%) outnumbered the neoplastic lesions (16.67%). Among the neoplastic lesions, benign tumours (11.9%) were more common than malignant tumours (4.76%) (Table1). Youngest patient affected was 19 years and oldest was 77 years. Non-neoplastic lesions and benign tumours were commonly seen in 3rd-5th decade while malignant tumours were seen in adult patient in 6th and 7th decade (Table 2). Males were predominantly

affected in non-neoplastic lesions and benign tumours. Malignant tumours showed female dominance (Table 3). Nasal obstruction was the most common complaint in all non-neoplastic and neoplastic lesions. Epistaxis was also common presentation in neoplastic lesions (Table 4). Overall, inflammatory nasal polyps were most common lesions (69.04%). These were further sub-classified as edematous or eosinophilic polyp (68.96%), fibroinflammatory polyp (21.83%) and polyp with seromucinous gland hyperplasia (9.19%). Fungal rhinosinusitis and rhinosporidiosis were other non-neoplastic lesions. Noninvasive fungal rhinosinusitis was seen in 60% cases and invasive fungal rhinosinusitis in 40% cases. Fungal ball was most common presentation (40%). Inverted papillomas were most common benign tumours followed by angiofibroma. Sinonasal undifferentiated carcinomas constituted 66.66% of malignant tumours.

Table 1: Distribution of lesions. Histopathological diagnosis I. Non-neoplastic lesions Inflammatory nasal polyp Fungal rhinosinusitis Rhinosporidiosis II. Benign tumours Inverted papilloma Angiofibroma Solitary fibrous tumour Lobular capillary hemangioma Osteoid osteoma Ossifying fibroma Giant cell reparative granuloma III) Malignant tumours Squamous cell carcinoma Sino-nasal undifferentiated carcinoma Neuroendorinetumour Total

No. of cases 105 87 15 3 15 6 4 1 1 1 1 1 6 1 4 1 126

Percentage (%) 83.33 69.04 11.90 2.38 11.90 4.76 3.17 0.79 0.79 0.79 0.79 0.79 4.76 0.79 3.17 0.79 100

Table 2: Age wise distribution of lesions. Age in years 1-10 11-20 21-30 31-40 41-50 51-60 61-70 71-80 Total

Non-neoplastic lesions (%) 0 7.6 30.5 25.7 22.9 7.6 3.8 1.9 100

Benign tumours(%) 0 20 13.3 26.7 20 6.7 13.3 00 100

Malignant tumours(%) 0 0 16.67 0 0 50.0 33.33 0 100

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Table 3: Gender wise distribution of lesions Sex Male Female

Non-neoplastic lesions (%) 57.1 42.9

Benign tumours(%) 80 20

Malignant tumours(%) 33.33 66.67

Non-neoplastic lesions (%) 84.76 55.23 18.09 22.85 37.14 11.42 0.95 4.76

Benign tumours(%) 80 53.3 6.7 53.3 13.3 0 6.7 0

Malignant tumours(%) 71.4 42.8 28.6 71.4 28.6 0 42.8 28.6

Table 4: Clinical features Clinical features Nasal obstruction Nasal discharge Headache Epistaxis Anosmia/ Hyposmia Breathlessness Facial swelling Eye related symptoms

Fig. 1a: Solitary fibrous tumor showing spindle cells arranged in fascicles with indistinct cytoplasmic margins and plump to spindle nuclei (H&E, X400).

Fig. 1b: Solitary fibrous tumor cytoplasmic positivity for CD34.

showing

diffuse

Fig 2a: Sinonasal undifferentiated carcinoma showing large tumor cells with scant cytoplasm, vesicular nuclei and prominent nucleoli (H&E, X400).

Fig. 2b: Sinonasal undifferentiated carcinoma showing strong pan-cytokeratin positivity.

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Fig. 3a: Neuroendocrine tumor, multiple fragmented, grayish white to tan tissue bits.

Fig. 3b: Neuroendocrine tumor, MRI showingtumour in sphenoid and ethmoidal sinus.

Fig. 3c: Neuroendocrine tumor showing rosette formation (H&E, X400).

Fig. 3d: Neuroendocrine chromogranin positivity.

Discussion

Non-neoplastic lesions were seen mainly in the 3rd-5th decade of life. Most common age group affected was 21-30 years (30.5%). They were least in the elderly age group. Humayun et al[5] (31.4%) and Jyothi Raj et al[11] (46.2%) also reported higher incidence of non-neoplastic lesions in 3rd decade. Parajuli et al[9] reported majority of non-neoplastic lesions in 2nd& 3rd decade. Males were predominantly affected with male to female ratio of 1.3:1. This was similar to Lathi et al[12] (1.3:1) and Jyothi Raj et al[11] (1.2:1). Nasal obstruction was the most common clinical presentation seen in 84.7% cases, followed by nasal discharge (55.2%). Humayun et al[5] also reported nasal obstruction as the most common feature (100%) followed by nasal discharge (82.5%).

We reported varieties of lesions involving the nose and paranasal sinuses, affecting all age groups and both sexes. No cases were seen in first decade. All patients presented with different symptoms, but nasal obstruction was the most common presentation. Imaging study reports were received in few cases. We divided these lesions into nonneoplastic and neoplastic. Majority of lesions reported were non-neoplastic (82%). In neoplastic lesions, benign tumours (11.8%) were more common than malignant tumours (5.5%). Parajuli et al[9] reported 80.4% non-neoplastic lesion, 12.8% benign tumours and 6.8% malignant tumours. Similarly, Kulkarni et al[10] reported 86.3% non-neoplastic lesion, 11.1% benign tumours and 2.6% malignant tumours. Thus, our study correlated well with these studies.

tumor

showing

strong

Out of 105 non-neoplastic lesions, nasal polyps were the most common lesions accounting 82.9%. This was similar

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to studies conducted by Lathi et al[12] (87.5%), Kulkarni et al[10] (69.3%) and Parajuli et al[9](89%). The most common age group affected was 21-30 years (36.8%). The peak age of presentation in Jyothi Raj et al,[11] Khan et al[8] and Modh et al[2]was also in the 2nd and 3rd decades of life. Male to female ratio was reported to be 1.2:1; similar to 1.3:1 in Jyothi Raj et al,[11] 1.7:1 in Khan et al[8] and 1.5:1 in Modh et al.[2]Most common clinical presentation was nasal obstruction (86.2%). Khan et al[8] also reported nasal obstruction as presenting symptom. Billroth (1864) described nasal polyps as neoplastic, but Zuckerlkandl considered them to be an inflammatory condition. Berdal (1959) was the first to introduce the practice of differentiating between benign and neoplastic conditions based on the histopathological classification.[13]Several theories have been proposed to explain the pathogenesis of nasal polyp which include allergy, vasomotor imbalance, Bernoulli phenomenon, super antigen, aspirin intolerance and others.[14] Davidsson and Hellquist[15] classified polyps histologically into four categories: edematous or eosinophilic polyps, fibroinflammatory polyps, polyps with seromucinous gland hyperplasia and Polyps with stromal atypia. Our study showed large percentage of eosinophilic polyps (69%) similar to Davidsson and Hellquist (86%). Computerised tomography (CT) scan reported features of sinusitis or nasal polyp. Fungal infections of nose and paranasal sinuses are increasingly recognized entity both in normal and immunocompromised individuals. Aspergillosis and Mucormycosis are the commonest of all fungal infections. [16] Though clinical presentation and radiological findings may provide diagnostic clue for each fungal sinusitis category, histopathological examination and classification of fungal rhinosinusitis into invasive or non-invasive disease is important with regards to treatment.[17] In the present study fungal rhinosinusitis was seen in the 4thâ&#x20AC;&#x201C;7th decade of life. Mean age of presentation was 50.7 years. Montane et al[18] reported 50 years mean age group and Soontrapa et al[19] reported 54.8 years mean age group. It was more common in males, with male to female ratio of 2:1. Male to female ratio in other studies were 1.8:1 in Navya et al[17] and 1.2:1 in Montane et al.[18] Most common clinical symptom in our study was nasal obstruction (86.7%), followed by nasal discharge (60%). Also in 33.3% of cases, there was history of diabetes mellitus. Wahid et al[20] and Soontrapa et al[19] also reported nasal obstruction as most common presenting symptom, 85% and 27.9% respectively. History of diabetes was seen in www.pacificejournals.com/apalm

20% and 30.2% of cases in Wahid et al[20] and Soontrapa et al[19] studies respectively. Imaging studies in few cases received, reported features of polyp or mucosal thickening. Rhinosporidiosis is a chronic granulomatous infectious disease, characterized by hyperplastic polypoid lesions of the mucous membrane. Ashworth, after a study of Rhinosporidium, proved that it was not a sporozoa, but belonged to the group phycomycetes in the sub-order of Chytridineae, and called it Rhinosporidiumseeberi, which has become its accepted name. Majority of cases are reported from India, Srilanka and Bangladesh. It usually presents as single or multiple, pedunculated or sessile masses, pink to deep red in colour, usually described as strawberry like appearance. They bleed easily with a history of nasal obstruction or epistaxis. We reported 3 cases of rhinosporidiosis, two in the age group of 21-30 years and one in 11-20 years age group. They presented mostly with nasal obstruction. Incidence of rhinosporidiosis in our study was similar to Nayak et al[21] (1.82%) and Lathi et al[12] (2.5%). Benign tumours were reported commonly in 4th decade (26.7%). Parajuli et al[9] and Lathi et al[12] reported benign tumours commonly in the 5th decade. Males were predominantly affected than females. Male to female ratio was 4:1. Lathi et al[12](1.7:1) and Jyothi Raj et al[11] (1.5:1) reported similar male predominance. Nasal obstruction was the most common clinical feature (80%), followed by nasal discharge (53.3%) and epistaxis (53.3%). Humayun et al[5] also reported nasal obstruction as most common symptom (66.7%). Nasal discharge and epistaxis was also common feature in Humayun et al[5]study. Inverted papillomas are two to five times more common in males and are found primarily in the 40â&#x20AC;&#x201C;70 year age group. These papillomas characteristically arise from the lateral nasal wall in the region of the middle turbinate or ethmoid recesses. Unilateral nasal obstruction is the most common presenting symptom. Grossly, these are pink, tan, or gray; non-translucent; soft to moderately firm polypoid growths with a convoluted or wrinkled surface. We reported 6 cases of inverted papilloma in our study. Majority of patients presented in the 4th& 5th decade of life (66.6%). Male to female ratio was 5:1. Nasal obstruction was the most common clinical presentation (83.3%), followed by epistaxis (50%) and nasal discharge (50%). Khan et al[8] reported 15 cases of inverted papilloma. The peak age of presentation was fifth decade of life and the male to female ratio was 3:1. Jaison et al[22] reported 5 cases of inverted papilloma. Most common age group affected was 5th& 6th decade and male to female ratio was 4:1. Most common clinical presentation was nasal obstruction eISSN: 2349-6983; pISSN: 2394-6466

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and epistaxis. CT scan findings in one case showed left maxillary, sphenoid, ethmoidal sinusitis with left nasal polyp and in one case Magnetic resonance imaging (MRI) suggested features of left sphenoid, maxillary sinusitis. Angiofibroma usually presents with nasal obstruction. The gross appearance of the neoplasm is of a lobulated, pink to purplish, smooth surfaced mass. We reported 4 cases of angiofibroma. Males were most commonly affected than females and male to female ratio was 3:1. Most common clinical symptoms were nasal obstruction (75%) and epistaxis (75%). Parajuli et al[9] reported 3 cases of angiofibroma with profuse recurrent epistaxis as chief complaint. Jaison et al[22] reported 5 cases of angiofibroma in first two decades of life and male to female ratio was 4:1. Solitary fibrous tumor was reported in a 50 year male with complaints of nasal obstruction and discharge. CT scan reported polypoidal mucosal thickening involving all the sinuses and nasal cavities. Immunohistochemically tumor cells showed diffuse cytoplasmic positivity for CD34 and CD99 (Fig. 1a & 1b).Other benign tumours included single cases of lobular capillary hemangioma, osteoid osteoma, ossifying fibroma andgiant cell reparative granuloma. Malignant tumours were seen in the age range from 1970 years. 71.4% of cases were seen in the 6th& 7th decade. Jyothi Raj et al[11] reported 62.5% of malignant lesions in the 6th decade. Parajuli et al[9] reported 60% cases of malignant tumours in the 5th-7th decade. Thus malignant tumours in our study were more common in elderly patients, similar to other studies. Malignant tumours were common in females. Male to female ratio was 1:2. Female dominance was also seen in studies conducted by Jyothi Raj et al[11] (1:1.7) and Bijjaragi et al[23] (1:1.6). Nasal obstruction (71.4%) and epistaxis (71.4%) were the most common symptoms, followed by nasal discharge, facial swelling, headache, loss of smell and eye related symptoms. Humayun et al[5] also reported nasal obstruction (100%) as most common symptom followed by epistaxis (75%). Sinonasal undifferentiated carcinoma typically presents as a rapidly enlarging tumor mass involving multiple sites of the sinonasal tract, often with evidence of extension beyond the anatomic confines. The pathogenesis still remains unknown. Epstein - Barr virus has been implicated as a potential pathogen.[24]The most common initial symptoms are epistaxis, facial pain, and nasal obstruction. We reported 4 cases of sinonsasl undifferentiated carcinoma. Two cases were reported in 6th decade, one in 7th decade and one in 3rd decade. Male to female ratio was 1:3. Nasal obstruction was most common presenting symptom in all cases. CT scan report was available in one case which

showed mass involving left maxillary, ethmoid, sphenoid sinus. Immunohistochemically tumor cells showed Pancytokeratin positivity in all cases (Fig. 2a &2b). Bijjaragi et al[23] reported two cases of sino-nasal undifferentiated carcinoma, one each in male and female. In study by Kalpana Kumari et al[25] malignant tumours were seen in 50% of the neoplastic cases and majority were sinonasal undifferentiated carcinomas (41%). Sinonasal squamous cell carcinomas occur most frequently in the maxillary sinus.[26] Symptoms include nasal obstruction; epistaxis; rhinorrhea; pain; paraesthesia; swelling of the nose or cheek or a palatal bulge; nasal mass; or, in advanced cases, proptosis, diplopia, or lacrimation.[26] We reported a single case of keratinizing squamous cell carcinoma in a 70 years female with complaints of nasal obstruction, nasal discharge, epistaxis and facial swelling. Neuroendocrine neoplasms are defined as epithelial neoplasms with predominant neuroendocrine differentiation. The clinical features of sinonasal neuroendocrine carcinoma are nonspecific and similar to those of other sinonasaltumours. Common presentations include nasal obstruction, epistaxis, facial mass, and/or facial pain.Â A single case of sphenoid sinus neuroendocrine tumour was observed in a 51 years male patient with complaints of headache. MRI suggested pituitary tumour involving sphenoid and ethmoidal sinus. Immunohistochemistry showed positivity for cytokeratin and chromogranin (Fig. 3a, 3b, 3c &3d).

Conclusion

It was found that varieties of lesion affect the nose and paranasal sinuses, which included non-neoplastic and neoplastic lesions. Non-neoplastic lesions outnumbered the neoplastic lesions. Inflammatory nasal polyps were most common lesion. Non-neoplastic lesions and benign tumours affected predominantly middle age group and neoplastic lesions were common in elderly patients. Males were predominantly affected but malignant tumours were seen more in females. Nasal obstruction was the most common symptom in all lesions. Imaging studies were not always conclusive. To conclude, sino-nasal masses or polyps can be nonneoplastic or neoplastic lesions and histopathological examination remains the mainstay in differentiating these lesions.

Reference 1.

Lingen MW. Head and neck. Chapter 16; In Kumar V, Abbas A K, Fausto N, Aster J C, eds. Robbins and Cotran Pathologic basis of disease, 8th ed. Elsevier: Haryana, India; 2010:751-2.

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Modh SK, Delwadia KN, Gonsai RN. Histopathological spectrum of sinonasal masses- A study of 162 cases. Int J Cur Res Rev 2013; 5(3): 83-91.

Hedman J, Kaprio J, Poussa T, Nieminen MM. Prevalence of asthma, aspirin intolerance, nasal polyposis and chronic obstructive pulmonary disease in a population-based study. Int J Epidemiol. 1999 Aug; 28(4): 717-22.

Barnes L, Tse LL, Hunt JL, Gensler BM, Curtin HD, Boffetta P. Tumours of the nasal cavity and paranasal sinuses. In: Leon B, John WE, Peter R, David S, editors. IARC WHO classification of tumours, pathology and genetics of head and neck tumours. Lyon: IARC Press:2005.9‑82.

Humayun AHM, ZahurulHuq AHM, Ahmed SMT et al. Clinicopathological study of sinonasal masses. Bangladesh J Otorhinolaryngol 2010;16:15–22.

Gupta N, Kaur J, Srinivasan R, Das A, Mohindra S, Rajwanshi A, et al. Fine needle aspiration cytology in lesions of the nose, nasal cavity and paranasal sinuses. ActaCytol 2011;55:135‑41.

Nigam JS, Misra V, Dhingra V, Jain S, Varma K, Singh A. Comparative study of intra-operative cytology, frozen sections, and histology of tumor and tumor-like lesions of nose and paranasal sinuses. J Cytol 2013;30:13-7.

Khan N, Zafar U, Afroz N, Ahmad SS, Hasan S. Masses of nasal cavity, paranasal sinuses and nasopharynx: a clinicopathological study. Indian Journal of Otolaryngology and Head and Neck Surgery 2006;58(3):259-63.

Parajuli S, Tuladhar A. Histomorphological spectrum of masses of the nasal cavity, paranasal sinuses and nasopharynx. J Pathol Nepal 2013;3:351–5.

14. Tikaram A, Prepageran N. Asian nasal polyps: a separate entity? Med J Malaysia 2013;68(6):445–7. 15. Davidsson A, Hellquist HB. The So-Called “Allergic” Nasal Polyp. ORL J Relat Spec 1993;55:30-5. 16. Joshi SR, Nagare M. Case report Fungal Rhinosinusitis ( Aspergillosis ) -3 cases. 2015;4(2):314–7. 17. Navya BN. Role of Histopathology in the Diagnosis of Paranasal Fungal Sinusitis. J Dent Med Sci 2015;14(1):97–101. 18. Montone KT, Livolsi V a., Feldman MD, Palmer J, Chiu AG, Lanza DC, et al. Fungal Rhinosinusitis: A Retrospective Microbiologic and Pathologic Review of 400 Patients at a Single University Medical Center. Int J Otolaryngol 2012;12:1–9. 19. Soontrapa P, Larbcharoensub N, Luxameechanporn T. Fungal rhinosinusitis : a retrospective analysis of clinicopathologic features and treatment outcomes at ramathibodi hospital. Southeast Asian J Trop Med Public Health 2010;41(2):442-9. 20. Fazal-I-Wahid, Adil Khan, Iftikhar Ahmad Khan. Clinicopathological profile of fungal rhinosinusitis. Bangladesh J Otorhinolaryngol 2012;18(1):48-54. 21. Nayak M, Roul B, Agrawal K, Nayak S. Clinicopathological study of lesions of sinonasal tract & Distribution of Sinonasal tract lesions in difefferent age groups & Sex. International Journal of Advanced Research 2015;3(4):726-733. 22. Jaison J, Tekwani DT. Histopathological lesions of nasal cavity, paranasal sinuses and nasopharynx. Annals of Applied Bio-Sciences 2015;2(2):40-6.

10. Kulkarni AM, Mudholkar VG, Acharya AS, Ramteke RV. Histopathological Study of Lesions of Nose and Paranasal Sinuses. Indian J Otolaryngol Head Neck Surg 2012;64(3):275–279.

23. Bijjaragi S, Kulkarni VG, Singh J. Histomorphological study of polypoidal lesions of nose and paranasal sinuses. Indian Journal of Basic and Applied Medical Research 2015;4(3):435-9.

11. Jyothi A Raj, Sharmila PS, Mitika Shrivastava, Mahantachar V, T Rajaram. “Morphological spectrum of lesions in the sinonasal region”. Journal of Evolution of Medical and Dental Sciences 2013;2,(37)7175-7186.

24. Mohammed AW, Bakshi J, Nada R. Sinonasal undifferentiated carcinoma with contralateral cavernous sinus involvement – A rare presentation. Curr Res Microbiol Biotechnol 2013;1(2):58–61.

12. Lathi A, Syed MMA, Kalakoti P, Qutub D, Kishve SP. Clinicopathological profile of sinonasal masses: a study from a tertiary care hospital of India. Acta Otorhinolaryngol Ital 2011;31(6):372–7.

25. Kalpana Kumari MK, Mahadeva KC. Polypoidal lesions in the nasal cavity. J Clin Diagn Res. 2013 Jun;7(6):1040-42.

13. Venkatarajamma K, Joshyam S, Gowda B, Zebanoorain. Clinic-Pathological Profile of Sinonasal Masses. Int J Gen Med Pharm 2015;4(2):7–18.

26. Santos MRM, Servato JPS, Cardoso SV, Faria PR De, Eisenberg LA, Dias FL, et al. Squamous cell carcinoma at maxillary sinus : clinicopathologic data in a single Brazilian institution with review of literature. Int J Clin Exp Pathol 2014;7(12):8823–32.

*Corresponding author: Dr. Prashant Shivaji Mane, Department of Pathology, Grant Government Medical College, Mumbai Maharashtra 400008. India Phone: +91 .9766855956 Email: prashantmane0791@gmail.com Date of Submission : 15.12.2016 Date of Acceptance : 01.04.2017 Financial or other Competing Interests: None. Date of Publication : 05.06.2017

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Original Article DOI: 10.21276/APALM.1218

Thrombocytopenia and Altered Platelet Indices as Potential Marker in Complicated Malaria Caused by Plasmodium Vivax: Cross Sectional Descriptive Study Bhavani K, Venkatraman J*, Roopa Urs A. N and Dhananjay S Kotasthane Dept. of Pathology, Mahatma Gandhi Medical college and Research institute, Pondicherry, India

ABSTRACT Background: Malaria is a major health problem with increased morbidity and mortality. Plasmodium falciparum and Plasmodium vivax are endemic infections in India and constitutes about 77% of the total malaria cases in Southeast Asia, commonly associated with hematological abnormalities. Hematological abnormalities that are observed in patients with malaria includes anemia and thrombocytopenia. Thrombocytopenia frequently complicates malarial infections and is usually noted in Plasmodium falciparum. In the present study we have tried to evaluate prognostic implications of altered platelet indices and factors associated with outcome of severe and complicated P. vivax malaria. Methods: This is cross sectional descriptive study of 60 patients with malaria caused by P. vivax who presented to Mahatma Gandhi medical college and research institute between june 2014 to April 2016.The smears positive for malaria exclusively caused by P.vivax infection were included in this study. Malaria was diagnosed by microscopic examination with both leishmain stained thin and giemsa stained thick smears. All blood cell counts were determined using automated equipment (ABX Pentra ES 60; Horiba) which provides MPV, PDW and PCT. Result: Out of 60 patients in the present study 56.7% were males and 43.3% were females, age of the patient ranges from 17-48 years with mean (SD) age of distribution were 32.10 (9.52).Complete blood count evaluation of all patients showed mean hemoglobin level of 9 g/dL. Mean hematocrit level of 26.68% (4.2%) and mean leukocyte count of 5866 (1170) cells/mm3. The mean platelet count was 95,247cells/ mm3. The MPV was 9.6 (0.96) Âľm, Mean PDW was 13.75% (0.89%) and mean PCT was 0.180% (0.033%) Conclusion: Our study concludes that history of fever associated with chills and rigor are sensitive indicator of malaria but lacks specificity. Platelet indices were altered during severe symptomatic malarial infection such as elevation of MPV and PDW with decreased PCT are the known potential risk factors associated with warning signs of acute malaria caused by plasmodium vivax. Even though these hematological parameters are useful predictors of acute malarial infection, identification of parasites and grading of parasitemia in peripheral smear were always gold standard for early treatment. Keywords: Plasmodium Vivax, Malaria, Severe Thrombocytopenia.

Introduction

Malaria is a major health problem with increased morbidity and mortality.[1] Plasmodium falciparum and Plasmodium vivax are endemic infections in India and constitutes about 77% of the total malaria cases in Southeast Asia, commonly associated with hematological abnormalities. [2,3] According to World health organization, malaria is the second most fatal communicable disease and is a public health problem in 90 countries of the world.[4] Each year there are more than 250 million cases of malaria, killing 1 to 3 million people.[1,5] Hematological abnormalities that are observed in patients with malaria includes anemia and thrombocytopenia.[5,6] Thrombocytopenia frequently complicates malarial infections and is usually noted in Plasmodium falciparum.[6] But it also frequently observed in vivax malaria but the exact mechanism has not been

elucidated and bleeding episodes are also rare in these patients.[7,8,9] In general, the underlying mechanisms of thrombocytopenia are found to be due to peripheral destruction, excessive sequestration of plateletsin spleen, excessive use of platelets associated with DIC, increased levels of cytokines, immunological destruction due to antiplatelet IgG and anti-oxidative mechanisms of thrombocytes were insufficient in malaria patients and caused oxidative stress.[11-15] The oxidative damage of thrombocytes might be important in the etio-pathogenesis of thrombocytopenia occurring in malaria.[10,11,12] In addition to reduction in number of platelets, functions of platelet is also compromised which is generally evident in platelet indices.[13] Platelet also plays important role in inflammation and depending upon severity of bacterial infection changes in platelet count and indices also has been

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Bhavani K et al. reported, further platelet activation also alters mean platelet volume (MPV) and platelet distribution width (PDW).[1417] Plateletcrit is also another marker for measurement of platelet biomass which combines mean platelet volume with absolute platelet count. MPV,PDW and PCT are considered as markers of platelet activation and altered in different clinical condition and they were also altered in malaria caused by vivax, however the relation between platelet indices and clinical outcome were controversial especially between altered PDW or PCT and severity of malaria caused by vivax.[17,18] In the present study we have tried to evaluate prognostic implications of altered platelet indices and factors associated with outcome of severe and complicated P. vivax malaria.[19,20]

Materials and Methods

This is cross sectional descriptive study of 60 patients with malaria caused by P. vivax who presented to Mahatma Gandhi medical college and research institute between june 2014 to April 2016. The smears positive for malaria exclusively caused by P.vivax infection were included in this study. Complete blood count and biochemical analysis was done for all the patients. Malaria was diagnosed by microscopic examination with both leishmain stained thin and giemsa stained thick smears. All blood cell counts were determined using automated equipment (ABX Pentra ES 60; Horiba) which provides MPV, PDW and PCT.

A-269 32.10 (9.52). The duration between onset of symptoms and diagnosis of malaria ranges from one to thirteen days. At the time of diagnosis 31.67% of patients had body temperature more than 41˚C. Sixteen patients (26.67%) had at least one warning sign of severe and complicated malaria including clinical and biochemical parameters. The mean parasitemia observed in sixty patients was 7397(SD 2285.82) parasites/mm3. No patient had potential drug history which interferes with platelet indices. Complete blood count evaluation of all patients showed mean hemoglobin level of 9 g/dL. Mean hematocrit level of 26.68% (4.2%) and mean leukocyte count of 5866 (1170) cells/mm3. The mean platelet count was 95,247cells/mm3. The MPV was 9.6 (0.96) µm, Mean PDW was 13.75% (0.89%) and mean PCT was 0.180% (0.033%)

Discussion

Out of 60 patients in the present study 56.7% were males and 43.3% were females, age of the patient ranges from 17-48 years with mean (SD) age of distribution were

India is considered in control phase of malaria with annual parasitic incidence of 0.7-1.6 million in 2014.[4] Many hematological parameters were useful in early diagnosis. In this study, specifically total leucocyte count and platelet counts were lower and significantly much lower in patients with warning signs and symptoms of complications as compared to the studies done by Kochar et al from northwestern India, Taylor et al northern-east papua and Saravu et al from Karnataka.[21,22,23] Anemia has been documented in our study population and nutritional status may also be the attributive cause for it. However associated hemoglobinopathies were not excluded. Many studies also noted poor correlation between anemia and malaria.[24,25,26] Studies from Delhi have reported 80.37% to 96% of P. vivax malarial infection with thrombocytopenia as compared to our study has showed 82-93.5%.[27,28] Pathogenesis of thrombocytopenia in malaria is due to peripheral destruction, splenic pooling or consumption coagulopathy but mast et al study has suggested thrombocytopenia in early malaria associated with Gplb shedding in absence of platelet activation and coagulopathy and is not at all due to reduction in megakaryocytes in marrow which are usually normal.[29] An inverse relationship was seen between parasite density and platelet counts and platelet counts returned to normal with treatment as similar to other studies.[30] Platelet indices show increase in mean platelet volume and platelet distribution width as platelet count decreases due to platelet aggregation leads to platelet dysfunction[30]. Chandra et al study says that MPV increased because of peripheral destruction of platelets and MPV decreased in cases of bone marrow parasitemia (due to hypoproduction).[31] However in this present study, MPV was increased in almost 90% of cases but bone marrow examination was done in few follow up cases with increased MPV to confirm marrow parasitaemia but all of

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Severe and complicated malaria was defined by using WHO predictors[4,5,6] or by using following haematological, biochemical and clinical parameters at the time of admission: Body temperature ≥41°C, dyspnoea, hypotension, serum creatinine level >1.5 mg/dL, hypoglycaemia, hyperbilirubinaemia, haemoglobin <7 g/dl, haematocrit <20% and primary infection lasts for more than 3 days also consider potential risk factor. The Mann- whitney non parametric test was used to analyse the association between platelet indices with warning signs of indicators of severe or complicated malaria caused by P. vivax. Spearman’s correlation coefficients was calculated to evaluate the relationships between platelet indices and parasitaemia. Multiple logistic regression analysis was constructed with each parameter as a dependent variable to analyze the independence of the associations between various severity of malaria and platelet indices. The level of significance wasp<0.05.

Result

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Table 1: Comparision between platelet indices and indicators of severity in the patients with Plasmodium vivax malaria MPV

First_Infection

Mean

Yes

10.46

1.07

14.41

1.35

0.17

0.03

9.34

0.66

13.45

0.26

0.18

0.03

0.000006880

0.000044049

0.168368355

Yes

9.67

0.97

13.84

1.01

0.18

0.03

9.78

0.95

13.48

0.28

0.19

0.04

p-value

severity

PCT

p-value

time_Onset1

PDW

Mean

0.690476231

0.170173984

0.549631323

Yes

10.71

0.99

14.68

1.31

0.18

0.03

9.33

0.63

13.42

0.26

0.18

0.03

p-value

0.000000032

0.000000090

0.953929067

Table 2: Clinical and laboratory characteristics of patients with malaria caused by Plasmodium vivax Variable

Frequency

Percent

Age1

<=20

21-40

>40

Female

Male

1.7

2-4

68.3

severity

Yes

Sex No_of_Days1

Parasitaemia

Mean

32.10

9.52

43.3

56.7

4.63

1.79

73.3

26.7

7397.00

2285.83

Table 3 : Multivariate logistic regression analysis of the associations between platelet indices, demographic variables, and severity indicators Variables

Odds Ratio

95% of Clearance

P value

MPV i.First time infection ii.Recurrent infection with severity

3.42 6.80

1.46 - 8.38 1.03 – 45.46

0028 0.046

PDW i.Recurrent Malarial infection ii.Presence of complicated signs of severity

0.63 5.83

0.77 – 0.93 2.20 – 41.36

0.002 0.017

Table 4 : Comparison of variation in platelet indices in post treatment Plasmodium vivax infected patients Mean (SD) of (n=41)

Mean (SD) of (n=6)

TLC

6.27 x 109 / L (1.08)

4.13 x 109 / L (0.46)

2.73 x 109/ L (1.01)

1.23 x 109/ L (0.83)

PDW

23.53 (1.31)

13.84 (1.07)

MPV

13.77 (0.66)

9.14 (0.33)

PCT

0.18 (0.03)

0.19 (0.14)

Parameters

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Fig. 1: Relationships between parasitic density and other variables in the patients with malaria caused by Plasmodium vivax.

them showed normal marrow poiesis and chronic ITP also ruled out. One study has mentioned MPV in malaria should be considered as predictor of acute infection by P. vivax.[32] Sushma et al study shows thrombocytopenia associated with increase in mean platelet volume and leucopenia were considered sensitive indicator for plasmodium vivax infection.[31] PCT was low in this study, since PCT is measure of platelet biomass (which is due to concominant thrombocytopenia associated with P.vivax). PCT has negative correlation with level of parasitemia as observed in other studies.[33] Change in platelet indices also influenced by warning signs of severe acute malaria such as longer duration of illness, hematological and biochemical parameters.[34] These changes are mainly due to increased giant platelets which are metabolically and enzymatically more active and involves in inflammatory process.[35] Initially in severe cases of malaria platelet shows hyperactivity followed by www.pacificejournals.com/apalm

hypoactivity. Hyperactivity is due to various aggregating agents like immune complexes, surface contact of platelet membrane to parasitized RBCâ&#x20AC;&#x2122;s and damage to endothelial cells which cause lyses of platelet inside the vessel and release its content which can cause even disseminated intravascular coagulation. Platelet distribution width was also increased due to variation in size of platelets (giant platelets) and also due to platelet aggregation.[29,34,35] Post treatment follow up was done in 47 cases out of which 41 patient showed almost normal to mild leucocytosis and platelet also returns to near normal MPV and PDW were also normal in range but Plateletcrit was not showed much difference from previous value. This suggested that malaria was the most likely cause which alters the studied platelet parameters.[33]

Conclusion

Our study concludes that history of fever associated with chills and rigor are sensitive indicator of malaria but eISSN: 2349-6983; pISSN: 2394-6466

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Altered Platelet Indices in Complicated P. Vivax Infection

lacks specificity. Hence thrombocytopenia (<150x109/l) and leucopenia (< 4x109/l) used as probable indicator for malarial infection. Platelet indices were altered during severe symptomatic malarial infection such as elevation of MPV and PDW with decreased PCT are the known potential risk factors associated with warning signs of acute malaria caused by plasmodium vivax. Even though these hematological parameters are useful predictors of acute malarial infection, identification of parasites and grading of parasitemia in peripheral smear were always gold standard for early treatment.

10. Fajardo L.F., Tallent C. Malarial parasites within human platelets. JAMA 1974;229:1205. 11. Katira B, Shah I Thrombocytopenia in Plasmodium vivax infected children, J Vect j,Borne Dis 43, September 2006, pp. 147–9. 12. Erel O., Vural H., Aksoy N., et al. Oxidative stress of platelets andThrombocytopenia in patients with vivax malaria.Clin Biochem 2001; 34(4):341- 4.

Acknowledgements

13. Greisenegger S, Endler G, Hsieh K, Tentschert S, Mannhalter C, Lalouschek W. Is elevated mean platelet volume associated with a worse outcome in patients with acute ischemic cerebrovascular events? Stroke. 2004;12:1688–1691.

Reference

15. Boos CJ, Beevers GD, Lip GY. Assessment of platelet activation indices using the ADVIATM 120 amongst “highrisk” patients with hypertension. Ann Med. 2007;12:72–78. doi: 10.1080/07853890601040063.

Authors sincerely thank Dr Koteeswaran G, Professor, Dept of Pathology, Mahatma Gandhi medical college and research institute, Puducherry for her constant support. Authors also acknowledge the immense help received from the scholars who articles are cited and included in references of this manuscript. 1.

Makkar, RPS, Mukhopadhyay S, Monga A, Gupta AK. Plasmodium Vivax Malaria Presenting With Severe Thrombocytopenia. The Brazilian Journal of Infectious Diseases 2002; 6 (5):263-265.

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Khan SJ, Khan FR, Usman M, Zahid S. Malaria can lead to Thrombocytopenia. Rawal MedJ 2008; 33: 183-55.

World Malaria Report 2009. WHO (cited on 2011, September 12). Available at: http://www.who.int/malaria/ world_malaria_report_2009/en/index.html. Last accessed on January, 2013.

Rodríguez-Morales AJ, Sánchez E, Vargas M, Piccolo C, Colina R, Arria M. Anemia and thrombocytopenia in children with Plasmodium vivax malaria. J Trop Pediatr. 2006;12:49–51. doi: 10.1093/tropej/fmi069

Patel U, Gandhi G, Friedman S, Niranjan S. Thrombocytopenia in Malaria. Journal of the national medical association 2004; 96 (9): 1212-1214.

Rizvi I, Tripathi DK, Chughtai AM, Beg M, Zaman S, Zaidi N. Complications associated with Plasmodium vivax malaria: a retrospective study from a tertiary care hospital based in western Uttar Pradesh, India. Ann Afr Med. 2013; 12:155–159. doi: 10.4103/1596-3519.117624. doi:10.4103/1596-3519.117624.

Jadhav UM, Patkar VS, Kadam NN .Thrombocyto-penia in Malaria - Correlation with Type and Severity of Malaria. JAPI 2004; 52: 615-618. Gupta NK, Bansal SB, Jain UC, Sahare K. Study of thrombocytopenia in patients of malaria. Trop Parasitol. 2013;12:58–61. doi: 10.4103/2229-5070.113914. doi:10.4103/2229-5070.113914

14. Mirsaeidi M, Peyrani P, Aliberti S, Filardo G, Bordon J, Blasi F, Ramirez JA. Thrombocytopenia and thrombocytosis at time of hospitalization predict mortality in patients with community-acquired pneumonia. Chest. 2010;12:416–420.

16. Jackson SR, Carter JM. Platelet volume: laboratory measurement and clinical application. Blood Rev. 1993;12:104–113. doi: 10.1016/S0268-960X(05)80020-7. 17. Becchi C, Al Malyan M, Fabbri LP, Marsili M, Boddi V, Boncinelli S. Mean platelet volume trend in sepsis: is it a useful parameter? Minerva Anestesiol. 2006;12:749–756. 18. Gasparyan AY, Ayvazyan L, Mikhailidis DP, Kitas GD. Mean platelet volume: a link between thrombosis and inflammation? Curr Pharm Des. 2011;12:47–58. doi: 10.2174/138161211795049804. 19. Lancé MD, Sloep M, Henskens YMC, Marcus MAE. Mean platelet volume as a diagnostic marker for cardiovascular disease: drawbacks of preanalytical conditions and measuring techniques. Clin Appl Thromb Hemost. 2012;12:561–568. doi: 10.1177/1076029612458147. 20. Ladhani S, Lowe B, Cole AO, Kowuondo K, Newton CRJC. Changes in white blood cells and platelets in children with falciparum malaria: relationship to disease outcome. Br J Haematol. 2002;12:839–847. doi: 10.1046/j.13652141.2002.03904.x. 21. Kochar DK, Das A, Kochar A, Middha S, Acharya J, Tanwar GS, Gupta A,Pakalapati D, Garg S, Saxena V, Subudhi AK, Boopathi PA, Sirohi P, Kochar SK. Thrombocytopenia in Plasmodium falciparum, Plasmodium vivax and mixed infection malaria: a study from Bikaner (Northwestern India). Platelets 2010; 21: 623- 627. 22. Taylor WR, Widjaja H, Basri H, Ohrt C, Taufik T, Tjitra E, Baso S, Fryauff D, Hoffman SL, Richie TL. Changes in the total leukocyte and platelet counts in Papuan and non Papuan adults from northeast Papua infected with acute Plasmodium vivax or uncomplicated Plasmodium falciparum malaria. Malar J 2008; 7:259.

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23. Saravu K, Docherla M, Vasudev A, Shastry BA. Thrombocytopenia in vivax and falciparum malaria: an observational study of 131 patients in Karnataka, India. Ann Trop Med Parasitol 2011; 105:593- 598.

Dongen-Lases EC, Sauerwein RW, van der Ven AJ. (2010). Thrombocytopenia in early malaria is associated with GP1b shedding in absence of systemic platelet activation and consumptive coagulopathy.Br J Haematol. 151: 495-503.

24. Erhart Laura M,Yingyuen Kritsanai,Chuanak Niphon. Hematologic and clinical indices of malaria in a semiimmune population of western Thailand.Am. J. Trop. Med. Hyg. 2004;70(1): 8–14.

30. Sushma N, Reddy MM, Vijayashree R, Padmavathy F, Begum R, Arudra P Haematological Parameters Including Platelet Indices in Vivax and Falciparum Malaria. Chettinad Health City Medical Journal 2014; 3(3): 95 – 100.

25. Maina Robert N, Walsh Douglas, Gaddy Charla. Impact of Plasmodium falciparum infection on haematological parameters in children living in Wester Kenya.Malaria Journal 2010; 9(Suppl 3):S4(1-11). 26. Richards MW, Behrens RH, Doherty JF. Short report: Hematologic changes in acute, imported Plasmodium falciparum malaria. Am. J. Trop. Med. Hyg.1998; 59(6): 859. 27. Sharma A, Khanduri U. How benign is benign tertian malaria? JVector Borne Dis 2009; 46: 141-144. PMid:18647661. 28. Singh H, Parakh A, Basu S, Rath B. Plasmodium vivax malaria : is it actually benign? J Infect Public Health 2011; 4: 91-95. 29. De Mast Q, de Groot PG, van Heerde WL, Roestenberg M, van Velzen JF, Verbruggen B, Roest M, McCall M, Nieman AE, Westendorp J, Syafruddin D, Fijnheer R, van

31. Chandra H, Chandra S, Rawat A, Verma SK. Role of mean platelet volume as discriminating guide for bone marrow disease in patients with thrombocytopenia. Int J Lab Hematol 2010; 32: 498- 505. 32. Chandra S, Chandra H. Role of haematological parameters as an indicator of acute malarial infection in Uttarakhand state of India. Mediterr J Hematol Infect Dis. 2013;12:e2013009. 33. Leal-Santos Fábio, Silva Soraya BR, Crepaldi Natasha P. Altered platelet indices as potential markers of severe and complicated malaria caused by Plasmodium vivax: a crosssectional descriptive study.Malaria Journal 2013; 12:462. 34. Jadhav UM, Patkar VS, Kadam NN. Thrombocytopenia in Malaria - Correlation with Type and Severity of Malaria. JAPI, Aug 2004; Vol. 52:615-618. 35. Bashawri Layla AM, Mandil Ahmed A, Bahnassy Ahmed A, Ahmed Mirghani A. Malaria: Hematological aspects. Annals of Saudi Medicine. 1979; 22(5)-6 ;54:961 76.

*Corresponding author: Dr Venkatraman J, Assistant Professor, Dept. of Pathology, ahatma Gandhi Medical college and Research institute, Pondicherry. India Phone: +91 9894460460 Email: vforvenkat2005@yahoo.co.in Date of Submission : 19.12.2016 Date of Acceptance : 01.03.2017 Financial or other Competing Interests: None. Date of Publication : 05.06.2017

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Original Article DOI: 10.21276/APALM.1284

Diagnostic Accuracy and Pitfalls in Fine Needle Aspiration Cytology Of Salivary Gland Lesions Crysle Saldanha*, Hilda Fernandes, Zeeshanali Fazalbhoy, Jayaprakash C S, Sumanth D and Reshma Kini Department Of Pathology, Father Muller Medical College, Mangalore, India

ABSTRACT Background: Fine Needle Aspiration cytology (FNAC) is an essential diagnostic method used to evaluate salivary gland lesions. However, at times, diverse morphological patterns and overlapping features between benign and malignant lesions becomes challenging and difficult to give a definitive diagnosis. Aim are to compare the findings of preoperative FNAC with their histopathological types and to discuss the causes for discordancy and identify the potential pitfalls in cytological diagnosis. Materials and Methods: An observational analytical study was carried out over a 4 year period to review the cases of patients with salivary gland lesions who underwent FNAC in a medical college, hospital. Taking histopathological diagnosis as gold standard, the cytological diagnosis of the cases was compared and the causes of discrepancies were evaluated. Sensitivity, specificity, accuracy, positive predictive value and negative predictive value was calculated. Results: In the present study, out of 137 cases, cytohistological correlation was available in 46 cases. Pleomorphic adenoma was the commonest lesion in the study. The diagnostic value of FNAC was: Sensitivity 66.7%, Specificity 97.4%, Positive Predictive Value 80%, Negative Predictive Value 95% and Diagnostic Accuracy 93.3%. False positive diagnosis was rendered in warthin’s tumor whereas false negative diagnosis was given in mucoepidermoid carcinoma. Conclusion: FNAC is useful in the preoperative diagnosis of salivary gland lesions. Pitfalls in cytologic diagnosis were due to errors in sampling, cystic lesions and interpretation of smears . Keywords: FNAC Salivary Gland , Diagnostic Accuracy, Pitfalls

Introduction

Materials and Methods

This study aims 1) To determine the diagnostic yield of FNAC of salivary gland lesions and compare it with histopathological diagnosis. 2) To discuss the causes for discordancy and identify the potential pitfalls in cytological diagnosis.

FNAC was performed using a 22 – 23 gauge needle attached to a disposable syringe under aseptic conditions after prior consent. The material was aspirated and smeared onto a clean glass slides and thin smears were prepared between two slides. The air dried and ethanol fixed smears were stained with MGG (May Grunwald’s Giemsa) and Pap (Papanicolau) stain. Forty six (46) patients underwent excision biopsies. After gross examination of specimens, the representative sections were processed and examined by H & E (Hematoxylin and Eosin) methods. Special stains were performed when necessary. Taking histopathological diagnosis as gold standard, the cytological diagnosis of the cases was compared and the causes of discrepancies was evaluated.

Various lesions of major and minor salivary glands, account for less than 3% of all head and neck tumors. Fine needle aspiration cytology (FNAC) is sensitive and specific technique as compared to incisional biopsy and frozen section in the diagnosis of salivary gland lesions. The main goal of FNA is to determine if a mass is inflammatory and/ or reactive, benign or malignant neoplasm and if possible, to render a specific diagnosis. FNA obviates surgery and is beneficial in preoperative information that may play a significant role in appropriate treatment to the patient. However, majority of the salivary gland lesions have varied morphological patterns and overlapping features between non-neoplastic, benign and malignant lesions thus making it difficult to give an accurate diagnosis .[1-6]

An observational analytical study was carried out over a 4 year period to review the cases of patients with salivary gland lesions who underwent FNAC in a medical college, hospital. The study was approved by the institution ethical and research committee.

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Sensitivity, specificity, accuracy, positive predictive value and negative predictive value was calculated.

Results

aspirate obtained was mucoid. On review of the smears the intermediate cells with eosinophilic cytoplasm was misinterpreted as oncocytic cells (Fig 2b).

Among the 137 patients who underwent FNAC, 75 (54.7%) were males and 62 (45.3%) were females (M: F ratio = 1.2:1). The most common gland involved was Parotid gland in 98 cases (71.5%), followed by submandibular gland in 38 cases ( 27.7 %) and a single case involved the minor salivary gland (0.7 %).

False positive cases were as follows: Case 03 was malignant Squamous neoplasm proved histologically as WT (Fig 3a). FNA showed necrotic debris which appeared as thick, granular material with few clusters and singley dispersed epithelial (squamous) metaplastic cells with atypia, mimicking squamous cell carcinoma (Fig 3b).

On FNAC, out of the 137 cases, 3 cases (2.18 %) were inadequate due to sparse cellularity, 59 cases (43. 06 %) were diagnosed as non-neoplastic, while among the 75 cases (54.74%) diagnosed as neoplastic, 65 cases (47.44%) were rendered benign while 10 cases (7.29 %) were malignant on cytology. In this study we analyzed 46 cases (33.57%) of salivary gland aspirates which had cytological and histological correlation of which one was excluded due to sparse cellularity. (Table 1).06 FNAC cases showed discordant diagnosis in specific typing of the lesion (Table 2).

Three cases remained benign on histology, however tumor type was changed which were as follows: Case 04 was WT misinterpreted as benign salivary gland cyst. Aspirated 3ml of thick material. FNA showed dirty background with normal acinar cells. Case 05 was pleomorphic adenoma which was incorrectly diagnosed as sialdenosis. The smear showed normal acinar and ductal epithelial cells. Case 06 was chronic sialdenitis incorrectly diagnosed as cellular pleomorphic adenoma. The smear showed abundant normal acinar cells, fibrillary fibrous stroma and occasional lymphocytes.

False negative cases were as follows : Case 01 was cytologically diagnosed as myoepithelioma (Fig 1a), subsequent histology revealed mucoepidermoid carcinoma with spindle cell component (Fig 1b). Case 02 was cytologically diagnosed as WT, while documented histologically as mucoepidermoid carcinoma (Fig 2a). The

Taking histology as the “gold standard, the diagnostic value of FNAC after excluding the inadequate cases was as follows: Sensitivity for diagnosis of malignant tumor was 66.7%, Specificity 97.4%, Positive Predictive Value 80%, Negative Predictive Value 95% and Diagnostic Accuracy 93.3%.

Table 1: Relation between FNAC diagnosis and final histopathologic diagnosis. CYTOLOGICAL DIAGNOSIS NON-NEOPLASTIC Epidermal cyst Sialdenosis Chronic sialadenitis Abscess Benign epithelial cyst BENIGN TUMORS Pleomorphic adenoma Warthin’s tumor Cystic lesion Myoepithelioma

NO.OF CASES 1 1 4 1 1 21 8 1 2

MALIGNANT TUMORS Mucoepidermoid carcinoma Adenoid cystic carcinoma Squamous cell carcinoma Low grade salivary gland neoplasm with atypical cells Squamous cell carcinoma /High grade Mucoepidermoid carcinoma

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1 1 1 1 1

HISTOLOGIC DIAGNOSIS 8 Epidermal cyst Pleomorphic adenoma Chronic sialadenitis Abscess Benign epithelial cyst 32 Pleomorphic Adenoma Chronic Sialdenitis Warthin’s tumor Mucoepidermoid carcinoma Warthin’s tumor Myoepithelioma Mucoepidermoid carcinoma with spindle cell component 5 Mucoepidermoid carcinoma Adenoid cystic carcinoma Warthin’s tumor Low- grade Mucoepidermoid carcinoma Metastatic squamous cell carcinoma (Parotid Lymph Node)

NO.OF CASES 1 1 4 1 1 20 1 71 1 1 1 1 1 1 1 1

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Table 2: 06 FNAC Cases of discordant diagnoses in specific typing of the lesion. Cases Age Sex Site Cytologic Diagnosis 01 58 M PAROTID Myoepithelioma 02 03 04 05 06

54 47 53 58 61

F M M M M

PAROTID PAROTID PAROTID PAROTID SUBMANDIBULAR

Warthin’s tumor. Squamous cell carcinoma Benign salivary gland cyst Sialdenosis. Cellular pleomorphic adenoma

Histopathological Diagnosis Mucoepidermoid carcinoma with spindle cell component Mucoepidermoid carcinoma Warthin’s tumor Warthin’s tumor Pleomorphic adenoma Chronic sialdenitis

Fig. 1a: Smear shows loosely cohesive spindle cells in a metachromatic fibrillary matrix misinterpreted as myoepithelioma (MGG ,10x ) Fig1b: Histological section showing mucoepidermoid carcinoma with spindle cell component (H&E,40x).

Fig. 2a: Case 2 shows FNA of mucoepidermoid carcinoma showing intermediate cells with eosinophilic cytoplasm misinterpreted as oncocytic cells of WT(Pap, 40x) Fig 2b: Histological section showing mucoepidermoid carcinoma with intermediate cells and mucous cells(H&E,40x).

Fig. 3a: FNA of WT, squamous metaplastic cells with atypia , mimicking squamous cell carcinoma (Pap,40x) Fig 3b: Histological section of WT showing cystic spaces with squamous metaplastic cells (H&E,40x) .Inset showing squamous metaplastic cells (H&E,100x).

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Discussion

FNAC of salivary gland lesions is very useful, quick, reliable and minimal invasive, however various interpretation challenges are encountered[1,21]. Sensitivity, specificity and accuracy of FNA diagnosis of salivary glands obtained in this study is comparable with the data obtained in similar studies done previously (Table 3). FNA of salivary lesions is difficult in cytology due to heterogeneity of benign and malignant tumors and the overlapping cytomorphological features which account for the indeterminate or “suspicious” diagnose[3,13]. False positive and false negative diagnoses were pointer towards problems and pitfalls in cytologic interpretation. The false negative results reported in literature ranged from 0-37% [2].In our work it was 4.44%, this was due to misdiagnosis of 2 cases , one case each of myoepithelioma and WT misdiagnosed as mucoepidermoid carcinoma. According to Orell et al [5] a definitive diagnosis of mucoepidermoid carcinoma requires the coexistence of mucin-secreting cells and cells with squamous differentiation. The challenge is not only in relation to cytodiagnosis but also in cytological typing[12]. Falsenegative diagnoses usually occur due to fluid causing dilution of tumour cells, inflammatory cells and debris obscuring the tumor cells. Rarely the bland-looking intermediate cells being misinterpreted as benign cells[6,19] as in our case . Spindle cell component in a mucoepidermoid carcinoma is however rare as seen in our case and only a few case reports have been reported. On review of the smears only spindle cell component was seen which led to an erroneous diagnosis of myoepithelioma. Neoplastic myoepithelium in salivary gland tumors, is prone to assume a spindle cell configuration and has been reported to undergo dedifferentiation[7]. The false positive rates reported in the literature ranged from 0-10%. In our series, it was 2.22%, this was due to misinterpertation of WT on FNAC as squamous cell carcinoma. WT have a characteristic cytomorphologic appearance represented by three main components oncocytes, lymphocytes, and the fluid background. Cytological difficulties can be divided into three areas:

1.) Absence of one or more diagnostic components; 2.) Squamoid pattern; and 3.) Mucinous metaplasia. The fluid imparts a dirty background appearance that may be confused tumor necrosis [8,10,15]. Mucoepidermoid carcinoma, squamous cell carcinoma, and oncocytoma are commonly misdiagnosed as WT. In the present case, FNA showed dirty necrotic background and squamous metaplastic cell clusters with atypia. However, oncocytes and lymphocytes were not visualized in the smears which was misleading and lead to the diagnosis of squamous cell carcinoma. It has been shown that metaplastic/reparative changes can occur in benign salivary gland neoplasms due to physical trauma induced by FNA which include squamous metaplasia, infarction, necrosis, subepithelial stromal hyalinization, acute/chronic hemorrhage, inflammation with multinucleated giant cells, granulation tissue with subsequent fibrosis, cholesterol cleft formation, pseudoxanthomatous reaction and microcystic degeneration[9]. Squamous metaplasia in our case could be attributed to previous FNA . In one case (case 4) WT , a non specific diagnosis of cystic lesion was made in cytology as the smears showed mainly fluid and benign acinar cells even on repeated aspiration. On histology it turned out to be WT with predominant cystic change. Histologi cally, WT characteristically consists of cystic and solid areas. The cystic area is lined by layers of tall columnar oncocytic luminal cells and flattened or cuboidal basal cells, while the stroma consists of lymphocytes[10-12].The cytological diagnosis of cystic salivary gland lesions is rather difficult due to the wide range of lesions that enter the differential diagnosis which include chronic sialadenitis, WT, acinic cell carcinoma, pleomorphic adenoma and mucoepidermoid carcinoma[4,21]. In one case (case 5) pleomorphic adenoma was reported as sialadenosis on FNA. The smear showed only acinar and benign ductal epithelial cells. The error was mainly due to sampling which highlights the importance of multiple sampling especially in a small sized lesion. In another case (case 6), a histologically proven chronic sialdenitis was misinterpreted as cellular pleomorphic adenoma on FNAC. This was mainly due to cellular

Table 3: Comparative analysis of sensitivity, specificity, accuracy of FNAC. Study No of Cases Sensitivity Specificity Present study Yadi Rama Raju etal14 StramandinoliRTetal16 Iqbal M et al 17 Rehman H et al18

46 75 79 49 50

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66.7% 83.3% 82.3% 62.5% 53.28%

97.4%, 97.7% 68.2% 96.97% 88.57%

Diagnositic Accuracy

Ppv

Npv

93.3%. 92% 87.7 % 96.4% 78%

80% 96.1% 68.2%

95% 89.8% 87.7%

72.7%

79.9%

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smears and lack of acini with only ductal epithelial cells. Usually clinical correlation solves the problem. Tumor like nodules caused by focal chronic inflammation is seen in chronic sialadenitis. Presence of epithelial cell aggregates associated with fibrillary fibrous stroma could be mistaken for pleomorphic adenoma, but the fragments of ductal epithelial cells are cohesive and stroma is not chondromyxoid[5] . Our experience shows that FNA cytology of salivary gland lesions is a valuable diagnostic method. Accuracy depends on experience, and this method provides superior advantages for the clinicians and the patients.

Conclusion

FNAC is recommended as a safe and reliable technique in diagnosis of salivary gland lesions. Despite of high sensitivity, there are certain pitfalls due to the misleading diagnostic yields. A cautious approach towards salivary gland lesions is recommended. Pitfalls in cytologic diagnosis were due to errors in sampling, cystic lesions and interpretation of smears.

References

Viguer JM, Vicandi B, Jiménez-Heffernan JA, López-Ferre P, González- Peramato P, Castillo C. Role of fine needle aspiration cytology in the diagnosis and management of Warthin’s tumour of the salivary glands. Cytopathology 2010; 21:164-9.

Chan JK, Tang SK, Tsang WY, Lee KC, BatsakiS JG. Histologic changes induced by fine-needle aspiration. Adv Anat Pathol 1996;3:71-90.

10. Veder LL, Kerrebijn JD, Smedts FM, Bakker MA. Diagnostic accuracy of fine-needle aspiration cytology in Warthin tumors. Head Neck 2010; 32: 1635-40. 11. Mukunyadzi P. Review of fine-needle aspiration cytology of salivary gland neoplasms, with emphasis on differential diagnosis. Am J Clin Pathol 2002; 118: 100-15. 12. Faur A, Lazăr E, Cornianu M, Dema A, Vidita CG, Găluşcan A. Warthin tumor: a curious entity: case reports and review of literature. Rom J Morphol Embryol 2009; 50: 269-73. 13. Desai P, Ami shah. Diagnostic Accuracy of Fine Needle Aspiration Cytology (FNAC) and Histopathology in Salivary Gland Lesions. IJSR, 2014; 4 (11):433-434. 14. Yadi1 RR, Chetri SR, Arasi NE, Anunayi J, Sreedhar V. Diagnostic accuracy of fine needle aspiration cytology in salivary gland neoplasms-2 year study. IJRHS 2015; 3 (1) :99-110.

Aan NL, Tanwani AK. Pitfalls in Salivary Gland FineNeedle Aspiration Cytology. International Journal of Pathology.2009;7(2):61-65.

15. Mukunyadzi P. Review of Fine-Needle Aspiration Cytology of Salivary Gland Neoplasms, With Emphasis on Differential Diagnosis Am J Clin Pathol. 2002;118:100-115.

Tahoun N, Ezza TN. Diagnostic Accuracy and Pitfalls of Preoperative Fine Needle Aspiration Cytology in Salivary Gland Lesions. Journal of the Egyptian Nat. Cancer Inst. 2008; 20(4):358-368.

16. Stramandinoli RT, Sassi LM, Pedruzzi PAG et al. Accuracy, sensitivity and specificity of fine needle aspiration biopsy in salivary gland tumours: A retrospective study. Med Oral Pathol Cir Bucal. 2010;15: 32-7.

Ersoz C, Uguz AH, Tuncer U, Soylu L, Kiroglu M. Fine needle aspiration cytology of the salivary glands: a twelve years’ experience. Aegean Pathology Journal. 2004;1:51–56.

17. Iqbal M, Anwar K, Ihsanullah, Mohammad J, Khan IA, Hussain G. The diagnostic value of fine needle aspiration cytology in masses of the salivary glands. JPMI 2011; 25: 73-7.

Sangeetha N, Karthika V, Latha S. Cytological analysis of salivary gland lesions with histopathological correlation. IJPBS.2013;3(4):122-128.

18. Rehman H, Khan MS, Wahid F, Ahmad I. A profile of parotid gland tumours from a tertiary care hospital in Peshawar. JPMI 2011;25158- 62.

Orell SR, Sterret GF, Whitaker D, editors. Fine Needle Aspiration Cytology, 5th ed. Edinburgh: Churchill Livingstone-Elsevier; 2005:53-69.

Al-Khafaji M, Nestok R, Katz L. Fine needle aspiration of 154 parotid masses with histololgic correlation. Cance Cytopathology.1998;84:153-159.

19. Klijanienko J, Vielh P. Fine-needle sampling of salivary gland lesions, II: cytology and histology correlation of 71 cases of Warthin’s tumor (adenolymphoma). Diagn Cytopathol.1997; 16:221-225.

Ide F, Mishima K, Saito I. Mucoepidermoid Carcinoma with Spindle Cell Change: A Low-grade Lesion Potentially Mistaken for Sarcomatoid Dedifferentiation. Head Neck Pathol 2008 ; 2(3): 227–230.

20. Elagoz SY, Gulluoglu MG, Imazbayhan DW, Ozer HS. The value of fine-needle aspiration cytology in salivary gland lesions. J Otolaryngol Head & Neck Surg. 2007; 69: 51-56. 21. Ameli F, Baharoom A, Nurismah, Akmal SN. Diagnostic challenges in fine needle aspiration cytology of salivary gland lesions. Malaysian J Pathol 2015; 37(1) :11 – 18 .

*Corresponding author: Dr Crysle Saldanha, Assistant Professor, Department Of Pathology, Father Muller Medical College, Mangalore-575002 India Phone: +91 9164323693 Email: cryslesaldanha@gmail.com Date of Submission : 15.01.2017 Date of Acceptance : 01.03.2017 Financial or other Competing Interests: None. Date of Publication : 15.06.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Original Article DOI: 10.21276/APALM.1340

Spectrum of Thyroid Lesions in A Tertiary Care Hospital using Bethesda System for Reporting Thyroid Cytopathology Mallikarjun A Pattanashetti1*, Ranjit P Kangle2 and Hema B Bannur2 Department of Pathology, S. Nijalingappa Medical College, Bagalkot, Karnataka, India Department of Pathology, KLE University’s Jawaharlal Nehru Medical College , Belagavi, Karnataka, India 1

ABSTRACT Background: Thyroid Fine Needle Aspiration (FNA) has been widely used as a first line investigation to assess thyroid nodules, as it is rapid, cost effective, safe and reliable. To bring uniformity and standardization in thyroid cytology reporting, “The Bethesda System for Reporting Thyroid Cytopathology” (TBSRTC) was introduced and it is gaining acceptance. This study has been undertaken to evaluate the reproducibility using TBSRTC system while reporting thyroid FNACs and to find out the utility of Bethesda system after correlating with the histopathology. Methods: A retrospective study was conducted in which 173 cases of thyroid aspirates were reclassified according to TBSRTC in to six categories by cytologist. Results: A total of 173 thyroid lesions were analysed. Category wise distribution of aspirates was Non diagnostic (ND) 9 (5.20%), Benign (BN) 145 (83.81 %), Follicular neoplasm (FN) 9 (5.20%), Follicular lesion of uncertain significance (FLUS) Nil, suspicious of malignancy(SM) 1(0.57 %) and malignant category 9 (5.20 %). Age and sex wise distribution was interpreted.Thyroid diseases were more prevalent in women and most common age group affected was 3rd to 4th decade. Out of 164 lesions which were satisfactory for evaluation,120 (73.17 %) were simple goitre , 15(9.14 %) were of thyroiditis, 6 (3.65%) were toxic goitre, 4 (2.43%) were thyroid cysts & 19 (11.57 %) were neoplasms of which benign included 1 (0.6%) Hurthle cell neoplasm and 8 (4.87 %) follicular neoplasm. Malignant lesions were 8 (4.87%) papillary carcinoma, 1 (0.6%) anaplastic carcinoma and 1(0.6%) medullary carcinoma. Conclusions: It was observed that standardized nomenclature of the Bethesda system has brought much needed clarity in thyroid FNAC reporting. Along with malignant category, the FLUS, FN and SM categories carry higher malignancy risk. Keywords: Thyroid FNAC; Bethesda System;

Introduction

Thyroid nodule is a common clinical condition and nearly 85 to 90% of them are benign lesions. [1,2] Thyroid FNA has been widely used as a firstline investigation to assess thyroid nodules, as it is rapid, cost effective, safe and reliable. [3] It is important that cytology report is unambiguous and clinically useful. It has been observed that thyroid FNAC smears terminologies vary significantly from one laboratory to other, sometimes from one cytologist to other in the same institution. This is creating confusion in some cases and has become an obstacle in sharing information amongst different institutions.[4,5] This issue of terminology related to thyroid cytology was addressed at National Cancer Institute (NCI) which hosted “NCI thyroid FNA state of the science conference” which led to the formation of “The Bethesda System for Reporting Thyroid Cytopathology” (TBSRTC).[6] The TBSRTC system is presently being widely used in US and several European Countries, and in India also it is gaining acceptance. This study has been undertaken to evaluate the reproducibility

using TBSRTC system while reporting thyroid FNACs and to find out the utility of Bethesda system as there is lack of such data in this region.

Materials and Methods

Details of all the thyroid FNAC cases done from Jan 2009 to November 2014 in a tertiary care hospital were retrieved from archives of cytology section. Experienced cytologist with experience in cytology reporting and self learned the different aspects of TBRTC, reviewed the slides. All the clinical details and available radiological and thyroid function test results which were noted down in the original cytology request forms were provided to cytologist. The FNA smears were reclassified in a double blinded fashion into six categories as per TBSRTC. The Six Categories are as below [7] a. Non diagnostic (ND); Smears were considered as nondiagnostic when a thyroid FNA sample failed to fulfill the recommended criteria for adequacy which

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are presence of a minimum of six groups of well visualized follicular cells, with at least ten cells per group, preferably on a single slide, absence of colloid or only blood. b. Benign (BN); Lesions were classified into this category if the smears showed features suggestive of colloid nodule, multinodular goiter, thyroiditis, as well as if the aspirate showed benign follicular cells only. c. Follicular lesion of undetermined significance (FLUS); smears that contain cells with architectural and/or nuclear atypia that is not sufficient to be classified as suspicious for a follicular neoplasm or suspicious for a malignancy. d. Follicular neoplasm (FN)/Suspicious for follicular neoplasm(SFN); Lesions were classified into this category if they were having high follicular cellularity with predominant microfollicle formations, scant colloid. Lesions exhibiting Hurthle cells predominantly were also included. e. Suspicious for malignancy(SM); Smears in this category were mainly cellular with crowded cell groups exhibiting nuclear and cytoplasmic pleomorphism with some occasional single atypical cells. In the context of suspicious papillary carcinoma rare presence of nuclear enlargement, grooves, overlapping and/or pseudoinclusions along with thick colloid were considered suspicious.

f. Malignant (MGT); Lesions were classified into this category if they were frankly malignant with type specification. The spectrum of all the thyroid lesions was classified as per the above six categories.

Result

A total of 173 thyroid lesions were analysed and categorized as per TBSRTC. Category wise distribution of aspirates was Non diagnostic (ND) 9 (5.20%), Benign (BN) 145 (83.81 %), Follicular neoplasm (FN) 9 (5.20%), Follicular lesion of uncertain significance (FLUS) Nil, Suspicious of malignancy (SM) 1 (0.57 %) and Malignant category 9(5.20 %) (Table 1). The various thyroid lesions in all the categories are described in Table 2. The female to male ratio in our study was 3.5 : 1. The youngest patient was 11 year female child with simple colloid goitre. The oldest patient was 80 years male patient with papillary carcinoma. Most common age group affected with thyroid lesions was 3rd to 4th decade and most common thyroid lesion was simple colloid goitre which was most commonly seen in 3rd to 4th decade. Least common age group affected was 71-80 years. Most common lesion in 7th to 8th decade was colloid goitre with cystic change as shown in Table 3. Most common thyroid malignancy was Papillary carcinoma seen most commonly in 6th to 7th decade. Most common Inflammatory thyroiditis was Hashimotoâ&#x20AC;&#x2122;s thyroiditis. The most common thyroid lesion in Pediatric age group was simple colloid goitre.

Table 1: Distribution of cases as per TBSRTC. Bethesda Diagnostic category I Nondiagnostic or unsatisfactory (ND) II Benign (BN) III Atypia of undetermined significance (AUS) or Follicular lesion of undetermined significance(FLUS) IV Follicular neoplasm or Suspicious for follicular neoplasm(FN /SFN) V Suspicious for malignancy (SM) VI Malignant (M) Total

Total 9 ( 5.20 %) 145 ( 83.81 %) 0 9 ( 5.20 % ) 1(0.57 %) 9 (5.20 %) 173

Table 2: Distribution of cases as per TBSRTC LESIONS NONDIAGNOSTIC OR UNSATISFACTORY BENIGN Simple Colloid goitre Colloid goitre with cystic change Thyroid cyst Toxic goitre Multinodular goitre Acute thyroiditis Granulomatous thyroiditis Lymphocytic thyroiditis Hashimotoâ&#x20AC;&#x2122;s thyroiditis

TBSRTC Category I

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Total 9 ( 5.20 %) 145 ( 83.81 %) 79 30 4 6 11 1 2 4 8

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LESIONS

TBSRTC Category

Total

III

ATYPIA OF UNDETERMINED SIGNIFICANCE OR FOLLICULAR LESION OF UNDETERMINED SIGNIFICANCE FOLLICULAR NEOPLASM OR SUSPICIOUS FOR A FOLLICULAR NEOPLASM

9 ( 5.20 % ) IV

Follicular neoplasm

Hurthle cell neoplasm

SUSPICIOUS FOR MALIGNANCY MALIGNANT Papillary Carcinoma Anaplastic carcinoma

1(0.57 %) 9 (5.20 %) 7 1

Medullary carcinoma

Total

173

Table 3: Age wise distribution of thyroid lesions. Age in years

Lesion

< 10

11- 20

21-30

31-40

41 - 50

51 -60

61-70

71 - 80

TOTAL

Simple Colloid goitre

Colloid goite with cystic change

Thyroid cyst

Toxic Goitre

Multinodular goitre

Acute thyroiditis

Granulomatous thyroiditis

Lymphocytic thyroiditis

Hashimoto’s thyroiditis

AUS / FLUS

Hurthle cell neoplasm

Follicular neoplasm

SUSPICIOUS FOR MALIGNANCY

Papillary Carcinoma

Anaplastic carcinoma

Medullary carcinoma

Total

173

NONDIAGNOSTIC OR UNSATISFACTORY BENIGN

FN / SFN

MALIGNANT

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Fig. 1: Smear shows abundant follicular epithelial cells in macrofollicles against background of thick and thin colloid suggestive of colloid goitre ( PAP stain – X 10).

Fig. 2: Smear shows cellular smears showing numerous microfollicles with scanty colloid in the background. (Giemsa stain – X 10).

Discussion

4). The various benign lesions (Class II) found in our study were Simple colloid goitre, Colloid goitre with cystic change, Thyroid cyst, Toxic goitre, Multinodular goitre, Acute thyroiditis, Granulomatous thyroiditis, Lymphocytic thyroiditis and Hashimoto’s thyroiditis. The class IV included Follicular neoplasm and Hurthle cell neoplasm. The malignant class VI included Papillary carcinoma, Anaplastic carcinoma and Medullary carcinoma However, percentage of BN is much higher accounting for 83% in our study may be because of the fact that along with the referral cases, our hospital caters to the needs of many direct hospital visitors without reference. Like in general population, benign lesions were maximum in our set up. Percentage of FLUS category was less as per TBSRT recommendations. Our study was compared with studies by Parikh et al [10] , Uma et al [11] and Bhatta et al.[12] It was found that most were benign lesions similar to all other studies. However our study had similar Non-diagnostic (ND) smears as study by Uma et al. The number of follicular neoplasms/ Suspicious for follicular neoplasms seen in our study is 5.2 % which is higher as compared all other studies which reported around 3.2 %. The malignant cases in our study is 5.2 % which varied widely from 2.5 % to 11.1 % in various other studies.

Fine needle aspiration cytology is considered standard diagnostic test for the diagnosis of thyroid lesions . There is a lack of uniformity which is a big hindrance in interpreting the reports of thyroid FNA. Institutional, personalized and descriptive terminologies without proper categorization is leading to confusion in the minds of treating physicians. TBSRTC system was introduced after the Bethesda meeting of cytopathologists, endocrinologists, surgical pathologists, radiologists and surgeons to put in place, a universal reporting system through which cytologists and physicians could understand each other and could help in predicting the prognosis by estimating the malignant potential of the individual category.[7,8,9] An attempt was made in the present study, to reclassify the 173 cases according to the new proposed six tier diagnostic classification system in reporting thyroid FNA results as Non diagnostic (ND) 9 (5.20%), Benign (BN) 145 (83.81%), Follicular neoplasm (FN) 9 (5.20%),Follicular lesion of uncertain significance (FLUS) Nil, Suspicious of malignancy (SM) 1 (0.57 %) and malignant category 9(5.20). TBSRTC also assists in calculating the malignancy risk for eash category which is essential for treatment decision. Benign category was maximum in our study similar to the other studies (Table Table 4: Comparison to other studies Lesions

Parikh et al

Uma et al 4

Bhatta et al

Our study

Non diagnostic / Unsatisfactory

Benign

207

381

145

FN / SFN Suspicious for malignancy

Malignancy

240

434

173

Total

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Conclusion

Fine needle aspiration cytology is standard diagnostic test for the diagnosis of thyroid lesions with a high diagnostic yield, accuracy, sensitivity & specificity. Fine needle aspiration cytology is a cost effective procedure that provides specific diagnosis rapidly with minimum complications. It was observed that standardized nomenclature of the Bethesda system is more systematic and brought much needed clarity in thyroid FNAC reporting. Along with malignant category, the FLUS, FN and SM categories carry higher malignancy risk. Close follow up of the patients and surgical intervention option has to be considered in FLUS, FN and SM categories. However, a prospective study with large number of cases for cytohistopathological correlation may be needed to improve our understanding of these TBSRTC categories.

Acknowledgement

The authors would like to thank the patients for their assistance in the study. W also thank Department of Pathology and Department of Surgery, Jawaharlal Nehru Medical College , Belagavi for their support.

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Cibas ES. Fine needle aspiration in the workup of thyroid nodules. Otolaryngol Clin North Am 2010;43:257-71.

Park JH, Kim HK, Kang SW, Jeong JJ, Nam KH, Chung WY, et al. Second opinion in thyroid fine needle aspiration biopsy by the Bethesda System. Endocr J.2012;59:205-12.

Baloch ZW, Li Volsi VA, Asa SL, Rosai J, Merino MJ, Randolph G, et al. Diagnostic terminology and morphologic criteria for cytologic diagnosis of thyroid lesions: A synopsis of the national cancer institute thyroid fine needle aspiration state of the science conference. Diagn Cytopathol 2008;36:425-37.

Cibas ES, Ali SZ. The Bethesda system for reporting thyroid cytopathology. Am J Clin Pathol 2009;132:658-65.

Richmond BK, O’Brien BA, Mangano W, Thompson S, Kemper S. The impact of implementation of the bethesda system for reporting thyroid cytopathology on the surgical treatment of thyroid nodules. Am Surg 2012;78:706-10.

Layfield LJ, Morton MJ, Cramer HM, Hirschowitz S.Implications of the proposed thyroid fine needle aspiration category of “follicular lesion of undetermined significance”: A five year multi institutional analysis.Diagn Cytopathol 2009;37:710-4.

References 1.

Sakorafas GH. Thyroid nodules; interpretation and importance of fine needle aspiration (FNA) for the clinician – Practical considerations. Surg Oncol 2010;19:130-9.

10. Parikh UR, Goswami H.M., Shah AM., Mehta N.P, Gonsai R.N. Fine Needle Aspiration Cytology (FNAC) Study of Thyroid Lesions (Study of 240 Cases) Gujarat medical Journal; 2012;67(2):25–30.

Redman R, Yoder BJ, Massoll NA. Perceptions of diagnostic terminology and cytopathologic reporting of fineneedle aspiration biopsies of thyroid nodules: a survey of clinicians and pathologists. Thyroid.2006;16:1003-8.

11. Bhatta S, Makaju R, Mohammad . Role of fine needle aspiration cytology in the diagnosis of thyroid lesions; Journal of Pathology of Nepal.2012;2:186-8.

Baqqa PK, Mahajan NC. Fine needle aspiration cytology of thyroid swelling: how useful and accurate is it? Indian Journal of Cancer.2010;47:437-42.

12. Uma Handa, Sukant garg, Harsh Mohan, Nitin Nagarkar .Role of fine needle aspiration cytology in diagnosis and management of thyroid lesions: A study on 434 patients.J Cytol.2008;25(1):13-8.

*Corresponding author: Dr. Mallikarjun .A .Pattanashetti, Plot No 295 , RS No 183, 3rd Main, 2nd Stage, Hanuman nagar , Belagavi – 590001, Karnataka, India Phone: +91 9739462156 Email: mallikarjun2030@gmail.com Date of Submission : 09.02.2017 Date of Acceptance : 13.03.2017 Financial or other Competing Interests: None. Date of Publication : 14.06.2017

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Original Article DOI: 10.21276/APALM.1355

Spectrum of Lymph Node Lesions by Fine Needle Aspiration Cytology: A Retrospective Analysis Shilpa Somashekar Biradar1 and Deepa Siddappa Masur2 * 1

Dept. of Pathology, Akash Institute of Medical Sciences and Research Centre, Devanahalli, Bangalore, Karnataka, India 2 Dept. of Pathology, S. N. Medical College, Bagalkot, Karnataka, India

ABSTRACT Background: Fine needle aspiration cytology (FNAC) is a veritable tool for the assessment and diagnosis of superficial lymph node enlargement. The cytologic patterns of lymph node fine needle aspirations exhibit a wide variation in different diseases. Lymphadenopathy is of great clinical significance and the underlying cause may range from a treatable infectious etiology to malignant neoplasms. The aim of the present study is to study and evaluate the patterns of various lymph node lesions on fine needle aspiration cytology

Methods: This retrospective study was conducted on 160 selected patients including all age groups and both sexes with lymphadenopathy who had undergone FNAC. We reviewed all the cases of lymphadenopathies. The cytomorphological features seen in the aspirate were critically analysed and correlated with their aetiology Result: Out of 160 cases, the most frequent cause of lymphadenopathy was found to be Reactive Lymphadenitis with 89 cases (55.62%). The next frequent diagnosis was Tuberculosis with 38 cases (23.75%) followed by malignant lymphoma in 8 cases (5%) and metastatic lymphadenopathy in 7 cases (4.37%). Conclusion: FNAC is a simple, safe, reliable, and inexpensive method in early detection of lymph node lesions, which has been proven in this study again. Keywords: Fine Needle Aspiration Cytology, Lymphadenopathy, Tuberculous Lymphadenitis, Reactive Lymphadenitis

Introduction

Fine needle aspiration cytology was thought as a means to confirm a clinical suspicion of local recurrence or metastasis of known cancer without subjecting the patient to further surgical intervention. The role of FNAC is not limited to neoplastic conditions. It has a valuable role in the diagnosis of inflammatory, infectious and degenerative conditions. The technique is minimally invasive and gives a speedy result [1].Lymph nodes are an important part of the immune system. Lymph nodes become enlarged in a wide spectrum of diseases, including infection and malignancy. Cytological examination of FNA smears can determine whether lymphadenopathy is due to reactive hyperplasia, infection, metastatic malignancy or malignant lymphoma [2] .The present study reports the results of FNAC of lymphadenopathies based on cytomorphology.

Materials and Methods

This was a retrospective study conducted in a tertiary hospital for a period of one year. The study was conducted on 160 patients including all age groups and both sexes who underwent FNAC for palpable lymphadenopathy, either single or multiple.

A brief clinical history followed by physical examination was done and the findings were noted. FNAC was performed under aseptic precautions using 22-24 Gauge needles attached to 10 ml syringes. The aspirated material was smeared on to the glass slides. The smears were then fixed in 95% ethyl alcohol and stained with Hematoxylin and Eosin stain and Papanicolaou stain. Leishman stain was done on air dried smears. Zeil-Neelson staining was done whenever required. The cytological diagnosis for each case was based on cytomorphology and available clinical information

Result

A total number of 160 patients were studied. Among them, 82(51.25%) patients were male and 78(48.75%) were female patients(Table 1). The age of the patients ranged from 3months to 80 years (Table 2). Cervical lymph nodes were enlarged in 111 of 160 cases (69.37%) followed by submandibular lymph nodes in 17 cases (10.62%), supraclavicular lymph nodes in 10 cases (6.25%), axillary in 8 cases (5%) ,inguinal in 5 cases (3.12%), submental in 4 cases(2.5%) (Table 3). Multiple lymph nodes were involved in 5 cases(3.12%). Five cases had inadequate material and were thus unsatisfactory for

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evaluation. Among 160 cases , maximum number of cases were recorded in age group less than 20 years. A major proportion of lymphadenopathies in this study were due to benign conditions (90.62%).Out of 160 cases, the most frequent cause of lymphadenopathy was found to be Reactive Lymphadenitis with 89 cases (55.62%). The next

frequent diagnosis was Tuberculosis (Figure 1) with 38 cases(23.75%) followed by malignant lymphomas (Figure 2) in 8 cases (5%) and metastatic lymphadenopathy in 7 cases (4.37%). Acute suppurative lymphadenitis was seen in 7 cases (4.37%) and granulomatous lymphadenitis in 6 cases(3.75%) (Table 4).

Table 1: Gender wise distribution of patients (n =160) Number of cases Male 82 Female 78

Percentage 51.25% 48.75%

Table 2: Age wise distribution of patients (n=160) Age group in years 0-20 21-40 41-60 61-80

Number of cases 75 60 17 08

Percentage 46.87 37.5 10.62 5

Table 3: Sites of lymph node involvement (n= 160). Site Number of cases Cervical 111 Axillary 08 Supraclavicular 10 Inguinal 05 Submandibular 17 Submental 04 Multiple lymph nodes 05

Percentage 69.37% 5% 6.25% 3.12% 10.62% 2.5% 3.12%

Table 4: Cytological diagnoses of lymph node aspirations(n=160). Diagnosis Number of cases Reactive Lymphadenitis 89 Tubercular Lymphadenitis 38 Malignant Lymphoma 09 Metastatic Lymphadenopathy 07 Acute Suppurative Lymphadenitis 06 Granulomatous Lymphadenitis 06 Inadequate 05

Percentage 55.62% 23.75% 5.62% 4.37% 3.75% 3.75% 3.12%

Fig. 1: Smear shows epithelioid cell granulomas and necrosis in the background (H & E stain ;400x).

Fig. 2: Smear shows clusters of atypical lymphoid cells from axillary lymph node FNAC (PAP Stain;400x).

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Spectrum of Lymph Node Lesions

Discussion

FNAC is the study of cellular aspirate obtained through a fine needle under negative pressure. The technique is relatively painless and economical. It can give unequivocal diagnosis in most of the conditions. FNAC is a simple, quick, and inexpensive procedure that is used to sample superficial masses. The procedure is performed in the outpatient clinic. Lymphadenopathy is an abnormal increase in size and altered consistency of lymph nodes. It is a clinical manifestation of regional or systemic disease and serves as an excellent clue to the underlying disease. In some cases, cervical lymphadenopathy may be the only clinical finding. This can be a clue for many underlying clinical conditions [3] . Our study highlights the spectrum of cytological findings of various lymphadenopathies on fine needle aspiration cytology. Majority of lymphadenopathies in this study were due to benign conditions (90.62%), which was in accordance with an earlier study, in which 86.4% of the lesions were benign [4] .The study conducted by Tilak [5] et al revealed that the lesion arising in lymph nodes can be found in patients of different age groups, ranging from an early to advanced age. This was correlated with our findings where we found that the youngest patient in the present study was 3 months old and the oldest one was 80 years old. These figures came in close comparison to other studies. In the present study, 75(46.87%) patients were in the age group of 0-20 years. Simlar to the observation of Gupta et al 52.26% [6] ,whereas in the study of Pandit AA et al[7] , most of the patients 146 ( 51.05%) were in the age group of 21-40 years. As compared to other studies, male predominance was noted in the current study. These findings are comparable with studies conducted by Gupta et al [6] and Khajuria et al [8]. The present study revealed that the most common group of lymph node involved were cervical nodes which is in accordance with other studies done by Kochhar et al [9], Pavithra et al [10].In the current study, Reactive lymphadenitis was the most common lesion and was reported in 55.62% cases. This result was comparable to other studies, where its incidence ranged from 18.9% to 42% [8,9,11,12,13,14 ] .The second common diagnosis in the present study was Tuberclar lymphadenitis accounting to 23.75% of cases. Low incidence of AFB positivity on ZN smears was noted in our study (15%) which is in accordance with the study done by Agarwal et al[15] (19.65%). This could be attributed to the compromised immune status or inadequacy of the cellular immune response. In this study, we considered the presence of scattered epithelioid cells with or without granulomas or only necrotic material with neutrophilic infiltration as tuberculous lymphadenitis, inspite of AFB being absent in these smears [16 ].Granulomatous

lymphadenopathies constituted 3.75% of cases. Acute suppurative lymphadenopathy was observed in 4.37% cases in our study which is in accordance with other studies done by Kochhar et al (4%) and Patra et al [17] (5.8%).Malignant lymphomas were less in our study constituting 5.62% of all the cases. Similar observations were seen in other studies[18,19,12,8] . Non –Hodgkin’s Lymphoma was reported in 5(3.12%) out of 9 lymphoma cases whereas 4(2.49%) cases of Hodgkin’s lymphoma were reported. Lymph node aspirates in 4.37% cases showed metastatic deposits predominantly squamous cell carcinoma. Similar results were obtained in the study conducted by Pavithra et al [10] .In the current study 3.12% of cases were unsatisfactory to report due to low cellular yield. Despite the limitations, FNAC provides a simple, reliable and convenient method for the initial management of cervical lymphadenopathy. FNAC has a valuable role in diagnosing neoplastic and metastatic lesions. It helps in detecting metastatic diseases and also gives the clue regarding the origin of the primary tumor.

Conclusion

FNAC is a very important diagnostic tool for diagnosing benign as well as malignant lesions. It is a simple, safe and inexpensive definite diagnostic procedure to render a diagnosis, especially in lymph node aspirates. Our study highlighted the various cytomorphological patterns of lymphadenopathies. We conclude that,the benign results should be interpreted in the context of clinical findings and if clinical malignancy is highly suspected, further evaluation is justified.

Reference 1.

Orell SR, Sterrett GF, Fine needle Aspiration Cytology.5th edition. Churchill Livingstone.2012

Bibbo M, Comprehensive Cytopathology. edition.W.B.Saunders Company.1991

Kataria P, Sachdeva M, Singh NK. FNAC as a diagnostic tool for the diagnosis of cervical lymphadenopathy. Bull Environ Pharmacol Life Sci 2012;1:72-5.

Ahmad S, Akhtar S, Akhtar K, Naseem S, Mansoor T. Study of Fine needle aspiration cytology in lymphadenopathy with special reference to acid-fast staining in cases of tuberculosis. JK science 2005;7 (1):1-4.

Tilak V, Dhadel AV, Jain R. Fine needle aspiration cytology of the head and neck masses. Ind J Pathol Microbiol.2002;45(1):23–30.

Gupta AK, Nayar M, Chandra M. Reliability and limitations of fine needle aspiration cytology of lymphadenopathies. An analysis of 1,261 cases. Acta Cytol 1991;35:777-83.

Pandit AA,Candes FP, Khubchandani SR. Fine needle aspiration cytology of lymph nodes.J Postgrad Med.1987;33(3):134-6

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Khajuria R, Goswami KC, Singh K, Dubey VK. Pattern of lymphadenopathy on FNAC in Jammu. JK Sci 2006;8:157-9.

cytology in worker population of Eastern zone of India. IJOH.2015;7(3):139-144.

Kochhar AK, Duggal G, Singh K, Kochhar SK. Spectrum of cytological findings in patients with lymphadenopathy in rural population of South Haryana, India â&#x20AC;&#x201C; Experience in a tertiary care hospital. Internet J Pathol.2012;13(2):8

15. Aggarwal P, Wali JP, Singh S, Handa R, Wig N, Biswas A. A clinicobacterial study of peripheral tuberculous lymphadenitis.J Assoc Physicians India. 2001; 49: 808-12.

10. 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):377-384 11. Mohanty R, Wilkinson A. Utility of fine needle aspiration of lymph nodes. IOSR J Dent Med Sci.20138(5);13-8. 12. Hirachand S, Lakhey M, Akhter J, Thapa B. Evaluation of fine needle aspiration cytology of lymph nodes in Kathmandu Univ Med J.2009;7:139-42. 13. Adhikari P, Sinha BK,Baskota DK. Comparison of fine needle aspiration cytology and histopathology in diagnosing cervical lymphadenopathies. Australas Med J.2011;4:97-9. 14. Mallick D, Nathprasad R, Gon S, Ghosh G.Spectrum of Lymph Node Lesions by fine needle aspiration

16. Paul PC, Goswami BK, Chakrabarti S, Giri A, Pramnik R. Fine needle aspiration cytology of lymphnodes â&#x20AC;&#x201C; an institutional study of 1448 cases over a five year period.J Cytol.2004;21:187-190. 17. Patra AK, Nanda BK, Mahapatra BVK, Panda AK. Diagnosis of lymphadenopathy by fine needle aspiration cytology. Indian J Pathol Microbiol.1983;26 :272-8. 18. Annam V, Kulkarni MH, Puranik RB. Clinicopathologic profile of significant cervical lymphadenopathy in children aged 1-12 years. Acta Cytol 2009;53:174-8. 19. Fatima S,Arshad S, Ahmed Z, Hasan SH.Spectrum of cytological findings in patients with neck lymphadenopathyexperience in tertiary hospital in Pakistan. Asian Pac J Cancer Prev.2011;12:1873-5.

*Corresponding author: Dr. Deepa S Masur #Assistant Professor, Dept. Of Pathology, S.N.Medical College, Bagalkot, Karnataka, India Email: drdns11s@gmail.com

Financial or other Competing Interests: None.

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Original Article DOI: 10.21276/APALM.1357

Role of Platelet Indices in Differentiating Hypoproductive and Hyperdestructive Thrombocytopenia Shaheena Parveen and Mourouguessine Vimal* Department of Pathology, Sri Manakula Vinayagar Medical College and Hospital, Puducherry, India

ABSTRACT Background: During evaluation of thrombocytopenic patients, it is essential to identify the etiology, whether it is due to hypoproduction or hyperdestruction which will have impact on the management. Aim of study is to evaluate the variation and relationship of platelet indices in hypoproductive and hyperdestructive thrombocytopenia patients. Methods: A cross sectional study for a period of 2 months on Patients with thrombocytopenia. Platelet count, Plateletcrit (PCT), Platelet Distribution Width (PDW) and Mean Platelet Volume (MPV) and relevant clinical details of the thrombocytopenic patients were collected and tested for statistical significance by unpaired t test. Results: This study included 120 patients of thrombocytopenia who were classified into hypoproductive (26 cases) and hyperdestructive (94 cases). The mean platelet count in hypoproduction group is 75.9 ± 36.4 and in hyperdestruction group is 79.6 ± 36.3 with a P value of 0.64. The mean MPV in hypoproduction group is 10.17 ± 1.3 and in hyperdestruction group is 12.3 ± 0.9 with a significant P value of 0.05. The mean PDW in hypoproduction group is 19.7 ± 5.4 and in hyperdestruction group is 19.3 ± 4.2 with a P value of 0.7. The mean PCT in hypoproduction group is 0.06 ± 0.03 and in hyperdestruction group is 0.08 ± 0.1 with a P value of 0.2. Conclusion: Mean platelet volume may provide useful information in discriminating the hypoproductive and hyperdestructive thrombocytopenia. Interpretation of platelet indices can help the thrombocytopenic patients in the initial management and can avoid invasive investigations. Keywords: Thrombocytopenia, Platelet Indices, Mean Platelet Volume, Plateletcrit, Platelet Distribution Width

Introduction

Thrombocytopenia is a significant finding in hospitalized patients which may be often missed if platelet parameters are not evaluated routinely. Platelet counts below 1,50,000 define thrombocytopenia, but they do not reveal the underlying pathology.[1]During evaluation of these patients, it is essential to identify the etiology, whether it is due to hypoproduction or hyperdestruction and this will have impact over the proper management of the patients. For a long time Bone marrow aspiration remained the gold standard method for evaluating the cause of thrombocytopenia. But this procedure is invasive, time consuming as well as carries an overt risk of bleeding diathesis in critical thrombocytopenia cases. Serology (For infectious diseases) , Platelet associated Immunoglobulin G(PAIgG) and Molecular markers for Disseminated Intravascular coagulation(DIC) are used in evaluating thrombocytopenic patients which are relatively costly.[2] Previously platelet count was the only vital information available about this small blood element. But recently automated blood cell analyzers have made it possible to measure various Platelet indices, such as Mean Platelet

Volume (MPV), Platelet Distribution Width (PDW), and Plateletcrit (PCT) with a simple Complete blood count and these parameters may provide some valuable information. [3] Recent years studies have come up to explore the utility of these parameters in routine clinical practice. This study attempts to find the usefulness of these platelet indices in the initial evaluation of patients with thrombocytopenia by assessing their variation in different clinical scenarios and to assess their sensitivity and specificity.

Aim: •

•

To evaluate the variation in platelet indices in establishing clinical correlation in patients presenting with thrombocytopenia. To study the relationship of platelet indices with respect to the underlying mechanism of thrombocytopenia.

Materials and Methods

This study is a cross sectional study for a period of 2 months in a tertiary care centre. Various Platelet parameters including Platelet count, Plateletcrit (PCT), Platelet Distribution Width (PDW) and Mean Platelet Volume

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(MPV) of the thrombocytopenic patients were collected from the routine laboratory blood investigations done in the Autoanalyser (ABX PENTRA DX 120) and documented. Correlation with routine peripheral smear findings of the respective cases was done. Relevant clinical details and available investigations including serological results of the patients were included. Inclusion criteria All adult Patients aged>18 years of both sexes with a platelet count of less than 1,50,000. Exclusion criteria1) Patients aged less than 18 years. & 2) Patient on antiplatelet drugs and other medications causing thrombocytopenia were excluded. Statistical analysis The Data collected wereanalysed using the software Statistical Package for Social Sciences (SPSS) program version 24 (IBN. Chicago, USA). Measurements of laboratory data platelet parameters of patients with thrombocytopenia in the two different hypoproductive and hyperdestuctive group were statistically tested by

unpaired t test. A p value less than 0.05 was considered statistically significant.

Results

This study included 120 patients of thrombocytopenia who were classified into hypoproductive and hyperdestructive group. In the hypoproductive we had 26 cases and in the hyperdestruction there were 94 cases. The distribution of cases in each subgroup and comparison of the distribution with similar studies were shown in table.1. The mean platelet count in the hypoproduction group is 75.9 ± 36.4 and in the hyperdestruction group is 79.6 ± 36.3 with a P value of 0.64. The mean MPV in the hypoproduction group is 10.17 ± 1.3 and in the hyperdestruction group is 12.3 ± 0.9 with a significant P value of 0.05. The mean PDW in the hypoproduction group is 19.7 ± 5.4 and in the hyperdestruction group is 19.3 ± 4.2 with a P value of 0.7. The mean PCT in the hypoproduction group is 0.06 ± 0.03 and in the hyperdestruction group is 0.08 ± 0.1 with a P value of 0.2. Mean values of different platelet indices and P-value in Hyper- destruction Immune thrombocytopenia and hypoproduction thrombocytopenia were shown in Table.2.

Table 1: The distribution of thrombocytopenia cases in each subgroup and comparison of the distribution with similar studies. Etiologies

Katti et al(23) Total cases (%)

Numbenjapon et al(24) Total cases (%)

Present study Total cases (%)

Hypoproduction Aplastic anemia

12 (11.8%)

Megaloblastic anemia

08 (8%)

4 (3.9%)

11 (9.2%)

Leukemia and MDS

06 (6%)

22 (21.6%)

2 (1.7%)

13 (10.8%)

4 (4%)

53 (52%)

3 (2.5%)

29 (29%)

26 (21.7%)

24 (24%)

8 (6.7%)

Others

Hyperdestruction ITP Dengue Scrub Typhus Malaria Chronic liver disease

3 (3%)

20 (16.7%)

Sepsis

4 (4%)

9 (8.8%)

6 (5%)

DIC Others

2 (2%)

19 (19%)

2 (2%)

31 (25.8%)

100

102

120

Total

Table 2: Mean values of different platelet indices and P-value in Hypoproduction thrombocytopenia and Hyperdestruction thrombocytopenia Platelet indices

Hypoproduction

Hyperdestruction

P Value

Platelet count (mean ± SD) (×10 /l)

75.9 ± 36.4

79.6 ± 36.3

0.64

MPV (mean ± SD) (fl)

10.17 ± 1.3

12.3 ± 0.9

0.05

PDW (mean ± SD) (fl)

19.7 ± 5.4

19.3 ± 4.2

0.7

PCT (mean ± SD) %

0.06 ± 0.03

0.08 ± 0.1

0.2

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Differentiation of Etiology of Thrombocytopenia by Platelet Indices

Discussion

Platelet indices include Plateletcrit (PCT), Platelet Distribution Width (PDW) and Mean Platelet Volume (MPV). Though these parameters have been available from the routinely used blood cell counters in the laboratory, their exact role in application to clinical diagnosis has still not been fully established.[4] Measurement of platelet indices in automated analysers has many advantages over manual estimation, as it is very simple, quick and inexpensive test which also eliminates the observer bias. [5, 6]Further in manual method, a delay between collection of blood and smear preparation, may change the platelet morphology and also artefactual increase in platelet diameter can occur because of increased adhesiveness with flattening of the platelets on the smears. [7] Numbenjapon et al[8]found that MPV was significantly higher in hyperdestruction group compared to hypoproductive thrombocytopenia. In hyper destructive thrombocytopenia, bone marrow compensates actively for the platelet loss and start releasing young larger platelets (“left shift”) which tend to decrease in size during its 7-10 days life span. [9]In our study also we found a significant low (10.17 ± 1.3) mean MPV in the hypoproduction group than in the hyperdestruction group (12.3 ± 0.9) Ntaios et al[10]found an increased MPV and PDW in Immune Thrombocytopenic Purpura (ITP). Kaito et al[2]similarly found a significant increase in MPV and PDW in ITP than in Aplastic anemia.Similarly few studies found a high PDW can also result in hyper destructive thrombocytopenia because of the release of heterogenous population of platelets which vary in their size (anisocytosis). [11, 12]Both MPV and PDW are reliable tests in hyperdestructive thrombocytopenia and considered as tests of 100% sensitivity and specificity for the diagnosis of ITP. [13,14]But in contrast, Tomito et al[15] found a low MPV in hyperdestructive thrombocytopenia and Nakadate H et al[16]and Baynes RD et al[17] found no significant difference in the MPV and PDW between the hyperdestructive and hypoproductive thrombocytopenia. Bashir AB et al[18]found significant differences in the MPV, PDW and PLT in patients with dengue infection and they suggested that these parameters can be used as probable indicators for dengue in endemic area. He also found a MPV <9 fl and high PDW >13fl had a considerable sensitivity for dengue fever. PCT is a representation of volume percent of platelets and its value is not altered by severity of thrombocytopenia of either hypoproductive or hyperdestructive etiology. Shah et al[19]found that increase in PDW and MPV. They also found increase in MPV in Ischemic heart disease patients.

In our study we did not find any significant difference in PDW between the two subgroups. But Shah et al[20]and Borkataky et al[6]found a higher PDW in hyperdestructive thrombocytopenia when compared to hypoproductive thrombocytopenia. Kaito et al [13]suggested that a PDW value of more than 17 fl and Ntaios et al[14]suggested a value between 15 and 17 fldiscrimate this two subgroups. But Xu et al[21]found a contrasting result of higher PDW values in the hypoproductive thrombocytopenia and attributed this as a result of significant dysplasia of hematopoiesis in the bone marrow in the hypoproductive group. The major disadvantage in these retrospective studies is that some had a smaller study population and other confounding factors that influenced the platelet volume. further the cut off values suggested by these studies have not been validated. [22, 23] Limitation of the study: The sample size in our study is small and further we had a limited number of cases 26 (21.7%) in the hypoproduction category. This may limit its application when applied to a larger patient community.

Conclusion

The Mean platelet volume may provide some useful information in discriminating the hypoproductive and hyperdestructive thrombocytopenia. Thus interpretation of this platelet indice can help the patients to avoid unnecessary invasive investigations like bone marrow aspiration and unnecessary platelet transfusion. Further studies with large number of cases in each subgroupsare needed to explore the role of this useful platelet index in thrombocytopenia and also to find the diagnostic roleof platelet indices various other diseases.

References 1.

Strauß G, Vollert C, von Stackelberg A, Weimann A, Gaedicke G, Schulze H. Immature platelet count: A simple parameter for distinguishing thrombocytopenia in pediatric acute lymphocytic leukemia from immune thrombocytopenia. Pediatr Blood Cancer. 2011 Oct 1;57(4):641–7.

Kaito K, Otsubo H, Usui N, Yoshida M, Tanno J, Kurihara E, et al. Platelet size deviation width, platelet large cell ratio, and mean platelet volume have sufficient sensitivity and specificity in the diagnosis of immune thrombocytopenia. Br J Haematol. 2005 Mar;128(5):698–702.

Park Y, Schoene N, Harris W. Mean platelet volume as an indicator of platelet activation: methodological issues. Platelets. 2002 Sep;13(5–6):301–6.

Giovanetti TV, do Nascimento AJ, de Paula JP. Platelet indices: laboratory and clinical applications. Rev Bras Hematol E Hemoter. 2011;33(2):164–5.

Beyan C, Kaptan K, Ifran A. Platelet count, mean platelet volume, platelet distribution width, and plateletcrit do not correlate with optical platelet aggegation responses in

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Parveen et al. healthy volunteers. J Thromb Thrombolysis. 2006 Dec 5;22(3):161–4. 6.

Borkataky S, Jain R, Gupta R, Singh S, Krishan G, Gupta K, et al. Role of platelet volume indices in the differential diagnosis of thrombocytopenia: a simple and inexpensive method. Hematology. 2009 Jun;14(3):182–6. Nelson RB, Kehl D. Electronically Determined Platelet Indices in Thro rn bocytopen ic Pa tien ts. Cancer. 1981;48:954–956.

8. Numbenjapon T, Mahapo N, Pornvipavee R, Sriswasdi C, Mongkonsritragoon W, Leelasiri A, et al. A prospective evaluation of normal mean platelet volume in discriminating hyperdestructive thrombocytopenia from hypoproductive thrombocytopenia. Int J Lab Hematol. 2008 Oct;30(5):408–14. 9.

Kottke-Marchant K, Corcoran G. The laboratory diagnosis of platelet disorders: an algorithmic approach. Arch Pathol Lab Med. 2002;126(2):133–146.

10. Ntaios G, Papadopoulos A, Chatzinikolaou A, Saouli Z, Karalazou P, Kaiafa G, et al. Increased values of mean platelet volume and platelet size deviation width may provide a safe positive diagnosis of idiopathic thrombocytopenic purpura. Acta Haematol. 2008;119(3):173–7. 11. Rajantie J, Javela K, Joutsi-Korhonen L, Kekomäki R. Chronic thrombocytopenia of childhood: use of noninvasive methods in clinical evaluation. Eur J Haematol. 2004 Apr 1;72(4):268–72. 12. Gardner FH, Bessman JD. Thrombocytopenia due to defective platelet production. Clin Haematol. 1983 Feb;12(1):23–38. 13. Kaito K, Otsubo H, Usui N, Yoshida M, Tanno J, Kurihara E, et al. Platelet size deviation width, platelet large cell ratio, and mean platelet volume have sufficient sensitivity and specificity in the diagnosis of immune thrombocytopenia. Br J Haematol. 2005 Mar;128(5):698–702. 14. Ntaios G, Papadopoulos A, Chatzinikolaou A, Saouli Z, Karalazou P, Kaiafa G, et al. Increased values of mean platelet

A-291 volume and platelet size deviation width may provide a safe positive diagnosis of idiopathic thrombocytopenic purpura. Acta Haematol. 2008;119(3):173–7. 15. Tomita E, Akatsuka JI, Kokubun Y. Differential diagnosis of various thrombocytopenias in childhood by analysis of platelet volume. Pediatr Res. 1980 Feb;14(2):133–7. 16. Nakadate H, Kaida M, Furukawa S, Ishii M, Higashihara M. Use of the Platelet Indices for Differential Diagnosis of Pediatric Immune Thrombocytopenic Purpura (ITP). Blood. 2008 Nov 16;112(11):4557–4557. 17. Baynes RD, Lamparelli RD, Bezwoda WR, Gear AJ, Chetty N, Atkinson P. Platelet parameters. Part II. Platelet volumenumber relationships in various normal and disease states. South Afr Med J Suid-Afr Tydskr Vir Geneeskd. 1988 Jan 9;73(1):39–43. 18. Bashir AB, Saeed OK, Mohammed BA, Ageep AK. Role of Platelet Indices in Patients with Dengue Infection in Red Sea State, Sudan. [cited 2015 Dec 5]; Available from: http:// www.ijsr.net/archive/v4i1/SUB15582.pdf 19. Shah AR, Chaudhari SN, Shah MH. Role of Platelet Parameters in Diagnosing Various Clinical Conditions. Hypertension. 2011;89:11–13. 20. Shah AR, Chaudhari SN, Shah MH. Role of Platelet Parameters in Diagnosing Various Clinical Conditions. Natl J Med Res. 2013;3(2):162–5. 21. Xu R-L, Zheng Z-J, Ma Y-J, Hu Y-P, Zhuang S-H. Platelet volume indices have low diagnostic efficiency for predicting bone marrow failure in thrombocytopenic patients. Exp Ther Med. 2013 Jan;5(1):209–14. 22. Leader A, Pereg D, Lishner M. Are platelet volume indices of clinical use? A multidisciplinary review. Ann Med. 2012 Dec;44(8):805–16. 23. Katti T, Mhetre S, Annigeri C. How far are the platelet indices mirror image of mechanism of thrombocytopeniamystery still remains? Int J Adv Med. 2014;1(3):200.

*Corresponding author: Dr Mourouguessine Vimal, No.21, Narmatha street, Vasanth nagar, Muthialpet, Puducherry – 605003 INDIA Phone: +91 9994083575 Email: drvimalm@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 16.02.2017 Date of Acceptance : 07.03.2017 Date of Publication : 14.06.2017

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Original Article DOI: 10.21276/APALM.1380

Cytological and Histopathological Correlation of Breast Lump: A 3 Year Study at Tertiary Care Center Sanjaykumar C. Chauhan, Ankur N. Sarvaiya* Dept. of pathology, GMERS Medical College, Himmatnagar, Gujarat, India

ABSTRACT Background: The fine needle aspiration cytology (FNAC) is an OPD procedure that is easy to perform, accurate, reproducible and cheap. Fine needle aspiration is a part of triple test to diagnose the breast lump. However a definitive conclusion should always be reached through histopathological examination Aim: The aim of our study is to correlate cytological diagnosis with histopathological diagnosis, to know clonicomorphologic spectrum of breast lump, and to figure out sensitivity, specificity, positive predictive value, negative predictive value and efficiency of FNAC. Methods: Total 244 cases who were subjected to FNAC and then subsequent biopsy was sent in each case were taken into consideration for analysis from tertiary care center in north Gujarat from February 2014 to January 2017 for 3 years. Results: In our study,4 were males and 240 were females(1.64% and 98.36% respectively). Maximum cases of breast lump were in the 2130 years age group. Out of total 244 cases 154(63.11%) were benign, 50(20.49%) cases were malignant, 08 (3.27%) were suspicious for malignancy and 32(13.11%) were other nonneoplastic. Out of total 244 cases, 241 were consistent on histopathology and 3 were inconsistent. Our study have Sensitivity, Specificity, Positive predictive value, Negative predictive value, and Efficiency to be 98.24%, 98.93%, 96.55%, 99.46% and 98.77% respectively. Conclusion: FNAC helps the surgeon on further accurate management of lump as it is sensitive and specific. FNAC results should be correlated with clinical findings and radiological investigations. Inconclusive results and on FNAC should always be confirmed with biopsy. Keywords: Cytology, Biopsy, Histopathology, Breast lump

Introduction

Breast lump is a common feature of most of the breast pathology. Many women in their life suffer from breast lump. Most of the breast lump cases are benign in nature.[1] This lump feature can cause an anxiety in the patients. Breast cancer is second commonest type of cancer after cervical cancer in India.[2] Mostly it also is presented as lump. The fine needle aspiration cytology (FNAC) is an OPD procedure that is easy to perform, accurate, reproducible and cheap.[3,4] Fine needle aspiration is a part of triple test. [5] Triple test consists of combined clinical examination, mammography and fine needle aspiration. However a definitive conclusion should always be reached through histopathological examination as it is universally accepted. Previously excisional biopsy was widely practiced, but preoperative evaluation by FNAC have number of benefits. [6,7] FNAC can be done with ultrasonographic guidance for better results.[8] It can also be used as an evaluation tool post lumpectomy.[9] It can also evaluate male breast , accessory breasts and axillary lymph nodes accurately.[10,11] In benign breast lesions triple test has reduced open biopsy rate.[12]

The aim of our study is to correlate cytological diagnosis with histopathological diagnosis, to know clonicomorphologic spectrum of breast lump, and to figure out sensitivity, specificity, positive predictive value, negative predictive value and efficiency of FNAC.

Materials and Methods

Total 244 cases who were subjected to FNAC and then subsequent biopsy was sent in each case were taken into consideration for analysis from tertiary care center in north Gujarat from February 2014 to January 2017 for 3 years. The study was retrospective in nature. Consent was taken for performing FNAC in every case. Findings about age, site, size, consistency, mobility, ulceration, pain, discharge, duration of lump, fixation to skin etc. were noted. Ultrasonography findings were noted wherever preformed. FNAC performed with 10 ml syringe and 23G needle after proper cleaning of site with spirit and antiseptic solution. Findings on aspiration were noted. Material taken on glass slide spread with pressure by another glass slide. Slides fixed with methanol and then stained with hematoxylin and eosin stain for cytology. Slides mounted with cover

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Chauhan et al. slip by DPX. Biopsies sent from surgical department were grossed, processed and wax blocks prepared. Sections were taken from it and stained with hematoxylin and eosin. FNAC findings have been concluded with different categories of diagnosis according to national cancer institute (NCI) guidelines[13]: benign, atypical, suspicious for malignancy, malignant and other nonneoplastic pathology. Subsequent histopathology findings were retrieved.

Results

Total 244 cases in which FNAC performed and subsequent biopsy received were taken for analysis. Table 1 shows age and sex wise distribution of these breast lump cases. In our study out of 244, 4 were males (1.64%) and 240 were females (98.36%). Maximum cases of breast lump were in the 21-30 years age group. Out of total 244 cases 154(63.11%) were benign, 50(20.49%) cases were malignant, 08 (3.27%) were suspicious for malignancy and 32(13.11%) were other nonneoplastic. In our study 154 cases were diagnosed as benign on cytology. Out of which 142 were fibroadenomas, 7 were fibrocystic disease, 2 were tubular adenoma, 2 were lactating adenoma and 1 was chronic non specific inflammation on histopathology. On FNAC reports, 50 cases were malignant.

A-293 On histopathology out of 50, 44 were infiltrating ductal carcinoma, 02 were infiltrating lobular carcinoma, 02 were medullary carcinoma and 2 were ductal carcinoma in situ. Eight cases were suspicious for malignancy, out of which 4 were infiltrating ductal carcinoma, 1 was ductal carcinoma in situ, 1 was mucinous carcinoma and 2 were sclerosing adenosis on histopathology. On other non neoplastic cases, 1 galctocele case was malignant on biopsy. In confirmed benign and malignant reports on cytology subsequent histopathology reports were consistent in all cases. Out of 8 suspicious for malignancy reports on cytology two turned out to be sclerosing adenosis (benign). Out of total 244 cases, 241 were consistent on histopathology and 3 were inconsistent. The chi square statistic is 3.0844. The p-value is 0.79047. It is significant at p<0.10 but it is not significant at p<0.05. So, FNAC is not the gold standard for diagnosing breast lump and it should be followed by biopsy in inconclusive results. Positive cases on statistics are malignant cases or suspicious for malignancy cases (58) and negative cases were benign cases and other cases (186). So our study have Sensitivity, Specificity, Positive predictive value, Negative predictive value, and Efficiency to be 98.24%, 98.93%, 96.55%, 99.46% and 98.77% respectively.

Table 1: Age and sex distribution of breast lump cases. Age (years) Males Males (%) 11-20 0 0 21-30 1 25 31-40 1 25 41-50 0 0 51-60 1 25 61-70 0 0 71-80 1 25 81-90 0 0 Total 4 100 Table 2: Cytological diagnosis Cytological diagnosis Benign Malignant Suspicious for malignancy Other (non neoplastic) Inflammatory Fibrocystic Galactocele Gynecomastia Nonspecific Total

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Females 40 84 48 28 18 16 04 02 240

Females(%) 16.66 35 20 11.66 7.5 6.66 1.66 0.83 100

Number of cases 154 50 08

Percentage 63.11 20.49 3.27

06 08 06 04 08 244

2.45 3.27 2.45 1.63 3.27 100

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Cytology of Breast with Cytomorphological Correlatation

Cytology

Number

Fibroadenoma

Scle. adenosis

Tubu. adenoma

IDC

ILC

DCIS

Mucinous ca

Medullary ca

Lacta. adenoma

Gynecomastia

Fibro. Breast d.

Granulomatous ma.

Chr.non specific

Total

Table 3: Cytological and histopathological correlation.

Benign

154

142

154

Malignant

Suspicious

Inflammatory

Fibrocystic

Galactocele

Gynecomastia

Non specific

Total

244

146

244

*Scle.=sclerosing, tubu.=tubular, IDC=infiltrating ductal carcinoma, ILC=infiltrating lobular carcinoma, DCIS=ductal carcinoma in situ, ca=carcinoma, Lacta.=lactating, Fibro.=fibrocystic, ma.=mastitis, chr.=chronic Table 4: Cyto histopathological comarison. Cytology diagnosis

Histopathology diagnosis

Number of cases

Consistent

Inconsistent

Total

Benign

154

154(100%)

00(00%)

154

Malignant

50(100%)

00(00%)

Suspicious of malignancy

06(75%)

02(25%)

Other

31(96.87%)

01(3.13%)

Total

244

241(98.77%)

03(1.23%)

244

Table 5: benign and malignant cases on cytology and histopathology. Benign (histopathology)

Malignant(histopathology)

Total

Benign(cytology)

185

186

Malignant (cytology)

Total

241

244

Table 6: Cytological diagnosis compared with previous published studies. Author Debra et al[14] Feichter G et al

[16]

Desouza rocha P et al[17] Singh K et al

[18]

Mohammad Q et al[15] Bukhari et al

[19]

Shrestha et al

[20]

Rupom TU et al[21]

Our study

Inadequate

Benign

Suspicious for malignant

Malignant

Other

Total

230(13.69%)

1019(60.65%)

300(17.85%)

131(7.79%)

1680

239(16.23%)

1003(68.13%)

49(3.32%)

181(12.29%)

1472

9(1.07%)

640(76.46%)

26(3.10%)

99(11.83%)

63(7.52%)

837

200(83.33%)

5(2.08%)

35(14.58%)

240

16(13.79%)

68(58.62%)

32(27.58%)

116

271(63.76%)

32(7.52%)

120(28.23%)

2(0.47%)

425

27(1.92%)

618(44.04%)

175(12.47%)

152(10.83%)

431(30.01%)

1403

3(0.57%)

431(82.25%)

17(3.24%)

72(13.74%)

4(0.76%)

524

154(63.11%)

8(3.27%)

50(20.49%)

32(13.11%)

244

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Chauhan et al.

A-295

Table 7: Comparison of accuracy on FNAC Sensitivity

Specificity

Positive predictive value

Negative predictive value

Efficiency

Kline TS et al[22]

89.5

92.5

85.33

91.63

Dominguez F et al[23]

93.49

95.73

93.49

95.73

98.75

99.3

Desouza rocha P et al[17]

93.8

98.21

92.7

97.40

Singh A et al

84.6

100

92.3

Khemka A et al

100

95.12

Bukhari et al

100

90.6

100

86.3

98.2

97.9

92.1

98.1

99.4

98.1

92.6

98.48

98.24

98.93

96.55

99.46

98.77

Author

Feichter et al[16] [24] [25]

[19]

Muhamed et al Rubin et al

[27]

Ishikawa et al Collaco et al Jan et al

[26]

[28]

[29]

[06]

Our study

Discussion

Conclusion

Our study have 63.11% benign, 3.27% suspicious for malignancy, 20.49% malignant and 13.11% other nonneoplastic cases on FNA cytology reports. These results have been compared with other previous studies in above table.

References

Fine needle aspiration cytology is globally recognized method as initial investigation and screening of breast lump. In our study fibroadenoma was commonest benign lesion and infiltrating duct carcinoma was commonest malignant lesion. These results are comparable with Debra et al[14] and Mohammad Q et al[15].

Table 7 shows comparison of statistical values of our study with various other previously published studies. A single case of galactocele was given on FNAC on the basis of aspiration and cytology findings. Patient was 40 years old and was having 6 month old child. 0.5 ml whitish fluid was aspirated on FNAC with 23G needle and 10 ml syringe. On ultrasonography finding, it was irregular hypoechoic mass lesion. So, biopsy was advised. Infiltrating ductal carcinoma was given on subsequent biopsy. Two cases were suspicious for malignancy on FNACs. One case was 38 years and other 46 years old. On palpation, it was hard, fixed and nonmobile swelling. On cytology, cellular pleomorphism and high nuclear to cytoplasmic ratio was present in few cells. So it was concluded as suspicious on FNAC. Biopsy was advised. On biopsy, it was sclerosis adenosis in both cases. www.pacificejournals.com/apalm

The fine needle aspiration cytology of breast lump is easy, valuable, cost effective, time saving and worldwide recognized method for initial investigation and screening of breast lump. It helps the surgeon on further management of lump as it is sensitive and specific. FNAC results should be correlated with clinical findings and radiological investigations. Inconclusive results on FNAC should always be confirmed with subsequent biopsy as histopathology is currently gold standard for diagnosis. 1.

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Rimm DL, Stastny JF, Rimm EB, Ayer S, Frable WJ: Comparison of the costs of fine needle aspiration and open surgical biopsy as methods for obtaining a pathologic diagnosis. Cancer; 1997; 81: 51-56.

Silverman JF, Lannin DR, Oâ&#x20AC;&#x2122;Brien K, Norris HT: The triage role of fine needle aspiration biopsy of palpable breast masses. Diagnostic accuracy and cost effectiveness. Acta Cytol; 1987; 31: 731-736.

Kaufmanz, Shpitz B, Shapiro M, Rona R, Lew S, Dinbar A. Triple approach in the diagnosis of dominant breast masses: combined physical examination, mammography and Fineneedle aspiration, J. SurgOncol 1994; 56: 254-7.

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Jan WA, NaikZada, Samieullah, Israr M. Comparison of FNAC and core biopsy for evaluating breast lumps. J coll physicians Surg. Pak. 2002; 12: 686-688.

Patrikar A, Maimoon S and Mahore S. Filarial granuloma in breast. Ind Jour Pat Mic 2008;23, 116 -122.

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20. Shrestha A, Chalise S, Karki S and Shakya G. Fine needle aspiration cytology in a palpable breast lesion. Journal of Pathology of Nepal. 2011; 1:131-135. 21. Rupom TU, Choudhary T. Study of Fine Needle Aspiration cytology of breast Lump of Breast Lump: Correlation of Cytologically Malignant Cases with Their Histological Findings. BSMMU J.2011; 4(2):60-64. 22. Kline TS, Joshi LP, Hunter SN. Fine needle aspiration of the breast: diagnoses and pitfalls.Cancer.1979; 44:1458-1464. 23. Dominguez F, Riera JR, Tojo S and Junco P. Fine needle aspiration of breast masses, an analysis of 1398 patients in a community hospital. Acta cytological.1997; 41(2):341-347. 24. Singh A, Haritwal A and Murali BM. Pattern Of breast Lumps and Diagnostic Accuracy Of Fine Needle Aspiration Cytology; A Hospital Based Study from Pondicherry, India. The Internet Journal of Pathology.2011; 11(2):1-14. 25. A Khemka, N Chakrabarti, S Shah, V Patel. Palpable Breast Lumps: Fine-Needle Aspiration Cytology versus Histopathology: a Correlation of Diagnostic Accuracy. The Internet Journal of Surgery. 2008 Volume 18 Number 1.

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27. Rubin J, Horiuchi K, Joy N, Haun W, Read R, Ratzer E, Fenoglio M. Use of FNAC for solid breast lesions is accurate and cost effective. Am J Surg 1997; 174: 694-6.

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*Corresponding author: Dr. Ankur N Sarvaiya, Assistant professor, dept. of pathology, GMERS Medical College, Himmatnagar. Gujarat, India Phone: +91 9974149292 Email: ankur.sarvaiya@gmail.com Date of Submission : 01.03.2017 Date of Acceptance : 01.04.2017 Financial or other Competing Interests: None. Date of Publication : 04.07.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Original Article DOI: 10.21276/APALM.1393

Variant Hemoglobin Spectrum By Cation Exchange High Performance Liquid Chromatography: A Study 0f 2035 Subjects Ankur N. Sarvaiya, Sanjaykumar C. Chauhan* Dept. of Pathology, GMERS Medical College, Himmatnagar, Gujarat, India

ABSTRACT Introduction: Hemoglobinopathies and thalassemia are hereditary disorders of hemoglobin (Hb) affecting mankind at prevalent regional level. Automated cation exchange high performance liquid chromatography is being increasingly used as the initial diagnostic method for identifying normal and abnormal hemoglobin variants. Methodology: Total 2035 sample received and studied. All samples run on cation exchange high performance liquid chromatography machine by BIO-RAD. Results: Total 386(18.96%) cases had abnormal hemoglobin fractions. Beta thalassemia major cases were 17(0.83%). Beta thalassemia intermedia cases were 4(0.19%). Sickle cell trait (heterozygous Hb S) cases were 96(4.71%). Double heterozygous for sickle cell-beta thalassemia cases were 14(0.68%). There were 06 cases (0.29%) of Hb D heterozygous. There were one each case of Hb E heterozygous and Hb E-beta thalassemia (0.04% each). Hb F was raised in 7 adult cases (0.34%). Conclusion: To conclude, cation exchange high performance liquid chromatography is less time consuming, cost effective, labor saving, reproducible, accurate, sensitive and specific method to detect hemoglobinopathies and thalassemia. Most of the abnormal cases are diagnosed with this method, with few inconclusive cases require further genetic and molecular workup. Keywords: Haemoglobinopathies, Thalassemia, Cation Exchange High Performance Liquid Chromatography, Screening

Introduction

Hemoglobin (Hb) is a conjugated protein of molecular weight 64,000. Haem group is attached to two pairs of polypeptide globin chains. Haem group binding with each of these chains is important for oxygen carrying capacity of hemoglobin and also it stabilizes the hemoglobin molecule. There are also many naturally occurring variants of hemoglobin (more than 1000) which are genetically determined, most are harmless and some of them have serious clinical consequences [1]. Hereditary disorders of hemoglobin result either from qualitative defect (structural alteration of a globin polypeptide chain) or quantitative defect(reduced synthesis of globin polypeptide chain). Examples of qualitative defects are Hb S, Hb D and Hb E etc. Examples of quantitative defects are alpha and beta thalassemia. Major causes of morbidity of all these are homozygous beta thalassemia and some alpha thalassemia [2] . 5% of world population is a carrier for hemoglobin disorders as per world health organization report [3]. In India 10,000 children every year are born suffering from thalassemia major, amounting to approximately 10% of total world numbers [4]. In India individuals with beta thalassemia are 3.5-15% in total population [5]. Almost 10 billion rupees per year is spent for thalassemia patients

in India. In India still the conventional methods are used in most of the places for diagnosing these defects. These conventional methods are family history, clinical features, hemoglobin level, red blood cell indices, red cell count, peripheral blood smear study, Hb A2 quantification, Hb F quantification, cellulose acetate electrophoresis at alkaline pH for hemoglobin and sickling test. Various drawbacks and shortcomings of these methods are that Hb S,G,D,Q and Lepore have same mobility on electrophoresis and Hb A2,C and E also have same mobility [6]. Double heterozygous states also are difficult to diagnose [6]. Clinical and laboratory features have low specificity. Parents with various heterozygous states can lead to offspring with double heterozygous or homozygous defects. Automated cation exchange high performance liquid chromatography is being increasingly used as the initial diagnostic method for identifying various normal and abnormal hemoglobin [7,8]. It is simple, easy, reliable, accurate yet cost effective method for early detection of normal and abnormal hemoglobin [9,10].

Material & Methods

Total 2035 sample received and studied from January 2015 to August 2016 that were sent for hemoglobin variant analysis

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Variant Hb Spectrum by Cation Exchange HPLC

in a tertiary care center in north Gujarat. Most of the cases were from North gujarat and Kutch area of Gujarat with some were from Rajasthan, Madhya Pradesh, Uttarpradesh and Bihar. Naked eye single tube red cell osmotic fragility test was done and observed for hemolysis. All the samples were run by automated hematology analyzer (cellenium trivitron, abacus3 and abacus5) and hemoglobin values and red cell indices were noted. The whole blood samples were taken in K3 EDTA (Ethylene Diamine Tetraacetic Acid) anticoagulant containing vacutte. Anticoagulated whole blood samples were analyzed with BIO-RAD VARIANT II (beta thalassemia short program) machine by BIORAD laboratories, United States of America. It runs on cation exchange high performance liquid chromatography principle. On each run, one calibrator and two controls with one blank were added initially. Acceptable area was between 1-3 million. Ranges outside this area had been rejected. All the data regarding clinical history, history of blood transfusion were recorded. Chromatogram results of samples printed. Specific defined windows are there from manufacturer from specific retention time and integrated peaks are accordingly assigned [11]. The retention time is the time taken from the sample injection up to the apex of elution peak [11]. Established ranges of elution of common hemoglobin variants are marked as â&#x20AC;&#x153;windowsâ&#x20AC;? (Table 1). Chromatogram result shows retention time, area, area percentage and concentration. Retention time not assigned comes as an unknown. Each sample takes somewhat around 6 minutes for result.

Results

Total 2035 samples received. Out of which 1186(58.28%) were male and 849(41.72%) were female. The overall sample patients age range was from 28 days to 74 years. The few upper age range was mostly grandparents of patients for parental screening. Total 386(18.96%) cases had abnormal hemoglobin fractions. Most common

defect was increased Hb A2. 3.9% was taken as a cutoff for beta thalassemia trait diagnosis (BTT). Total 216 cases were diagnosed with beta thalassemia trait (raised HbA2). Peripheral blood smear findings were microcytic hypochromic red blood cells, anisocytosis, and target cells. Red blood cell count is increase in most cases. The Hb A2 retention time was between 3.50 to 3.75 min. Table 1 shows retention times for all predefined windows. Mentzer index was less than 13 in 179 out of 216 cases. Mentzer index is a ratio of mean corpuscular volume in fL divided by the red blood cell count in Millions per micro Liter. Beta thalassemia major cases were 17(0.83%). Beta thalassemia intermedia cases were 4(0.19%). All the thalassemia major cases were first presented in their first two years. Peripheral blood smear findings of these cases were severe anemia, severely microcytic and hypochromic red blood cells, moderate to severe anisopoikilocytosis, target cells and nucleated red blood cells. Sickle cell anemia (homozygous Hb S) patients had Hb S range from 72-89% and total 19 (0.93%) cases were there. Sickle cell trait (heterozygous Hb S) cases were 96(4.71%). Hb S in them was from 32 to 38%. Double heterozygous for sickle cell-beta thalassemia cases were 14(0.68%). There were 06 cases (0.29%) of Hb D heterozygous. D window is displayed in HPLC. Retention time was between 4.03 to 4.21 min. There were one each case of Hb E heterozygous and Hb E-beta thalassemia (0.04% each). There is a peak of Hb E in A2 region. Retention time was 3.58 min. Hb F was raised in 7 adult cases (0.34%). Provisional diagnosis of hereditary persistence of fetal Hb was made in each case with an advice for molecular confirmation. Various abnormal hemoglobin defects in our study are shown in table 2 and figure 1. Various hematological parameters are shown in table 3.

Table 1: Window time and retention time of predefined parameters of BIO-RAD VARIANT II. Peak name

Window(min)

Retention time(min)

F window

1.00-1.30

1.15

P2 window

1.30-1.60

1.45

P3 window

1.60-1.90

1.75

A0 window

1.90-3.30

2.60

A2 window

3.30-3.90

3.60

D window

3.90-4.30

4.10

S window

4.30-4.70

4.50

C window

4.90-5.30

5.10

P2 and P3 are associated with Hb A

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Table 2: Sex wise distribution of hemoglobinopathies. Hemoglobinopathies

Male

Female

Total (percentage)

Beta thalessemia trait

119

216(10.61%)

Beta thalassemia major

17(0.83%)

Thalassemia intermedia

04(0.19%)

Hb S homozygous

19(0.93%)

Hb S heterozygous

96(4.71%)

Hb S- beta thalassemia

14(0.68%)

Hb D heterozygous

06(0.29%)

Hb E heterozygous

01(0.04%)

Hb D-beta thalassemia

02(0.09%)

HbS-Hb D

01(0.04%)

Hb D-Hb E

01(0.04%)

Hb E-beta thallesemia

01(0.04%)

HPFH

07(0.34%)

TOTAL

203

183

386(18.96%)

Table 3: Hematological parameters of normal and different hemoglobinopathies. Parameter

Normal

BTT

Thal major

Hb S Homo.

Hb S Hetero.

Sickle- ß thal

Hb D hetero.

HPFH

Hb(g/dl)

10.6±2.9

10.2±2.4

4.86±2.23

6.38±2.1

10.1±2.9

7.68±2.3

10.9±2.9

12.3±2.8

RBC

4.55±0.9

5.62±0.82

2.36±1.32

3.26±0.8

4.2±0.36

3.86±1.0

4.7±1.25

4.6±0.9

PCV

31.4±4.7

30.4±3.62

15.2±2.91

21.2±5.23

30.6±7.1

24.1±7.3

35.9±10.8

39.1±4.5

MCV

76.7±12.71

59.91±7.1

61.9±7.2

76.1±8.6

70.6±5.1

70.4±6.34

75.5±20.4

81.1±7.5

MCH

26.4±3.85

20.3±2.58

21.62±4.2

24.7±3.2

21.6±4.0

23.4±2.1

24.7±4.1

25.6±3.4

MCHC

31.21±2.1

30.4±1.98

31.1±4.62

31.8±2.3

30.9±2.3

32.4±1.4

32.4±2.4

31.5±2.3

18±4.7

18.4±3.8

20.32±5.2

22.5±4.1

21.6±4.3

21.68±3.9

18.4±3.64

17.6±2.8

Hb A

89.12±4.65

86.3±4.3

20.4±19.8

5.4±2.3

58.1±4.3

6.3±2.21

51.68±5.27

85.2±3.5

Hb A2

2.63±0.61

5.15±0.72

3.4±1.2

3.54±0.86

3.4±0.9

5.58±1.21

2.4±0.38

2.7±0.51

Hb F

0.65±0.36

1.4±0.5

81.2±14.3

15.9±6.89

1.9±3.21

2.31±7.4

0.7±0.2

9.4±3.4

Hb S

74.5±8.4

32.6±5.6

69.4±8.93

Hb D

36.7±6.8

RDW-CV

*Numbers are mean ± standard deviation. Hb=hemoglobin, RBC=red cell count, PCV= packed cell volume, MCV= mean corpuscular volume, MCH= mean corpuscular hemoglobin, MCHC= mean corpuscular hemoglobin concentration, RDW= red cell distribution width, BTT= beta thalassemia trait, Thal major= beta thalassemia major, Homo=homozygous, Hetero=heterozygous, HPFH=hereditary persistence of fetal hemoglobin

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Variant Hb Spectrum by Cation Exchange HPLC Fig. 1: Column chart shows frequency of hemoglobinopathies.

*BTT= beta thalassemia trait, Thal major= beta thalassemia major, thal inter=thalassemia intermedia, Homo=homozygous, Hetero=heterozygous, s- ß = sickle cell beta thalassemia, D- ß =Hb D beta thallesemia, S-D= sickle cell HbD, D-E= Hb D Hb E, E- ß = Hb E beta thalessemia, HPFH=hereditary persistence of fetal hemoglobin

Discussion

In India and Mediterranean belt, still thalassemias and hemoglobinopathies are very common causes of morbidity and also exert burden on expenditure. To reduce the burden accurate and reliable screening procedure should be there. The diagnosis of hemoglobinopathies and thalassemia is required to explain hematological abnormalities, identify abnormality in pre symptomatic period, preconceptional screening, screening of fetus to offer termination of pregnancy, and to confirm a presumptive diagnosis [12]. Naked eye single tube red cell osmotic fragility (NESTROFT) and red cell indices are helpful tests that aid in diagnosis but the result must have been supported by confirmatory test as suggested by Chakrabarti et al [13], Gorakshakar et al [14], Dangi et al [15] and other studies [16]. Yousafzai et al stated that NESTROFT is easy and simple but it is very much error prone, red cell indices also are lacking in sensitivity and specificity [16]. We have found in our study that sensitivity and specificity of mentzer index are 60.4 and 91%. Both of these are lacking in sensitivity and specificity so not used for diagnostic purpose exclusively. So far, some of the common investigations done for thalassemia and hemoglobinopathies are Hb electrophoresis by cellulose acetate at alkaline pH, acid electrophoresis, Hb F quantification by alkali denaturation and Hb A2 quantification by chromatography. These all methods have certain limitations; these methods are totally dependent on the performer’s expertise. So variability of the result is there. Even with the same performer, the bands are

not exactly positioned on the same area (reproducibility is low). To differentiate between hemoglobins that have same electrophoretic mobility is difficult and the control with known variant hemoglobin or multiple stored known controls is required to be run every time with patient’s sample. It is very difficult to get a single control for comparison with all the variant hemoglobin in it [17]. It is also nearly impossible to run many samples in a single run. These methods are also time consuming. Compound heterozygous or double heterozygous states and unusual hemoglobin variants are all clinically important to diagnose, so exact identification of them is very important [18,19] . With single electrophoretic method none out of these variants can be identified precisely [20]. Cation exchange high performance liquid chromatography has high sensitivity, specificity and also is reproducible compared to hemoglobin electrophoresis [21]. Total 386 cases (18.96%) were diagnosed with some hemoglobin variants. Out of which beta thalassemia trait cases were 216(10.61%). This proves antenatal screening of value to prevent potential offspring with thalassemia major. Borderline Hb A2 requires further investigation before reaching out on conclusion. Studies have suggested both iron deficiency anemia and megaloblastic anemia may have an effect on level of Hb A2; iron deficiency anemia can mildly reduce and megaloblastic anemia can mildly increase [22,23,24,25]. Different studies by Sachdev et al [26], Rao et al [27], Dolai et al [28] and Mukhopadhyay et al [29] have taken 2600, 800, 35413 and 10407 cases respectively for their study.

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Sarvaiya et al. Sachdev et al [26], Jain et al [30] and Mukhopadhyay et al [29] found 12.57%, 29.3 % and 14.5% abnormal hemoglobin fractions respectively. Sachdev et al [26], Rao et al [27], Dolai et al [28] and Mukhopadhyay et al [29] found beta thalassemia trait cases to be 8.9 %,18.1 %,10.38 % and 5.6 % respectively. Our findings are comparable to these different Indian studies.

Conclusion

To conclude, cation exchange high performance liquid chromatography is less time consuming, cost effective, labor saving, reproducible, accurate, sensitive and specific method to detect hemoglobinopathies and thalassemia. It should be used as a screening tool. Most of the abnormal cases are diagnosed with this method; with few inconclusive cases require genetic and molecular studies.

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Hardison R, Miller W. (Updated) Globin gene server. Available at: www.globin.cse.psu.edu.

Kutlar F. Diagnostic approach to hemoglobinopathies. Hemoglobin. 2007 Jan 1;31(2):243-50.

WHO executive board EB118/5, 118th Session Report by the Secretariat on Thalassaemia and other haemoglobinopathies: Prevalence of Haemoglobinopathies. 11 May 2006:1-8.

Varawalla NY, Old JM, Sarkar R, Venkatesan R, Weatherall DJ. The spectrum of β⁪thalassaemia mutations on the Indian subcontinent: the basis for prenatal diagnosis. British journal of haematology. 1991 Jun 1;78(2):242-7. Balgir RS. The genetic burden of hemoglobinopathies with special reference to community health in India and the challenges ahead. Indian J Hematol Blood Transfus. 2002;20(1):2-7. Gupta PK, Kumar H, Kumar S, Jaiprakash M. Cation exchange high performance liquid chromatography for diagnosis of haemoglobinopathies. Medical Journal Armed Forces India. 2009 Jan 31;65(1):33-7.

Wild BJ, Stephens AD. The use of automated HPLC to detect and quantitate haemoglobins. International Journal of Laboratory Hematology. 1997 Sep 1;19(3):171-6.

Higgins TN, Ridley B. Tentative identification of hemoglobin variants in the Bio-Rad VARIANT II Hb A 1C method. Clinical biochemistry. 2005 Mar 31;38(3):272-7.

Phelan L, Bain BJ, Roper D, Jury C, Bain K. An analysis of relative costs and potential benefits of different policies for antenatal screening for beta thalassaemia trait and variant haemoglobins. Journal of clinical pathology. 1999;52(9):697-700.

A-301 12. Working Party of the General Haematology Task Force of the British Committee for Standards in Haemotology. Guideline: The laboratory diagnosis of haemoglobinopathies. British Journal of Haematology. 1998;101:783-92. 13. Chakrabarti I, Sinha SK, Ghosh N, Goswami BK. BetaThalassemia Carrier Detection by NESTROFT: An Answer in Rural Scenario?. Iranian Journal of Pathology. 2012 Jan 1;7(1):19-26. 14. Gorakshakar AC, Colah RB. Is RBC discrimination index suitable for differentiating between α-and β-thalassemias?. Indian journal of human genetics. 2011 Sep 1;17(3):115. 15. Dangi CB, Sajid M, Sawke GK, Ambhore J. Sickle cell hemoglobinopathies in district Bhopal. Indian journal of human genetics. 2010 May 1;16(2):100. 16. Yousafzai YM, Khan S, Raziq F. Beta-thalassaemia trait: Haematological parameters. J Ayub Med Coll Abbottabad. 2010;22:84-6. 17. Joutovsky A, Hadzi-Nesic J, Nardi MA. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60000 samples in a clinical diagnostic laboratory. Clinical chemistry. 2004 Oct 1;50(10):1736-47. 18. Somervaille T. Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. Journal of the Royal Society of Medicine. 2001; 94(11):602-603. 19. Dash S, Huisman TH. Hb [A. sub. 2] in subjects with Hb D. Clinical chemistry. 1998 Nov 1;44(11):2381-3. 20. Bain BJ. Haemoglobinopathy diagnosis. 2nd ed. Oxford: Blackwell Publishing Ltd; 2006. 21. Ou CN, Rognerud CL. Diagnosis of hemoglobinopathies: electrophoresis vs. HPLC. Clinica chimica acta. 2001 Nov 30;313(1):187-94. 22. El-Agouza I, Abu Shahla A, Sirdah M. The effect of iron deficiency anaemia on the levels of haemoglobin subtypes: possible consequences for clinical diagnosis. Clinical & laboratory haematology. 2002 Oct 1;24(5):285-9. 23. Madan N, Sikka M, Sharma S, Rusia U. Haematological parameters and HbA2 levels in beta-thalassaemia trait with coincident iron deficiency. Indian journal of pathology & microbiology. 1998 Jul;41(3):309-13. 24. Bencaiova G, Burkhardt T, Krafft A, Zimmermann R. Screening for β-thalassaemia trait in anaemic pregnant women. Gynecologic and obstetric investigation. 2006 Mar 1;62(1):20-7.

10. Riou J, Godart C, Hurtrel D, Mathis M, Bimet C, BardakdjianMichau J, Préhu C, Wajcman H, Galactéros F. Cationexchange HPLC evaluated for presumptive identification of hemoglobin variants. Clinical chemistry. 1997;43(1):34-9.

25. Gupta AD. Abrogation of macrocytosis in pernicious anemia by β-thalassemia does not mask the diagnosis of vitamin B12 deficiency. American journal of hematology. 2002 Sep 1;71(1):61-2.

11. Bio–Rad VARIANT II, beta thalassemia short program. Instruction Manual. 2003:10.

26. Sachdev R, Dam AR, Tyagi G. Detection of Hb variants and hemoglobinopathies in Indian population using HPLC:

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report of 2600 cases. Indian journal of pathology and microbiology. 2010 Jan 1;53(1):57. 27. Rao S, Kar R, Gupta SK, Chopra A, Saxena R. Spectrum of haemoglobinopathies diagnosed by cation exchange-HPLC & modulating effects of nutritional deficiency anaemias from north India. Indian Journal of Medical Research. 2010 Nov 1;132(5):513. 28. Dolai TK, Dutta S, Bhattacharyya M, Ghosh MK. Prevalence of hemoglobinopathies in rural Bengal, India. Hemoglobin. 2012 Feb 1;36(1):57-63.

29. Mukhopadhyay D, Saha K, Sengupta M, Mitra S, Datta C, Mitra PK. Spectrum of hemoglobinopathies in West Bengal, India: a CE-HPLC Study on 10407 subjects. Indian Journal of Hematology and Blood Transfusion. 2015 Mar 1;31(1):98-103. 30. Jain BB, Roy RN, Ghosh S, Ghosh T, Banerjee U, Bhattacharya SK. Screening for thalassemia and other hemoglobinopathies in a tertiary care hospital of West Bengal: implications for population screening. Indian journal of public health. 2012 Oct 1;56(4):297.

*Corresponding author: Dr. Sanjaykumar C. Chauhan, Dept. of pathology, GMERS Medical College, Himmatnagar, Gujarat, India Phone: +91 09687626937 Email: drsanjaychauhan@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 06.03.2017 Date of Acceptance : 31.03.2017 Date of Publication : 04.07.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Original Article DOI: 10.21276/APALM.1405

Histopathological Spectrum of Non-Neoplastic Uterine Cervical Lesions in a Tertiary Care Centre Priyadarshini. D* and Arathi. C.A Department of Pathology, Shridevi Institute of Medical Sciences And Research Hospital, Tumakuru, India

ABSTRACT Background: Uterine cervix in our routine hysterectomies and biopsies from gynecological specimens constitutes major portal for nonneoplastic lesions. Routine histopathological study of suspicious cases enhances early detection of these uterine cervical lesions. Methods: 250 cases of uterine cervical non-neoplastic lesions were evaluated either from hysterectomy or cervical biopsy specimens. The purpose of this study is to analyze and determine the frequency and histomorphological patterns of non-neoplastic cervical lesions at the tertiary care center and to study various metaplasias of the endocervical epithelium. The cervical lesions were subjected to detailed gross and microscopic examination and further classified into various non-neoplastic lesions Result: Our study showed that 48 % of cases featured chronic nonspecific cervicitis. The commonest encountered endocervical epithelial lesions were chronic polypoidal endocervicitis (20%) and squamous metaplasia (36%) and the uncommon lesions included micro glandular adenosis (3.2%), endocervical glandular hyperplasia (4%), diffuse laminar endocervical glandular hyperplasia(0.8%) tunnel clusters (0.4%) and mesonephric rests (0.4%). A majority of the ectocervical lesions includes Koilocytic changes, exocytosis, Suprabasal bulla and prolapse changes like hyperkeratosis and parakeratosis. Conclusion: In the present study we emphasized mainly about nonneoplastic uterine cervical lesions among which Chronic non-specific cervicitis is the most confronted lesion in histopathological specimens. However, there are many lesions that appear to be exuberant and can be misdiagnosed to be malignant. On the basis of this, a detailed histomorphological study of the nonneoplastic lesions of the cervix was taken up and further categorized into various lesions which can cause serious morbidities and detailed histopathological study is considered as the gold standard. Keywords: Non-neoplastic, Cervicitis, Endocervix, Histopathology, Hyperplasia

Introduction

In the gynecological specimens, uterine cervix acts as a gateway to a distinctive array of non-neoplastic as well as neoplastic lesions. These non-neoplastic cervical lesions are most common in the women of reproductive age group. Accordingly, the cervical specimens should be analyzed with utmost care as these lesions can lead to significant morbidity in women. The majority of the histopathological specimens are from gynecological department and includes predominantly inflammatory and some tumor likes nonneoplastic lesions. Therefore, the histopathological evaluation is of paramount importance in diagnosing nonneoplastic cervical lesions. In our routine reporting of nonneoplastic lesions acute and chronic inflammatory lesions of infective and noninfective etiology are considered, but the nonneoplastic tumor-like lesions such as cervical tunnel clusters, mesonephric and diffuse laminar hyperplasia, endometriosis, and micro glandular endocervical hyperplasia which simulates neoplasia are not much emphasized, hence identification of

these lesions require familiarity in their histopathological features which is a prerequisite for proper management. Chronic cervicitis is the most common uterine cervical lesion in the reproductive age group occurring between 25 to 55 years of age linked to sexual activity and also in postmenopausal women because of reduction in immunity and hormonal replacement therapy.

Materials and Methods

This is a prospective study of 250 cases of nonneoplastic cervical lesions. The study includes 250 nonneoplastic cervical lesions which were analyzed from either hysterectomy or cervical biopsy specimens undertaken in the Department of Pathology at Shridevi institute of medical sciences and Research Hospital, Tumakuru. The hysterectomy specimens were subjected to detailed gross and microscopic examination, The specimens were fixed, processed and sections were cut from paraffin-embedded blocks and stained with the routine with Hematoxylin and eosin.

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Non-Neoplastic Uterine Cervical Lesions

Uterine cervix sections were studied in detail and identified various nonneoplastic lesions and further categorized into inflammatory, metaplasias and epithelial changes.

Results

Our study includes 250 cases of nonneoplastic cervical lesions categorized into the variety of lesions. The commonest ectocervical lesions are Koilocytic change (27.2%) and prolapse changes (9.6%) characterized by epidermidization with hyperkeratosis and parakeratosis, with rare cases of exocytosis and Suprabasal bulla. The most common endocervical lesions are chronic non specific cervicitis (48%) followed by squamous metaplasia (36.8%), chronic papillary endocervicitis (31.2%), nabothian cyst (32%), chronic polypoidal endocervicitis

(20.08%), few cases of endocervical polyps (7.2%), endocervical glandular hyperplasia (4%)microglandular Adenosis (3.2%), and The rarely reported cases are tubal metaplasia, Follicular cervicitis, Diffuse laminar endocervical hyperplasia, Endometriosis, Cervical tunnel clusters and Mesonephric rests. (Table 1) The microscopic images shows important histopathological features of each nonneoplastic lesions (Fig 1-6) The most frequent age group presented with these nonneoplastic cervical lesions are most common in reproductive age between 25 to 50 years and the chief complaints were pain abdomen, abnormal uterine bleeding, white discharge per vagina, uterine prolapse and post coital bleeding.

Table 1: Various categories and percentage of non neoplastic uterine cervical lesions Sl No.

Non neoplastic lesions

No of cases /250

Percentage

Chronic cervicitis

120/250

48%

Squamous metaplasia

92/250

36.8%

Nabothian cyst

80/250

32%

Chronic papillary endocervicitis

78/250

31.2%

Koilocytic change

68/250

27.2%

Chronic polypoidal endocervicitis

52/250

20.8%

Prolapse changes

24/250

9.6%

Endocervical polyp

18/250

7.2%

Endocervical glandular hyperplasia

10/250

Microglandular Adenosis

8/250

3.2%

Tubal metaplasia

7/250

2.8%

Intestinal metaplasia (goblet cell metaplasia)

4/250

1.6%

Follicular cervicitis

2/250

0.8%

Diffuse laminar endocervical glandular hyperplasia

2/250

0.8%

Suprabasal bulla

2/250

0.8%

exocytosis

2/250

0.8%

Cervical tunnel clusters

1/250

0.4%

Mesonephric rests

1/250

0.4%

Endometriosis

1/250

0.4%

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Fig. 1: Photomicrograph showing 1a-Chronic inflammatory cells (H and E 10x). 1b-Follicular cervicitis :Lymphoid follicles(H and E 10x). 1c-Chronic papillary endocervicitis:Papillary infoldings (H and E 10x). 1d-Chronic polypoidal endocervicitis: Polypoidal mucosal folds (H and E 10x).

Fig. 2: Photomicrograph showing: 2a-Tubal metaplasia (Hand E 40x). 2b- Goblet cell metaplasia (H & E 10x). 2c-Squamous metaplasia (H & E 10x). 2d- Nabothian cyst(H & E 10x)

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Fig. 3: photomicrograph showing ; 3a-Endocervical polyp (H & E 10x). 3b- endocervical glandular hyperplasia(H & E 10x).

Fig. 4: Photomicrograph showing;4a- Koilocytic change with exocytosis (H & E 10x). 4b- Suprabasal bulla with cervical prolapse changes (H & E10x).

Fig.5: Photomicrograph showing; 5a-Cervical tunnel clusters (H & E 10x). 5b- Mesonephric rests (H & E 10x).

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FIG: 6a-Microglandular adenosis (H & E 10x). 6b-Diffuse laminar endocervical glandular hyperplasia (H & E 10x).

Discussion

Uterine cervix forms the major portal for the non-neoplastic lesions in the routine histopathology specimens. There are significant numbers of non-neoplastic lesions, which are of immense importance to the clinician and the pathologist as these lesions are notably overlooked, diagnosis and approach towards these lesions are greatly neglected. Naveen Kumar B J et.al[1] depicted that there is poor cervical cytology correlation with subsequent cervical biopsy reports. Accordingly, for the diagnosis of nonneoplastic uterine cervical lesions, histopathological examination still remains the gold standard. Reddy SD et.al[2] described that nonneoplastic lesions of uterine cervix configure bountiful quantum of gynaecological specimens in the histopathological section. The commonest lesion is â&#x20AC;&#x153;chronic cervicitisâ&#x20AC;? displaying chronic inflammatory cells. Our study also reported chronic non specific cervicitis is the most confronted lesion. The most frequent symptom patient presents with non neoplastic cervical lesions are Abnormal Uterine Bleeding, pain abdomen, white discharge per vagina and postcoital bleeding and other non specific chronic inflammatory diseases. Olutoyin G et.al[3]reported that 82% of nonneoplastic lesions of the cervix occur predominantly in sexually active age group and most commonly inflammatory in nature. They may be acute or chronic; each of these can be either infective or non-infective cause. Sexually transmitted diseases, chronic Granulomatous inflammation and viruses especially Human papilloma virus and Herpes simplex virus persistently infect the cervix. Chemical irritants, douching, local trauma and www.pacificejournals.com/apalm

foreign bodies like intrauterine contraceptive devices, pessaries and tampons can cause secondary infections. Pallipady A et.al[4] stated in their study that Koilocytic change is considered as the histological hallmark of the human papilloma virus infection and was diagnosed in 3.84% among all the cervical specimens during the study period compared to which our study shows 27.2% of koilocytosis. Follicular cervicitis was observed in 14 cases, out of which 8 cases were in the 4th decade of life. We had only 2 cases of follicular cervicitis in the 3rd decade. Suprabasal bulla with acantholytic squamous cells was rare lesions seen only in 2 cases both of these were associated with severe chronic cervicitis which is almost similar to our study. Deepa Hatwal et.al[5] reported 315 cases of nonneoplastic lesions of the cervix from Uttarakhand and showed that 85% were chronic inflammatory lesions implies more for the symptom duration along with heavy chronic inflammatory infiltrate and also stated that cervical squamous metaplasia is a physiological change during puberty, reproductive years, menopause and most commonly encountered during a routine histopathological examination. Nonneoplastic cervical glandular lesions studied were Nabothian cyst, cervical tunnel clusters, diffuse laminar endocervical glandular hyperplasia, mesonephric rests, micro glandular hyperplasia and various metaplasias. Nabothian cysts: Cervical gross examination of the cervix revealed multiple mucin-filled cysts which on microscopic examination shows cysts lined by columnar to flattened endocervical-type cells without atypia and mitotic activity. [7]Â Cervical Tunnel Clusters: Richard J et.al[6] reported in detail about endocervical glandular lesions and described eISSN: 2349-6983; pISSN: 2394-6466

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Non-Neoplastic Uterine Cervical Lesions

that Cervical tunnel clusters are frequently multifocal and are approximately 2 mm in diameter and can be greater than 4cm in aggregate and 1.5 cm in thickness. Type A clusters are inconspicuous aggregates of tubules that otherwise resemble the mucosal folds cut in various planes. Type B tunnel clusters are different from usual endocervical glands having complex architecture often composed of dilated or cystic tubules arranged in lobular units. We reported 0.4% of tunnel clusters. Diffuse Laminar Endocervical Glandular Hyperplasia: The first case of diffuse laminar endocervical glandular hyperplasia was reported in 1991, which is very rare Pseudoneoplastic endocervical glandular lesion with the typical histologic finding of a diffuse laminar proliferation of tightly packed hyperplastic endocervical glands with sharp demarcation from underlying stroma and no deeper than an inner third of cervical wall from the underlying stroma. This lesion can be confused easily with minimal deviation adenocarcinoma which is characterized by an irregular proliferation of glands deep in the cervix. [11-12] Mesonephric Rests: We reported a single case of mesonephric rests. They also stated that the mesonephric rests may mimic adenocarcinoma and can progress to mesonephric carcinoma.[1] Microglandular Hyperplasia: Microglandular hyperplasia is the more common pseudoneoplastic lesions showing a peculiar and distinctive form of polypoid hyperplasia of the endocervix which mimicks well-differentiated endometrial adenocarcinoma and is also common in young women taking oral contraceptive pills. Microglandular endocervical hyperplasia is frequently associated with endocervical squamous metaplasia, explained due to some commune etiologic factors.

of ciliated, intercalated (peg), and secretory cells, more characteristically found in the fallopian tube. Adenocarcinoma in situ has to be differentiated from atypical tubal metaplasia due to its degree of atypia in which the glands are lined by similar tubal type cells, but are crowded with larger hyperchromatic and pseudostratified nuclei but mitoses and apoptosis are less when compared to insitu lesions. [20] Endometriosis: Endometriosis, Endocervical hyperplasia, and endocervical polyp are nonneoplastic tumor-like conditions sometimes misinterpreted as neoplastic lesion thus leading to inappropriate and aggressive treatment. Endometriosis presents with excessive vaginal bleeding resulting in severe anemia and its complications.[1-2] We reported only a single case of cervical endometriosis

Conclusion

Nonneoplastic uterine cervical lesions are the most common cervical lesions followed by malignancies in the study population. Chronic non-specific cervicitis is most commonly reported followed by papillary endocervicitis and other lesions, seen in reproductive age group presenting with abdominal pain, pelvic inflammatory disease, abnormal uterine bleeding and uterine prolapse. Thus, categorization of the cervical non-neoplastic lesions with their histopathological findings and familiarity are necessary for recognizing the lesions and for better management of the patient and can avoid further morbidity and complications.

Acknowledgements

We thank our Medical Director Dr. Raman M Hulinaykar, Our beloved Principal Dr. S D Desai and Mr. Arun B R for their constant support and encouragement in completing this research study.

To differentiate between microglandular hyperplasia and clear cell carcinoma is based on architectural and cytological changes like papillary pattern of growing without squamous metaplasia in clear cell carcinoma along with big cells with hobnailing pattern, hyperchromatic nuclei and frequent mitoses. [13-14-15] We reported 3.6 % cases of microglandular hyperplasia.

Abbreviations

Kumar BJ, Annam V. Clinico-Pathological Study of Non-Neoplastic Lesions of Uterine Cervix with their Histopathological Categorization. International Journal of Science and Research. 2013: 2319-7064.

Squamous Metaplasia: The authentic recognition of squamous metaplasia on histopathology is important and can avoid an unnecessary diagnosis of CIN (cervical intraepithelial neoplasia). Our study reported 36% of squamous metaplasia compared to 14.9% mentioned in their study.[5]

Reddy SD, Rani MS, Rao KS. Clinico-histopathologic study of nonneoplastic uterine cervical lesions. Int J Med Sci Public Health. 2016: 5(8);1536-1539.

Olutoyin G, Omoniyi-Esan OG, Osasan SA, Ojo OS. Nonneoplastic diseases of the cervix in Nigeria. A histopathological study. Afr Health Sci 2006:6;76-80.

Pallipady A, Illanthody S, Vaidya R, Ahmed Z, Suvarna R, Metkar G. A Clinico-Morphological Spectrum of the Non Neoplastic Lesions of the Uterine Cervix at AJ Hospital,

Tubal Metaplasia: Tubal metaplasia tends to occur more commonly in upper endocervix and composed

H & E â&#x20AC;&#x201C; Hematoxylin and Eosin

References

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Priyadarshini. D et al. Mangalore: Journal of Clinical and Diagnostic Research. 2011:5(3); 546-550. 5.

Deepa H, Neha B, Arvind K, Sheela C, Sachan B. Spectrum of Nonneoplastic Lesions of Uterine Cervix in Uttarakhand. National Journal of Laboratory Medicine. 2016;1-5.

Richard J, Zaino M.D. Glandular Lesions of the Uterine Cervix: Mod Pathol 2000:13(3);261–274.

Jayadeep G, Suman LK, Veena S, Sumit G. Clinicopathological Evaluation of Nonneoplastic and Neoplastic Lesions of Uterine Cervix. Imperial Journal of Interdisciplinary Research (IJIR).2016:2(4).

Nwachokor FN, Forae GC. Morphological spectrum of nonneoplastic lesions of the uterine cervix in Warri, Nigeria. Niger J Clin Pract.2013:16(4);429-32.

Matos E, Lotia D, Amestoy G, Herrera L, Prince MA, Moreno J, et al. Prevalence of human papillomavirus infection among women in Concordia, Argentina: A population based study. Sex Transm Dis.2003;30;593-9.

10. Jyothi V, Manoja V, Reddy KS. A clinicopathological study on cervix.2015:4(1)3; 2120-2126 11. Nucci MR. Symposium Part III: Tumor-Like Glandular Lesions of the Uterine Cervix. Int J Gynecol Pathol. 2002: 21(4); 347-359. 12. Louis AD, Thomas CK, Joel CW, Christopher MZ, John CE, Mildred RC et.al. Diffuse Laminar Endocervical Glandular Hyperplasia A Case Report. Int J Gynecol Cancer 2009:19;1091-1093.

A-309 13. Florescu M, Simionescu C, Georgescu CV, Marinescu M. Histopathologic aspects in microglandular hyperplasia of endocervix. Morphol-Embryol.2004:181–184 14. Simionescu C, Margaritescu CL, Georgescu CV, Mogoanta L, Marinescu AM. Pseudo-tumoral lesions of the cervix. Rom J Morphol Embryol 2005:4;239-47. 15. Medeiros F, Bell DA. Pseudoneoplastic lesions of the female genital tract. Arch Pathol Lab Med. 2010:134(3);393-403. 16. Younis MT, Iram S, Anwar B, Ewies AA. Women with asymptomatic cervical polyps may not need to see a gynaecologist or have them removed: an observational retrospective study of 1126 cases. Eur J Obstet Gynecol Reprod Biol. 2010: 150(2):190-4. 17. Ozumba BC, Nzegwu MA, Anyikam A. Histological patterns of gynaecological lesions in Enugu, Nigeria. A five year review. Adv Biores.2011;2:132-36. 18. Nigatu B, Gebrehiwot Y, Kiros K, Eregete W. A five year analysis of histopathological results of cervical biopsies examined in a pathology department of a teaching hospital (2003‑2007). Ethiop J Reprod Health 2010:4;52‑7. 19. Fatima Q, Verma S, Bairwa NK, Gauri LA. Spectrum of Various Lesions in Cervical Biopsies in North West Rajasthan: A Prospective Histopathological Study. Int J Med Res Prof.2017: 3(1); 104-11. 20. Kay J P, Robert A. Current concepts in cervical Pathology. Archives of Pathology & Laboratory Medicine.2009:133; 729-738.

*Corresponding author: Dr. Priyadarshini. D. #108, A Block, Sai Gangotri Apartments, Ullal main road, Opp Bharath Petroleum, Muneshwaranagar, Bengaluru – 560056, INDIA Phone: +91 7829765527 Email: priyadarshinid.arun@gmail.com Date of Submission : 12.03.2017 Date of Acceptance : 06.04.2017 Financial or other Competing Interests: None. Date of Publication : 04.07.2017

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Original Article DOI: 10.21276/APALM.1411

Use of Mean Platelet Volume as an Initial Diagnostic Marker in Evaluation of Dengue Fever Nabila Afsar*, Idrees Akhtar Afroze, Shahmeen Humaira and Zakia Abid Department of Pathology, Deccan College of Medical sciences, Hyderabad, Telengana. India

ABSTRACT Background: Dengue fever is the most rapidly spreading mosquito-borne viral disease. Four main characteristic manifestations of dengue illness are (i) continuous high fever lasting 2-7 days; (ii) haemorrhagic tendency as shown by a positive tourniquet test, petechiae or epistaxis; (iii) thrombocytopoenia (platelet count <100Ă&#x2014;109/l); and (iv) evidence of plasma leakage manifested by haemoconcentration (an increase in haematocrit 20% above average for age, sex and population), pleural effusion and ascites, etc. Recently, novel platelet indices such as MPV, PDW, and P-LCR have been investigated as prospective platelet activation markers .Platelet volume, a marker of platelet function and activity is measured as mean platelet volume (MPV) by hematology analyzer. Method: The study was done with the aim to assess whether mean platelet volume has any diagnostic importance in the initial evaluation of dengue fever . 45 adult patients were diagnosed with Dengue NS1 antigen positivity and underwent routine complete blood counts on automated hematology analyser HORIBA Pentra ES60. Result: A total of 76 cases ranging from 6 months to 50 years of age were reported as antigenically positive for dengue indicating acute infection, out of which 45 were adults ranging from 12 to 50 years of age. 34 were male and 11 were female. Infants and children below 12 years of age were not included in this study. 55 controls were studied which included 31 males and 24 females. The mean platelet volume was found to be significantly higher in dengue cases when compared to controls. MPV was also noted to be significantly higher in patients with platelet count below the normal biological reference range of 1.5 lakhs ,when compared to dengue patients with normal platelet counts. Conclusion: To conclude, high MPV indicates platelet activation and may be used as an initial marker to suspect dengue fever in a case of thrombocytopenia. Keywords: Dengue Fever, MPV, Mean Platelet Volume

Introduction

Dengue fever is the most rapidly spreading mosquitoborne viral disease in the world. An estimated 50 million infections per years occur across approximately 100 countries. Incidence has increased 30-fold with increasing geographic expansion with potential for further spread[1] .It is transmitted mainly by Aedes aegypti mosquito and also by Aedes albopictus. All four serotypes can cause the full spectrum of disease from a subclinical infection to a mild self limiting disease, the dengue fever (DF) and a severe disease that may be fatal, the dengue haemorrhagic fever/dengue shock syndrome (DHF/ DSS). The WHO 2009 classification divides dengue fever into two groups: uncomplicated and severe, though the 1997 WHO classification is still widely used. [2] The 1997 classification divided dengue into undifferentiated fever, dengue fever (DF), and dengue haemorrhagic fever (DHF). Four main characteristic manifestations of dengue illness are (i) continuous high fever lasting 2-7 days; (ii) haemorrhagic tendency as shown by a positive tourniquet test(Hess test or Rumpel-Leed capillary fragility test),

petechiae or epistaxis; (iii) thrombocytopoenia (platelet count <100Ă&#x2014;109/l); and (iv) evidence of plasma leakage manifested by haemoconcentration (an increase in haematocrit 20% above average for age, sex and population), pleural effusion and ascites, etc. Recently, novel platelet indices such as MPV, PDW, and P-LCR have been investigated as prospective platelet activation markers .Platelet volume, a marker of platelet function and activity is measured as mean platelet volume (MPV) by hematology analyzers. Mean platelet volume is derived from impedence platelet size distribution curve and is highly dependent on technique of measurement, length and storage of blood sample prior to testing. In normal subjects, the MPV is inversely proportional to platelet count. MPV is decreased in megaloblastic anemia and bone marrow depression. It is increased in patients at risk of and following myocardial infarction and cerebral infarcts and also gives evidence of inherited macrothrombocytopenia.[3] When platelet production is decreased, young platelets become bigger and more active, and MPV levels increase.

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Increased MPV indicates increased platelet diameter, which can be used as a marker of production rate and platelet activation. During activation, platelets’ shapes change from biconcave discs to spherical, and a pronounced pseudopod formation occurs that leads to MPV increase during platelet activation.[4]

fever and healthy controls were studied and analysed with the calculation of mean, median, standard deviation and range on Microsoft excel sheet and the statistical significance was established using the student T test to ascertain the P value.

Result

The study was undertaken with the aim of establishing if any relationship is seen to exist with Mean Platelet Volume in cases of dengue fever and to ascertain if there is any correlation with age and gender related difference.

45 adult patients were reported as antigenically positive for dengue indicating acute infection, ranging from 12 to 50 years of age. 34 were male(75.5%) and 11 were female (24.5%). Infants and children below 12 years of age were not included in this study. 55 controls were studied which included 31 males(56.4%) and 24 females(43.6%). Table 1.

Materials and Methods

A retrospective case control study of dengue cases was carried out in the Department of Pathology of Owaisi Hospital and Research Centre, Deccan College of Medical Sciences which is a tertiary care teaching hospital in Hyderabad. It was conducted during the seasonal peak of July 2016 to October 2016.

86.7% of Dengue fever cases are young patients below 30 years of age with 44.4% cases seen in 21to 30 years of age and 42.2% cases seen in the age group of 12 to 20 years. (Table 2)

45 adult patients were diagnosed with Dengue NS1 antigen positivity and underwent routine complete blood counts on automated hematology analyser HORIBA Pentra ES60. Samples were collected in K3 EDTA vacutainers and processed within 2 hour of collection. Samples of 55 controls including 31apparently healthy non smoker adult male and 24 female controls were also collected and processed as above.

A statistically significant increase in MPV was noted in cases of dengue in age groups of 12-20 and 21-30 years compared to the control group. The ages 31 to 50 years were analysed as a single group owing to the low number of cases and controls and no statistical significance was noted in this group probably due to the low number of cases though the mean was higher in dengue fever group.(Table 3)

Inclusion criteria: All adult NS1 positive cases of dengue undergoing routine haematological investigation.

The mean platelet volume was found to be significantly higher in dengue cases (MPV=8.84±1.05 fl) when compared to controls (8.2±0.78 fl). (Table 4)

Exclusion criteria: Dengue negative patients and infants and children with antigen positivity were excluded to rule out any age related difference in platelet parameters.

MPV was also noted to be significantly higher in dengue patients with thrombocytopenia (MPV=9.08±1.08 fl) , when compared to dengue patients with normal platelet counts (MPV=8.1±0.68 fl).(Table 5)

Data was collected retrospectively from the laboratory records. Platelet count and MPV of cases of dengue Table 1: Sex wise distribution of cases and controls. Gender

Cases

Controls

Male

75.5

56.4

Female

24.5

43.6

Total

100

Table 2: Age and gender wise distribution of cases and controls. AGE

CASES

CONTROL

Male

Female

Total

Male

Female

Total

12-20

15 (33.3%)

4 (8.9%)

19 (42.2%)

20 (36.4%)

44 (80%)

21-30

14 (31.1%)

6 (13.3%)

20 (44.4%)

7 (12.7%)

31-40

1 (2.2%)

4 (7.3%)

41-50

4 (8.9%)

1 (2.2%)

5 (11.1%)

Total

34 (75.5%)

11 (24.5%)

45(100%)

31 (56.4%)

24 (43.6%)

55(100%)

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MPV A Diagnostic Marker of Dengue Fever

Table 3: Age wise distribution of MPV in dengue cases compared to controls. CASES MPV in CASES (fl) CONTROL Age Mean SD Range Age 12-20 (n=19) 8.85 1.10 7.3-10.8 12-20 (n=44) 21-30 (n=20) 9 0.94 7.6-10.6 21-30 (n=7) >30(n=6) 8.25 0.99 7-9.6 >30 (n=4)

MPV in CONTROLS(fl) Mean SD Range 8.23 0.84 6.5-10.5 8.29 0.48 7.7-9.1 7.88 0.35 7.3-8.2

Table 4: Statistical evaluation in cases of dengue versus normal controls MPV(fl) Cases(n=45) Control(n=55) Mean 8.84 8.2 Median 8.8 8.2 SD 1.05 0.78 Range 7-11.1 6.5-10.5 Table 5: MPV according to platelet count in dengue cases. Platelet count <150x103 / MPV(fl) mm3 (n=34) Mean 9.08 Median 9.15 SD 1.08 Range 7-11.1

Discussion

Dengue fever was noted to be predominant in males in the present study which is consistent with study done by Martha Anker et al who found a male predominance in the reported number of incident dengue cases among persons 15 years or older in several Asian countries.[5] Three independent studies from epidemics in India done by Agarwal et al., Ray et al., and Wali et al., also found nearly twice the number of male patients infected with dengue compared to females, M:F being 1.9:1, 1:0.57, and 2.5:1, respectively.[6],[7],[8],[9] Dengue fever was seen to be more rampant in younger age group. Cecilia D observed that the 21–30 years age group was most affected by dengue throughout the 6 years. [10] This was consistent with our study with 86.7% dengue fever patients being under 30 years of age. Normal platelet size (mean platelet volume) varies between 7.5 and 10.5 fl. Some patients with severe thrombocytopenia have little or no bleeding, and show morphologically large platelets on blood smears. This information has spawned the idea that some individuals have hyperfunctional platelets that compensate for low numbers by their increased effectiveness.[11] A literature has developed that supports the idea that young platelets, presumably those recently released from bone marrow , are larger and more dense, contain more of certain proteins(i.e. PF4), and exhibit some alteration in function as compared to smaller platelets.[12, 13] These observations may explain the findings of significant increased MPV(9.08 fl±1.08 fl) in cases of dengue fever with thrombocytopenia when

Platelet count >150x103 / mm3 (n=11) 8.1 8.1 0.68 7.3-9.9

P value 0.04 0.02 0.46 T test

P value = 0.0015

T test P value = 0.001946

compared to MPV(8.1 fl±0.68 fl) in dengue fever cases with normal platelet count. Basu et al suggested that DV-2 inhibits in vitro megakaryopoiesis and induces apoptotic cell death in a subpopulation of early megakaryocytic progenitors which may contribute to thrombocytopenia in dengue disease.[14] Ghosh et al showed that DV-2 may directly interact with and activate platelets and thus may be responsible for thrombocytopenia.[15] This study may explain the findings of the present study with MPV being significantly higher in cases of dengue fever when compared to controls due to the activation of platelets causing a rise in MPV. Budak YU discussed that when platelet production is decreased, young platelets become bigger and more active, and MPV levels increase. Increased MPV indicates increased platelet diameter, which can be used as a marker of production rate and platelet activation. During activation, platelets’ shapes change from biconcave discs to spherical, and a pronounced pseudopod formation occurs that leads to MPV increase during platelet activation.[16] This analogy also explains the high MPV noted in the present study. However, other studies were found to show conflicting results. Prakash G. M. found no significant difference was observed in Mean between MPV at the time of minimal platelet counts and at discharge in dengue cases except in dengue fever cases.[17] This was correlated with study done by Dewi et al [18] and Wiwanitkit et al[19] who suggested that MPV is not decreased in patients with dengue haemorrhagic fever and appears to be similar to that of healthy population. Bashir AB et al found significant

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differences in the MPV, PDW and PLT in patients with dengue infection, low platelet count, MPV, and PDW may be used as probable indicators for dengue in endemic area. Low MPV <9 fl and high PDW >13fl shows considerable sensitivity for dengue fever.[20] Kritika Sharma et al showed that MPV showed no significant correlation with severity, serology & treatment outcome, thus excluding its role in dengue cases. Mean platelet volume was not found to be an important prognostic parameter in dengue fever cases[4].

Conclusion

Dengue fever had a higher incidence in males and in individuals younger than 30 years of age. MPV was significantly higher in patients of Dengue fever when compared to healthy controls. It was found to be significantly higher in dengue cases with low platelet count. This suggests that dengue virus causes activation of platelets leading to their subsequent increase in size and MPV. It could also be related to previous treatment history which may be stimulating new platelet formation. To conclude, high MPV indicates platelet activation and may be used as an initial marker to suspect dengue fever in a case of thrombocytopenia. However, considering the small sample size, further studies may be undertaken during future epidemics to evaluate a larger population in order to use MPV as an initial marker available in a routine hematology analyser report to avoid unnecessary serological testing

Reference 1. 2. 3. 4.

WHO. Dengue hemorrhagic fever: Diagnosis, treatment, prevention and control. Geneva: WHO press; 2009:3-106. Dengue: Guidelines for diagnosis, treatment, prevention, and control in sub-Saharan Africa and 13 countries in South America. Geneva: World Health Organization; 2009. WHO. Dacie and Lewis Practical Hematology 11th edition:47-48 Sharma K, Yadav A. Association of Mean Platelet Volume with Severity, Serology & Treatment Outcome in Dengue Fever: Prognostic Utility J Clin Diagn Res. 2015; 9(11): EC01–EC03 Anker M, Arima Y. Male-female differences in the number of reported incident dengue fever cases in six Asian countries. Western Pacific Surveillance and Response Journal. 2011, 2(2):17-23. doi:10.5365/wpsar.2011.2.1.002 Guha-Sapir D, Schimmer B. Dengue fever: New paradigms for a changing epidemiology. Emerg Themes Epidemiol 2005;2:1.

8. 9. 10. 11. 12. 13. 14.

15.

16.

17.

18. 19. 20.

Agarwal R, Kapoor S, Nagar R, Misra A, Tandon R, Mathur A, et al. A clinical study of the patients with dengue hemorrhagic fever during the epidemic of 1996 at Lucknow, India. Southeast Asian J Trop Med Public Health 1999;30:735-40. Ray G, Kumar V, Kapoor AK, Dutta AK, Batra S. Status of antioxidants and other biochemical abnormalities in children with dengue fever. J Trop Pediatr 1999;45:4-7. Wali JP, Biswas A, Handa R, Aggarwal P, Wig N, Dwivedi SN. Dengue haemorrhagic fever in adults: A prospective study of 110 cases. Trop Doct 1999;29:27-30. McMillan R. Therapy for adults with refractory chronic immune thrombocytopenic purpura. Ann Intern Med 1997;126:307-314 Cecilia D. Current status of dengue and chikungunya in India. WHO South-East Asia Journal of Public Health | JanMar 2014 | 3 (1) 22-27 Karpatkin S. Heterogeneity of human platelets. VI. Correlation of platelet function with platelet volume. Blood 1978;51:307-316 Wintrobe’s Clinical Heamatology, 11th edition: 632 Basu A, Jain P, Gangodkar S, Shetty S, Ghosh K. Dengue 2 virus inhibits in vitro megakaryocytic colony formation and induce apoptosis in thrombopoietin-inducible megakaryocytic differentiation from cord blood CD34+ cells. FEMS Immunol Med Microbiol. 2008;53:46–51. Ghosh K, Gangodkar S, Jain P, Shetty S, Ramjee S, Poddar P, et al. Imaging the interaction between dengue 2 virus and human blood platelets using atomic force and electron microscopy. J Electron Microsc. 2008;57:113–8. 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. Prakash GM, Anikethana G V. Use of mean platelet volume and platelet distribution width in predicting trend in platelet count and bleeding risks in patients of dengue fever. Int J Adv Med 2016;3:611-3. Dewi YP. Mean Platelet Volume (MPV): Potential Predictor of Disease Severity in Dengue infection. In proceeding of: International Dengue Symposium. 2013 conference paper. Wiwanitkit V. Mean platelet volume in the patients with dengue hemorrhagic fever. Platelets. 2004;15(3):185. Bashir AB, Saeed OK, Mohammed BA, Ageep AK. Role of Platelet Indices in Patients with Dengue Infection in Red Sea State, Sudan. International Journal of Science and Research 2015;4:1573-1576

*Corresponding author: Dr. Nabila Afsar, 5-5-203/3, Patel Nagar, Nampally, Hyderabad, 500001, Telengana, India Phone: +91 9848020386 Email: nabila_dr@yahoo.com

Financial or other Competing Interests: None.

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Date of Submission : 14.03.2017 Date of Acceptance : 11.04.2017 Date of Publication : 04.07.2017

eISSN: 2349-6983; pISSN: 2394-6466

Original Article DOI: 10.21276/APALM.1346

Chitotriosidase Enzyme Activity and MiRNA-146a Expression and Their Value as Potential Biomarkers of Subclinical Atherosclerosis in Type 2 Diabetes Mellitus Hala Mourad Demerdash1*, Radwa Mohamed ElSharaby2 and Yasser Mohamed Abdel Raouf3 Clinical Pathology Department, Faculty of Pharmacy, Alexandria University Hospitals, Pharos University in Alexandria, Egypt 2 Clinical Pathology Department, Faculty of Medicine, Tanta University. Egypt 3 Department of Internal Medicine, Faculty of Medicine Tanta University, Egypt

ABSTRACT Background: Type 2 diabetes T2DM is a common disease with increased mortality and morbidity due to vascular complications. Carotid Intimal-Medial Thickness (CIMT) is being used as a marker to assess subclinical atherosclerosis.MicroRNA (miR-146a) is a new discoveredbiomarker that regulates endothelial cell function and vascular inflammation, together with Chitotriosidase (CHIT1)enzyme being involved mainly in the immune and inflammatory responses. This study aimed to investigate an assumed association between CHIT1 activity and miR-146a gene expression in type 2 diabetes patients for detection of endothelial dysfunction and subclinical atherosclerosis, in association with CIMT. Material and Methods: 30 control subjects (group I) and 100 patients diagnosed as T2DMclassified according to CIMT results; group II (38 patient) with CIMT value less than 0.7mm and group III (62patients) with CIMT value more than 0.7mm. Plasma CHIT1 activity was measured fluorometricallyand quantitative Real Time PCR (qPCR) for miR-146a. Routine laboratory parameters such as blood glucose, lipid profile, glycated hemoglobin were also measured. Results: revealedCHIT1activity was significant high(p < 0.001) in group II and III diabetic patients compared to group I, also it waspositively correlated with CIMT and other parameters as glycemic control, lipid profile, duration of disease and blood pressure. On the other hand, plasma miR-146a was significantly reduced (p < 0.001) in group III and was negatively correlated with CIMT and other parameters. Conclusion: IncreasedCHIT1activity may be due to the associated changes in the relative gene expression of miRNA 146a in patients with increased CIMT above 1mm. Moreover, evaluation of microRNA 146a gene expression can be used as useful biomarker for detection of endothelial dysfunction and development of atherosclerosis in T2DM. Keywords: atherosclerosis; Chitotriosidase; MicroRNAs; Carotid Intimal-Medial Thickness. Abbreviations: T2DM,Type 2 diabetes; ED,Endothelial dysfunction; CHIT1, enzyme chitotriosidase; CIMT, carotid intimaâ&#x20AC;&#x201C;media thickness; HbA1C, glycated hemoglobin

Introduction

Type 2 diabetes (T2DM) is a chronic metabolic disease, thatresults from insulin resistance and or deficiency. It is associated with many metabolic abnormalities, such as obesity, hypertension, and dyslipidemia. However, pathologic hallmark of T2DM involves the vascular complications due to long term hyperglycemia [1]. Endothelial dysfunction (ED)contributes to the pathological progression ofvascular complications in T2DM.ED occurs as consequence of combination of several elements including genetic predisposition, systemic inflammation and cardiovascular risk factors [2]. The endothelium is a key regulator of vascular function, and ED tends to be the initial event in macrovascular complications such as atherosclerosis inT2DM. Mechanisms of ED include the down-regulation of

endothelial nitric oxide synthase levels, oxidative stress, activation of inflammatory pathways and differential expression of vascular endothelial growth factor and other inflammatory mediators [3]. A large body of evidence designates that inflammation is important in initiation of atherogenesis and development of subclinical atherosclerosis, manifested by increased carotid artery intima-media thickness and increased number of carotid plaques, in patients with T2DM [4]. The enzyme chitotriosidase (CHIT1) belongs to the glycoside hydrolase family 18. It is one of the most abundant and indicative proteins secreted by activated macrophages and neutrophil-speciďŹ c granules. It has an active role in the immune response, being implicated in the activation and polarization of macrophages, propagating

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Demerdash et al. chronic inflammation, as well as activation of other immune cells such as T helper cells [5]. MiRNAs are a group of approximately 20 to 22 nucleotides noncoding RNAs and have been proven to regulate gene expression at the post-transcriptional level. In addition, they can directly stimulate or inhibit target gene transcription by directly binding to promoter regions, RNAactivation phenomena. miR-146a is involved in modulation of the inflammatory pathways in T2DM [6] Measurement of the carotid intima–media thickness (CIMT) has been recognized as a powerful method for identifying subclinical atherosclerosis, and increased thickness of the intima– media complex reflects local generalized atherosclerosis [7].The objective of this study was to evaluate respective values of miRNA-146a and CHIT1andtheir association with CIMT, being a valid marker of atherosclerosis in T2DM patients.

Materials and Methods

This cross-sectional study was carried out on 30 control subjects and 100 T2DM patients. Subject’s selection was undertaken in collaboration between departments of Internal Medicine and Radiology, Tanta University Hospitals. The research protocol was performed in accordance Declaration of Helsinki and approved by the Medical Ethics Committee of the Faculty of medicine, Tanta University, from April 2015until April 2016. Informed written consent was obtained from all subjects included in the study. Inclusion criteria were diabetes type 2 patients on diabetes therapy including insulin and/or oral hypoglycemic drugs more than five years. Exclusion criteria included type 1 diabetes, patients with history of familial dyslipidemia, hypothyroidism, liver or kidney disease, Cushing’s syndrome, myocardial infarction during the last 6 months. After an overnight fasting,9 ml blood was taken from every investigated subject. 4 ml of EDTA whole blood, centrifuged at 4000 rpm for 10 min at 4 °C, plasma was separated and stored at −80 °C for estimation of plasmachitotriosidase enzyme activity (CHIT1) andmiR- 146a gene expression. 2 ml of EDTAwholeblood for Glycated Hemoglobin (Hb A1C) was determined directly (without measurement of total hemoglobin) by turbidimetric inhibition immunoassay (Roche, Basel, Switzerland). 3 ml of blood in a plain sterile tube and centrifuged at 4000 rpm for 10 min at 4 °C; serum was separated Fasting blood glucose (FBG) and the rest of sample stored at −20 °C for estimation of lipid profile. Also, 2ml of blood were collected in a plain sterile tube after twohours for post-prandial blood glucose (PPBG) determination. www.pacificejournals.com/apalm

A-315 Both FBG and PPBG were estimated by a glucose analyzer 2 (Beckman Instruments, Fullerton, CA) based on the glucose oxidase method.Lipid profile; Serum total cholesterol (T.C), serum triglyceride, high density cholesterol (HDL-C) and low density cholesterol (LDL-C) were determined with kits from SENTINEL CH. (via principle Eugenio 5-20155 MILAN-ITALY). Plasma Chitotriosidase activity(CHIT1):was measured fluorimetricallyby enzymatic hydrolysis of the fluorimetric substrate 4-methylumbelliferyl-β-D-NN,N’triacetylchitotriose (Sigma-Aldrich ChemieGmBH, Taufkirchen, Germany) and release of the fluorescent molecule 4-methylumbelliferone (4MU) which was read in a Microfluor 2 ® plate by a fluorimeter (BIO-TEK 5SynergyHT; Biotek Instruments Inc., Winooski, VT). Plasma miR-146a gene expression quantification by real-time PCR (RTq-PCR): Extraction of miRNA by miRNeasyMiniKit (supplied by Qiagen, Valencia, CA, USA) Reverse transcription was performed for cDNA synthesis using the miRNeasy plasma Reverse Transcription Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The expression miR-146a was evaluated by qRTPCR analysis according to the manufacturer’s protocol. miR-146a Forward primer:5’TGAGAACTGAATTCCATGGGTT-3’ while miR-146aReverse primer:5’-ATCTACTCTCTCCAGGTCCTCA-3. Reference gene U6 small nuclear RNA (snRNA) primers, Forward primer:5’-CTCGCTTCGGCAGCACA-3’, while Reverse primer:5’-AA CGCTTCACGAATTTGCGT-3’. For real-time PCR, the cDNA template was mixed with SYBER Green Master Mix (Qiagen, Valencia, CA, USA). PCR array plate enriched with forward and reverse miRNAspecific primers. Real-time PCR reactions were performed using an Applied Biosystems 7300HT analyzer.The cycle threshold (CT) is defined as the number of cycles required for the fluorescent signal to cross the threshold in realtime PCR. The quantification of miRNAs gene expression was reported as the ΔCT value, which was calculated by subtracting the CT values of miRNA Reference gene from the CT values of the target miRNAs as following: The comparative miR-146a Expression as Measured by: (ΔΔCt) = (Ct miR-146a normal - Ct RNU6 normal) - (Ct miR-146a RA- Ct RNU6 RA). The Relative expression in the target gene was calculated based onthe estimated mean difference (2)-ΔΔCt . Measurement of Carotid Intima–Media Thickness (CIMT): CIMT was determined using a standardized protocol; patient was examined in supine position with eISSN: 2349-6983; pISSN: 2394-6466

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Chitotriosidase and miRNA-146a in type 2 Diabetes.

head turned 45º opposite to the side being tested. CIMT measurement was done using high resolution B-mode (9MHz) ultrasound (Toshiba SSA370A, Japan). The CIMT was defined as distance between edges of the lumen-intima and the media-adventitia echos, in a plaquefree section. CIMT measured bilaterally, CIMT values ≤0.7 mm indicated normal CIMT and values > 0.9 mm indicated increased CIMT[8]. All measurements were performed blinded for patients’ clinical data by the same experienced sonographer.

groups; group IIincluded 38 T2DM patients(with CIMT values less than 0.7mm)none of them had history of transient ischemic attacks or ischemic stroke, while group III included 62 T2DM patients (with CIMT values of 0.9 mm or more). The disease duration was significantly higher in group III (11.43±3.1 years) compared to group II (7.8± 2.25 years). Studied groups revealed no significant difference as regard age; in which group, I was 44.87± 6.03 years, group II was 43.92 ± 3.94 yearsand group III was 44.22±3.66years. Also, male to female ratio in group I was 1.5:1 (18:12), in group II 2.2:1.0, (26:12) while in group III 1.6:1.0 (38:24) there was no statistically significant difference between the males to females ratio in studied groups.Table (1) shows patientsbiochemical data and blood pressure of studied groups.

Statistical Analysis: Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. Qualitative data were described using number and percent. Quantitative data were described using range (minimum and maximum), mean, standard deviation and median. Significance of the obtained results was judged at the 5% level.

The carotid intima thickness (CIMT) was significantly higher in group III compared to both group II and I(table 2). There was a significant increase in CHIT1 enzyme activity in group II and III compared to group I. The level of miR-146a gene expression was significantly increased in group III more than group I and II(table 3). Correlation between CIMT values and studied biomarkers was performed; results revealed a positive correlation between CIMT and CHIT1 and negative correlation with miR-146a gene expression (table 4).

The used tests were 1- ONE WAY: Analysis of Variance (ANOVA), was performed for comparison between more than two sample 2- Correlation coefficient; to determine the association between each two studied variables.

RESULTS

The significant parameters which had an effect on CIMT were duration of diabetes CHIT1 enzyme activity and miR-146a gene expression and HbA1C level (table 5).

Studied subjects were classified into three groups; group I were 30 healthy control subjects;diabetic patients were divided into groups based on results of CIMT into 2

Table 1: Blood pressure and Biochemical data of the three studied groups Group III (N=62)

129.31±11.8b

149.54±21.44c

22.3

0.001*

193.61±45.23

226.77±65.9c

22.6

0.001*

8.62±2.1

8.22

0.013*

15.6

0.0052*

Group II (N=38)

Group I (N=30) FBG (mg/dl)

87.9 ± 3.9a

PPBG (mg/dl) Hb A1C

112.67±7.67

Systolic BP (mm Hg)

114.1±5.89a

6.41±1.42

Diastolic BP (mm Hg) T.C (mg/dl) T.G (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl)

7.94±1.96

123.7 ± 14.6b

131.2 ± 15.7b

76.4 ± 4.2

85.1 ± 7.8

89.8±6.4

2.65

0.122

153.50±15.08 a 107.62±14.94 a 49.57±4.13 a

225.46±3.16 b 140.95±16.40 b 40.95±4.43 b

235.82±9.05b 181.80±8.01 c 43.71±6.26ab

62.25 44.98 8.2

0.0001* 0.0001* 0.0026*

84.87±8.15 a

129.32±6.13 b

138.50±7.76b

74.2

0.0001*

* Statistically significant at (P ≤ 0.05) Data are expressed as mean ± SD; p < 0.05 was considered statistically significant.

Table 2: Carotid intima thickness (CIMT) in the three studied groups. Group I 0.60±0.07

CIMT (mm) F p

Group II 0.76±0.048 22.6 0.001*

* Statistically significant at (P ≤ 0.05)

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Group III 1.14±0.09

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Table 3: biomarkers of endothelial dysfunction (ED) in the three studied groups. Group I

Group II

Group III

CHIT1 enzyme activity

193.647±17.40a

313.78±8.65b

335.85±20.29 b

49.8

0.0001*

miR-146a

0.93±0.12a

4.36±0.77b

7.79±1.02 c

32.6

0.001*

* Statistically significant at (P ≤ 0.05).

Table 4: Correlation between CIMT and Endothelia dysfunction biomarkers. Group II

CIMT

Group III

CHIT1 enzyme activity

0.801

0.001*

0.599

0.016*

miR-146a

-0.521

0.023*

-0.562

0.011*

* Statistically significant at (P ≤ 0.05).

Table (5): Multiple logistic regression analysis using different biologically important variables as independent variables, and CIMT as the dependent variable. Model

Unstandardized Coefficients

Standardized Coefficients

Sig.

-3.21

.001*

.106

.32

.065

3.165

.026*

-.015-

-.046-

.961

.025

.442

.626

.161

1.179

.241

.004

.006

.011

0.980

0.013

.100

.086

.135

.893

0.049

.167

.189

.293

.770

CHIT1 enzyme activity

0.026

.0017

.825

7.11

.0001*

miR-146a

0.052

.006

.323

4.003

.016*

Hb A1C

0.107

.201

.425

3.25

.021*

Std. Error

Constant

-0.622

.228

age

-0.103

.104

Duration of disease

-0.063

.033

T.C.

-0.0013

.006

0.0016

.0001

HDL-C

0.005

.004

LDL-C

-0.0028

T.C./HDL LDL/HDL

Beta -.051-

a. Dependent Variable: CIMT

Discussion

Pathogenesis of atherosclerosis necessitates comprehensive understanding of the proteins secreted in the vessel wall by macrophages and the molecular mechanisms that regulate gene expression that participate in this process.In this study, we observed a relationship between two biomarkers; CHIT1 enzyme activity and miR-146a gene expression in assessment of subclinical atherosclerosis in view of CIMT being an establishedindicator of cardiovascular risk.

Several studies reported CIMT values below 0.7 mm is considered normal, while CIMT of 1 mm or more is always associated with anenhanced absolute risk of Chronic Heart Disease [9,10]. Diabetic patients were classified according to CIMT value wherein CIMT >0.9mm was considered as increased CIMT (indicating vascular hypertrophy) [10] . In this study, results revealed that diabetic patients (group III) displayed significant increase in CIMT values compared to other groups; group I and II patients had CIMT values of 0.60±0.07and0.76±0.048mm respectively, while group III patients had CIMT value of 1.14±0.09mm. Raised CIMT values were explained by deposition of lipid-laden macrophages in the wall of blood vessel which promoted the expression of different genes that stimulated the inflammatory process in atherogenesis. This process consequently resulted in arterial stiffness and thickening of the intima and media

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Type 2 diabetes (T2DM) is associated with increased morbidity and mortality due to vascular complications. Hyperglycemia results in low-grade inflammation and formation of advanced glycosylated end-products (AGE). Endothelial dysfunction as consequenceof chronic inflammation evokes premature onset of atherosclerosis in T2DM [2].

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of the arterial wall [11]. Group III patients had an extended duration of diabetes 11.43±2.1 years and poor glycemic control as determined by HbA1C compared to group II patients where shorter disease duration was 7.8± 2.25 years and better glycemic control. These results came in concordance with Butt et al and Sibal et al.; they concluded upsurge of CIMT value was influenced by duration of disease and glycemic control [9, 10]. Results of present study revealed that total cholesterol and LDL-cholesterol were significantly increased in diabetic patients compared to control group with no significant difference between group II and III.Regression analysis was done and detected lack of relationship between CIMT and parameters of lipid metabolism in T2DM. Likewise, Byrkjeland et al found no correlation between in LDLcholesterol and CIMT, inferring that increase in LDLcholesterol did not influence CIMT progression [12]. In contrast, Kota et al reported that glycemic parameters as FBG, and HbA1C collectively with lipid parameters were all significantly higher in diabetic patients with increased CIMT values[11]. As regards Systolic BP it was significantly elevated in group II and III compared to group I, with no significant difference observed between group II and III.Similarly, Perumal et al reported that hypertension had a significant positive correlation with CIMT in T2DM [13]. Consequently, univariate analysis of present work discloses that hyperglycemia, raised systolic BP, increased T-cholesterol and LDL-cholesterol are all risk factors that predispose to the development of atherosclerosis, over prolonged period more than 5 years as perceived in group III. Our results demonstrated significant increase in CHIT1 activity in group II and III compared to group I, with no significant difference between group II and III. Increased activity observed in T2DM patientshad a significant positive relationship with CIMT. It implied that CHIT activity may exhibit a key role in vascular inflammation, generation of Reactive oxygen species, and endothelial dysfunction, triggering the progression of atherosclerosis [14]. CHIT1 has transglycosidase activity, with resultant formation of chitotetraose while chitin being degraded; Chitotetraose acts as signaling molecule that plays an important regulatory role on the immune system [15]. In accordance, CHIT1 activity is increased in patients with atherosclerosis and is correlated with the severity of the disease [16,17]. Furthermore, Artieda et al confirmed that CHIT1 is upregulated in response to inflammatory stimuli, and is responsible for macrophagesactivation and polarization. As macrophages shift toward M1–M2 phenotype; M1

phenotype, CHIT1 becomes implicated in phagocytosis, and induction of the adaptive immune response. While in M2 cells they promote phagocytosis of cellular debris, and coordinate tissue repair through the production of extracellular matrix proteins [18]. Also, Boot et al. established a relationship between increased CHIT1 activity and lipidladen macrophages in atherosclerotic vessel wall [19]. In addition, CHIT1 is expressed during dendritic cell (DC) differentiation and maturation.DCs arelocalized in the intima of atherosclerotic vessels and they are activated by oxidized LDL-cholesterol (oxLDL) in atherosclerotic plaque. Also, intense expression of CHIT1 was identified in mature DCs indicating itsvital role in inflammation [20]. CHIT1 is expressed in neutrophils upon stimulation with interferon (IFN) γ, tumor necrosis factor (TNF)-α [16]. Żurawska-Płaksej et al reported involvement of neutrophilderived chitinasesas novel group of molecules, which may contribute to metabolic derangement and inflammatory pathways in the course of T2DM particularly vascular complications [21]. In this work, elevated CHIT1 activity in diabetic groups was coupled with poor glycemic control as evident by elevated FBG, PPBG levelsas well as HbA1C. In addition, prolonged duration of disease particularly in group III patients may suggest its participation in the course of diabetes. On the contrary, Ramanathan et al. proposed that CHIT1 is a major protein product of activated macrophages, and increase of CHIT1 activity may be generated by natural age-related chronic macrophage activation, not an inflammatory condition [22]. Several miRNAs have emerged as possible regulators of endothelial cell function in diabetic microangiopathy; miR146a is upregulated in endothelial cells upon stimulation by pro-inflammatory mediators, nuclear factor NF-κB [23]. Yang et al found that miR-146a decreases lipid uptake and cytokine release in oxLDL-stimulated macrophages by inhibiting activation of toll-like receptor 4 signaling pathway, suggesting it may have a protective role against the development of atherosclerosis [24] miR-146a gene expression was evaluated in this work; its level was significantly reduced in group III relative to both group II and I. In other words, its level was significantly decreased in group III patients with CIMT level above 1 mm. Reduced miR-146a expression in group III is characteristically considered a warning of a proinflammatory condition. Moreover, results demonstrated an inverse relationship between miR-146a expression and CIMT, while positive relationship between CIMT and Hb A1C and CHIT1 activity. In agreement with our results, Balasubramanyam et al displayed an inverse relationship between miR-146a expression and glycated hemoglobin,

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tumor necrosis factor receptor-associated factor (TRAF)-6, and NF-kB mRNA levels and circulatory levels of TNF-α and IL-6 [25]. Also, Boldin et al supposed that targeted miR146a deletion in mice leads to unwarranted production of proinflammatory cytokines (TNFα, IL-6) [26].

Avogaro A, Albiero M, Menegazzo L, Kreutzenberg SD, Fadini GP. Endothelial dysfunction in diabetes—the role of reparatory mechanisms. Diabetes Care. 2011;34(2):285–90.

Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001; 414:813–20.

In addition, Rong et al reported miR-146a overexpression is caused by increased plasma heme oxygenase-1 concentrations and other anti-oxidant enzymes [27]. Furthermore, downregulation of miR-146a in peripheral blood in diabetic patients in response to hyperglycemic, suggested that it may be implicated in impaired proangiogenic effect and disturbed cellular metabolic control, which could be underlying molecularmechanisms for T2DM pathogenesis [28,29].

Mudau M, Genis A, Lochner A, and Strijdom H. “Endothelial dysfunction: the early predictor of atherosclerosis”. Cardiovascular Journal of Africa. 2012; 23 (4): 222–231.

Di Rosa M, Malaguarnera G, De Gregorio C, Drago F, and Malaguarner L. Evaluation of CHI3L-1 and CHIT-1 Expression in Differentiated and Polarized Macrophages. Inﬂammation. 2013; 36(2):482-492.

Accordingly, over expression of miR-146 in endothelial cells inhibits the inflammatory response prompted by IL-1β and on the contrary inhibition of miR-146a exacerbates the inflammatory response to IL-1β [28]. In addition, miR-146 atargets two serine/threonine kinases, interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), upon activation they become associated with the interleukin-1 receptor (IL-1R) and are therefore responsible for IL-1-induced upregulation of NF-kB. This binding results in suppression of NF-κB’s target genes; such as the interleukins IL-6, IL-8, IL-1β, and TNF alpha (TNF-α) [29, 30].

Sheetz MJ, King GL.Molecular understanding of hyperglycemia’s adverse effects for diabetic complications. JAMA2002; 288: 2579–2588.

Nomura K, Hamamoto Y, Takahara S, Kikuchi O, Honjo S, Ikeda H, Wada Y, Nabe K, Okumra R, Koshiyama H: Relationship between carotid intima media thickness and silent cerebral infarction in Japanese subjects with type 2 diabetes. Diabetes Care 2010;33(1):168–70.

Gayathri R, Chandni R, Udayabhaskaran V. Carotid artery intima media thickness in relation with atherosclerotic risk factors in patients with type 2 diabetes mellitus. J Assoc Physicians India 2012;60:20–4.

Butt MU, Zakaria M. Association of common carotid intimal medial thickness (CCA-IMT) with risk factors of atherosclerosis in patients with type 2 diabetes mellitus. J Pak Med Assoc. 2009;59(9):590-593.

Conclusion

10. Sibal L, Agarwal SC, Home PD. Carotid intima-media thickness as a surrogate marker of cardiovascular disease in diabetes. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy. 2011; 4:23-34.

On the basis of these significant alterations in miR-146a and CHIT1 activity in group III patients with increased CIMT values, it is probable to conclude that as T2DM progresses; metabolic disturbances develop allowing for activation of the inflammatory response in those patients. Data obtained indicate potential role of CHIT1 enzyme in prediction and diagnosis of endothelial dysfunction and its increased activity may be due to the change in the relative gene expression of miR-146aas displayed by increased CIMT. However, further studies are needed to establish the diagnostic value (specificity and sensitivity) of both biomarkers in early detection of atherosclerosis in diabetic patients.

Acknowledgements

We kindly appreciate the support of Professor Dr Aml S Bendary (Prof of Clinical Pathology at Faculty of Medicine Tanta University) in revising it critically for important intellectual content.

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*Corresponding author: Hala Demerdash, 75 Ismail Serry Street, Smouha-Sidi Gaber District-Zip code 21523, Alexandria, Egypt. Phone: +2034258618, +201229501257 Email: demerdashh@yahoo.com

Financial or other Competing Interests: None.

Date of Submission : 10.02.2017 Date of Acceptance : 01.04.2017 Date of Publication : 04.07.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Original Article DOI: 10.21276/APALM.1174

A Study of Histological Types of Leprosy Along with Clinico-Histopathological Correlation in a Tertiary Centre from North Maharashtra Region Maya S Vasikar*, Bharati M. Patil. and Rajesh Y. Thakur Dept of Pathology, Shri Bhausaheb Hire Govt. medical College, Dhule, Maharashtra, India

ABSTRACT Background: To achieve WHO new global strategy of 2016-, To decrease the case load of leprosy, early and proper classification of leprosy for proper treatment and avoiding disability due to leprosy. Aim- Aim of this study was to find out the incidence of various histological types of leprosy in North Maharashtra. To find out which is the common type and to know the importance of histopathological examination in diagnosis of leprosy and its correlation clinically. Methods: A retrospective study of five years from Jan 2011 to Dec 2015 was conducted in the department of Pathology Government Medical College Dhule. Clinical diagnosis was correlated with histopathological report. Results: Sixty seven clinically diagnosed cases were included in the study. From sixty seven cases two cases on histopathological and on AFB (Fite Ferraco) stain were negative for leprosy. The Male to Female ratio was 0.8:1, maximum cases belonged to age group 21-30 years, and clinically hypo-pigmented hypo aesthetic macules were commonly seen. Clinically as well as histopathological Borderline Tuberculoid leprosy was common, it also showed maximum parity. A case of Histoid leprosy was too diagnosed on HPE which clinically was diagnosed as lepromatous leprosy. Erythema nodosum leprosum two cases were diagnosed clinically as well as histopathological in this period. Fite Farraco stain was positive in all cases of borderline lepromatous and lepromatous leprosy. Conclusion: A combination of clinical diagnosis, histopathological examination along with AFB stain is essential for proper treatment of the patient and for achieving leprosy free world. Keywords: Leprosy, Macules, Histopathology, Fite Farraco Stain.

Introduction

Leprosy is a chronic infectious disease caused by Mycobacterium Leprae. It can affect any age and both sex. Leprosy was a major public health problem of India in the last century. Hence in 1955 the leprosy control programme was initiated with single drug Dapsone. In 1982 it was switched to multidrug therapy following the recommendation of WHO [1].There was dramatic decline in number of leprosy patient in the world. In 1993, the National leprosy Elimination programme (NLEP) was initiated by WHO. The goal of the programme was to decrease the prevalence rate of leprosy below 1 case/10,000 population. India has achieved this goal in December 2005 by achieving the prevalence rate of leprosy 0.95 cases/10,000 populations [2]. In March 2006 it is further declined to 0.84/10,000 population [3]. The focus of WHO was to eliminate leprosy at global level by the end of 2000 year. In 2015 a total of 2, 10758 new leprosy cases were reported from 106 countries in all WHO regions, representing a 21% drop from the number of cases

reported in 2005[4]. India has 60% of the entire global case load, followed by Brazil and Indonesia [4]. In 2016 WHO has launched a new global strategy. The global leprosy strategy 2016-2020:â&#x20AC;?Accelerates towards a leprosy free world.â&#x20AC;? It aims to reinvigorate efforts for leprosy control and to avoid disability, especially among children affected by the disease in endemic countries [5]. To achieve WHO new global strategy and to decrease the global case load, it is essential to have an early, proper diagnosis, by clinical and, histopathological correlation, so a complete treatment according to the type of leprosy can be given. Hence the present study was undertaken to know which type of leprosy is common in North Maharashtra and clinico-histopathological correlation was done to avoid mislabelling of a case. Thus it will help in avoiding disability as well as grievance to the patient and to the society.

Material and Methods

A retrospective study of five years from January 2011 to December 2015 was conducted in our department. We

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received sixty seven skin biopsies of clinically diagnosed leprosy cases in the five year study span. The biopsy specimens were processed as per standard procedure, sections were stained with haematoxylin-eosin and FiteFaraco stain was done for demonstration of acid fast bacilli. Ridley-Jopling classification was used to classify leprosy. The histopathology slides along with its AFB (Fite Farraco) stain were reviewed. Clinical data and clinical diagnosis done by dermatologist were retrieved from the histopathological requisition form.

On histopathological examination, the most common histological type was borderline Tuberculoid (BT) leprosy35 %( fig 1), followed by borderline lepromatous (BL) 20.8%, Tuberculoid (TT) leprosy 13%, lepromatous (LL) leprosy 11.9 %( fig 2), indeterminate leprosy (IL) 10.4%. From sixty seven cases, two cases diagnosed as indeterminate leprosy and Borderline Tuberculoid leprosy, on histopathological showed no evidence of leprosy. They were diagnosed as perivascular dermatitis Fite Farraco stain for demonstration of Acid fast lepra bacilli was done in all cases. It was positive in all the histopathological diagnosed BL and LL types of leprosy cases (fig 3). It was negative in IL, TT and 9 cases of BT leprosy.

Results

The study shows that 30(44.7%) were male, while 37(56.9%) were female. The male to female ratio was 0.8:1. The majority of patient belonged to 21-30 years (26.8%), followed by 11-20 years (25.4%). The age group of patient ranged from 9 years to 83 years. Clinically the most common presentation was hypo-aesthetic, hypo-pigmented macules 65%, followed by plaque 30%, nodules 3%, Erythematous lesion 2%.The highest number of cases clinically diagnosed were borderline Tuberculoid (BT)56.7%,followed by borderline lepromatous(BL)13.4%,lepromatous leprosy(LL)11.9%,indetermine leprosy(IL) and Tuberculoid leprosy(TT) both 7.4% and two cases of erythema nodusum leprosum2.9%. Table 1: Clinico- histopathological co-relation. HPE TYPE NO.of cases IL IL 7 2 BT 24 1 TT 9 1 BL 14 LL 8 Histoid 1 ENL 2 Chr infl 2 1 TOTAL 67 5

BT 5 22 4 6

1 38

Fig. 1: Borderline Tuberculoid Leprosy (400X H&E)showing granuloma of epithelioid cells and lymphocytes in dermis.

Analysis of clinical diagnosis & histopathological types was done (Table1). Maximum parity of 91% was seen in borderline Tuberculoid leprosy, followed by lepromatous leprosy 62%. ). Two cases of Erythematous nodosum leprosum were diagnosed clinically and they were confirmed by HPE. Both the cases were of lepromatous leprosy, on treatment. A case of lepromatous leprosy was diagnosed as Histoid leprosy on histopathology (fig 4). The patient was a male aged 42 years. Histoid leprosy showed bundles of acid fast bacilli in spindle shaped cells. TT 1 4

6 3

2 5 1

ENL

Parity 28% 91% 44% 42% 62%

100%

Fig. 2: Lepromatous Leprosy, early stage (400X H&E)Thinning of epidermis, macrophages with pink, granular cytoplasm in dermis.

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Fig. 3: Fite Farraco stain : positive for Acid fast bacilli (100X) in fig 2 case.

Fig. 4: Histoid Leprosy- interlacing bands of spindle shaped cells.

Discussion

with IL type of leprosy with hypopigmented patch on face in whom sensory deficit is difficult to illicit [9].

Leprosy, Hansenâ&#x20AC;&#x2122;s disease is an infectious and highly curable disease. In the present study, the male to female ratio was 0.8:1. A slight increase in female preponderance was seen. While male preponderance was seen in studies conducted by M Giridhar (2012) [6], Moorthy(2001)[7]. The explanation provided was inhibition on the part of females for reporting and treatment due to social taboos and stigma [6, 7]. In our setting we can say that as years passed health awareness in females has increased due to mass media. Maximum cases (26.8%) belonged to young, active age group 21 to 30 years. Majority of the studies showed maximum cases in the same age group (41%-23.5%) [6, 7, 8, 9] .Least numbers of cases (2.9%) were below the age of 10 years in present study. This is explained due to of longer incubation period of lepra bacilli [10] .The eldest case in our study was 84 years female while 9 year boy was the youngest case. Again explaining that increased awareness in mother resulting in early diagnosis in a 9 year old boy. Hypo pigmented, hypoaesthetic macules were the commonest clinical lesions (65%) seen in our study, and in these lesions, features of IL, BT, TT leprosy were frequently found. While in plaque (30%), nodules (2%), features of BL, LL, and Histoid leprosy were seen. Similar result was obtained in a study by Vargas Ocampo and Francisco 2004[11], Shivmurthy V 2013[12]. Two cases clinically diagnosed as IL and BT leprosy, on histopathological examination were diagnosed as perivascular dermatitis. Similar finding was seen by Manander U et al [13]. While M Giridhar et al[6] diagnosed Lupus Vulgaris and vitiligo in two clinical diagnosed cases of leprosy. The discrepancy is explained due to misinterpretation and over diagnosis of hpopigmented macule as leprosy. Misdiagnosis is common in children www.pacificejournals.com/apalm

BT was most common clinical (56.7%) and histopathological (35%) type of leprosy in our study. Similar findings were seen in other studies too [12, 13, 14,]. The clinical and histopathological correlation was seen in 41 cases (61%) in our study. While Kumar et al[15] found 60.6% in the year 2000, Pandya[16] et al found 58% in the year 2008, Mathur[17] et al found 80.4% in 2011, Giridhar[6] et al found 60.23% in 2012, Rizvi[9] et al found 70% in the year 2015, Banushree C S[18] found highest 79.44% in the year 2016. In our study maximum parity was seen in BT (91%), followed by LL (62%), which co-related with Manandhar u et al [13]. While B Mehta et al[8], Moorthy et al[7], Banushree et al[18] found maximum parity in lepromatous leprosy. While Alia Rizvi[9] et al found maximum parity in Tuberculoid leprosy. Indicating maximum parity in polar type of leprosy. One case of TT and one case of BT leprosy showed histological features of indeterminate leprosy. Similar findings were noted by other authors too [13, 19, 20].Five cases of BL leprosy were classified as BT leprosy. Bhatia A S[19] et al and Sehgal V N[20] also noted similar findings. Histoid leprosy is considered as a variant of lepromatous leprosy. It shows male preponderance and the average age of diagnosis is between 21 and 40 years [21]. Our case was too clinically diagnosed as lepromatous leprosy, the patient was a male aged 42 years and showed nodular presentation. Two cases of Erythema Nodosum leprosum were clinically and histopathological diagnosed. Both the cases were of Lepromatous leprosy on treatment. The end of National leprosy eradication programme (NLEP) in India indicates the elimination of leprosy as a public health problem. But eISSN: 2349-6983; pISSN: 2394-6466

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numbers of newly diagnosed leprosy cases are increasing in private and teaching hospitals [16, 17].

Conclusion

Though clinical diagnosis of types of leprosy can be done, to avoid disparity and for proper treatment histopathological examination and Fite Farraco stain is essential. Thus a definitive diagnosis and proper treatment will help us to achieve the WHO global leprosy strategy 2016-2020: Accelerating towards leprosy free world.

References 1.

Rao P N: Recent advances in the control programs and therapy of leprosy. Indian J Dermatology Venerology Leprology.2004; 70: 269-76.

Special Correspondent, India achieves leprosy eradication target. The Hindu News paper 2005, Jan 31, p 15.

National leprosy eradication programme-Progress report for the year 2006, central leprosy division. Directorate General of health services, Nirbhan Bhavan, New Delhi; 2006.

WHO Global leprosy update on the 2014 situation. Weekly epidemiology record, 2014; 36:389-400.

Global leprosy programme. Global leprosy strategy 20162020; 2016: 7.

Giridhar M, Arora G. Clinico-histopathological concordance in leprosy: A Clinical, Histopathological and Bacteriological study in 100 cases. Indian Journal leprosy.2012; 84:217-225.

Moorthy BN, Kumar P, Chatura KR, Chandrasekhar HR, Basavaraja PK. Histopathological correlation of skin biopsies in leprosy. Indian journal of Dermatology Venerology Leprology.2001; 67:299-301.

8. Mehta B, Desai N, Khar S. Clinico-pathological co-relation in leprosy. International journal of dermatology.2012; 9(1):1-5. 9.

Rizvi AA, Sharma YK, Dash K et al: An epidemiology and clinico-histopathological study of leprosy in semi urban area under Pimpri Chinchwad Municipal Corporation in Pune district Maharashtra. Medical journal of Dr D Y Patil university.2015; 8(5):609-613.

10. Fine PE. Leprosy: the epidemiology of a slow bacterium. Epidemiology Rev. 1982; 4: 161-188. 11. Vargas-Ocampo and Francisco. Analysis of 6000 skin biopsies of the National leprosy control Programme in Mexico. Int J Lepra-other Mycobact Dis 2004 59;28-35. 12. Shivamurthy V, Gurubasavraj H, Sastry SP, Kumar P: Histomorphological study of leprosy. Afr J Medical health science.2013;12: 68-73. 13. Manandhar U, Adhikari RC, Sayami G: Clinicohistopathological correlation of skin biopsies in leprosy. Journal of Pathology Nepal.2013; 3:452-8. 14. Tiwari M, Ranabhat S: Clinico-histopathological correlation of leprosy. A retrospective study of skin biopsy specimens in Chitwan Medical Science Research and Practise 2015, 2 (1): 8-11. 15. Kumar A, Negi SR, Vaishnav K. A study of clinichistopathological correlation of leprosy in a tertiary care hospital in western district of Rajasthan. J Res Med Den Sci 2014; 2:43-8. 16. Pandya AN, Tailor H J. Clinico histopathological correlation of leprosy. Indian J Dermatology Venerology Leprology 2008; 74:174-6. 17. Mathur MC, Ghimre RBK, Shrestha P, Keda SK. Clinicopathological correlation in leprosy. Kathmandu Univ Med J 2011; 9: 248-51. 18. Banushree CS, Bhat RV. Clinicopathological correlation of Hansenâ&#x20AC;&#x2122;s disease, a retrospective study of skin biopsies. Indian Journal of Pathology and Oncology, 2016; 3(3): 491-495. 19. Bhatia AS, Katach K, Naryanan R B. Clinical and histopathological correlation of leprosy. Int J Lepra-other Mycobact Dis 1977; 45:278-80. 20. Sehgal VN, Sardana K, Dogra S: The imperatives of leprosy treatment in the pre and post global leprosy elimination era: appraisal of changing the scenario to current status. J Dermatology treatment. 2008; 19: 82-91. 21. Annigeri SR, Metgud SC, Patel JR: Lepromatous leprosy of Histoid type: A case report Indian Journal Medical Microbiology. 2007; 25.

*Corresponding author: Dr Maya Vasaikar, Jaihind college Road, Behind Modern Nursery, opposite DR. Morey hospital. Deopur, Dhule, 424002, Maharashtra, India. Phone: +91 8806052553 Email: mayavasaikar@yahoo.co.in Date of Submission : 21.11.2016 Date of Acceptance : 05.04.2017 Financial or other Competing Interests: None. Date of Publication : 05.07.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Original Article DOI: 10.21276/APALM.1175

Study of Histomorphological Characteristics and it’s Correlation with Clinical, Biochemical, Serological and Immunohistochemical Parameters in Incidentally Detected Hepatitis B Patients Rakesh Kumar Gupta1, Puja Sakhuja2*, Shahajad Ali2, Sidharth Srivastava3, Barjesh Chand Sharma3 and Amarender Singh Puri3 1 Department of Pathology, Chattisgarh Institute of Medical Sciences, Bilaspur, Chattisgarh, India. Departments of Pathology Govind Ballabh Pant Institute of Postgraduate Medical Education and Reseach, New Delhi, India. 3 Departments of Gastro-enterology Govind Ballabh Pant Institute of Postgraduate Medical Education and Reseach, New Delhi, India. 2

ABSTRACT Background: India lies in intermediate endemicity zone for hepatitis B virus (HBV) infection and constitutes the second largest global pool of HBV infection worldwide. Hepatitis B has a varied clinical presentation ranging from clinically asymptomatic state to cirrhosis and hepatocellular carcinoma. A significant liver injury can occur, without accompanying elevation in alanine transaminase (ALT) and HBV DNA levels, especially in incidentally detected asymptomatic hepatitis B subjects (IDAHS). Hence, a role of liver biopsy to be incorporated with other investigations is debatable, but important to initiate antiviral therapy. We explored correlation between histomorphological outcomes with various clinical, biochemical, serological and immunohistochemical parameters in IDAHS. Methods and Material: Total 113 patients were consecutively selected over a period of 4.5 years. Serological work-up for HBsAg, AntiHBeAg, Anti-HBeAb, Anti-HBcAb, and HBV DNA levels were done as per resources. A liver biopsy was done in each patient after a written consent. Ishak’s scoring system was used to assess histological parameters. Immunohistochemistry (IHC) was done for HBsAg and HBcAg. Appropriate statistical tests were applied. Results: The mean age of the patients was 30 years with a male to female ratio of 3:1. A higher necro-inflammatory activity (NIA >3) correlated with high ALT (>40 U/l), HBV DNA (>105 copies/ml) and fibrosis (F ≥2). HBeAg-positive patients had significantly higher NIA and HBV DNA levels. Anti-HBeAb delineated association with ALT (≤40 U/l) and low HBV DNA but more severe fibrosis (F≥2). Steatotic changes were noted in 52.2% biopsies. IHC for HBsAg and HBeAg showed positivity in 82.7% and 39.2% of cases respectively with a significant correlation between membranous pattern of HBsAg staining and serum HBV DNA levels. Conclusion: IDAHS represent tip of the iceberg of major HBV infection reservoir. A liver biopsy is a useful additional tool with other parameters to further tailor the therapy in such asymptomatic patients. Keywords: Hepatitis B Virus, Hepatitis B Surface Antigen, Hepatitis B e Antigen, Viral DNA, Alanine Transaminase.

Introduction

Worldwide prevalence of hepatitis B virus (HBV) varies significantly among different population. In India, prevalence of hepatitis B surface antigen (HBsAg) in general population ranges from 2% to 7%, which lies in intermediate endemicity zone.[1] Of the estimated 360 million worldwide chronic carrier, India accounts for about 50 million, forming the second largest global pool of chronic HBV infections.[2] In India transmission is mostly acquired through childhood horizontal spread due to sub-optimal hygiene and crowded living conditions and perinatal transmission of infection from mother to infants is not an important route.[1] Hepatitis B disease has a varied clinical presentation ranging from clinically asymptomatic

state to the development of cirrhosis and hepatocellular carcinoma depending upon the phase. Patient may present (a) in a state of immune tolerance, (b) HBeAg-positive chronic HBV, (c) HBeAg-negative chronic HBV, or (d) as an inactive HBsAg carrier.[3, 4] Chronic HBV (both HBeAgpositive and HBeAg-negative) patients have a relatively higher chance of developing various complications.[5] All major liver organizations, such as the American association for the study of liver diseases (AASLD), the European association for the study of the liver (EASL), and the Asian-Pacific association for the study of the liver (APASL), consistently recommend therapy for patients with liver damage and complications.[6, 7] However, these recommendations for treatment of chronic hepatitis B

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patients are not always appropriate to apply in most of the developing countries.[8] Although incidentally-detected asymptomatic HBsAgpositive subjects (IDAHS) appear healthy at the time of presentation, varying proportions have evidence of liver disease on biopsy.[9] Though ALT, HBV DNA levels and HBeAg status determination serve as important means to predict the extent of disease in these patients, nevertheless a liver biopsy plays an important role to ascertain amount of injury and also decision making to plan antiviral treatment.[10-12] The limited number of liver biopsies in IDAHS patients, more so in developing countries and the scarce literature on correlation between various histological indices with serological and viral markers provided us insight for this study. We started this study with an aim to correlate various measurable histological events with serological and viral parameters in IDAHS patients to guide clinicians in deciding appropriate antiviral therapy.

Methods and Materials

This paper is approved by departmental review board. Total 137 consecutive patients presented in the liver clinic of Gatroenterology outpatient department of the institute over a period of 4.5 years from January, 2010 to June, 2014 were included in the study. All patients were apparently healthy without any features of liver-related diseases during their first visit to the hospital. These patients came to the hospital for a routine check-up before foreign trip (as asked by embassy officials for visa), during pregnancy, after household contacts, health personnel after accidental exposure, referred from blood bank etc. All the patients who found to be HBsAg positive were asked to follow different public health measures. Also, patients were requested to seek HBsAg testing after 6 months and to visit in case of any illness. When HBsAg was detected 6 months after the first test, the patients were regarded as being chronically infected with HBV, and enrolled. Patients with the past history of chronic liver disease or decompensation of liver function, alcohol consumption more than 20 g/day, hepatotoxic drug, any systemic illnesses such as diabetes mellitus, and those having coinfection with HIV or hepatitis C virus were excluded from the study. Physical parameters such as body mass index and waist hip ratio was measured in each patient at the time of registration. Ultrasonography report, if available was also included. Biochemical and Serological Tests: Routine haemogram, lipid profile, serum insulin, fasting blood sugar and liver

function tests were done on all patients. The cutoff for the upper limit of normal (ULN) was ALT > 40 U/l. HBsAg was assessed using a commercial ELISA kit (Diasorin, Fallugia, Italy). HBeAg and anti-HBe antibody were tested using an ELISA kit (Abbott Labs, Chicago, Ill., USA). Serum HBV DNA was quantified using an RT-PCR kit (Amplicon HBV Monitor Assay, Roche Molecular Systems, Calif.,USA). The lower limit of detection was 250 copies of HBV DNA/ml. A level of >105 copies/ml was considered as active/replicative infection. Liver biopsy: A prior written informed consent was taken from all the patients before liver biopsy. Percutaneous liver biopsies from all the patients were obtained under local anesthesia using 16G Tru-Cut needles (Cardinal Health, McGaw Park, Ill, USA). The liver biopsies at least 1 cm long with ≥6 portal triads were included. The liver biopsies were fixed in 10% buffered formalin and four micron sections were stained with hematoxylin and eosin (H&E), masson’s trichrome and orcein stains. Necro-inflammatory activity (NIA) and fibrosis stage were assessed using the Ishak’s scoring system. Extent of hepatitis was graded as mild (NIA≤3), and significant (NIA≥4), while fibrosis was staged as follows: F0, no fibrosis; F1, mild portal fibrosis with/without septa; F2, marked portal fibrosis with/without septa; F3, portal fibrosis with occasional P-P bridging septa; F4, portal fibrosis with numerous bridging septa; F5, marked bridging fibrosis with occasional nodules; F6, cirrhosis. Immunohistochemistry (IHC) was performed on 4µ thick formalin fixed, paraffin-embedded sections using antibodies directed against HBsAg (Neomarker) and HBcAg (Neomarker). Antigen retrieval for HBsAg was done in a microwave oven using citrate buffer at pH 6.0 and streptavidin biotin conjugate immunoperoxidase method was used. For each batch, appropriate positive controls were taken and for negative controls primary antibodies were omitted. Statistical Analyses: Data were analyzed using SPSS version 17 software package (SPSS, Inc., Chicago, IL). Statistical analyses were performed using Chi square and Fisher exact tests for categorical variables. Student t test or 1-way analysis of variance was used for group comparisons of parametric quantitative data. Differences were considered significant at p< 0.05.

Results

Baseline characteristics of patients (Table 1). Of the 137 patients, 113 were found eligible after putting exclusion criteria’s. The mean age of the patient was 30 years; 84 (74.3%) were male and the other 29 (25.7%) were female. HBeAg status did not revealed significant correlation with age. The median level of serum ALT was 47 U/l (range 11–

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790 U/l). ALT was higher than the cut off for the ULN in 65.6% subjects. Of the 113 patients, liver biopsies showed variable amount of portal inflammation in all, ground glass hepatocytic changes in 74 (65.4%), and lobular inflammation in 92 (81.4%) (Table 2). Both ground glass changes with concurrent lobular inflammation were noted in 57 (50.4%) patients. Interface activity, focal lymphoid aggregate and ductal injury was noted in 8 (7%), 13 (11.5%) and 7 (6.1%) biopsies respectively. Abdominal ultra-sonography reports were available in 24 patients, 5 (20.8%) of which showed abnormal echotexture. High NIA delineated significant correlation with high ALT levels, HBV DNA and fibrosis (Table 3). HBeAg status was known in 90 patients, of which 43 (47.7%) were HBeAg-positive. Anti-HBe antibody (Anti-HBeAb) levels were investigated in 58 patients, 30 (51.7%) of which showed positivity (Table 4). Anti-HBeAb positivity was more common in patients with low ALT (≤40 U/l) and HBV DNA levels (<105 copies/ml) but more severe fibrosis (F≥2). Anti HBc antibody (Anti-HbCAb) was measured only in 17 patients, 12 (70.5%) of which were positive.

Comparatively higher HBV DNA levels were noted in HBeAg-positive against HBeAg-negative patients (71.4 vs. 28.6%, p < 0.0001) (Table 5). Similarly, a higher NIA was seen among HBeAg-positive patients. Also, in HBeAg-positive group, 21 (48.8%) patients had both higher serum ALT and HBV DNA levels, in comparison to HBeAg-negative group, 6 (12.8%) and the difference was statistically significant (p value <0.0001). Steatotic changes were noted in 59/113 (52.2%) patients, which varied considerably ranging from 5% to 85% of the hepatocytes with a meadian of 10%. Steatosis amount and pattern did not showed differences with HBeAg status. IHC for HBsAg and HBcAg showed positivity in 67/81(82.7%) and 33/84 (39.2%) of the biopsies respectively (Figure 1). Both the antibodies showed three pattern of the staining including cluster, diffuse and scattered. In addition to cytoplasmic positivity, HBsAg also showed membranous staining in 24/67 (35.8%) of the biopsies. HBcAg revealed both nuclear and cytoplasmic positivity in 10/33 (39.2%) of the cases. The membranous pattern of HBsAg showed a positive correlation with the serum levels of HBV DNA (p=0.018).

Table 1: Baseline characteristics of the hepatitis B patients Parameters

HBeAg

Anti-HBeAb

Anti-HBcAb

ALT levels

HBV DNA levels

Ultrasound

Values No of patients

113

Age, years

29.9±11.9

Male

74.3%

Female

25.7% positive

47.8%

negative

52.2%

positive

51.7%

negative

48.3%

positive

70.6%

negative

29.4%

Normal

34.4%

Raised

65.6%

≤105 (copies/ml)

41.2%

>105 (copies/ml)

58.8%

Normal echotexture

79.2%

Coarse echotexture

20.8%

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Table 2: Histopathological characteristics, Ishak’s staging and grading and immunohistochemistry (HBsAg & HBcAg) of the liver biopsies in the hepatitis B patients. Parameters

Values Portal inflammation

100%

Ground glass changes

65.5%

Lobular inflammation

81.4%

Interface activity

7.1%

Lymphoid aggregate

11.5%

Duct injury

6.2%

NIA score Fibrosis stage

Steatosis (%) (n=59)

1-3

51.9%

48.1%

0-1

65.9%

≥2

34.1%

1-9%

37.3%

10-30%

44.0%

>30%

18.7%

HBsAg positive (Total/C/C+M/M)

82.7%/64.1%/32.8%/2.9%

HBcAg positive (Total/N/C+N)

39.2%/69.6%/30.3%

‘NIA’- necro-inflammatory activity, ‘C’- cytoplasmic, ‘M’- membranous, ‘N’- nuclear Table 3: Correlation of NIA with ALT levels, serum HBV DNA and fibrosis. Parameters

NIA (≤3)

NIA (≥4)

P value

ALT (≥1ULN)

54.1%

78.4%

0.0001

HBV DNA (≥10 copies/ml)

44.1%

66.7%

0.04

HBeAg+

22.9%

71.9%

0.0001

Fibrosis stage (High ≥2)

10.7%

65.8%

0.0001

Anti-HBeAb+

70.0%

45.5%

0.09

Table 4: Comparison of histological, serological and virological parameters between HBeAg-positive and HBeAg-negative patients. Parameters

HBeAg-positive

HBeAg-negative

Normal

28.9%

46.7%

No of patients (n=90) ALT levels HBV DNA levels

Raised

71.1%

53.3%

≤105 (copies/ml)

14.6%

66.7%

>10 (copies/ml)

85.4%

33.3%

1-3

32.3%

75%

67.7%

25%

0-1

60.0%

73.0%

≥2

40.0%

27.0%

1-9%

74.4%

63.8%

10-30%

23.3%

23.4%

>30%

2.3%

12.8%

Positive

29.6%

80.0%

Negative

70.4%

20.0%

NIA score Fibrosis stage Steatosis (%) (n=46) Anti-HBeAb

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P value 0.98 0.0001 0.0001 0.26 0.17

0.0001

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Table 5: Comparison of histological, serological and virological parameters between AntiHBeAb-positive and AntiHBeAbnegative patients Parameters

AntiHBeAb positive

AntiHBeAb negative

Normal

48.3%

18.5%

Raised

51.7%

81.5%

≤105 (copies/ml)

63.0%

7.1%

>10 (copies/ml)

37.0%

92.9%

1-3

58.3%

33.3%

41.7%

66.7%

0-1

56.0%

89.5%

≥2

44.0%

10.5%

No of patients (n=58) ALT levels HBV DNA levels NIA score Fibrosis stage Steatosis (%) (n=29) HBeAg

1-9%

46.7%

35.7%

10-30%

46.7%

50.0%

>30%

6.7%

14.3%

Positive

28.6%

79.2%

Negative

71.4%

20.8%

P value

0.01 0.0001 0.10 0.01

0.72

0.0001

Fig. 1: Photomicrographs showing cytoplasmic and membranous immunopositivity for HBsAg (A) and nuclear & cytoplasmic positivity for HBcAg (B) (X 200 each).

Discussion

India is the second largest global pool of chronic hepatitis B (CHB) carriers. Because of socio-economic hurdles, millions of these patients are unaware of HBV infection and serve as an important source of reservoir. There are no effective screening system and treatment guidelines for these patients. Since these patients are asymptomatic, they do not seek medical attention till complications develop. We endeavor to find significance of each histological change in the liver biopsy including portal inflammation, ground glass change, lobular inflammation, lymphoid aggregate, duct injury and steatosis separately. We found www.pacificejournals.com/apalm

ground glass change in ~65% of the biopsies which is a characteristic feature in CHB. Interestingly, in this study, we found steatosis in 52% (n=59) of the biopsies, out of which 62% had steatosis ≥10% and 11 patients among them with >30% steatosis, which is more characteristic of chronic hepatitis C (CHC). However, no significant association was found between extent of steatosis and NIA. The higher positivity rate in liver biopsies for HBsAg (82.7%) than for HBcAg (39.2%), explain selective secretion of HBsAg and, inactivation of more immunogenic portions like core genes during the viral genome integration, thus evading immunologic attack and elimination.[13] eISSN: 2349-6983; pISSN: 2394-6466

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Role of Liver Biopsy in Incidentally Detected Hepatitis B Patients

Mukhopadhya et al described scattered, cluster and sheet patterns of staining both for HBsAg and HBcAg, which were also seen in this study.[14] Earlier, membranous staining of HBsAg has been reported to be associated with active viral replication and disease activity.[15] We found a significant positive correlation of membranous HBsAg staining with higher serum levels of HBV DNA (p=0.018). In our study, 21 (18.5%) patient had high ALT (above ULN), along with HBV DNA (>105) and NIA (≥4) which is higher than 8.5% described in study by Mahtab et al.[12] This is the first study which explored the role of other immunological factors such as Anti-HBeAb and AntiHBcAb in the pathogenesis of CHB. Anti-HBeAb showed a positive correlation with normal ALT and low HBV DNA levels (<105 copies/ml) but with distinctly higher fibrosis stage (F ≥2) (p <0.016). Viral load, HBeAg status, ALT levels and NIA are the important indicators of liver damage and serve to guide treatment.[6, 7] In this study, we found a good correlation between these parameters. The extent of liver injury however does not always correlate with the ALT and HBV DNA levels.[16] Also, since there is a complex interplay between HBV, hepatocytes and host immune system, these markers fluctuate markedly in the course of disease.[12] ALT levels are also influenced by body mass index (BMI), metabolism and time of measurement.[17, 18] Earlier, studies supported that even in immunotolerant phase and with relatively normal ALT levels significant liver fibrosis and necro-inflammatory changes can occur.[19, 20] Recently, Kumar et al showed that IDAHS display variable amount of liver injury with normal level of biochemical (ALT), virological (HBV DNA) and immunological (HBeAg) markers.[11] In India and other developing nations, HBV infection mostly acquired in early childhood, reflecting a longer period of insult to hepatocytes in comparison to developed countries, where most of the HBV infections trigger in adult life. Early onset of infection, itself explain more likelihood of severe liver injury than western population. So, EASL, and AASLD recommendations, based on western population with main consideration of biochemical and virological factors to commence antiviral therapy, should not be extrapolated in the developing world. About 10% patients with HBV DNA levels <105 copies/ml showed significant fibrosis (F≥2). Earlier, also patients with <105 copies/ml have shown to carry risk for progression to cirrhosis or hepatocellular carcinoma.[21, 22] So, the cut-off of >105 copies/ml for HBV DNA levels may not be justified in many patients.

So, we encourage and support EASL & Lok and McMahon recommended treatment guidelines based on serum ALT levels, HBV DNA levels, and HBeAg status but a liver biopsy can be corroborated to further tailor the therapy. Rather, a personalized approach, considering regional, demographic, racial and socio-economic factors, may benefit maximum in these patients. Also, there is a need to sensitize healthcare cadre and governments towards the epidemic level of HBV infection in developing countries, and develop a global system of screening programme to curtail the dreadful further spread of this infection.

Acknowledgment:

We would like to thanks Dr Tanu Anand for her support in statistical analysis.

References: 1.

Elizabeth W. Hwang, Ramsey Cheung. Global Epidemiology of Hepatitis B Virus (HBV) Infection. N A J Med Sci. 2011;4(1):7-13.

Datta S. An overview of molecular epidemiology of hepatitis B virus (HBV) in India. Virol J. 2008;5:156.

Ganem D, Prince AM. Hepatitis B virus infection-natural history and clinical consequences. N Engl J Med. 2004;350(11):1118-29.

Fattovich G, Bortolotti F, Donato F. Natural history of chronic hepatitis B: special emphasis on disease progression and prognostic factors. J Hepatol. 2008;48(2):335-52.

Keeffe EB, Dieterich DT, Han SH et al. A treatment algorithm for the management of chronic hepatitis B virus infection in the United States: 2008 update. Clin Gastroenterol Hepatol. 2008;6(12):1315-41.

Lok AS, McMahon BJ. Chronic hepatitis B: update 2009. Hepatology. 2009;50(3):661-2.

Liaw YF, Leung N, Kao JH et al. Chronic Hepatitis B Guideline Working Party of the Asian-Pacific Association for the Study of the Liver. Asian-Pacific consensus statement on the management of chronic hepatitis B: a 2008 update. Hepatol Int. 2008;2(3):263-83.

Akbar SM, Hiasa Y, Mishiro S, Onji M. Treatment of hepatitis B virus-infected patients: utility of therapeutic recommendations in developing countries. Expert Opin Pharmacother. 2009;10(10):1605-14.

Chandra R, Kapoor D, Agarwal SR, Malhotra V, Sakhuja P, Sarin SK. Profile of asymptomatic chronic HBV infection in India. Indian J Med Res. 2002;116:50-7.

10. Papatheodoridis GV, Manesis EK, Manolakopoulos S et al. Is there a meaningful serum hepatitis B virus DNA cutoff level for therapeutic decisions in hepatitis B e antigennegative chronic hepatitis B virus infection? Hepatology. 2008;48(5):1451-9. 11. Kumar M, Sarin SK, Hissar S et al. Virologic and histologic features of chronic hepatitis B virus-infected asymptomatic

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Gupta et al. patients with persistently normal ALT. Gastroenterology. 2008;134(5):1376-84. 12. Al-Mahtab M, Rahman S, Akbar SM, Kamal M, Khan MS. Clinical use of liver biopsy for the diagnosis and management of inactive and asymptomatic hepatitis B virus carriers in Bangladesh. J Med Virol. 2010;82(8):1350-4. 13. Thomas HC. Immunological mechanisms in chronic hepatitis B infection. Hepatology. 1982;345:886-89. 14. Mukhopadhya A, Ramakrishna B, Richard V, Padankatti R, Eapen CE, Chandy GM. Liver histology and immunohistochemical findings in asymptomatic Indians with incidental detection of hepatitis B virus infection. Indian J Gastroenterol. 2006 ;25(3):128-31. 15. Chu CM, Liaw YF. Membrane staining for hepatitis B surface antigen on hepatocytes: a sensitive and specific marker of active viral replication in hepatitis B. J Clin Pathol. 1995;48:470-3. 16. Prati D, Taioli E, Zanella A et al. Updated definitions of healthy ranges for serum alanine aminotransferase levels. Ann Intern Med. 2002;137(1):1-10. 17. Piton A, Poynard T, Imbert-Bismut F et al. Factors associated with serum alanine transaminase activity in healthy subjects: consequences for the definition of normal

A-331 values, for selection of blood donors, and for patients with chronic hepatitis C. MULTIVIRC Group. Hepatology. 1998;27(5):1213-9. 18. Kim HC, Nam CM, Jee SH, Han KH, Oh DK, Suh I. Normal serum aminotransferase concentration and risk of mortality from liver diseases: prospective cohort study. BMJ. 2004;328(7446):983. 19. Wang C, Deubner H, Shuhart M et al. High prevalence of significant fibrosis in patients with immunotolerance to chronic hepatitis B infection. Hepatology. 2005;42(Suppl 1):A573. 20. Nguyen MH, Trinh H, Garcia RT et al. Significant histologic disease in HBV-infected patients with normal to minimally elevated ALT levels at initial evaluation. Hepatology. 2005;42(Suppl 1):A593. 21. Werle-Lapostolle B, Bowden S, Locarnini S et al. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology. 2004;126(7):1750-8. 22. Iloeje UH, Yang HI, Su J, Jen CL, You SL, Chen CJ. Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer-In HBV (the REVEAL-HBV) Study Group. Predicting cirrhosis risk based on the level of circulating hepatitis B viral load. Gastroenterology. 2006;130(3):678-86.

*Corresponding author: Dr Puja Sakhuja, Head and Professor, Department of Pathology, Room no 327, 3rd floor, Academic block, Jawahar lal nehru marg, G B Pant Institute of Postgraduate Medical Education and Research, New Delhi, Pin 110002. INDIA Phone: +91 9718599073 Email: pujasak@gmail.com Date of Submission : 21.11.2016 Date of Acceptance : 30.04.2017 Financial or other Competing Interests: None. Date of Publication : 05.07.2017

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Original Article DOI: 10.21276/APALM.1203

Bacteriological Analysis Including Antimicrobial Susceptibility Pattern of Blood Stream Infections in Tertiary Care Hospital Iqbal M Desai1, Hardik Kamlesh Bhavsar2*, Sachin M Darji2 and Jitendrakumar S. Parmar3 Department of Microbiology, Gujarat Research and Medical Institute. Rajasthan Hospitals. Ahmedabad. India 2 Department of Microbiology, GCS Medical college, Hospital and Research centre. Ahmedabad. India 3 Dept of Pathology, Gujarat Research and Medical Institute. Rajasthan Hospital, Ahmedabad. India

ABSTRACT Background: Blood stream infections cause significant morbidity and mortality worldwide. Illness associated with blood stream infection ranges from self-limiting infections to lifethreatening sepsis that require rapid and aggressive antimicrobial treatment. Rational and correct use of antibiotic requires understanding of common pathogens and their drug resistance pattern in the community as well as hospital. Methods: A retrospective study was conducted on the microbial profile of isolates of blood culture and their antimicrobial sensitivity pattern in a tertiary care hospital. Blood culture samples received from January to December 2015 in Microbiology department of the laboratory were enrolled in the study. Result: Positivity of the blood culture was found to be 16.66% (312/1872). Among the isolates, Gram negative organisms were 66.76% (203/300) while gram positive organisms were 32.33% (97/300) including 45 (15%) fungal isolates which were of various candida species. Klebsiella species was the most commonly isolated organism (27.3%) followed by E.coli (18.6%) and Acinetobacter species (8.3%). Staphylococcus aureus was isolated in 6% cases. Colisitin was found to be showing 100% sensitivity for Klebsiella, E.coli, Acinetobacter and Pseudomonas. Vancomycin was found to be sensitive for all Staphylococci isolated from blood. Ampicillin failed to show significant sensitivity against any of the above mentioned organisms. Conclusion: Specific antibiotic usage strategies to be prepared and implemented in the form of antibiotic usage policy like antibiotic restriction, combination therapy and usage according to the standard antibiotic susceptibility testing are needed for each tertiary care hospital to prevent emergence and spread of drug resistance. Keywords: Antibiotics, Blood Stream Infection, Bacteria, Antimicrobial Susceptibility.

Introduction

Blood stream infections cause significant morbidity and mortality worldwide and are among the most common healthcare associated infections. Microorganisms present in circulating blood whether continuously or intermittently are a threat to every organ in the body. Approximately, 200,000 cases of bacteremia and fungemia occur annually with mortality rates ranging from 20-50%. [1]

like Klebsiella, Enterobacter, Salmonella, Pseudomonas, Acinetobacter, etc.

Neonatal sepsis is a significant cause of neonatal morbidity and mortality in the newborn, particularly in preterm and low birth weight infants. According to World Health Organization (WHO) estimates, neonatal sepsis remains the major cause out of five million neonatal deaths per year. [2]

Illness associated with blood stream infections range from self- limiting infections to life-threatening sepsis that requires rapid and aggressive anti-microbial treatment. The incidence of blood stream infections in patients has been reported to correlate with the increasing use of central venous catheters, patient illness (e.g., oncology, burn, trauma and high risk nursery), and other predisposing factors including intensive care unit (ICU) stay, lapses in hand washing and non adherence to infection control practices of medical staff. Respiratory, genitourinary, and intra abdominal foci are often identifiable foci of blood stream infections. [3]

Since early 1950s, there has been a striking increase in incidence of bacteraemia caused by members of enterobacteriaceae and other gram negative bacteria. Escherichia coli which was reported to be common in the past is being replaced by other multidrug resistant bacteria

Nowadays, bacterial drug resistance is an important problem and due to wide variations in bacterial drug resistance, results of studies and reports in one region or in one period of time are not necessarily true for other regions or periods of time. They are related with a series of

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

M Desai et al. social, environmental and technological changes. [4], [5] The isolated bacteria are numerous and their associated diseases need urgent and invasive management with antimicrobial agents. Rational and correct use of these agents requires understanding of common pathogens and drug resistance pattern in the region. [6] The varying microbiological pattern of septicemia warrants the need for an ongoing review of the causative organisms and their antimicrobial susceptibility pattern. A study was conducted to identify the bacteriological profile and their antibiotic susceptibility patterns by analyzing the data on the blood culture isolates of a tertiary care hospital.

Materials and Methods

In this retrospective study, 1872 blood samples from patients clinically suspected of having bacteremia, were collected during the period of January to December 2015 in a tertiary care hospital. Processing of the samples was done at the Microbiology Department of the same hospital. 8-10 ml and 4-6 ml blood was collected using aseptic precautions by the trained staff nurses from adult and pediatric patients, respectively. Blood was transferred into respective BACTEC aerobic and BACTEC Ped plus blood culture bottles, at the blood collection site. Inoculated blood culture bottles were transferred to Microbiology laboratory immediately where they were loaded into BACTEC machine according to operational guideline for BACTEC. Positive bottles were identified by the machine and were then inoculated onto 5% sheep blood agar and MacConkey agar. Organism was identified using Gram stain technique, colony morphology and various biochemical tests.[7] Antibiotic susceptibility testing of the isolated organisms was performed using Kirby Bauerâ&#x20AC;&#x2122;s disc diffusion method on Mueller Hinton agar plates.[8] The antibiotic discs that were used to identify the susceptibility pattern of the bacterial pathogens along with their concentrations are: Ampicillin (10 mcg), Amikacin (30 mcg), Cefotaxime (30 mcg), Ceftazidime (70 mcg), Cefepime (30 mcg), Cefoperazone + sulbactum (70 mcg), Levofloxacin (5 mcg), Cotrimoxazole (trimethoprim /sulphamethoxazole 1.25/23.75 mcg), Erythromycin (10 mcg), Gentamicin (10 mcg), Imipenem (10 mcg), Linezolid (30 mcg), Piperacillin + tazobactum (100/10 mcg), Colistin (10 mcg) and Vancomycin (30 mcg). The data obtained were tabulated and analyzed to identify the common causative pathogens of blood stream infections and the antibiotics to which the identified organisms were sensitive and resistant. The results obtained were expressed by descriptive statistics.

A-333 December 2014, of which 312(16.66%) were identified as culture positive samples. The gender distribution of positive samples was found to be 278 (89.1%) males and 34(10.89%) females. The positive samples belonged to 245 (78.52%) adults (age range >18years) comprising of 221(90.20%) male and 24 (9.79%) female patients, 15 (4.80%) adolescents (age range 13-18years) which included 14 (93.33%) males and 1 (6.66%) female patients. 37 (11.85%) children (age range 1-12 years) which included 31(83.78%) males and 6(16.21%) female patients. 15(4.80%) infants (age range <1year) which included 11 (73.33%) males and 4 (26.66%) female patients. Out of 312 positive cultures, 300 (96.15%) showed bacterial growth. Out of those 300 isolates, 97 (32.33% ) were grampositive which included candida which is fungus and 203 (67.66%) were gram-negative.The most commonly isolated gram-positive bacteria were Staphylococcus aureus in 22 (42.3%), Enterococci in 12 (23.07%), Streptococcus in 3 (5.76%) out of total gram positive bacterial isolates i.e.52. The most prevalent gram-negative bacteria found in positive cultures were Klebsiella pneumoniae in 82 (40.39%) cultures, followed by Escherichia coli in 56 (27.58%), Salmonella typhi 25 (12.31%), Pseudomonas aeruginosa in 20(9.85%), Acinetobacter baumannii in 12 (4%) culture specimens out of all isolated gram negative bacteria. [Figure 1]. The results of antibiotic drug sensitivity of gram positive bacteria showed that Staphylococcus aureus was highly sensitive to Vancomycin (100%) and Linezolid (100%) followed by Levofloxacin (68.18%). Enterococci also were highly sensitive to Linezolid (100%) and Vancomycin (75%) followed by Levofloxacin (41.6%). Streptococci were found to be highly sensitive against Ampicillin (100%). So of all the antibiotics, Vancomycin and Linezolid were found to be most active against Staphylococcus aureus and Enterococci. [Table 2].

A total of 1872 blood culture samples were sent to Microbiology lab during the period of January to

The results of antibiotic drug sensitivity of gram negative bacteria showed the following results. Klebsiella, E. coli and Pseudomonas showed 100% sensitivity towards Colistin. Acinetobacter also showed maximum sensitivity towards Colistin (83.33%). Klebsiella showed maximum sensitivity to Imipenem (79.26%) after Colistin; followed by Piperacillin+Tazobactum (43.90%), Cefoparazone+Sulbactum (36.02%) and Amikacin (30.48%). All isolated Klebsiellae were resistant to Cefotaxime. E. coli also showed maximum sensitivity towards Imipenem (92.85%) after Colistin, followed by Amikacin (78.57%), Piperacillin+Tazobactum (64.28%), Cefoparazone+Sulbactum (60.71%).E coli

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Result

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showed least sensitivity towards Cefotaxime (8.92%). Levofloxacin however, was sensitive in 35.71% isolates. Acinetobacter showed maximum sensitivity towards Colistin (83.33%) and Imipenem (66.66%) followed by Cefoparazone+Sulbactum and Amikacin (33.33% each). Piperacillin+Tazobactum showed sensitivity of 25% and Levofloxacin showed 16.66% sensitivity. Cefotaxime or Cefepime did not show any sensitivity against Acinetobacter isolates. Pseudomonas aeruginosa showed

maximum sensitivity towards Imipenem (80%) after Colistin (100%), followed by Piperacillin + Tazobactum and Cefoparazone + Sulbactum (75% each). Amikacin showed 60% sensitivity, same as Levofloxacin. Cefepime showed 20% sensitivity while Cefoparazone could show only 10% sensitivity. Salmonella typhi showed 100% sensitivity against cephalosporins like Cefotaxime. Levofloxacin however was either resistant or intermediately sensitive for 60% Salmonella isolates.[ Table 1]

Gentamicin

cotrimoxazole

Cefipime

Cephotaxim

Cefoparazon+sulbacta m

Piperacillin+tazobacta m

Ampicilin

Colistin

Levofloxacin

Amikacin

Imipenem

Table 1: Antimicrobial susceptibility for various gram negative bacterial isolates in percentage out of total number of isolates of the organism.

Klebsiella

E.coli

Acinetobacter

Pseudomonas aeruginosa

Salmonella typhi

Salmonella paratyphi A

Amikacin

Levofloxacin

Ampicilin

Piperacillin+tazobacta m

Cefoparazon+sulbact am

cefoparazon

cefipime

Linezolid

Vancomycin

Clindamycin

Erythromycin

Cotrimoxazol

Table 2: Antimicrobial susceptibility of gram positive bacterial isolates in percentage out of total number of the organism isolated.

Staphyl ococcus aureus

Staphyl ococcus CONS

Enteroc occi

Streptoc occus pyogenu s

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M Desai et al.

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Fig. 1: Number of various isolates from positive blood cultures.

Discussion

Bacteriological profile and the antimicrobial susceptibility are constantly evolving. Study of bacteriological profile with antibiotic susceptibility pattern plays an important role in effective management of bacteremia cases. Many studies have been undertaken to determine the organisms responsible for blood stream infections all over the world. Results have varied in different centers and different parts of the world. Many factors e.g. socioeconomic, geographic, use of ventilators, and administration of different antibiotics etc., play a very important role in explaining these differences. The results of the retrospective study conducted in our tertiary care hospital demonstrated that 312 (16.66%) out of 1872 total blood samples screened were positive for the presence of micro-organisms of which 255 (85%) were bacteria and 45 (15%) were fungi. A study conducted by Mehta et al also reported similar frequency of positive blood culture as 16.4%.[9]

many as four blood cultures may be needed. Similar comment was made by other investigators that more than three blood cultures are needed for 99% test sensitivity. In India, variation might be due to the fact that most of the patients are given antibiotics before they come to the tertiary care hospital and other reason is that in most of the cases self medication is very common as the antimicrobials are available over the counter.

In contrast to above reports, the study done by Khanal et al [8] and Sharma [10] reported high frequency of positive blood cultures accounting for 44%, 33.9% and 20.2% respectively. Whereas studies by Anbumani et al[11] and Arora[12] reported lower frequency of positive blood cultures accounting for 7.89% and 9.94% ,respectively.

In most of the studies, gram-negative bacilli have taken over the gram-positive organisms, especially in hospital settings. Mehta et al12 have reported the incidence of 80.96% culture positivity for gram-negative and 18% for gram-positive organisms which is similar to our findings. Our study revealed that gram-negative bacteria were predominant (66.76%). Klebsiellae were reported to be the most common gram-negative organisms isolated from blood stream infections in many studies like present study. In the present study, we also observed a high frequency of E. coli. (18.66%). The high occurrence of non-lactose fermenters especially Pseudomonas spp.(6.66%) and Acinetobacter spp (4%). is of concern. Both of these bacteria are associated with a high degree of resistance to antibiotics. Another important finding of this study was high rate of Salmonella isolation (8.33%).

The variation in blood culture positivity is related to different factors such as the number and amount of blood cultures taken for screen as reported by Lee et al.[13] They believed that for achieving a detection rate of >99% as

The incidence of gram-positive organisms is 32.33%, including Candida, in our study.Gram positive bacterial isolates were 17.33%. Among the total gram positive bacterial isolates (n=52), the most common was

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Staphylococcus aureus (42.3%), followed by Enterococcus faecalis (23.07%) Staphylococcus seems to be emerging as the dominant organism in blood stream infections with 45% MRSA isolates. Here the percentage were calculated out of total gram positive bacteria isolated. Similar trend has been reported in the data from the west over the last two decades. Nosocomial infection due to Staphylococcus aureus constitutes a major part of the total annual nosocomial infections. [14] Enterococcus spp. also, was observed as an important pathogen in our study (23.07%;n=52). They form part of the normal gastrointestinal and female genitourinary tract flora. Over the past decade, Vancomycin resistant enterococci (VRE) have emerged as a leading cause of nosocomial infections due to its spread by direct patient to patient contact and by direct transmission via hospital personnel, environmental surfaces and hospital equipment. In our study, 25% of Enterococci were VRE. A study by CDC also reported high prevalence of VRE (29%), similar to our study.[15]

and Linezolid are highly active against gram positive organisms causing blood stream infections. However, it should not be expected that this activity continues for a long time. Therefore it is advisable to continuously evaluate the sensitivity-resistance pattern of isolates in each region so as to make a rational use of antibiotics. The present study provided much needed information on the prevalence of bacterial pathogens in blood stream infections and their antibiotic sensitivity patterns. The study identified both gram positive and gram negative bacteria were responsible for blood stream infections and most of them were multi drug resistant. The main forces driving the increase in antimicrobial resistant bacteria are poor infection control practices and inappropriate use of antibiotics. Specific antibiotic utilization strategies like antibiotic restriction, combination therapy and antibiotic recycling may help to decrease or prevent the emergence of resistance and antibiotic usage according to the standard antimicrobial susceptibility testing may reduce the incidence of blood stream infections.

In the present study, Candida isolates were seen in 25 (15%) cases. This epidemiologic trend has also been observed in many studies in different parts of the world. [16] . According to the surveillance data from US Center for Disease Control and Prevention (CDC), Candida accounts for 12% of all hospital acquired Blood stream infections.[17]

Acknowledgements

Most of the gram negative bacilli in the study were multi drug resistant. The most common resistance was seen to Ampicillin in all isolated gram negative bacteria. Other studies have also reported multidrug resistance for their isolated gram negatives. Colistin, Amikacin, Imipenem and piperacillin + tazobactum were found to be most effective antibiotics for all gram negative bacterial isolates including non- fermenters. Salmonella typhi was found to be highly sensitive to Ceftriaxone. Among the grampositive organisms, high resistance was noted against Ampicillin. An increased ampicillin resistance of 64%, 87% was also reported by Guha et al [18] and Karki et al [19] ,respectively in their studies. Resistance to third and fourth generation cephalosporins (cefotaxime, cefixime and cefepime) was also observed with Staphylococcus aureus and Enterococcus faecalis in the present study. This could be due to the abundant use of these drugs; especially third generation cephalosporin’s in hospitals, as reported by Nathisuwan et al.[20] The study also showed that Staphyloccocus aureus was found to be highly sensitive to vancomycin & linezolid.

Beekman SE, Chapin KC. Epidemiology and outcome of nosocomial and community on set blood stream infection. J clin microbiol 2003; 41: 3655-60

WHO. Perinatal Mortality. Report No: WHO/FRH/ MSM/967. Geneva: WHO, 1996.

Karlowsky JA, Jones ME, Draghi DCVolturo GA. Prevalence and antimicrobial susceptibilities of bacteria isolated from blood cultures of hospitalized patients in the United States in 2002. Annals of clinical microbiology and antimicrobials 2004;3:7.

Reingold AL. Antibiotic resistance patterns of bacterial isolates from blood in San Francisco country, California, 1996- 1999. Emerg Infect Dis 2007; 8: 195-201.

Cohen ML. Epidemiological factors influencing the emergence of antimicrobial resistance. Liba Found Symp 1997; 207: 223 – 231.

Graham C. Bacteremia and antibiotic resistance of its pathogens reported in England and wales between 1990 and 1998: Trend analysis. Br Med J 2000; 320: 213 – 216.

Cruickshank K, Duguid JP, Marmion BP. Test for sensitivity to antimicrobial agents. In: Medical Microbiology. Churchill Livingstone, 1980; 190-209.

Khanal B, Harish BN, Sethuraman KR, Srinivasan S. Infective endo carditis: Report of prospective study in an Indian Hospital. Trop Doct 2002; 32:83-85.

Conclusion

Overall, present results indicate that Colistin and Imipenem are highly active against gram negative and Vancomycin

We would like to acknowledge that all the samples were collected and processed in “Gujarat Research and Medical Institute, Rajasthan Hospital, Ahmedabad.” We also want to thank the institute for allowing us to conduct the study.

Reference

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Mehta MP, Dutta, V Gupta. Antimicrobial susceptibility pattern of blood isolates from a teaching hospital in North India. Jpn J Infect Dis 2005;58:174-176.

10. Sharma PP, Halder D, Dutta AK. Bacteriological profile of neonatal septicemia. Ind Pediatr 1987;24:1011-1017. 11. Anbumani N, Kalyani J, Mallika M. Original research distribution and antimicrobial susceptibility of bacteria isolated from blood cultures of hospitalized patients in a tertiary care hospital. Indian Journal for the practicing doctor 2008;5(2). Available at: http;//www.indmedica.com/journals. php?journalid+3&issueid=1658&action=article (1of 9). 12. Arora U, Devi P. Bacterial profile of blood stream infections and antibiotic resistance pattern of isolates. J K Sci 2007; 9:186- 190. 13. Lee A, Mirrett S, Reller LB, Weinstein MB. Detection of blood stream infections in adults: How many blood cultures are needed? J Clin Microbiol 2007; 45:3546-48. 14. Roy, I. ,A.Jain,M.Kumar and S.K.Agarwal. Bacteriology of neonatal septicemia in a tertiary care hospital of Northern India. Indian J.Med.Microbiol 2002;20:156-159.

A-337 15. Centers for Disease Control and Prevention National Nosocomial Infection Surveillance (NNIS) System Report, data summary from January 1992 through June 2004, issued October 2004. Am J Infect Control. 2004;32:470-85. 16. Bassetti M, Righi E, Costa A, et al. Epidemiologic trends in nosocomial candidemia in intensive care, BMC infect dis. 2006;6:21 17. Hidron Al, Edwards JR, Patel J et al. Antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centre for Disease Control and Prevention 2006-2007. Infect. Control Hosp Epidemiol. 2008;29:996-1011 18. Guha DK, Jaspal D, Das KMS, Guha RA, Khatri RL, Srikumar R. Outcome of neonatal septicemia:A clinical and bacteriological profile. Indian Pediatr 1978;15:423-27. 19. Bassetti M, Righi E, Costa A, et al. Epidemiologic trends in nosocomial candidemia in intensive care, BMC infect dis. 2006;6:21 20. Nathisuwan S, Burgess DS, Lewis II JS. Extended spectrum - lactamases: Epidemiology, Detection and treatment. Pharmacotherapy 2001;21:920-928.

*Corresponding author: Dr Hardik K Bhavsar, Department of Microbiology, GCS Medical College, Opp. D.R.M office, Naroda road, Ahmedabad, Gujarat, India Phone: +91 9099084835 Email: bhavsar.dr.hardik@gmail.com Date of Submission : 13.12.2016 Date of Acceptance : 03.05.2017 Financial or other Competing Interests: None. Date of Publication : 05.07.2017

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Case Report DOI: 10.21276/APALM.1121

Diagnosis of Bony Metastasis of Renal Cell Carcioma at a Rare Site on Fine Needle Aspiration Cytology: A Rare Case Report Mohanvir Kaur, Deepika, Vijay Kumar Bodal* and Vikram Jassal Department of pathology, Government Medical College, Patiala, India

ABSTRACT Fine needle aspiration cytology (FNAC) is cheap, non invasive and time saving procedure in diagnosis and management of patients in developing countries. Metastasis of renal cell carcinoma (RCC) to distant sites and organs at time of presentation is not infrequently encountered in the setting of FNAC for initial diagnosis. Here, we present a case of metastatic RCC to right clavicle initially diagnosed on FNAC and was confirmed on radiological examination of abdomen and histopathological examination. Keywords: FNAC, Metastasis, Renal Cell Carcinoma, Right Clavicle

Introduction

Renal cell carcinoma is generally a tumor of adults (average age at diagnosis: 55-60 years). [1]Approximately one-third of patients with renal cell carcinoma already have distant metastasis at the time they seek medical attention. [2] The most common sites of distant metastasis are lungs and skeleton. The bones most commonly involved are pelvis and femur, but there is also a predilection for the sternum, scapula and small bones of the hands and feet. [3,4] Metastasis can also develop in the adrenal gland, liver, skin, soft tissue, central nervous system, ovary and almost any other site.[5] Renal cell carcinoma is actually notorious for metastasizing to the most unusual places such as nasal cavity, oral cavity, larynx, parotid, thyroid, heart, bladder, testis, prostate and pituitary gland.[6] These metastasis are often solitary, at least at the clinical level.[7] Because of this and the fact that primary tumor is often clinically silent, these metastasis tend to be confused with primary tumors of the organs in which they lodge.

Case Report

A 50 year female, presented to the orthopaedics out patient department of our institution with complaint of swelling and pain in right supraclavicular region since 2 months, along with difficulty in over head abduction [Figure-1]. On local examination, a swelling measuring 5x6cm in size was present over right clavicle; firm in consistency with local raised temperature. On X-ray examination, an osteolytic lesion was seen involving the lateral half of right clavicle with mild soft tissue bulge query neoplastic, infective, inflammatory [Figure-2]. Routine investigations showed increased ESR=65mm/hr, CRP= 1.0 mg/dl. Ultrasonography of right clavicle was done which revealed a heterogenous mass (? neoplastic) with predominant hypo-

echoeic echo pattern measuring 6.0x9.7 cm in size with increased vascularity on color doppler. Patient was referred to pathology department for FNAC of the clavicular mass. On examination a pulsatile swelling measuring 5x5 cm size was present on lateral side of right clavicle. FNAC of the clavicular mass was done and hemorrhagic material was aspirated. Smears were stained with May Grunwald Giemsa (MGG) stain. On microscopic examination blood mixed smears showed low cell yield with epithelial cells forming clusters, papillaroid structures and papillae with focal fibrovascular core [Figure-3]. The cells were medium sized with foamy vacuolated cytoplasm and single small hyperchromatic nuclei.[Figure-4]. Keeping in view presence of lytic lesion possibility of metastatic deposits of renal cell carcinoma from morphology was kept. On further investigating the patient, ultrasound and CT scan abdomen was done which revealed right renal mass [Figure-5]. Nephrectomy was done. Specimen was sent to the other institution to confirm the diagnosis of clear cell renal cell carcinoma.[Figure-6], hence confirming the diagnosis of metastatic deposits of renal cell carcinoma on histopathologically.

Discussion

Renal cell carcinoma is a tumor with an unpredictable clinical course and behavior. Metastases have been reported to develop 17 years or more after the primary lesion is removed. [8] Renal cell carcinoma accounts for approximately 3 % of all the cancer cases. [9] RCC is the third most common infraclavicular tumor to metastasize to head and neck region

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Fig. 1: Photograph of patient showing swelling in right clavicular region.

Fig. 2: X-ray right side shoulder joint showing an osteolytic lesion involving the lateral half of right clavicle.

Fig. 3: Blood mixed smears showing epithelial cells forming papillaroid structures and papillae with focal fibrovascular core (MGG, 100x).

Fig. 4: Blood mixed smears showing medium sized cells with foamy vacuolated cytoplasm and single small hyperchromatic nuclei (MGG, 400 x).

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Fig. 5: CECT whole abdomen revealed large lobulated mass arising from upper, middle pole and pelvis of right kidney measuring 9cm x8cm x7.3 cm.

Fig. 6: Smear showing malignant epithelial cells lying in tubules and sheets, exhibiting pleomorphism with sharply outlined cell borders with clear cytoplasm (H & E,400x)..

preceded only by breast and lung. Metastasis solely to head and neck region occurs in only 1% patients with primary RCC and usually affect thyroid, nose, paranasal sinuses and oral cavity.[10]29-57% of patients develop metastasis by the time tumor is diagnosed. Only <1% of patients with bone metastasis manifested clavicular RCC metastasis. Thus clavicular metastasis as the initial presentation of RCC is extremely rare. Bony metastasis from RCC are purely lytic, expansile and highly vascular. [11]

Local resection without sacrifice of the vital structures is the treatment of choice depending on the site of presentation. This may provide a chance of cure of the head and neck metastasis and avoid associated morbidity that may occur if the lesion is left untreated. [17]

Clavicular symptoms may be manifested before the diagnosis of primary tumours, such as RCC. Clavicular fracture may be the first symptom of tumor metastases to bones. Distinguishing pathological fractures from clavicular fractures due to other causes may help diagnose the primary tumors. [12] Characteristically, the tumor is slow-growing and encapsulated in its early stages and thus asymptomatic. When a patient with a clinically asymptomatic renal cell carcinoma has signs and symptoms referable to a localized lesion, the final diagnosis depends on histologic and cytologic evaluation of a biopsy specimen. [13] Microscopically it can be confused with other clear cell carcinomas. Hughes et al. have described prominent vascularity to be an important feature of RCC to distinguish it from other clear cell tumors.[11] Fuhrman nuclear grade used in histology can be applied in cytology smears.[14] Cells of RCC contain intra cytoplasmic fat, hence Oil Red O staining of air dried smears can be used to distinguish RCC from other clear cell tumors.[15]While metastasis are associated with poor outcome, solitary bone lesions have increased associated survival when compared to multiple bony lesions or a combination of bone and other organs.[16] www.pacificejournals.com/apalm

Conclusion

The case is being presented due to its rare site of metastasis, lytic expansible and highly vascular nature of the lesion which can be misdiagnosed and confused with other clear cell carcinomas. Early diagnosis may increase survival and avoid associated morbidity without sacrifice of vital organs.

Acknowledgements

We are thankful to Dr. Sarbhjit Kaur, Assistant Professor, Department of Gynaecology, Government medical college, Patiala, for helping us in preparing and writing this article.

Reference 1.

Cohen HT, McGovern FJ. Renal cell carcinoma. N Engl J Med. 2005; 353: 2477-2490.

Holland JM. Cancer of the kidney. Natural history and staging. Cancer .1973; 32: 1030-1042.

Gurney H, Larcos G, McKay M, Kefford R, Langlands A. Bone metastasis in hypernephroma. Frequency of scapular involvement. Cancer.1989; 64: 1429-1431.

Troncoso A, Ro JY, Grignon DJ, Han WS, Wexler H, Von Eschenbach A, Ayala AG. Renal cell carcinoma with acrometastasis. Report of two cases and review of the literature. Mod Pathol .1991; 4: 66-69.

Insabato L, De Rosa G, Franco R, Dâ&#x20AC;&#x2122;Onnofrio V, Di Vizio D. Ovarian metastasis from renal cell carcinoma: a report of three cases. Int J Surg Pathol. 2003; 11: 309-312.

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Leung CS, Srigley JR, Robertson AR. Metastatic renal cell carcinoma presenting as solitary bleeding prostatic metastasis. J Urol Pathol. 1997; 7: 127-132.

Radley MG, McDonald JV, Pilcher WH, Wilbur DC. Late solitary cerebral metastasis from renal cell carcinoma. Report of two cases. Surg Neurol.1993; 39: 230-234.

Coppa JF, Oszczakiewicz M. Parotid gland metastasis from renal carcinoma. Int Surg 1990; 75: 198-202.

Novick AC, Campbell SC. Renal tumors. Campbellâ&#x20AC;&#x2122;s Urology.2002; 8: 2672-731.

10. Huang HC, Chang KP, Chen TM, Wu KF, Ueng SH. Renal cell carcinoma metastasis in the head and neck. Chang Gung Med J.2006; 29: 59-65. 11. Setlik DE, McCluskey KM, McDavit JA. Best cases from the AFIP: Renal cell carcinoma manifestations - solitary bone metastasis. Radiographics. 2009; 29: 2184-9. 12. Yan K. Jin W. Huan L. Peng G. Jian FX. He-Le F. Pathological clavicular fracture as first presentation of renal cell carcinoma. Cancer Biol. Med 2015; 12: 409-12.

13. Wahner DL, Sebo TL. Renal cell carcinoma:Diagnosis based on metastatic manifestation. Mayo Clin Proc 1997; 72:935-41 14. Hughes JH, Jensen CS, Donnelly AD, Cohen MB, Silverman JF, Geisinger KR et al. the role of fine needle aspiration cytology in the evaluation of metastatic clear cell tumors. Cancer. 1999; 87: 380-9. 15. Wan MW, Husain SN. Fine needle aspiration cytology of metastatic renal cell carcinoma â&#x20AC;&#x201C; A case report. Med Health. 2006; 1: 75-80. 16. Parada SA, Franklin JM, Uribe PS, Manoso MW. Renal cell carcinoma metastasis to bone after 33 year remission. Orthopaedics. 2009; 32: 446. 17. Yeh HC, Yang SF, Ke HL, Lee KS, Huang CH, Wu WJ. Renal cell carcinoma presenting with skull metastatsis: a case report and literature review. Kaoh J Med Sci 2007; 23:475-78.

*Corresponding author: Dr Vijay Kumar Bodal, Department of pathology, Government Medical College, Patiala, India Phone: +91 9814714946 Email: vijay_bodal@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 22.10.2016 Date of Acceptance : 10.03.2017 Date of Publication : 29.05.2017

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Case Report DOI: 10.21276/APALM.1132

Hamartomatous Nodule, Sertoli Cell Adenoma in Complete Androgen Insensitivity Syndrome with Wolffian/Müllerian Duct Remnants: An Unusual Case Report Prashant Vijay Kumavat1*, Chetan S Chaudhari1, Anita Padmanabhan1, Nitin M Gadgil1, Sangita S Margam1, Ganesh R Kshirsagar1 and Unnati D Rathod2 Dept. Of Pathology, LTMMC and LTMGH Sion, Mumbai, India Dept. Of Microbiology, Sir JJ Hospital, Byculla, Mumbai, India

ABSTRACT Complete Androgen insensitivity syndrome is a disorder of hormone resistance characterized by a female phenotype with an XY karyotype and testes producing age-appropriate normal or higher concentrations of androgens. We present a case of 26 year old, unmarried phenotypically female, with left inguinal swelling and amenorrhea. MRI finding revealed bilateral inguinal masses, uterus cervix was not visualized and hypoplatic vagina was noted. Karyotyping revealed her genotype as 46 XY. Hormonal investigation showed testosterone, estradiol and LH were increased and FSH was within normal limits. Patient underwent laparoscopic bilateral gonadectomy with left open hernia repair. Histopathology examination revealed hamartomatous nodule, sertoli cell adenoma, leydig cell hyperplasia, which are more pronounced as age advances as result of absent activity of androgen. Fallopian tube, underdeveloped vas deference, Wolffian/Müllerian cysts lined by cuboidal epithelium was also noted which may be reminiscent of Wolffian/ Müllerian structure. Immunostaining for PLAP and CD 117 were negative. The clinical, MRI, laboratory and histopathology findings confirmed diagnosis of complete androgen insensitivity syndrome. Keywords: Complete Androgen Insensitivity Syndrome, Gonads, Hamartoma, Müllerian, Wolffian.

Introduction

Complete androgen insensitivity syndrome (CAIS) is a female phenotype with a male karyotype (46, XY) which results from inactivating mutation in the androgen receptor (AR) gene.[1] The targeted response to testosterone or dihydrotestosterone is annulled due to mutation. As a result, male external genitalia differentiation and Wolffian duct development do not occur correctly. Sertoli cells of normally developed gonads produces anti-Mullerian hormone which regresses Mullerian duct. Residual Müllerian structures exist approximately in one third of patients.[2] Development of the gonads is normal, and serum androgen level is comparable with that of a normal male. Most of the patients with complete androgen insensitivity typically presents either at puberty with primary amenorrhea or before puberty with masses in the inguinal canal that are subsequently identified as testes. Breast development occurs because of the aromatization of the excess testosterone into oestrogen and pubic hair tends to be sparse or absent. External genitalia are that of female with short vagina and absent uterus, cervix, or fallopian tubes. After development of secondary sexual features, testes either in the inguinal canals or in the pelvis should be removed, because gonadal tumours are known to develop in 5 % of cases. [3] We report a case of complete

androgen insensitivity syndrome in twenty six year old unmarried female but found to have a 46, XY genotype, with hamartomatous nodule, sertoli cell adenoma and residual Müllerian/Wolffian tissue.

Case report

A young unmarried, 26 year old female, presented to surgery OPD with left inguinal swelling increasing on straining and coughing, since 4 years. She was amenorrheic but never consulted for this complaint to physician. She had past history of right inguinal hernia operated four years back. MRI finding revealed bilateral inguinal masses,on right side, 2.3 × 1.1 cm mass anterior to external iliac vessel and similarly on left side, and 2 × 1 cm anterior to external iliac vessel (Figure 1 A, B).Uterus cervix was not visualized and a small tubular structure was noted between urethral and anal opening, suggestive of hypoplatic vagina. She has not attained menarche but has attained thelarche (breast development), 12 years back. On local examination atrophic vagina and clitoris was present and axillary, pubic hair growth was absent. Karyotyping was advised and revealed her genotype as 46 XY. Hormonal investigation showed, testosterone level of 251.4 ng/dl (increased), Follicle stimulating hormone (FSH) level of 9.88 mIU/ ml (normal), Luteinizing hormone (LH) of 82.84 mIU/ ml (increased), estradiol of 413 pg/ml (increased) and

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progesterone of 0.19 ng /ml (decreased). Intraoparative findings revealed absence of ovaries and uterus and bilateral small atrophied gonads were present. Widened left sided deep inguinal ring, with left sided gonads partially herniating was observed.Patient underwent laparoscopic bilateral gonadectomy with left open hernia repair. Grossly, we received left inguinal mass measuring 6×3×2 cm, with cut section showing grey white nodule measuring 1.2×1.0cm and right sided inguinal mass measuring 5×3×2 cm with cut surface showing grey white in appearance. (Figure 1C). Histopathology examination of both masses showed hamartomatous nodule of sertoli and leydig cells, which is well circumscribed and composed of sertoli cells tubules and leydig cell in between tubule and leydig cell

hyperplasia (Figure 2 A). At many places fibrotic stroma with atrophic seminiferous tubule were noted (Inset Figure 2 A). Areas of ovarian like stroma were noted (Inset Figure 2B). Sertoli cell adenoma showing Somniferous tubules having little fruitless lumens and typically containing just sertoli cells were also noted (Figure 3 A, B). There was no evidence of spermatogenesis in any of the structures and no apparent malignant changes. Fallopian tube, underdeveloped vas deference, and Wolffian/ Müllerian duct cysts lined by cuboidal epithelium were also noted (Figure 3 C, D). IHC revealed negative immunostaining for PLAP and CD 117(Figure 3 E, F). The clinical, MRI, laboratory and histopathology findings confirmed the diagnosis of complete androgen insensitivity syndrome

Fig. 1: 1A MRI Scan - 2.3 × 1.1 cm inguinal mass anterior to external iliac vessel on right side. 1B MRI – 2 × 1 cm on left side anterior to external iliac vessel.

Fig. 1C: Gross- Left mass measures 6×3×2 cm, cut sections showed grey white nodule measuring 1.2×1cm. Right sided mass measures 5×3×2 cm and cut section showed grey white.

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Fig. 2: 2A- Well circumscribed Hamartomatous nodule of sertoli and leydig cells and leydig cell hyperplasia (H and E 100x) Inset - At many places fibrotic stroma with atrophic seminiferous tubule were noted. 2B- Inset- Areas of ovarian like stroma was noted.

Fig. 3: 3A- Sertoli cell adenoma - Seminiferous tubules have little fruitless lumens and just sertoli cells (H and E 100x). 3B- High power of 3A (H and E 400x). 3C- Fallopian tube like structure. 3D â&#x20AC;&#x201C; underdeveloped vas deference and Wolffian/ MĂźllerian duct cysts lined by cuboidal epithelium. 3E- CD 117 negative. 3F- PLAP negative.

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Discussion

Androgen insensitivity syndrome (AIS) is an X-linked, male pseudo hermaphroditism also known as testicular feminization syndrome, in which clinically phenotypically patient is female but shows 46 XY genotype. The frequency of this syndrome was estimated about 0.05% (~70/140000).[4] In post pubertal patients, the most frequent cause for consultation is primary amenorrhea; however, in prepubertal patients, AIS is often diagnosed during the investigation of inguinal hernia.[5] There are 3 types of ASI: complete, mild and incomplete. The estimated prevalence of CAIS is about 1:20,000-64,000 male births.[6] In most of the cases CAIS, phenotypically female present with normal female external genitalia like a short blind ending vagina but there will be an absence of Wolffian duct derived structures like vas deferens and seminal vesicles epididymis and prostate. Although pubic and axillary hair are sparse or absent but breast development is recognised at puberty. [6] The histologic pattern of testes removed from adult patients with AIS is similar to that of many cryptorchidic, infantile or immature testes. [7] Although some differences regarding testicular histology between the complete and incomplete forms of AIS were reported in the first studies [6] later studies of a wide series of patients concluded that there are no histologic differences in the testicular pattern between the 2 forms. Rutgers and Scully reported two histopathological testicular patterns in AIS, namely, hamartomas (hamartomatous nodules) and sertoli cell adenomas which are seen in our case also. [7] Hamartomas consisted of solid tubules filled with immature sertoli cells, prominent leydig cells, and rare fascicles of smooth muscle. Multiple hamartomas occurred in 63% of AIS cases (bilateral in 40% of cases). Sertoli cell adenomas consisted of small tubules that were filled by immature sertoli cells and scant leydig cells and occurred in 23% of cases. In the present case, hamartomatous nodule was seen with sertoli cell adenoma and leydig cell hyperplasia. We propose that the development of hamartomas in AIS is not neoplastic proliferation of sertoli cells but it results from malformative process of the congenitally hypoplastic seminiferous tubules. Regression of the Müllerian structures occurs (the fallopian tubes, uterus, and upper portion of the vagina) because of AMH (Anti- Müllerian hormone) secreted by sertoli cells of the testis. The incomplete regression of Müllerian remnants can be due to 1) Deficiency of secretion of AMH, 2) AMH although secreted enough but it’s not functional, 3) Lack of response by the Müllerian tissue to AMH because of the high oestrogen levels caused by the

conversion of testosterone to oestrogen in the androgen insensitive foetus, 4) Early testicular descent that removes the Müllerian structures out of the effective range of the AMH. [6, 8, 9] There is a possibility of common link between androgen insensitivity syndrome and defective action of AMH, this can be suggested as there are persistence of the Müllerian remnants in patients diagnosed with complete androgen insensitivity syndrome. [10] In one of the studies from Netherlands (one of the largest databases compiled) of the families examined revealed 3 of 7 CAIS families had female siblings with differing Müllerian remnants. [11] One third of the case showed fallopian tube which is also noted in our case.[7] One more interesting fact seen in our case is presence of underdeveloped vas deference that may be reminiscent of Wolffian duct. Wolffian/Müllerian duct cysts lined by cuboidal epithelium was also noted which may be reminiscent of that structure. Ovarian like stroma was present in our cases.[12] So this is very rare case showing finding of CAIS with discordant Wolffian/ Müllerian remnants, offering another example and more studies should be initiated and evaluated to study the cause leading to the residual tissue. Some histopathological changes seen in our case that is Hamartomatous nodule, sertoli cell adenoma and leydig cell hyperplasia develop during puberty as a consequence of the almost or entirely absent activity of androgens in complete androgen insensitivity syndrome.[12]Decrease or absent germ cells, tubular atrophy, stromal atrophy occur early in childhood which may be due to abnormal location (inguinal) of the gonads.[12] Intratubular germ cell neoplasia, can be diagnosed only if at least one cross section of seminiferous tubule contains a homogenous population of atypical germ cell with angulated nuclei.[12] This particular feature was absent in our case, which may be due to absolute loss of abnormal germ cells in adulthood when gonads would have been retained and a failure of progression of the pre-invasive lesions into an invasive cancer. In CAIS both these above mentioned mechanism may be due to lack of androgen action because of which there is risk of tumour development in patients with partial AIS as compared to CAIS (15% versus 0.8% in complete androgen insensitivity syndrome according to Cools et al). [13] Due to alteration in androgen receptor gene which causes end organ resistance to the testosterone, leading to testosterone and LH levels are usually elevated in AIS.[6] In AIS excess of testosterone is converted to oestrogen in periphery which causes increase levels of estradiol, while FSH levels are normal. This suggests that regulation of FSH secretion is maintained by the combine action of estradiol and gonadal hormone like Inhibin. [14]

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Kumavat et al. In conclusion Hamartomatous nodule, sertoli cell adenoma, leydig cell hyperplasia is more pronounced as age advances as result of absent activity of androgen. This is a very rare case showing finding of CAIS with discordant Wolffian/Müllerian remnants, and more studies should be initiated and evaluated to study the cause leading to the residual tissue. Lack of androgen theory would correlate with significantly higher risk for tumor development in patient with partial androgen insensitivity syndrome as compared to complete androgen insensitivity syndrome.

References 1.

Hughes I, Davies J, Bunch T, Pasterski V, Mastroyannopoulou K, MacDougall J. Androgen insensitivity syndrome. The Lancet. 2012;380(9851):1419-1428.

2. Nichols J, Bieber E, Gell J. Case of sisters with complete androgen insensitivity syndrome and discordant Müllerian remnants. Fertility and Sterility. 2009;91(3):932.e15-932.e18. 3.

Chen C, Chen S, Wang T, Wang W, Hwu Y. A frame shift mutation in the DNA-binding domain of the androgen receptor gene associated with complete androgen insensitivity, persistent müllerian structures, and germ cell tumours in dysgenetic gonads. Fertility and Sterility. 1999;72(1):170-173. D Farhud D, Zarif Yeganeh M, Sadighi H, Zandvakili S. Testicular Feminization or Androgen Insensitivity Syndrome (AIS) in Iran: a Retrospective Analysis of 30-Year Data. Iranian Journal of Public Health. 2016;45(1):1-5. Bangsbøll S, Qvist I, Lebech P, Lewinsky M. Testicular feminization syndrome and associated gonadal tumours in Denmark. Acta Obstetricia et Gynecologica Scandinavica. 1992;71(1):63-66. Quigley C, Bellis A, Marschke K, El-Awady M, Wilson E, French F. Androgen Receptor Defects: Historical,

C-77 Clinical, and Molecular Perspectives. Endocrine Reviews. 1995;16(3):271-321. 7.

Rutgers J, Scully R. The Androgen Insensitivity Syndrome (Testicular Feminization). International Journal of Gynaecological Pathology. 1991;10(2):126-144.

Heller D, Ranzini A, Futterweit W, Dottino P, Deligdisch L. Müllerian remnants in

complete androgen insensitivity syndrome. International journal of fertility. 1992;37(5):283.

10. Ulloa-Aguirre A, Mendez J, Chavez B, Carranza-Lira S, Angeles A, Perez-Palacios G. Incomplete regression of Müllerian ducts in the androgen insensitivity syndrome. Fertility and Sterility. 1990;53(6):1024-1028. 11. Damiani D, Mascolli M, Almeida M, Jaubert F, Fellous M, Dichtchekenian V et al. Persistence of Mullerian Remnants in Complete Androgen Insensitivity Syndrome. Journal of Paediatric Endocrinology and Metabolism. 2002;15(9):1553-1556. 12. Boehmer A, Brüggenwirth H, Van Assendelft C, Otten B, Verleun-Mooijman M, 13. Niermeijer M et al. GenotypeversusPhenotype in Families with Androgen Insensitivity Syndrome. The Journal of Clinical Endocrinology & Metabolism. 2001;86(9):4151-4160. 14. Kaprova-Pleskacova J, Stoop H, Brüggenwirth H, et al. Complete androgen insensitivity syndrome: factors influencing gonadal histology including germ cell pathology. Modern Pathology. 2014;27(5):721-30. 15. Cools M, Drop S, Wolffenbuttel K, Oosterhuis J, Looijenga L. Germ Cell Tumours in the Intersex Gonad: Old Paths, New Directions, Moving Frontiers. Endocrine Reviews. 2006;27(5):468-484. 16. Schmitt S, Knorr D, Schwarz H, Kuhnle U. Gonadotropin regulation during puberty in complete androgen insensitivity syndrome with testicles in situ. Hormone Research. 1994;42(6):253-256.

*Corresponding author: Dr Prashant Vijay Kumavat, Room no 1303, building no 27, hawre city, kasarvadvali, Thane (w) 400615 Email: drkumavat_83@rediffmail.com

Financial or other Competing Interests: None.

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Date of Submission : 13.11.2016 Date of Acceptance : 12.02.2017 Date of Publication : 29.05.2017

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Case Report DOI: 10.21276/APALM.1137

Vimentin Positive Primary Thyroid Diffuse Large B-Cell Lymphoma: Cytological Diagnosis of Two Cases Swati Bhardwaj*, Charanjeet Ahluwalia and Ashish Kumar Mandal Department of Pathology, Vardhman Mahavir Medical College & Safdarjung Hospital, New Delhi, India

ABSTRACT Primary thyroid lymphomas are rare tumours of thyroid accounting for 5% of all thyroid tumours. Diffuse large B cell lymphoma is one of the most common type of primary thyroid lymphoma. Here we are presenting two rare cases of primary thyroid lymphoma. The first case is of a sixty year old female who presented with a rapidly enlarging thyroid mass, causing compressive symptoms. Radiological investigations revealed deviation and compression of trachea by the mass. Fine needle aspiration was performed, and routine as well as liquid based cytology smears were prepared. Smears revealed features of diffuse large B cell lymphoma of thyroid. Immunocytochemistry was performed that confirmed the diagnosis, and also revealed vimentin positive nature of the neoplasm, that has been shown to confer some degree of resistance to chemotherapy. A tru cut biopsy was performed that reconfirmed the diagnosis, and also helped in determining Ki67 proliferation index of the tumour that determines the aggressive nature of non-Hodgkin’s lymphomas. The second case is of a seventy year old woman who presented with a rapid enlargement of thyroid over a period of three months, leading to difficulty in breathing and dysphagia. Fine needle aspiration was performed that showed features of immunoblastic type of DLBCL. The diagnosis was confirmed by immunocytochemistry, which also showed vimentin positivity. These two cases are important for their rarity, especially being vimentin positive, diagnosed on cytology with the aid of immunocytochemistry. Keywords: Thyroid, Cytology, FNAC, Lymphoma, Diffuse Large B Cell Lymphoma, Immunocytochemistry.

Introduction

Case Reports

Fine needle aspiration cytology plays a vital role in diagnosing thyroid lesions. Since biopsy has a minor role to play in thyroid, the onus lies on FNA alone to establish a diagnosis for adequate patient management. The management of DLBCL thyroid is non-surgical and relies on chemo-radiotherapy. Therefore, it is important to evade surgery by establishing diagnosis on FNA beforehand.

Fine needle aspiration was performed from two sites, owing to the large size of the swelling. Conventional smears were prepared and material for immunocytochemistry was fixed in cold acetone. Liquid based cytology was

Primary thyroid lymphoma is a rare entity and accounts for only 5% of all thyroid malignancies and approximately 3% of all non-Hodgkin’s lymphoma. [1, 2] There is an overall female predominance with a female: male ratio of 3:1. [3, 4] They typically occur in middle to old aged individuals. [5] Most thyroid lymphomas are of B cell origin. [6]The most common presentation of thyroid lymphoma is a rapidly enlarging, painless goitre. Other symptoms such as dyspnoea, dysphagia, and hoarseness may arise as a result of the pressure effects of the mass. Rarely, stridor or superior vena cava obstruction can occur. Cervical lymphadenopathy is present in the majority of cases. [7] The underlying pathogenesis of primary thyroid lymphoma is not exactly known, but is attributed to chronic antigenic stimulation leading to the development of intra-thyroid lymphoid tissue as is seen in Hashimoto’s thyroiditis. This also explains why the risk of development of primary thyroid lymphoma is 40-80 times higher in patients of Hashimoto’s thyroiditis. [4]

CASE 1: A sixty year old female with no previous history of thyroid complaints, presented with a rapidly enlarging thyroid swelling, over a period of three months (Figure-1). It was accompanied by pressure symptoms of dysphagia. There was no history of any hyperthyroid or hypothyroid symptoms. On examination, the patient had anterior neck swelling measuring 11x7x6 cm. The swelling moved little with deglutition owing to its large size. There was no palpable cervical lymphadenopathy. The patient had normal vitals, normal general physical examination and no tremors were noted. Ultrasound neck showed bilateral thyroid lobe enlargement. There was decreased echo pattern in both lobes with multiple echogenic strands, with few subcentimetric lymph nodes in level III. Features were suggestive of thyroiditis (Hashimoto’s disease). The patient then underwent CT scan of the neck that revealed diffuse enlargement of the thyroid gland, compressing and displacing the larynx, vocal cord, and cricothyroid membrane (Figure-2). Few enlarged lymph nodes were noted on the left side of the neck, measuring 7mm to 9mm.

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Bhardwaj et al. also done. Conventional Giemsa stained smears were moderately cellular, showing monomorphic cells present mainly singly and a few small dyscohesive clusters against a haemorrhagic background (Figure-3a). The individual cells were large, exhibiting high degree of pleomorphism, high nucleo: cytoplasmic ratio, and scant cytoplasm. Most cells showed prominent nucleoli. Brisk mitoses, including atypical forms were noted. A differential diagnosis of diffuse large B cell lymphoma and anaplastic carcinoma were kept. Immunocytochemistry was performed to establish a definitive diagnosis. The cells were positive for LCA, Bcl6, and Vimentin and negative for CK (Figure-3b-3d). The smears also showed the presence of TTF-1 positive cells confirming the origin from thyroid. Thus, a diagnosis of diffuse large B cell lymphoma was established. A tru cut biopsy was performed to reconfirm the diagnosis. Histopathology revealed features of diffuse large B cell lymphoma, which was confirmed on immunohistochemistry. (Figure-4). The patient was started on chemo radiotherapy and is doing well now.

Fig. 1: Diffuse anterior neck swelling, case 1.

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C-79 CASE 2: A seventy year old lady, housewife by profession presented with a rapidly enlarging thyroid swelling that increased in size over a period of five months (Figure 5a). There was history of difficulty in swallowing, and breathing present since past 1 month. There was no history of hyperthyroid or hypothyroid symptoms. The patient also had loss of weight over the past one month. On examination, the swelling was soft to firm, measuring 9.5X10 cm. It moved little with deglutition. There was no cervical lymphadenopathy. Ultrasound showed diffuse thyroid swelling, involving both lobes. CECT neck revealed diffuse thyroid enlargement, compressing and displacing trachea to one side. Retrosternal extension was also present. There was no cervical lymph node enlargement. FNAC was performed from two sites, owing to the large size of the swelling. Smears were hypercellular, showing lymphoid cells scattered singly. The cells exhibited moderate degree of pleomorphism and a high mitotic rate. (Figure 5b) Immunocytochemistry was performed, that showed the cells to be LCA, and BCL6 positive. They were negative for CK and CD20. These cells were also positive for Vimentin. Thus a diagnosis of diffuse large B cell lymphoma, immunoblastic type was made.

Fig. 2: CT picture shows tracheal displacement by thyroid swelling

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Vimentin Positive Primary Thyroid Diffuse Large B-cell Lymphoma

Fig. 3a :Giemsa stained smears shows scattered large, pleomorphic lymphoid cells with prominent nucleoli and brisk mitoses, 20X. Figures 3b, 3c, 3d: Immunocytochemistry showing positivity for LCA, Bcl 6 and Vimentin respectively, 10X.

Fig. 4a: H&E stained tru cut biopsy showing monomorphic lymphoid cells with high degree of pleomorphism, 20X. Figure 4b, 4c, 4d, 4e: Immunohistochemistry showing positivity for Ki67, Bcl-2, MUM-1 and CD-20 respectively, 20X.

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Bhardwaj et al.

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Fig. 5a: Diffuse anterior neck swelling, case 2. Figure 5b: Case 2, Giemsa stained smear showing high cellularity of lymphoid cells with prominent nucleoli and mitotic figures, 20X.

Discussion

Diffuse large B cell lymphomas conventionally undergo CHOP chemotherapeutic regimen. In about 40% cases of DLBCL, development of resistance to CHOP regimen causes treatment failure. [9] Among the various causes investigated, vimentin positivity was found to be one implicated in causing greater tumour invasiveness and resistance to therapy. Maxwell et al have described that activation of an Akt-14-3-3Îś signalling pathway plays a role in promoting a multidrug-resistant phenotype associated with a vimentin-dependent invasive behaviour in DLBCL cells. [9] Thus our cases, are examples of uncommonly observed vimentin positivity, indicating poor response to chemotherapy.

In the first case, the diagnosis had already been established on cytology, thus guiding the clinical decision against surgical management. However, a tru cut was done for a reconfirmation and for determining the ki67 proliferation index of the tumour. According to literature, a higher Ki67 proliferation index indicates a poor prognosis due to aggressive disease course. [8] In our case, the ki67 index was about 50%. The mean Ki67 proliferation index differs by type of lymphoma. A cut off value of 45% can help differentiate indolent from aggressive Non Hodgkin lymphoma. [8] In diffuse large B cell lymphoma, a cut off value of 75% can distinguish patients with a good and bad prognosis, when combined with low IPI score and bulky disease. [8]

Both of the patients were treated with a combination of chemo and radiotherapy and are doing well.

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These two cases present an example of a rare thyroid neoplasm, diagnosed primarily by FNAC and reconfirmed on histopathology. On FNA smears, DLBCL of thyroid can be difficult to distinguish from anaplastic carcinoma thyroid. Both present with a rapidly enlarging thyroid swelling clinically, with a somewhat overlapping cytological picture of large, atypical cells with large, pleomorphic nuclei, and prominent nucleoli. It is essential to clearly and accurately distinguish the two as both require drastically different treatment. While anaplastic carcinoma is treated by radical surgery, DLBCL does not need surgery as radiotherapy and chemotherapy suffice. Differentiation between the two can be done by cytology and immunocytochemistry.

Conclusion

Cytology combined with other ancillary techniques such as immunocytochemistry plays a very important role in diagnosing thyroid lesions. This case report highlights the diagnostic importance of FNAC in diagnosing even rare lesions such as thyroid lymphoma. The use of immunocytochemistry enabled accurate typing of lymphoma, thereby facilitating early treatment with minimal intervention. These two cases are also important for their rarity, and uniqueness.

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Vimentin Positive Primary Thyroid Diffuse Large B-cell Lymphoma

Reference

Green LD, Mack L, Pasieka JL. Anaplastic thyroid cancer and primary thyroid lymphoma: A review of these rare thyroid malignancies. J Surg Oncol.2006;94:725-736.

Gupta N, Nijhawan R, Srinivasan R, Rajwanshi A, Dutta P, Bhansaliy A, Sharma SC. Fine needle aspiration cytology of primary thyroid lymphoma: a report of ten cases. Cytojournal. 2005;20:21.

Graff-Baker A, Roman SA, Thomas DC, et al. Prognosis of primary thyroid lymphoma: Demographic, clinical, and pathological predictors of survival in 1,408 cases. Surgery. 2009;146:1105-1115.

Sasai K, Yamabe H, Haga H, et al. Non-Hodgkin’s lymphoma of the thyroid. A clinical study of twenty two cases. Acta Oncol.1996;35:457-462.

Li J, Hu R, Liao AJ, Shi HY, Yan W, Liu ZG. Ki67 proliferative index in non-Hodgkin’s lymphoma and its clinical significance. Zhongguo Shi Yan Xue Ye XueZaZhi. 2011;19(4):935-9.

Maxwell SA, Cherry EM, Bayless KJ. Akt, 14-3-3 , and vimentin mediate a drug-resistant invasive phenotype in diffuse large B-cell lymphoma. Leuk Lymphoma. 2011 May;52(5):849-64.

Ansell SM, Grant CS, Habermann TM. Primary thyroid lymphoma. SeminOncol. 1999;26:316-323.

Pederson RK, Pederson NT. Primary non-Hodgkin’s lymphoma of the thyroid gland: A population based study. Histopathology.1996;28:25-32. Foppiani L, Secondo V, Arlandini A, Quilici P, Cabria M, Del Monte P. Thyroid lymphoma: a rare tumor requiring combined management.Hormones (Athens). 2009; 214-8.

*Corresponding author: Dr. Swati Bhardwaj, 289, Sector-38, Gurgaon-122001, Haryana, India. Phone: +91 0124-2200289, +919811859747. Email: swat.bhardwaj@yahoo.com

Financial or other Competing Interests: None.

Date of Submission : 03.11.2016 Date of Acceptance : 03.04.2017 Date of Publication : 29.05.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Case Report DOI: 10.21276/APALM.1147

Cytology of Extrapulmonary Tuberculosis of Masseter Muscle: A Rare Case Report R. Ramya*, G. Barathi and D. Prathiba Department of Pathology, Sri Ramachandra University, Chennai, India

ABSTRACT Tuberculosis (TB) is a major health hazard Worldwide. India bears a huge burden of worldâ&#x20AC;&#x2122;s TB, according to World Health Organzation (WHO) statistics. The most common form of presentation is pulmonary tuberculosis. Extra Pulmonary presentation of TB in masseter muscle is extremely rare. We present a case of 26 year old male with right sided facial swelling, Fine Needle Aspiration Cytology (FNAC) of which revealed TB of masseter muscle. FNAC is a simple,cost effective out patient procedure that can aid in the early diagnosis of orofacial lesions such as extra pulmonary Tuberculosis. The purpose of this paper is to state the significance of early diagnosis of orofacial Tuberculosis and the crucial role played by FNAC in the diagnosis. Keywords: Masseter muscle, Extra pulmonary, Tuberculosis, Cytology

Introduction

Tuberculosis has been the alarming World health problem for many centuries. India holds a huge burden of Worlds TB[1].Pulmonary tuberculosis is the most common form of presentation. Extra pulmonary presentations can occur in Lymph nodes, Gastrointestinal tract, Liver, Genitourinary system, Central nervous system, Skin, Bones etc[2]. TB occurring in orofacial region is very rare[3]. Tuberculosis of the facial muscle is extremely rare, accounting to less than 1% of all TB cases[2]. The real challenge lies in the early diagnosis of such rare cases.Herein we report a case of extra pulmonary TB of masseter muscle diagnosed primarily by FNAC.The prompt onset of treatment has led to complete resolution of the disease.Hence the role of FNAC for orofacial lesions can be very vital for the early diagnosis of TB.

Fine Needle Aspiration of the lesion yielded 4.5ml of thick turbid pus. Cytology revealed epithelioid granuloma and dense inflammatory cells, composed predominantly of neutrophils and macrophages in a necrotic background(Figure 3 & 4). Special stains for Acid Fast Bacilli (AFB) was positive. This confirmed the diagnosis of tuberculosis (Figure 5). The patient was diagnosed of Tuberculosis by a simple FNAC procedure. The patient underwent anti tuberculous treatment for 6 months. He responded well to the treatment. His swelling subsided completely and he was disease free after 6 months. On repeated follow up, the patient was symptom free.

Case Report

A 26 year old male presented to the Pathology department with right sided face swelling of one month duration, which was associated with pain during swallowing. There was no history of fever or any other constitutional symptoms. On examination, the swelling was cystic and dumbbell shaped, measuring 7x4 cm. It was soft in consistency, non-fluctuant, with normal overlying skin, extending from nasolabial fold to epicanthus of right eye (Figure 1). Complete Blood Count (CBC) was within normal limits. Erythrocyte Sedimentation Rate(ESR) was 15mm/hr. Mantoux test was not performed. MRI of head and neck revealed a well circumscribed bilobed cystic lesion in the right masseter muscle extending up to right zygomatic region. There was no bony erosion noted(Figure 2). HIV status of the patient is Non reactive.

Fig.1: Patient with dumbbell shaped swelling in the right side of the face.

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Extrapulmonary Tuberculosis of Masseter Muscle

Fig. 2: MRI of head and neck showing well circumscribed cystic lesion in right side (Arrow).

Fig. 3: FNAC of swelling-MGG 200X showing granuloma with inflammatory cells in the background.

Fig. 4: FNAC of swelling-H &E 200X showing inflammatory cells in a necrotic background.

Fig. 5: AFB stain-Oil Immersion Field showing Acid Fast Bacilli (Circled).

Discussion

the most common form of presentation[8]. Extra pulmonary Tuberculosis (EPTB) refers to TB involving organs other than the lungs[11]. They constitute about 15% to 20% of all TB cases affecting immunocompetent people[4]. EPTB is on the rise worldwide. EPTB is often seen in immunocompromised patients, especially associated with HIV patients[12].

Tuberculosis has been haunting the mankind for a very long period. The reigns of the most cruel dictators have met its end, but Tuberculosis is a mere disease which still reigns this world from time immemorial. It has indeed evolved with a fierce rage towards mankind. TB is an alarming health hazard worldwide, with an estimated 9 million new cases and 1.5 million deaths every year[1]. TB is a chronic infectious granulomatous disease which can involve almost any part of the body.The causative organism for this dreadful disease is Mycobacterium tuberculosis also known as Kochâ&#x20AC;&#x2122;s bacillus. Pulmonary Tuberculosis is

TB of head and neck region comprises about 10% of all EPTB[12]. Orofacial tuberculosis is indeed a rare form of presentation and it can occur in sites such as gingiva, tongue, fausial pillars, buccal mucosa, temperomandibular joints and muscles of mastication[3]. Tuberculous oral lesions accounts for 0.05 to 5% of all TB cases[2,8].

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Ramya et al. The musculoskeletal TB accounts for nearly 3% of all EPTB[7]. Tuberculosis in masseter muscle is an extremely rare presentation, accounting to less than 1% of EPTB occurring in orofacial region[3]. TB of masseter muscle can cause trismus or present with submasseteric abscess[6,7]. Majority of these lesions are secondary lesions occuring mainly due to reactivation of latent primary infection such as cutaneous TB like scrofuloderma and spreading to sites like masseter muscle[6].Since there is no specific pathognomonic signs for TB in such cases, diagnosis can be very crucial[3]. In our case, the patient had no signs to point towards TB, except for the tender swelling in his right cheek. Orofacial lesions can occur in the form of ulcers, fissures, tuberculomas or nodules[5].These lesions may be painful or painless, solitary or multiple, irregular, well circumscribed ulcer[5,9].The main differential diagnosis for such lesions should be Tuberculosis. The diagnosis of these pauci bacillary lesion is often overlooked as it has variable modes of presentation[3,6].India being one of the capitals for TB, any suspicious lesion should always be investigated for TB. FNAC of these lesions are mandatory as it can guide us towards the cause. FNAC could be crucial in the initial diagnosis of diseases like Tuberculosis. FNAC proves to be less invasive and rapid in diagnosis for such cases. If FNAC is inconclusive due to the absence of granuloma or negative for AFB in the smear, diagnosis can be established by histopathological and microbiological study of tissue specimen[4]. Confirmation of the diagnosis can also be done by sending the material aspirated by FNAC for Polymerase Chain Reaction (PCR) analysis. In our case, cytology showed definite granuloma, followed which Acid fast bacilli was demonstrated by AFB staining. Thus clinching the early diagnosis of Tuberculosis by mere FNAC.

Conclusion

Extra pulmonary tuberculosis detection can be very challenging to the medical world .In Tuberculosis prevalent country like India, its always better to suspect TB if there is a long standing lesion in the head and neck region. Any orofacial lesion should undergo FNAC, as it can be the initial step towards diagnosis. EPTB occurring in the orofacial region,such as masseter muscle is extremely rare. FNAC being a simple cost effective test, when done

C-85 meticulously along with a thorough search for AFB on smear, can point to the definitive diagnosis. For patients who cannot afford ancillary molecular testing, FNAC & AFB staining of smears can prove to be a boon for early diagnosis of the disease. Definitive diagnosis of such cases of TB is vital, in order to initiate timely treatment regimen and to prevent complications.

Acknowledgements

We place our sincere thanks to the patient who was utmost cooperative till date.

Reference 1.

World Health Organization. Global tuberculosis report 2015. World Health Organization; 2015. 2. Jain P, Jain I. Oral manifestations of Tuberculosis: step towards early diagnosis. J Clin Diagn Res. 2014 Dec 1;8(12):18-21. 3. Andrade NN, Mhatre TS. Orofacial tuberculosis—a 16-year experience with 46 cases. Journal of Oral and Maxillofacial Surgery. 2012 Jan 31;70(1):e12-22. 4. Kishore DN, Geetha NT, Umashankara KV, Rai KK. Submasseteric Tuberculous Lesion of Mandible: Report of a Case and Review of the Literature. Case reports in dentistry. 2014 Jun 22;2014. 5. Prabhu SR, Daftary DK, Dholakia HM. Tuberculous ulcer of the tongue: report of case. Journal of oral surgery (American Dental Association: 1965). 1978 May;36(5):384-6. 6. Mascarenhas S, Tuffin JR, Hassan I. Tuberculous submasseteric abscess: case report. British Journal of Oral and Maxillofacial Surgery. 2009 Oct 31;47(7):566-8. 7. Yadav S, Madan K. Tuberculous ‘lock jaw’. BMJ case reports. 2015 Jul 9;2015:bcr2015211183. 8. Kamala R, Sinha A, Srivastava A, Srivastava S. Primary tuberculosis of the oral cavity. Indian Journal of Dental Research. 2011 Nov 1;22(6):835. 9. Kannan S, Thakkar P, Dcruz AK. Tuberculosis masquerading as oral malignancy. Indian journal of medical and paediatric oncology: official journal of Indian Society of Medical & Paediatric Oncology. 2011 Jul;32(3):180. 10. Sharma SK, Mohan A. Tuberculosis: From an incurable scourge to a curable disease-journey over a millennium. 11. Lee JY. Diagnosis and treatment of extrapulmonary tuberculosis. Tuberculosis and respiratory diseases. 2015 Apr 1;78(2):47-55. 12. Pandurang K, Shenoy VS, Bhojwani K, Alva A, Prasad V, Gandla S. Tuberculosis in the head and neck in India: down but not yet dead. J Mycobac Dis. 2014;4(148):2161-1068.

*Corresponding author: Dr Ramya. R, 6/4, Shreshtariverview apt, Woodcreek county, Nandampakkam, Chennai-89. India Phone: +91 9962500343 Email: sungeeth.ramya@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 07.11.2016 Date of Acceptance : 09.03.2017 Date of Publication : 29.05.2017

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Case Report DOI: 10.21276/APALM.1181

Echinococosis lung: A Cyto-Histopathogical Study in Two Cases Rimi Pandey1*, Nivedita Yadav1, Padam Kumari Agarwal1, Suruchi Shukla2, Mustafa Ali2, and Pooja Singh1 Pathology department. Vivekananda Polyclinic & Institute of Medical Sciences. Lucknow, India Microbiology Department. Vivekananda Polyclinic & Institute of Medical Sciences. Lucknow, India 1

ABSTRACT Echinococcosis is a parasitic infestation caused by the cestode of the genus Echinococcus. Man serves as an intermediate host and becomes infected by accidental ingestion of eggs from soil, water or by direct contact with the definitive host (dog). The present communication deals with two cases of echinococcosis of the lung. The presenting chief complaints were of breathlessness and dry cough. Routine haematological investigations revealed relative lymphocytosis in both the cases. However X-ray chest and CT showed a mass in the left lung in one case and a mass in the right lung in another case. For further evaluation CT guided fine needle aspiration cytology (FNAC) was performed in case one and the final diagnosis of hydatid cyst lung was established. In another case surgery was performed. Histopathological examination, established the diagnosis of echinococcosis lung. Both the patients were treated accordingly and they responded well to antihelminthic regime of therapy. Keywords: Echinococcosis Lung, Lung Mass, Cytology, Hydatid cyst,

Introduction

Hydatidosis also known as Echinococcosis remains a serious health hazard in endemic countries, like India. It is a zoonotic disease where the parasite completes its lifecycle involving definitive host (dogs). The man is infested by accidental ingestion of larval forms of Echinococcus. In the present cases, there was no history of contact with any of the animals, responsible for transfer of the infection to human beings. In humans, hydatid disease commonly involves the liver (70%) and the lungs (25%)[1]. In the later case, the children are more commonly infected as compared to adults. In the present communication, both the cases were young boys. Presumptive diagnosis of hydatid cyst was suspected on imaging techniques, however cytopathological or histological confirmation was considered essential prior to therapy. FNAC may lead to rupture of the cysts, resulting in hypersensitive/anaphylactic reaction of mild to moderate degree and also no reaction at all[2,3]. But, this procedure is still being carried out routinely, at some centres to confirm the diagnosis[3], since FNAC is an economical and simple technique and well accepted procedure. In the present cases, one of them was finally diagnosed on FNAC alone, whereas surgery was performed in another case, followed by histopathological confirmation.

Case Reports

Case 1: A 14-years-boy attended the outpatient department of a tertiary care hospital with chief complaints of loose stools, breathlessness and dry cough since last 3 months.

Routine investigations were within normal limits except for relative lymphocytosis. X-ray chest showed a mass near the pericardium. It was confirmed on CT scan, which revealed a massive lesion in left side thorax lying in relation to the pericardium showing homogeneous enhancement and specks of calcification, probably neoplastic in nature. CT guided FNAC was performed to confirm the diagnosis. Multiple slides were prepared, some of them were wet fixed in 95% alcohol for Papâ&#x20AC;&#x2122;s and H:E stain and others were kept air dried for MGG stain. All the slides were studied and reported by cytopathologist. On microscopic examination the cytosmears were covered by necrotic debris and showed neutrophilic and lymphocytic infiltrate. Reactive squamous epithelial cells (fig1a), multinucleated giant cells (fig1b) and proliferated capillaries (fig1c&d) were present. On careful screening occasional refractive lanceolate hooklets (fig1e & f) were present embedded in the necrotic debris. In another slides irregular thin fragments of bluish enucleated membranous structures covered by inflammatory cells (fig1g) were present. The surface was lined by thin germinal type of cells (Fig 1h), but laminations were not clear in cytosmears. To rule out the possibility of any associated infection, special stains for microorganisms were carried out, such as for tuberculosis, the cytosmears were stained with ZiehlNeelsonâ&#x20AC;&#x2122;s stain, which was negative for acid fast bacilli. But the spines were seen more prominently as AFB

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Yadav et al. negative structures (fig1g). Also the Grocottâ&#x20AC;&#x2122;s stain was performed to rule out any associated fungal infection. No fungal elements were present, but spines were better seen in the background of inflammatory exudate (fig1h). In one of the smear one scolex (fig1i) was also seen. Thus based on these findings final diagnosis of hydatid cyst (echinococcosis) lung was given. Later on, serology and repeated stool examination was advised which came out as negative. The patient was given a course of anti-helmithic antibiotics (Albendazole, Metrogyl and Ceconorm). He responded well as observed after two months of treatment. His repeat chest X-ray revealed only pneumonitis. No mass was seen. Case 2: An 8- year- old boy presented to the hospital with chief complaints of dry cough and respiratory distress since 2 days. Cough increased on sitting position and relieved on lying down. A year back he had a history of chest trauma following a fall after which he developed hematemesis. On examination of the chest, there were decreased breathing movements on the right side of chest and decreased breath sounds on the same area. He did not respond to conventional medication and started having frequent episodes of cough and respiratory distress. He was referred to this tertiary care centre for further management.

C-87 His chest x-ray revealed a cystic lesion in right lung (fig.2a). HRCT chest showed a large well defined cystic space, occupying lesion in the right lung along the interlobar fissure (fig.2b). A differential diagnosis of encysted pleural effusion or a hydatid cyst was made. Hematological investigation revealed mild leukocytosis only. Thoracoscopic excision of the cyst along with adjoining lung tissue from right middle lobe of lung was performed. Both the specimens were submitted for histopathological examination for confirmation of diagnosis. Post operatively antibiotics were started. He tolerated the anti-helmithic drugs well. Post-op x-ray revealed air in the cyst. The child did well after the operation and had no fresh complaints. Histopathological Examination: An opened up thin white glistening cyst measuring 9x3x1 cm. was received. (fig.2c). Another piece of adjoining lung tissue measured 2x0.6x0.5 cm. in size. Both the specimens were thoroughly examined and multiple pieces were submitted for microscopic examination. Histopathological examination revealed that the cyst wall was composed of laminated acellular structure, lined by germinal epithelium. Tissue from the lung revealed congested alveoli occupied by thin pinkish bits of hyadatid cyst wall and multiple scolexes of hydatid (fig.2e&f).

Fig. 1: (a-g) Cytosmears: (a) Multi-nucleated foreign body type of giant cells (H: E X 40). (b) Multinucleate giant cells with phagocytic leucocytes (H: E X 40). (c) Proliferated capillaries (H: E X 10), Higher magnification (inset, H: E X 100). (d) Necrotic background with bi-nucleate macrophage and a capillary (H: E X 40). (e) Membranous structures and necrotic debris (MGG X 10). (f) Hooklet lying on the necrotic background (H: E X 40). (g) Hooklets (Grocott X 100). (h) Scolex (MGG X 100).

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Echinococosis lung

Fig. 2(a-f):(a) Chest x-ray showing cystic lesion in right lung (b) HRCT chest showing large well defined cystic space along the inter-lobar fissure (c) Gross specimen: Opened up cyst (d)Histopathology: Laminated acellular cyst wall lined by germinal layer (H: E X 40). (e) Multiple scolex along the congested bronchial wall (H: E X 10); Multiple scolex (inset, H: E X 40). (f) Congested alveoli occupied by multiple scolex of hydatid wall (H: E X 10); scolex (inset, H: E X 100).

Discussion

Hydatid cyst is an infectious disease which is most commonly caused by the cestode Echinococcus granulosus and rarely by Echinococcus multilocularis[1]. In lung

hydatidosis one lobe, usually the basal one of the right lung is involved in 72% of the cases.[3] Our case no. 1 had a mass along the pericardium in the left lung, while the case no. 2 had cyst in the middle lobe of the, right lung which are not

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Yadav et al. common sites. The disease in the lung is common in the first two decades of life. Incidentally, both the present cases were young boys. The signs and symptoms depend on the size and the site of the lesion. The common presenting symptoms are cough, chest pain, and breathlessness. The case no.1, presented with chest pain and breathlessness while the other presented with chest pain and respiratory distress following chest trauma. He was asymptomatic before the trauma, but his chest symptoms were aggravated after trauma to the chest wall. Skouti et.al in 2015 reported a similar case of hydatid cyst following chest trauma. [4] Routine investigations in both the cases revealed leucocytosis with a normal eosinophil count. Haematological and biochemical tests are not reliable indicators to establish the diagnosis of hydatid disease. Casoni’s test and repeated stool examination were negative in the present cases. Radiological studies e.g. Plain X-ray films, Ultrasonography, Computed Tomography (CT), and Magnetic Resonance Imaging (MRI) are of great value in diagnosing and detecting the Echinococcal cysts and also determining the anatomic extent of the disease. Histological/cytological confirmation is mandatory before the treatment, especially surgery[6] Cytological findings have complementary role in the management of patients, particularly those with negative serology and normal eosinophil count. The finding of protoscolices, hooklets or hydatid membranes on cytosmears are the hallmarks of the diagnosis of an Echinococcus cyst. [7, 8] In our first case, FNAC confirmed the diagnosis of Echinococcus and the patient was treated accordingly. Another important feature in the cytosmears in our case no.1 was extensive tissue reaction, the knowledge of these changes is essential to avoid false positive diagnosis of a neoplasia or hamartoma. Possibility of various differential diagnoses like fungal infection, granulomatous lesions etc. was also ruled out by studying the smears for microbiological infections on special staining for fungus and acid fast bacilli. Anaphylaxis, which is a recognized complication, was not observed in the present case, as reported in earlier studies [8,9].The patient was given a

C-89 course of anti-helminthic drugs to which he responded well and his symptoms almost disappeared on follow up after about a year. The second child is doing well after surgery. No complications were noted in the case even after two years follow up. A word of caution, that the Hyadatidosis should be taken into account in paediatric patients of both the sexes, especially the boys presenting with pulmonary symptoms, irrespective of the side and the involved lobe of the lung by a unilateral pulmonary cystic/mass lesion. After radiological suspicion of a mass or an encysted pleural effusion in any lobe, a guided FNAC is mandatory to confirm the diagnosis before instituting medical/ surgical treatment for the proper treatment and better prognosis of the patients.

References 1.

2. 3. 4. 5. 6.

7. 8.

Eckert J, Schantz P, Gasser R,et al. Geographic distribution and prevalence. In: Eckert J, Gemmell MA, Meslin FX, Pawlowski ZS, eds. WHO/OIE Manual on Echinococcosis in Humans and Animals: a Public Health Problem of Global Concern. Paris: World Organization for Animal Health, 2001: 100-141. Eckert J, Deplazes P. Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern. ClinMicrobiol Rev. 2004; 17: 107–135 Ahmad S, Jalil S, Saleem Y, et al. Hydatid cysts at unusual sites: reports of two cases in the neck and breast. J Pak Med Assoc 2010; 60: 232-4. Sokouti M, Shokouhi B, Sokouti M et al. Giant Pulmonary Hydatid Cyst and Trauma in a 9 Year-Old Child: A Case Report. Open Respir Med J. 2015; 9: 67–69. Basavana GH, Siddesh G, Jayaraj BS, et al. A ruptured hydatid cyst of the lung. JAPI.2007; 55:141-45. Tekinbas C, Turedi S, Gunduz A, Erol M. Hydatid cyst disease of the lung as an unusual cause of massive hemoptysis: a case report. Journal of Medical Case Reports .2009; 3:21. Mitra S, Kundu S, Das S, Mukherjee S. Mediastinal hydatid disease: an unusual presentation. Indian J Chest Dis Allied Sci.2010; 52: 245-47. Das DK, Bhambhani S, Pant CS. Ultrasound guided fine needle aspiration cytology: diagnosis of hydatid disease of the abdomen and thorax. Diagn Cytopathol 1995 March; 12 (2): 173-6. Agarwal P K, Husain N, Singh B N. Cytologic findings in aspiratedhydatid fluid. Acta Cytol 1989; 33: 652-654.

*Corresponding author: Dr Rimi Pandey, M-8, sector-i, Near Prabhat Chauraha, Jankipuram, Lucknow (226021) India Phone: +91 9554464552 Email: rimi.may26@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 26.11.2016 Date of Acceptance : 02.04.2017 Date of Publication : 29.05.2017

eISSN: 2349-6983; pISSN: 2394-6466

Case Report DOI: 10.21276/APALM.1240

Postpartum TTP-HUS Syndrome: A Rare Autopsy Case Report in a Tertiary Care Hospital

Ganesh R. Kshirsagar, Chetan S. Chaudhari*, Prashant Vijay Kumavat, Nikita Patel and Nitin M. Gadgil Dept of Pathology, LTMMC & LTMGH, Sion, Mumbai, India

ABSTRACT Thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are the two main disorders included under Thrombotic Microangiopathy. 1 in 25,000 pregnancies present with these rare disorders, mostly seen after uncomplicated gestation and delivery. A diagnostic pentad for TTP was given by Amorosi & Ultmann in 1966: thrombocytopenia, microangiopathic hemolytic anemia, neurologic symptoms and signs, renal functional abnormalities and fever without other explanation. HUS has features of TTP along with acute renal failure.12%-31% of TTP patients are women during pregnancy or postpartum period. Pathological diagnosis requires hyaline thrombi in terminal arterioles and capillaries. We present a case of a 28 years old primigravida female, with 38 weeks of gestation, who was apparently alright earlier and presented with prolonged second stage of labour. However, day 2 postpartum she developed fever, breathlessness, loose motions and vomiting. She also developed pallor, icterus and petechial haemorrhages.Laboratory investigations revealed low platelet count, elevated bilirubin (direct more than indirect), elevated serum creatinine, and mildly raised hepatic transaminases. Day 5, she developed anuria and grade 3 dyspnoea and succumbed to death. Complete autopsy was performed .Histopathology on sections from kidneys revealed glomerular capillaries and arterioles showing platelet-fibrin thrombi and diffuse thickening of glomerular capillary wall with double contour of glomerular basement membrane. Lungs showed lobar pneumonia. Cause of death given was acute renal failure with lobar pneumonia with HUS-TTP in a postpartum female. TTP-HUS is a rare disorder and although some clinical features may suggest diagnosis, histopathological examination of renal specimen and applying special stains like PAS stain, silver stain and MSB (Mauritus,Scarlet,Blue) stain for fibrin, confirms the diagnosis. Keywords: Postpartum, TTP-HUS, Autopsy, Platelet-fibrin Thrombi

Introduction

Thrombotic Microangiopathy (TMA), where the characteristic pathogenesis is occlusive micro vascular thrombosis, clinically manifests as thrombocytopenia, microangiopathic hemolytic anemia, and variable features of organ ischemia. [1]Thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are the two primary syndromes under TMA.There are several distinct pathophysiologic mechanisms responsible for these syndromes. However it is difficult to distinguish between TTP and HUS. TTP was first described by Moschcowitz in 1924. HUS has triad of thrombocytopenia, anemia and renal insufficiency .TTP has pentad of thrombocytopenia, anemia, neurologic deficit, renal dysfunction and fever. [2, 3] HUS-TTP occurring postpartum is a rare entity, but if it manifests it is most commonly seen in early postpartum primiparous women with mean age of 27 years, after an uneventful uncomplicated pregnancy and delivery (no pre-eclampsia, hemorrhagic shock, DIC or sepsis). [3] It is characterized by acute onset renal failure, thrombocytopenia, and thrombotic microangiopathic hemolytic anemia, mildly elevated liver function tests, normal coagulation profile parameters of PT, aPTT and

increased serum Lactate Dehydrogenase levels. Risk factors include decrease in platelet count, fibrinolytic activity and prostaglandin (PGI-2) production. Other risk factors are deficiency of vWF-cleaving protease (ADAMTS 13), increase in fibrinogen, factor VIIa , VIII, vWF, plasma thrombomodulin levels plasminogen activator inhibitor-I and hypercoagulability. [3] Histopathological examination of renal biopsy often reveals dramatic destruction of the renal cortex .Glomerular thrombosis is characteristic, with an enlarged appearance that suggests capillary congestion rather than ischemia. Extension of thrombosis into the afferent arteriole is common. Mesangial changes appear to be uncommon. The tubules are often atrophic, may show necrosis, and frequently contain hyaline casts and red blood cells. [3,4]

Case Report

A 28 years old primigravida with 38 weeks of gestation, apparently alright earlier, was referred to our institute in view of prolonged second stage of labour. On day 2 postpartum, she developed fever without chills, breathlessness, loose motions and 10-15 episodes of vomiting. Her general condition was poor with pallor, icterus, petechial hemorrhages and ecchymoses all over

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Yadav et al. body. Clinical examination showed mild hepatomegaly. Laboratory investigations showed low Hb (8.3gm/ dl),elevated WBC count(14300/cu.mm), low platelet count (14000/cu.mm), elevated total/direct bilirubin (18.6/11.2 mg/dl), mildly elevated SGOT (144U/L) and SGPT (88U/L), raised serum creatinine (2.8 mg/dl), raised LDH (857 U/L). All viral markers were negative. On day 5, patient developed anuria and grade IV dyspnoea. Despite all resuscitative measures patient did not survive. A complete autopsy was performed. On gross examination, both kidneys were mildly enlarged and cut surface showed rim of peripheral cortical necrosis (Fig 1A). Liver was enlarged and yellowish. Lungs showed features of lobar pneumonia. Other organs did not show any remarkable gross features. For histopathological examination, Hematoxylin –Eosin stain (H & E), and special stains like Periodic Acid Schiff (PAS) stain, silver stain and MSB (Mauritus, Scarlet, Blue) stain for fibrin were used. H & E stained sections from kidneys revealed glomerular capillaries and arterioles showing platelet – fibrin thrombi and fragmented RBCs without vasculitis (Fig 1B).PAS stain and MSB (Mauritus, Scarlet, Blue) stain for fibrin showed glomerular capillary thickening, fibrinoid necrosis of endothelium and fibrin thrombi in capillaries and afferent renal arterioles (Fig 2 A,B,C). Silver staining showed diffuse thickening of glomerular capillary wall with double contours of Glomerular Basement Membrane (Fig 2D). Correlating clinical features, gross and microscopic findings, cause of death was given as acute renal failure with lobar pneumonia with HUS-TTP in a postpartum female.

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Discussion

HUS-TTP occurs commonly in women, especially among pregnant ones. In pregnancy, risk period is near term and during postpartum period. [4] This period also carries profound risk for thrombotic events and other pregnancy associated syndromes like preeclampsia, eclampsia, and HELLP syndrome (hemolysis, elevated liver enzymes and low platelets). These conditions may also present with thrombocytopenia, microangiopathic hemolytic anemia, neurologic symptoms, and renal insufficiency, as a result of which it is difficult in making their distinction from HUS-TTP. ADAMTS13 enzyme, a plasma reprolysin-like metalloprotease, is necessary for the proteolytic cleavage of von Willebrand Factor (vWF) under various conditions of oxidative stress. [4] Deficiency or impaired activity of this enzyme leads to TTP.The activity of ADAMTS13 progressively decreases during the course of pregnancy. During pregnancy, there is physiological increase in vWF concentrations .It has been shown that ADAMTS13 activity inversely correlates with plasma von Willebrand factor concentrations. [4] This may be the mechanism behind decreased levels of ADAMTS13 during pregnancy. The characteristic pathologic feature of both the disorders is the formation of platelet thrombi in microvasculature. Immune mediated injury or apoptosis of vascular endothelium causes endothelial damage which releases large amount of abnormal ultra large vWF multimers. This triggers formation of platelet thrombi, following which there is tissue ischemia/infarction and stress on RBCs passing through capillaries causing fragmentation.

Fig. 1: (A)Gross:Examination of cut surface of kidney shows rim of renal cortical necrosis.(B) Glomerular capillaries and arterioles showing platelet –fibrin thrombi and fragmented RBCs without vasculitis (H & E:100X ,inset:400X).

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Echinococosis lung

Fig. 2: PAS stain (A & B) and fibrin stain (C) showed glomerular capillary thickening, fibrinoid necrosis of endothelium and fibrin thrombi in capillaries and afferent renal arterioles. Silver staining (D) showed diffuse thickening of glomerular capillary wall with double contours of Glomerular Basement Membrane.

In this case, histopathology of the kidney showed that glomerular capillaries, glomerular infundibulum, afferent arterioles, terminal part of small interlobular arteries are involved (sparring venous circulation). There were amorphous, eosinophilic, hyaline plateletfibrin thrombi and fragmented RBCs without vasculitis. Diffuse edematous thickening and duplication of glomerular capillary wall (double contours) were seen. Tubules showed acute necrosis, RBCs and hyaline casts with edematous and fibrous interstitium. Mild mononuclear and RBCs infiltration noted near cortical necrosis. Electron microscopy in postpartum HUS-TTP showed fluppy electron dense material in expanded sub endothelial zone. Immunofluorescence showed granular fibrinogen deposition in sub endothelial zone in glomeruli and afferent arterioles.

Acute kidney injury is severe in women with pregnancy associated HUS-TTP and 76% women progress to end stage chronic kidney disease.[5]TMA associated maternal mortality has declined in recent years to 10-20%. However ,increased perinatal mortality rate (30-80%) has been reported ,reasons being growth restriction and placental infarction caused by thrombosis of decidual arteries.[5]Treatment in cases of HUS-TTP includes early plasmapheresis,steroids,antiplatelet therapy and hemodialysis (80-90% survival in acute cases).[5,6]

Conclusion

The occurrence of preeclampsia and related syndromes, the hypercoaguable state that occurs in late pregnancy and postpartum, and the progressively decreasing concentration of ADAMTS13 that occurs during late pregnancy may combine to increase the risk for the occurrence of HUS-

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TTP. The prognosis in pregnancy is poor. Although clinical manifestations are highly suggestive of the diagnosis of HUS-TTP, histopathology using routine and special stains is highly confirmatory and provides new insights in the pathogenesis of HUS-TTP.

George JN. The association of pregnancy with thrombotic thrombocytopenic purpura- hemolytic uremic syndrome. Curr Opin Hematol 2003, 10: 339–344.

Zheng XL. Structure-Function and regulation of ADAMTS13 protease. J Thromb Haemost. 2013; 11(01): 11-23.

Reference

Xheng XL, Sadler JE. Pathogenesis of Thrombotic Microangiopathies. Annu Rev Pathol.2008; 3: 249–277.

Machado S, Figueiredo N, Borges A, Pais MS. Acute kidney injury in pregnancy: a clinical challenge. J Nephrol 2012; 25(01):19-30.

Fakhouri F, Roumenina L, Provot F, Sallee M, Caillard S, Couzi L et al. Pregnancy-Associated Hemolytic Uremic Syndrome Revisited in the Era of Complement Gene Mutations. J Am Soc Nephrol. 2010; 21(5): 859–867.

Furlan M, Lammle B. Heamolytic-uraemic syndrome and thrombotic thrombocytopenic purpura-new insights into underlying biochemical mechanisms. Nephrol Dial Transplant 2000; 15: 1112-1114.

*Corresponding author: Dr. Chetan Sudhakar Chaudhari, 11, Jagannath darshan, M.D. Keni Road, Near RBI Quarters, Bhandup Village (E), Mumbai, India Phone: +91 9819133606 Email: drchetanchaudhari26@yahoo.co.in Date of Submission : 31.12.2016 Date of Acceptance : 18.02.2017 Financial or other Competing Interests: None. Date of Publication : 29.05.2017

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Case Report DOI: 10.21276/APALM.1265

Extra abdominal Umbilical Vein Varix Causing Stillbirth: A Case Report Luis Humberto Cruz Contreras1, Haneen Adnan Al-Maghrabi2*, Sareni Chávez Martínez1 1 Department of Pathology, Mother and Child Hospital, Irapuato, Guanajuato, México Department of Pathology, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia

ABSTRACT Stillbirth continues to be one of the most devastating events for patients and doctors. Efforts for determining the cause of death should always be made, to prevent endangering future pregnancies and obstetric complications. A comprehensive approach that includes autopsy, placental examination and ancillary studies such as microbiology and toxicology are the best way of establishing the cause of stillbirth. Careful examination of the placenta and umbilical cord is imperative since they are associated with common causes of stillbirth. Here we present a case of umbilical vein varix that presented with associated complications that resulted in intrauterine fetal death. Keywords: Stillbirth, Umbilical Vein Varix, Extra Abdominal

Introduction

The death of a formed fetus is one of the most emotionally devastating events for parents and clinicians.[1] The estimated stillbirth rate worldwide is around 3.2 million per year with the vast majority of the cases occurring in developing countries.[2] Fetal death that occurs before the stage of legal viability is called late spontaneous abortion or miscarriage while fetal death after the stage of legal viability is called intrauterine fetal death or stillbirth.[3] The death is indicated by the fact that after separation from the mother the fetus does not breathe or show any other evidence of life, such as beating heart, pulsation of the umbilical cord, or definite movement of voluntary muscles. [3] Once fetal death has occurred delivery may be safely accomplished either medically or surgically and expectant management is a safe alternative for interested patients. It is often difficult to determine a “certain” cause of death. [1] Common causes and risk factors for fetal death include chromosomal abnormalities, genetic syndromes, infections, placental abnormalities, fetal–maternal hemorrhage, maternal diseases such as diabetes and hypertension, antiphospholipid syndrome, thrombophilias, and abnormalities of multiple gestations.[1] Over 50% of fetal deaths are related to placental causes.[4] Autopsy, placental examination, and ancillary studies such as microbiology and toxicology are the best way of determining the cause of stillbirth. Efforts should be made at determining the cause since subsequent pregnancies may be at increased risk for fetal death and obstetric complications.[1] Statistics and knowledge gained from post-mortem examinations are an essential part of clinical practice, which facilitates

the research of stillbirth and is a crucial step towards its prevention.[3]

Case Report

We present the case of a 31-year-old women in her third pregnancy (Gravida 3, Para 1, abortus 1) without any relevant personal and family history, she had been attending prenatal care for low-risk pregnancies adequately, with a total of 6 visits to the clinic, all of them reported a pregnancy with normal evolution. She presented to the obstetric emergency department at 38 gestational weeks referring that she stopped perceiving fetal movements. Physical examination and ultrasound demonstrated intrauterine fetal death. The Patient underwent labor induction for vaginal delivery. Delivery was accomplished a day after she presented to our institution. A female stillbirth product was obtained. Amniotic fluid was referred as bloody by the attending gynecologist. The umbilical cord was cut and both Fetus and Placenta and were sent to the pathology department for post-mortem examination. The patient had no further complications. In pathology department, we received a female term stillbirth with no external malformations. The umbilical cord had been previously sectioned from the placenta. The segment attached to the abdominal wall (9 cm length) had avariable diameter, was ulcerated and hemorrhagic (Fig1). Serial sections of this segment revealed two normal arteries and an abnormal umbilical vein with irregular wall and a dilated lumen to a maximum of 1.9 cm, with progressive thinning of the surrounding Wharton’s Jelly and areas with ulceration. Additionally, an umbilical cord hematoma

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(1.9 cm greatest dimension) was found in close proximity to the dilated umbilical vein which was compressed. Placental findings were prominent vascular congestion

and focal hemorrhage (Fig. 2 and 3).The rest of the post mortem examination revealed only generalized vascular congestion. No other abnormalities were found.

Fig. 1: Gross examination of the external aspect of umbilical cord (fetal insertion) which showsdilated and hemorrhagic outer surface.

Fig. 2: Serial sections of the cord showing progressive dilatation of the umbilical vein (left to right, top to bottom) with progressive thinning of surrounding Wharton Jelly with ulceration and associated cord hematoma.

Fig. 3: Two cord sections (close-up) showing in detail the umbilical vein dilatation and cord hematoma(*).

Discussion

The umbilical cord enables the fetus to develop, grow, adapt, survive andborn healthy. Some umbilical cords abnormalities are well-established factors for gestational complications and impairment of fetal wellbeing. [5] Umbilical vein varix , a localized dilatation of vein diameter can present either intra-abdominal or extraabdominal (intra-amniotic).[6] Beraud and his colleagues defined the intra-abdominal umbilical vein varix according

to two criteria; umbilical vein diameter greater than or equal to 9mm, and an increase in the subhepatic segment diameter greater than 50% of the intrahepatic segment diameter.[7] This focal intra-abdominal vein dilatation is diagnosed by ultrasonography as dilated segment at the axial section of the abdomen. Thus, it is essential to examine the intra-abdominal portion of the umbilical vein. It is believed that the intra-abdominal segment can be examined and visualized easily in comparison with

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Extra abdominal Umbilical Vein Varix

the extra-abdominal vein varix. Ultrasound examination of the extra-abdominal vein varix usually shows normal umbilical vein study with no increase in diameter. Some cases might lead to hematoma and surface ulceration like our case. These changes might lead to bradycardia and caesarean section should be considered. As Fung and his group found in their studies that intra-abdominal varix of the umbilical vein implies a higher incidence of adverse perinatal outcomes, especially the fetal complications like anemia, chromosomal abnormalities, and intrauterine fetal death.[8] Cardiovascular anomalies can be seen frequently associated with trisomy 21 and 18. Other reported anomalies are trisomy 9, triploidy and mosaics like Turner syndrome.[8] Thus, careful ultrasound cardiac examination is required. Most of the literature had reviewed intraabdominal umbilical vein dilatation, but only a few studies report cases of extra-abdominal umbilical vein varix. [6] Due to the extreme difficulties in diagnosis at the prenatal period, it is usually made as post-natal diagnosis and confirmed by histopathology exam. Sadly, the majority of cases were diagnosed retrospectively during autopsy of antepartum dead fetus.[9] Moreover, there is an increase in the risk of stillbirth, fetal heart failure, thrombosis, and hydrops fetalis.[8, 10] Kanenishi and his colleagues report 13 cases of extra-abdominal intra-amniotic umbilical vein varices, in which half of them were diagnosed after birth. [11] This shows how difficult it is to diagnose it before birth. Intra-amniotic umbilical vein varix is associated with high frequency of umbilical vein thrombosis, antepartum fetal death, fetal anomalies and neonatal coagulation problems. [11] In these cases, the rate of caesarean sections is elevated with a good fetal outcome. Most of the studies in the literature emphasis the importance of prenatal diagnosis and regular follow up in order to establish the appropriate treatment, once the fetal lung is mature. However, the diagnosis of cases with extra-abdominal intra-amniotic umbilical vein varices remains a big diagnostic challenge by ultrasound studies. There are some risk factors that can lead to umbilical cord mechanical compression and blood ectasias, these include umbilical cord length, long cord (more than 70 cm) or short cord (less than 35 cm), reduced diameter (less than 8.0 mm), and excessive cord twisting (more than 0.3 cm/loop).[5] These might lead to serious complications like arterial or venous thrombosis, hematoma and ulceration.[5, 12, 13] Ulceration of the umbilical cord is one of the complications that can result in alteration of fetal heart rate. Many theories had been developed to explain the

cases of umbilical cord ulceration. These include vascular theory, reflux theory, and epithelial abnormality.[13] The first theory is related to vascular changes and ischemic changes which will lead to surface ulceration. The second theory is related to chemical material reflux like gastric and intestinal digestive enzymes effects. And the third theory is related to the abnormal epithelial changes like the bulla formation.[10] Other related causes of umbilical cord ulceration include smoking, patent omphalomesenteric duct, absence of Wharton’s jelly and injury by fetal nail.[13]

Conclusion

Extra-abdominal intra-amniotic umbilical vein varix is an uncommon anomaly. There are numerous reports in the literature about intra-abdominal vein varix which can be easily detected by ultrasound examination. Sadly, most of the extra-abdominal vein varix are detected in the post-natal period. Delivery of the fetus, as soon as lung maturation is confirmed, is recommended to reduce umbilical cord complication and fetal distress.

References 1.

Silver RM., et al., Work-up of stillbirth: a review of the evidence. American journal of obstetrics and gynecology, 2007. 196(5): 433-444.

Abraham A., et al., Umbilical Cord Haematoma Causing Still Birth-A Case Report. Journal of clinical and diagnostic research: JCDR, 2015. 9(12): QD01.

Lehtonen, T., et al., Causes of Stillbirth in Turku, Finland, 2001–2011. Pediatric and Developmental Pathology, 2017. 20(1):5-15.

Boyd, T.K., et al., The Stillbirth Classification System for the Safe Passage Study: Incorporating Mechanism, Etiology, and Recurrence. Pediatric and Developmental Pathology, 2016: p. 1093526616686251.

Oliveira, G.H.d., et al., Intrauterine thrombosis of umbilical artery-case report. Sao Paulo Medical Journal, 2016 (AHEAD).

Crespo, V., Variz venosa umbilical intramniótica. Reporte de un caso. Ginecol Obstet Mex, 2015. 83:356-362.

Beraud, E., et al., Umbilical vein varix: Importance of anteand post-natal monitoring by ultrasound. Diagnostic and interventional imaging, 2015. 96(1): 21-26.

Fung, T., et al., Fetal intra‐abdominal umbilical vein varix: what is the clinical significance? Ultrasound in obstetrics & gynecology, 2005. 25(2): 149-154.

Cruise, K.R. and G. Rouse, Klippel-Trenaunay-Weber syndrome complicated by extrafetal umbilical vein varix. Journal of Diagnostic Medical Sonography, 2002. 18(5):317-320.

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Contreras et al. 10. Sepulveda, W., et al., Fetal prognosis in varix of the intrafetal umbilical vein. Journal of ultrasound in medicine, 1998. 17(3): 171-175. 11. Dussaux, C., et al., Umbilical vein thrombosis: to deliver or not to deliver at the time of diagnosis? Clinical case reports, 2014. 2(6):271-273.

C-97 12. Viora, E., et al., Thrombosis of umbilical vein varix. Ultrasound in Obstetrics and Gynecology, 2002. 19(2): 212-213. 13. Maheshwari, B., et al., Umbilical cord ulceration: An underdiagnosed entity. Obstetrics & Gynecology Science, 2016. 59(5):388-392.

*Corresponding author: Haneen Adnan Al-Maghrabi, Department of Pathology, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia Email: haneen.almaghrabi@hotmail.com

Financial or other Competing Interests: None.

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Date of Submission : 07.01.2017 Date of Acceptance : 24.02.2017 Date of Publication : 29.05.2017

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Case Report DOI: 10.21276/APALM.1074

Water Clear Parathyroid Adenoma: A Report of 2 Cases Subhash C Yadav*, Kritika Singh and Pragati Sathe Department of Pathology, Seth G. S. Medical College, Parel, Mumbai, India

ABSTRACT Hyperparathyroidism occurs mainly due to parathyroid hyperplasia, adenoma and rarely carcinoma. Most of the adenomas are composed of chief cells. Water clear cell adenoma is a rare phenomenon. We are sharing two cases of parathyroid adenomas composed entirely of water clear cells and require high degree to suspicion to identify them as they have low endocrinological activity. Keywords: Water Clear Cell, Parathyroid Adenoma, Rare, Low Endocrinologic Activity

Introduction

Primary hyperparathyroidism, a parathyroid abnormality can be divided into three histological types: adenoma, hyperplasia and carcinoma with relative frequencies as 77.9%, 14.6% and 4.8% respectively. Solitary adenoma is the most common cause of primary hyperparathyroidism. Multiple adenomas can occur, but are extremely rare. Most of these adenomas are composed of chief cells or mixed cells. Oxyphil adenomas are rarer than chief cell adenomas. Water clear cell adenomas are even rarer causes of parathyroid adenoma with approximately 10 cases reported in the literature till now. We report two cases of primary hyperparathyroidism due to water clear cell adenomas which one should be aware of.[1-3]

Case Report

CASE 1: A 65- year- old diabetic female, presented with generalised bodyache and inability to stand from sitting position since 3 months. She underwent modified radical mastectomy followed by chemotherapy for right breast carcinoma a year back. Patient was evaluated and found to have hypercalcemia (11.9 mg/dl) with low serum phosphorus levels (2.1 mg/dl) and high serum parathyroid hormone (PTH) levels (254pg/ml). Bone scan did not suggest metastasis. Contrast enhanced computed tomography of neck showed 13x10 mm left inferior parathyroid adenoma with scintigraphic evidence of methoxyisobutylisonitrile (MIBI) avid lesion in the corresponding area. Subsequently, left inferior parathyroidectomy was done. Grossly, the received specimen measured 2x1.5x0.8cm and weighed 1.81 gms. CASE 2: A 28- year-old female, presented with five month history of left hip pain. There was no history of renal calculi, fracture, irregular menses or a significant family history. Magnetic resonance imaging showed a 6x2.5x1.8cm well defined, lytic lesion involving left proximal femoral

metadiaphysis suggestive of fibrous dysplasia. Laboratory tests revealed elevated serum calcium levels (11.9 mg/dl), low serum phosphorus levels (2.4mg/dl) and markedly increased serum PTH level of 483pg/ml. Triple phase venous computed tomography scan showed a right inferior parathyroid adenoma and MIBI confirmed uptake in the same region. The right inferior parathyroid gland was excised. Grossly, the gland measured 3cm×1cm×0.5cm weighing 2.47grams. Microscopic examination in both cases revealed encapsulated parathyroid neoplasms composed of tumour cells arranged in acinar pattern with few nests and sheets. (Fig 1a,1b) Monomorphic cells had basally located nuclei and abundant supranuclear clear cytoplasm. Focal anisonucleosis noted. (Fig 2a,2b,2c) Lesion was devoid of mature adipose tissue and a rim of compressed normal parathyroid gland tissue was seen at the periphery. The cytoplasm of these cells were positive for periodic acid Schiff (PAS) stain. (Fig 2d) There was no evidence of capsular or vascular invasion. Based on these histological findings, diagnosis of water-clear adenoma of parathyroid gland was given. Post operatively, calcium levels of both patients dropped to normal. Presently, they are on oral calcium supplementation and have been advised regular follow up.

Discussion

Primary hyperparathyroidism is a common endocrine disorder with an incidence of 21.6 per 1,00,000 person – years. Elevated serum calcium levels without associated symptoms are seen in approximately 80% of newly diagnosed cases of primary hyperparathyroidism.[2] Parathyroid adenomas, usually single however multiple adenomas, either unilateral or bilateral have been described and sometimes it is difficult to distinguish multiple adenomas from hyperplasia. Patients with multiple adenomas have relatively higher PTH levels.[3] Histologically, parathyroid

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Fig. 1a: Low power view showing an encapsulated parathyroid neoplasm (HE 100X). Fig. 1b: The tumour cells are predominantly arranged in acinar pattern with few nests and sheets (HE 100X).

Fig. 2a, 2b & 2c: High power view showing benign parathyroid neoplasms with predominant acinar pattern composed of monomorphic cells with basally located nuclei and abundant supranuclear watery clear cytoplasm (HE 400x) Fig. 2d: The cytoplasm of these cells were PAS positive indicating presence of glycogen (PAS 400X).

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Water Clear Parathyroid Adenoma

adenomas are composed of functioning parathyroid tissue containing single population of cells, consisting primarily of chief cells but can be sometimes mixed.[2] Described by Kovack’s, water clear cell parathyroid adenoma are very rare neoplasm with vacuolated to optically clear cytoplasm due to vesicles containing glycogen. It is not difficult to differentiate water clear cell adenoma (WCCA) from water clear cell hyperplasia (WCCH) as WCCH involves all the glands and WCCA is usually solitary. However, due to unequivocal distribution, sometimes it may be difficult to differentiate.[1,2] WCCH is also extremely rare however, unlike chief cell hyperplasia, it is not associated with any endocrine syndromes nor does it have a malignant potential connotation.[4] WCCH with primary hyperparathyroidism was first described in 1934 by Albright et al. For reasons unknown, there has been a steady decline in the incidences of WCCH & WCCA from 6.8% to 4.9%, and finally to less than 1%.[5-6] Water clear cells are not seen in normal parathyroid gland and its presence is usually associated with hyperfunctioning of gland. The proportion of these clear cells increases with advancing age and they probably represent transformed chief cells. Classically, WCCH starts in the superior parathyroid gland first and then involves the inferior parathyroid gland.[4] Electron microscopy done by Kovac, showed prominent glycogen deposits in the cytoplasm and cellular structures called annulate lamellae with rail like configuration. Holzman & Lange described numerous membrane limited vacuoles 1-5µ in diameter with occasional dense body, containing a finely particulate thread-like material. None of the authors demonstrated these findings in the normal parathyroid cells. The five theoretical possibilities for the origin of these vacuoles are dilated golgi bodies, dilated sacs of endoplasmic reticulum, swollen mitochondria, de novo produced by the cell or pinocytic or lysosomal vesicles.[3,5] Water clear cell type has most histologic features similar to a regular adenoma i.e. circumpscription, presence of a thin fibrous capsule and presence of a rim of relatively normal extra-capsular parathyroid tissue. Unlike its name, the WCCs are seldom entirely ‘‘clear’’ and are often variably

vacuolated, foamy, granular and clear.[1,6] The differential diagnosis considered are chief cell adenoma with extensive clear cell change. However, the entire tumor should not be replaced by clear cells and the clear cells of chief cell adenoma contains glycogen, which is PAS diastase sensitive. According to one report by Grenko et al. the water clear cell adenoma is negative for glycogen. Other differential diagnoses that can be considered are metastatic renal cell carcinoma, paraganglioma and clear cell change in salivary gland tumor like acinar cell carcinoma and mucoepidermoid carcinoma. Immunohistochemistry should be therefore done in suspected cases.[1] Despite high levels of parathyroid hormones, serum calcium levels are not markedly increased in cases of WCCA until they reach a large size indicating that these adenomas have low endocrinological activity as suggested by Kanda et al.[2]

Conclusion

The aim of this report is to draw attention to this uncommon variant of parathyroid adenoma seen in two cases composed entirely of water clear cells that has low endocrinologic potential and hence needs high level of suspicion.

Reference 1.

Liang Y. Mojica W. Chen F Water-Clear Cell Adenoma of Parathyroid Gland: A Case Report and Literature Review: N A J Med Sci. 2010;3(4):194-198

Piggott R. Water P. Ashraf J. Colesky F. Kerin M.J. Waterclear cell adenoma: A rare form of hyperparathyroidism Int J Surg Case Rep. 2013; 4(10): 911–913

Kazuko K. Yosuke O. Takahisa T , et. Al. A rare case of primary hyperparathyroidism with clear cell adenoma: Endocrine journal 2004;51: 207-212,

Ezzat T, Maclean G, Parameswaran R, et al. Primary hyperparathyroidism with water clear cell content: the impact of histological diagnosis on clinical management and outcome. Annals of The Royal College of Surgeons of England. 2013;95(3):e60-e62.

Roth SI. The ultrastructure of primary water-clear cell hyperplasia of the parathyroid glands. Am J Pathol. 1970;61(2):233-48

Kuhel WI, Gonzales D, Hoda SA, Pan L, Chiu A, Giri D, DeLellis RA. Synchronous water-clear cell double parathyroid adenomas a hitherto uncharacterized entity? Arch Pathol Lab Med. 2001;125(2):256-9

*Corresponding author: Dr. Yadav Subhash C., Department of Pathology, Seth G.S.Medical College, Parel, Mumbai-400012. India. Phone: +91 9833844678 Email: drsubhashyadav23@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 01.10.2016 Date of Acceptance : 15.03.2017 Date of Publication : 29.05.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

Letter to Editor DOI: 10.21276/APALM.1410

Epithelioid Glioblastoma, A Newly Described Rare Variant of Common Tumor Describing Immunohistochemical Expression Pattern Rakesh Kumar Gupta1*, Neha Garg2, Vineeta V Batra1 and Lalit Garg3 Departments of Pathology, G B Pant Institute of Postgraduate Medical Education and Research, New Delhi India. 2 Department of Pathology, Geetanjali Medical College and Hospital, Udaipur, Rajasthan, India. 3 Departments of Radiodiagnosis, G B Pant Institute of Postgraduate Medical Education and Research, New Delhi, India 1

Dear Sir,

Epithelioid glioblastoma (Ep-GBM) has recently been recognized as a distinct variant of glioblastoma (GBM), which is included in the 2016 revised World Health Organization (WHO) classification of central nervous system (CNS) tumors.[1] Together with giant cell glioblastoma and gliosarcoma, it is kept under the broad category of IDH-wildtype glioblastoma. Ep-GBM is characterized by large cells with eccentric nuclei, vesicular chromatin, prominent nucleoli and abundant amount of eosinophilic cytoplasm. In addition, they may also show glandular, ductular and squamoid pattern expressing both GFAP and epithelial markers (cytokeratins) thus closely mimicking with the metastatic carcinomas.[2] Hence, they should be carefully distinguished from metastatic carcinomas of unknown primaries. In contrast to typical GBM, they possess a higher predilection for children and younger adults, and usually present as superficial cerebral mass. Epithelioid GBMs frequently shows BRAF-V600E mutation.[3] Rhabdoid glioblastoma is a close histological mimicker of Ep-GBM, however it is differentiated by the loss of expression of INI-1.[4] A 48-years old male presented to the Emergency Department of Neuro-surgery with the sudden onset of multiple episodes of vomiting, headache and weakness in the right half of body. On examination, patient was conscious, oriented with a normal Glasgow coma score. The power was 4/5 in all the four limbs. Haemogram, serum electrolytes, kidney and liver functions were within normal limits. A Non contrast computer tomography scan was done which revealed a large intra-axial heterogenous hyperdense mass lesion with significant peri-lesional edema and midline shift (Figure 1A). The magnetic resonance imaging showed large intra-axial based mass lesion in left para-falcine region in parieto-occipital lobe showing CSF cleft surrounding the tumor brain interface with extensive peri-lesional white matter edema, intra-

lesional hemorrhage and mass effect (Figure 1B-1F). A radiological diagnosis of high grade glial tumor with intra-lesional bleed was made. A left parieto-occipital flap craniotomy with complete excision of tumor was done. Pathological findings: The tumor was comprised of large epithelioid cells arranged in sheets with perivascular clustering displaying eccentric nuclei with vesicular chromatin, prominent nucleolei and abundant amount of eosinophilic cytoplasm (Figure 2). Frequent bi and multinucleation, necrosis and atypical mitotic figures were noted. The cells showed diffuse positivity for GFAP, S-100, epithelial membrane antigen (EMA), CK20 and vimentin (Figure 3). Tumor showed retention of INI-1 expression, while IDH-1 was negative. BRAF-V600E mutation analysis was not performed due to unavailability of resources. Based on morphology and immunohistochemistry a final diagnosis of IDH-wild type Ep-GBM was made. The first case of Ep-GBM was reported by Kepes et al followed by few subsequent case reports. Ep-GBM possesses a poor outcome with survival less than the median survival time of 14.6 months in conventional GBM.[5] Most of the reported cases of Ep-GBM occurred denovo as a primary lesion. However, Alexandrescu et al showed that Ep-GBM and epithelioid pleomorphic xanthoastrocytoma (PXA) shares many molecular features such as BRAF-V600E mutation, wild type IDH, retained INI-1 expression and frequent loss of ODZ3 deletion.[6] Similarly, Tanaka et al also described a case of Ep-GBM arising in a patient diagnosed with PXA 13-years after the treatment.[7] Considering the above two examples it seems that Ep-GBMs are closely related with PXA which is a low grade glial tumor in comparison to conventional GBMs. However, since Ep-GBM has poor outcomes than typical GBM as well as uncertain response to temozolamide, this hypothesis is questionable and needs further evaluation. In our case, patient was referred for chemotherapy and later lost to follow-up.

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Gupta et al.

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Fig. 1: (A) Axial NCCT image showing large intra-axial heterogenous hyperdense mass lesion with significant peri-lesional edema and midline shift. MRI images (B-F), B (Sagittal T1), C (Axial T2), D (Coronal FLAIR), E (Post Contrast), F (Gradient sequence) showing a large heterogenous signal intensity intra-axial lesion in left parietal region in para-falcine location. Lesion shows heterogeneous post contrast enhancement with central non enhancing area suggesting necrosis. There is blooming on gradient sequence suggestive of hemorrhage. Significant peri-lesional edema and midline shift noted.

Fig. 2: Photomicrographs showing a) A tumor comprising of large epithelioid cells arranged in sheets with area of necrosis (red arrow) and frequent bi and multi-nucleation (black arrow) (HEx100), b) Many atypical mitotic figures (arrow heads) (HEx200) and c) The cells displaying eccentric nuclei with vesicular chromatin, prominent nucleolei and abundant amount of eosinophilic cytoplasm (arrow) (HEx200).

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Epithelioid Glioblastoma

Fig. 3: The photomicrographs showing immunohistochemical expression in tumor cells a) GFAP diffuse cytoplasmic and fibrillary (IHCx200), b) CK20 membranous (IHCx200), c) EMA membranous (IHCx200), d) S-100 both nuclear and cytoplasmic staining (IHCx200), e) vimentin cytoplasmic (IHCx200) and retention of INI-1 positivity (arrow showing positive internal control in endothelial cell) (IHCx200).

References 1.

Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK et al. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathol. 2016;131(6):803-20. Tanaka S, Nakada M, Hayashi Y, Nakada S, SawadaKitamura S, Furuyama N, et al. Epithelioid glioblastoma changed to typical glioblastoma: the methylation status of MGMT promoter and 5-ALA fluorescence. Brain Tumor Pathol. 2011;28(1):59-64. Kleinschmidt-DeMasters BK, Aisner DL, Birks DK, Foreman NK. Epithelioid GBMs show a high percentage of BRAF V600E mutation. Am J Surg Pathol. 2013;37(5):685-98. Kleinschmidt-DeMasters BK, Alassiri AH, Birks DK, Newell KL, Moore W, Lillehei KO. Epithelioid versus

rhabdoid glioblastomas are distinguished by monosomy 22 and immunohistochemical expression of INI-1 but not claudin 6. Am J Surg Pathol. 2010;34(3):341-54. 5.

Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ et al. European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups. National Cancer Institute of Canada Clinical Trials Group. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352(10):987-96.

Alexandrescu S, Korshunov A, Lai SH, Dabiri S, Patil S, Li R et al. Epithelioid Glioblastomas and Anaplastic Epithelioid Pleomorphic Xanthoastrocytomasâ&#x20AC;&#x201D;Same Entity or First Cousins? Brain Pathol. 2016;26(2):215-23.

Tanaka S, Nakada M, Nobusawa S, Suzuki SO, Sabit H, Miyashita K et al. Epithelioid glioblastoma arising from pleomorphic xanthoastrocytoma with the BRAF V600E mutation. Brain Tumor Pathol. 2014;31(3):172-6.

*Corresponding author: Dr Rakesh Kumar Gupta, Senior Resident, Department of Pathology, G B Pant Institute of Postgraduate Medical Education and Research, New Delhi -110002 Phone: +91 9718599076 Email: rakesh.newjobi@gmail.com Date of Submission : 14.03.2017 Date of Acceptance : 12.04.2017 Financial or other Competing Interests: None. Date of Publication : 28.05.2017

Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 3, May-June, 2017

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