APALM 3.4 (2016) by PaGe

eISSN: 2349-6983 pISSN: 2394-6466

Annals of Pathology and Laboratory Medicine October-December. 2016; Vol. 3, Issue 4

Cover design: Dr Prashant

DOI : 10.21276/apalm

www.apalm.org

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

Annals of Pathology and Laboratory Medicine Co-Editor in Chief

Dr Rajan Chopra King Fahad Hospital, Hufof, Saudi Arabia 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 Amado Ona Tandoc III Research Institute for Tropical Medicine, Muntinlupa City, Philippines 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

Dr Sarah Iqbal Ch Faculty of Pathology King Edward Medical University, Lahore, Pakistan Dr Jerad M Gardner Asst Prof, Pathology and Dermatology, University of Arkansas for Medical Sciences, Little Rock, AR, United States Dr Naila Atif Associate Prof., Histopathology, Central Park Medical College, Lahore, Pakistan

Published by: Pacific Group of e-Journals (PaGe) Kotla Market, Mayur Vihar, New Delhi (India) Website: www.pacificejournals.com www.apalm.org E-mail: contactus@pacificejournals.com

Advisory Editors

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

Annals of Pathology and Laboratory Medicine Disclaimer

Annals of Pathology and Laboratory Medicine (APALM) is an international, Double-blind peer-reviewed, indexed, open access, online and print journal with an Impact Factor (IFJ): 2.8952 and IC Value (ICV 2014): 67.16 published by ‘Pacific group of e-Journals’ (PaGe), an ISO 9001:2008 Certified academic publishing house.

APALM is the top ranking Indian Journal in the field of Pathology and Laboratory Medicine, with the highest IC Value (67.16) amongst the Indian Journals in the field of Pathology and Laboratory Medicine that are indexed with Index Copernicus.

Publication Frequency Four per year, i.e. Quarterly.

•

Original Article Hepatic Expression of Nitric Oxide Isoforms and Serum Nitrites/Nitrates in Chronic

A249-A258

Evaluation of RAGE (Receptor for Advanced Glycation End-products) Expression in Gastric Carcinoma of Egyptian patients in Relation to Helicobacter pylori Infection Tarek Aboushousha, Afkar Badawy, Mona Moussa, Zeinab Omran, Ahmed-Hazem Helmy, Magdy Youssef

A259-A268

Utility of latex agglutination test in the diagnosis of acute pyogenic meningitis Shilpa Dayanand Putta, Smita Deshpande, Renu Bharadwaj

A269-A275

Platelet Volume Indices: Silver linings for vascular complications in diabetes mellitus Mitakshara Sharma, Sanjeev Narang, S K Nema

A276-A283

Study of serum magnesium levels in Type 2 Diabetes mellitus at tertiary medical centre, Karnataka, India Vidya Baleguli, Kishan Prasad HL, Kishan Prasad HL, U Sanjana Rao, U Sanjana Rao, Chandrika Rao, Chandrika Rao, Suchetha Kumari, Suchetha Kumari

A284-A288

In any condition and due to any reason, if any Educational/Research Institution (College or University) denies accepting the research paper published in APALM, it will not be the responsibility of the Editor, Publisher or the Management.

An Insight Into Pituitary Fossa Lesions: A single institutional experience Purwa Rangrao Patil, Bhavana Madhukar Bharambe, Divyaja Sondankar

A289-A295

Clinicopathological study of primary focal segmental glomerulosclerosis: A new vision of all variants Sakhi Anand, Aminder Singh, Mary Mathew

A296-A305

All the legal disputes related to PaGe are subject to Delhi Jurisdiction only

Correlation Of Histopathological, Biochemical & Clinical Spectrum Of Chronic Hepatitis B: A New Look In To Old System Aminder Singh, Bhavna Garg, Neena Sood, Ajit Sood

A306-A313

Emergence of Chikungunya infection in North India Krunal D Mehta, Shruti Malik, K P Singh, T N Dhole

A314-A319

Apoptosis and micronucleus in cervical pap smears: promising assays to increase the diagnostic value of the test. Amoolya Bhat, Vijaya C, Padmasri R

A320-A328

Utility of modified bleach method technique for the demonstration of acid fast bacilli in the diagnosis of Tuberculous lymphadenopathy in comparison to routine Ziehlneelsen staining Manika Khare, Manju Kaushal

A329-A332

1. Thoughts, Language and examples in published research papers are entirely of Authors of Research Papers. It is not necessary that both Editor and Editorial Board are satisfied by the research paper. The responsibility of the matter of the research paper/ article is entirely of author. Scientific knowledge is ever changing. As new research and experiences broaden our knowledge, old concepts may be required to be looked into afresh. The Authors and editors of the material herein have consulted sources believed to be reliable in their efforts to provide information that is complete and in accord with the standards accepted at the time of publication. However, in view of the possibility of human error by the authors, editors, or publisher, nor any other who has been involved in the preparation of the work warrants that the information contained herein in every aspect accurate or complete, and they are not responsible for any errors or omissions or the results obtained from the use of such information. Readers are advised to confirm the information contained herein with other sources.

APALM publishes original, peer-reviewed articles for pathologists and clinical laboratory scientists. APALM is a specialized journal in the field of Pathology and Laboratory Medicine which, inter alia, includes Histopathology, Cytopathology, Hematology, Clinical Pathology, Forensic Pathology, Blood Banking, Clinical Bio-Chemistry, Medical Microbiology (Bacteriology, Virology, Mycology, Parasitology), etc.

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Contents

Only the First author is entitled to receive the print copies of all co-authors.

Hepatitis C With or Without Schistosomiasis Afkar Abdel-GhanyBadawy, Olfat Hammam, Mona Moussa, Raafat Atta, Tarek Aboshousha, Noha Said, Nihal El-Assaly, Nawal El-Badrawy

III

Annals of Pathology and Laboratory Medicine Disclaimer

Publication Frequency Four per year, i.e. Quarterly.

•

Original Article Hepatic Expression of Nitric Oxide Isoforms and Serum Nitrites/Nitrates in Chronic

A249-A258

A259-A268

Utility of latex agglutination test in the diagnosis of acute pyogenic meningitis Shilpa Dayanand Putta, Smita Deshpande, Renu Bharadwaj

A269-A275

Platelet Volume Indices: Silver linings for vascular complications in diabetes mellitus Mitakshara Sharma, Sanjeev Narang, S K Nema

A276-A283

A284-A288

An Insight Into Pituitary Fossa Lesions: A single institutional experience Purwa Rangrao Patil, Bhavana Madhukar Bharambe, Divyaja Sondankar

A289-A295

Clinicopathological study of primary focal segmental glomerulosclerosis: A new vision of all variants Sakhi Anand, Aminder Singh, Mary Mathew

A296-A305

All the legal disputes related to PaGe are subject to Delhi Jurisdiction only

Correlation Of Histopathological, Biochemical & Clinical Spectrum Of Chronic Hepatitis B: A New Look In To Old System Aminder Singh, Bhavna Garg, Neena Sood, Ajit Sood

A306-A313

Emergence of Chikungunya infection in North India Krunal D Mehta, Shruti Malik, K P Singh, T N Dhole

A314-A319

Apoptosis and micronucleus in cervical pap smears: promising assays to increase the diagnostic value of the test. Amoolya Bhat, Vijaya C, Padmasri R

A320-A328

A329-A332

Contents

Only the First author is entitled to receive the print copies of all co-authors.

Hepatitis C With or Without Schistosomiasis Afkar Abdel-GhanyBadawy, Olfat Hammam, Mona Moussa, Raafat Atta, Tarek Aboshousha, Noha Said, Nihal El-Assaly, Nawal El-Badrawy

III

Case Report

Seroprevalence of Transfusion transmitted infections by using 4th generation Enzyme- linked immunosorbent assay kit: A 3 year study in a tertiary health care centre of Delhi Mohammad Jaseem Hassan, Sabina Khan, Zeeba Shamim Jairajpuri, Safia Rana, Salamah Parveen Imteyaz, Sujata Jetley

A333-A339

Association of thyroid disorders with the hormones of anterior pituitary in female infertiltiy Rakhee Yadav, Sanjiv Kumar Bansal, Busi Karunanand, Eram Hussain Pasha, Birendra Kumar Yadav, Smita Prasad Shastry

A340-A346

Thyroid cytology evaluation based on the Bethesda system with clinico-morphological correlation Dimple Mehrotra, Anita A M, Sainath K Andola, Anuradha G Patil

A347-A355

“Bacteriological profile and antimicrobial sensitivity pattern of clinical isolates from patients attending tertiary care hospital” Santosh Kotgire, Sunil Hatkar, Sufia Siddique, A B Deshmukh, Uzma Afreen, Sayyed Mariya

A356-A361

A Unique case of Multiple Endocrine Neoplasm-1 Syndrome satisfying all six WHO criteria Rushabh Jitendra Shah, Samruddhi Dilip Rajpurkar

C170-C174

A rare case of leukemia cutis in T-cell acute lymphoblastic leukemia (T-ALL) in an elderly patient Anshu Gupta, Sadhana Tiwari, Tanima Dwivedi

C175-C180

Autoimmunization in Thalassemia: A case report with review of literature Sangeeta Pahuja, Geetika Sharma, P. Lalita Jyotsna, Richa Chauhan

C181-C185

Membranous Nephropathy: A Rare Association With Renal Vein Thrombosis Ankur Mittal, Ruchi Gupta, Pavneet Selhi, Harpreet Kaur, Neena Sood, Jasvinder Sandhu

C186-C189

Brunner’s gland hamartoma- A rare cause of upper gastro-intestinal bleed: report of two cases emphasising pathogenesis Rakesh Kumar Gupta, Ravindra Kumar Saran, Shivakumar Varakanahalli, Amarender Singh Puri

C190-C193

An extremely rare distant non communicating uterine horn mimicking parasitic leiomyoma: a diagnostic dilemma Abhijit Das, Nidhi Verma, Sompal Singh, Namrata Nargotra, Rakesh Deepak

C194-C198

Primary sino-nasal T cell lymphoma in a young female: A rare case report Rumpa Das, Gorakh Nath, Sangita Bohara, Ritu Sharma, Aarti B Bhattacharya, Vivek Gupta

C199-C202

Cytological diagnosis of fibromatosis coli: a rare presentation of infantile neck swelling Abhijit Das, Namrata Nargotra, Ila Tyagi, Ritu Arora, Sompal Singh

C203-C206

Primary Splenic Lymphoma Presenting as Splenic Abscess: A Rare Entity Anshu Jain, Noora Saeed, Veena Maheshwari, Abdul Bari

C207-C211

Persistent eosinophilia in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma and TP53 deletion is a potential predictor of variant Richter’s transformation Madhu M Ouseph, James N Butera, Rogers C Griffith, Dariusz Stachurski, Diana Olguta Treaba

C212-C216

Balantidium coli in urine microscopy: A mere coincidence or more. Pratibha Mane, Jyoti Sangwan, Ashok Kumar Malik

C217-C219

Cholesterol granuloma of paratesticular tissue: report of a rare case Amoolya Bhat, Vijaya C, D H Biradar, Safarulla ., Suraksha B Rao

C220-C223

Unusual Cause of Splenomegaly: Splenic Cyst Anitha Chakravarthy, Kishan Prasad HL, Chandrika Rao, Jayaprakash Shetty

C224-C227

Type I (Non-neuronopathic) Gaucher’s disease with bone marrow involvement: a rare case report. Mallikarjun Adiveppa Pattanashetti, Ramesh Yashwantrao Chavan

C228-C232

Acute Oxalate Nephropathy in a young boy due to ingesting Averrhoa bilimbi; case report and literature review of an under-recognized cause of tropical renal disease Niranthi Ruwini Perera, Hiranya Dulanjalie Tennekoon, Randula Ranawaka

C233-C235

Letter to editor Nucleic amplification testing: Individual verses Minipool, what to choose? Rateesh Sareen

Published by: Pacific Group of e-Journals (PaGe) Kotla Market, Mayur Vihar, New Delhi (India)

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

E-mail: contactus@pacificejournals.com

L15-L16

Case Report

A333-A339

A340-A346

Thyroid cytology evaluation based on the Bethesda system with clinico-morphological correlation Dimple Mehrotra, Anita A M, Sainath K Andola, Anuradha G Patil

A347-A355

A356-A361

A Unique case of Multiple Endocrine Neoplasm-1 Syndrome satisfying all six WHO criteria Rushabh Jitendra Shah, Samruddhi Dilip Rajpurkar

C170-C174

A rare case of leukemia cutis in T-cell acute lymphoblastic leukemia (T-ALL) in an elderly patient Anshu Gupta, Sadhana Tiwari, Tanima Dwivedi

C175-C180

Autoimmunization in Thalassemia: A case report with review of literature Sangeeta Pahuja, Geetika Sharma, P. Lalita Jyotsna, Richa Chauhan

C181-C185

Membranous Nephropathy: A Rare Association With Renal Vein Thrombosis Ankur Mittal, Ruchi Gupta, Pavneet Selhi, Harpreet Kaur, Neena Sood, Jasvinder Sandhu

C186-C189

C190-C193

An extremely rare distant non communicating uterine horn mimicking parasitic leiomyoma: a diagnostic dilemma Abhijit Das, Nidhi Verma, Sompal Singh, Namrata Nargotra, Rakesh Deepak

C194-C198

Primary sino-nasal T cell lymphoma in a young female: A rare case report Rumpa Das, Gorakh Nath, Sangita Bohara, Ritu Sharma, Aarti B Bhattacharya, Vivek Gupta

C199-C202

Cytological diagnosis of fibromatosis coli: a rare presentation of infantile neck swelling Abhijit Das, Namrata Nargotra, Ila Tyagi, Ritu Arora, Sompal Singh

C203-C206

Primary Splenic Lymphoma Presenting as Splenic Abscess: A Rare Entity Anshu Jain, Noora Saeed, Veena Maheshwari, Abdul Bari

C207-C211

C212-C216

Balantidium coli in urine microscopy: A mere coincidence or more. Pratibha Mane, Jyoti Sangwan, Ashok Kumar Malik

C217-C219

Cholesterol granuloma of paratesticular tissue: report of a rare case Amoolya Bhat, Vijaya C, D H Biradar, Safarulla ., Suraksha B Rao

C220-C223

Unusual Cause of Splenomegaly: Splenic Cyst Anitha Chakravarthy, Kishan Prasad HL, Chandrika Rao, Jayaprakash Shetty

C224-C227

Type I (Non-neuronopathic) Gaucher’s disease with bone marrow involvement: a rare case report. Mallikarjun Adiveppa Pattanashetti, Ramesh Yashwantrao Chavan

C228-C232

C233-C235

Letter to editor Nucleic amplification testing: Individual verses Minipool, what to choose? Rateesh Sareen

Published by: Pacific Group of e-Journals (PaGe) Kotla Market, Mayur Vihar, New Delhi (India)

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

E-mail: contactus@pacificejournals.com

L15-L16

Original Article Hepatic Expression of Nitric Oxide Isoforms and Serum Nitrites/ Nitrates in Chronic Hepatitis C With or Without Schistosomiasis Afkar Abdel-Ghany Badawy1, Olfat Hammam1, Mona Moussa1, Raafat Atta2, Tarek Aboshousha1, Noha Said Helal1*, Nihal El-Assaly3 and Nawal El-Badrawy2 Department of Pathology, Theodor Bilharz Research Institute, Giza, Egypt. Department of Gastroenterology & Hepatology, Theodor Bilharz Research Institute, Giza, Egypt. 3 Department of Clinical Chemistry, Theodor Bilharz Research Institute, Giza, Egypt. 1

Keywords: Inducible nitric oxide synthase, Endothelial nitric oxide synthase, Chronic hepatitis C, Schistosomiasis, Immunohistochemistry.

ABSTRACT Background: Role of nitric oxide (NO) in pathogenesis of chronic viral hepatitis is not fully understood but it seems that its overproduction is responsible for the pathological changes under inflammatory conditions. Methods: This study was undertaken to evaluate hepatic expression of inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) as well as assessment of serum nitrates/nitrites representative to NO release in Egyptian chronic hepatitis C (CHC) with or without schistosomiasis and their relation to histopathology in 72 core liver biopsies from CHC patients. Hepatic sections were immunohistochemically stained by antibodies of iNOS and eNOS. Results: In control livers, iNOS was detected in hepatocytes and localized mainly in periportal zone of liver acinus, while eNOS was uniformly distributed in hepatocytes as well as in sinusoidal and vascular endothelium. In diseased livers, both isoforms overexpressed in a diffuse distribution pattern, eNOS translocated to hepatocytic nuclei, and iNOS consistently labeled portal tract inflammatory cells. Over expression of iNOS and serum level of NO correlated with hepatitis activity and fibrosis, while that of eNOS correlated with Schistosomal coinfection. Conclusions: Chronic hepatitis C is accompanied by significant iNOS up-regulated expression and increased serum level of NO that reflects disease severity. Schistosomal coinfection can be considered as a risk factor for haemodynamic disturbance. Further studies are required to determine whether iNOS inhibitors could be useful in reducing liver disease severity and improve the benefits of antiviral therapies.

*Corresponding author: Noha Said Helal, Pathology Department, Theodor Bilharz Research Institute, El-Nile Street, Warrak El-Hadar, Imbaba, PO Box 30, Giza 12411, Egypt. Phone: +20 235401019 Email: nohasaidhelal@yahoo.com

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

A-250

Nitric Oxide Isoforms in Chronic Hepatitis C

Introduction

World Health Organization (WHO), 2015 reported that 130-150 million people globally have chronic hepatitis C infection and approximately 500 000 people die each year from hepatitis C-related diseases.Egypt has the highest prevalence of hepatitis C virus (HCV) in the world, estimated nationally at 14.7%,[2]especially genotype 4a.[3] [1]

Schistosomiasis is a parasitic disease caused by blood flukes (Trematodes) of the genus Schistosoma.[4]According to WHO, 2012[5],the disease affects at least 240 million of people in the world. In Egypt viral hepatitis along with Schistosoma mansoni infection is the major cause of chronic liver disease and liver cirrhosis.[6] Hepatic inflammation, steatosis and fibrosis are either direct consequences of hepatitis B and C virus infection or indirect damage determined by the subsequent inflammation and oxidative stress.[7]An important mediator of chronic inflammatory processes in liver cells could be the nitric oxide(NO).[8]This free radical is produced from L-arginin during the conversion of citrulin in a reaction catalyzed by nitric oxide synthase (NOS).[9]NO is produced by 2 constitutive isoforms of the enzyme NOS (endothelial or type 3 NOS/eNOS and neuronal or type 1NOS/nNOS) and 1 inducible isoform (iNOS or type 2NOS).[10] The results of reports investigating NOS isoforms in hepatic tissue are somewhat controversial.[11]In healthy livers, iNOS is not thought to be expressed constitutively. However, it is readily up-regulated in the liver under a number of disease conditions, including ischemia-reperfusion injury, hepatic fibrosis, cirrhosis and regeneration.iNOS is also up-regulated in vitro in hepatocytes and Kupffer cells in response to endotoxins and cytokines alone or in combination.[12]The availability of specific antibodies directed against iNOS has prompted attempts to understand their cellular distribution in the liver, and how that may affect the pathogenesis of liver dysfunction.[12] In normal liver, small quantities of NO are generated by eNOS in order to maintain perfusion in liver sinusoids by influencing vascular tonus or permeability. NO could also regulate leukocytes adhesion to liver sinusoids endothelium and inhibits platelets adhesion and aggregation.[13] Nitric oxide is considered to exert a hepato-protective action against tissue injury and cytotoxic effects of invading microorganisms, parasites and tumor cells.[14, 15] Nitric oxide is involved in the control of programmed cell death. Its effects on apoptosis depend on its concentration in one hand and the cell types in the other. Low concentrations block apoptosis via inhibition of the main mediators of cell death-caspases (caspase-3 and -8),

while higher concentrations are toxic via the formation of reactive products like peroxynitrite and dinitrogen trioxide.[16]However any situation that causes uncontrolled, prolonged and/or massive production of NO by iNOS may result in liver damage; leading to inflammation and even tumor development.[17]Nitric oxide plays an active role in the progression of liver fibrosis and hepatocellular damage in chronic viral hepatitis.[18, 19] In cirrhosis, the primary factor leading to portal hypertension is an increase in intra-hepatic resistance to blood flow. Many vasoactive substances contribute to the development of portal hypertension. Among these, nitric oxide is the key mediator that paradoxically regulates the sinusoidal (intrahepatic) and systemic/splanchnic circulation.[20] NO is considered a powerful endogenous vasodilator,[21]and the NOS isoform involved in this seems to be mainly eNOS.[22] The intracellular NO quickly forms nitrate and nitrites which are the stable end product that can be estimated in plasma, urine, and other body fluids as ascitic fluid.[23] This study aims to reveal the role of NO in pathogenesis of chronic liver diseases through demonstration of hepatic immunoexpression of iNOS and eNOS in Egyptian CHC with or without schistosomiasisas well as estimation of circulating NO in these disease entities.

Material and Methods

Patients and Controls This study was conducted on 72 patients with chronic liver disease admitted to Department of Gastroenterology and Hepatology, Theodor Bilharz Research Institute (TBRI), Giza, Egypt.Patients were subjected to thorough clinical examination and abdominal ultrasound (Hitachi EuB515A).Core liver biopsies were obtained from patients by percutaneous ultrasound-guided Menghini needle for histopathological and immunohistochemical studies. Serological Investigations 1. Liver Function Tests: Asparate aminotransferase (AST), Alanine aminotransferase (ALT) activities, and bilirubin were determined in serum according to the method described by Reitman &Frankel.[24] 2. Circulating schistosomal antigen and anti: schistosomal antibodies were done according to Demerdash et al[25]and Engvall & Perlman[26] respectively. 3. Hepatitis viral Markers were Detected Including: Hepatitis B surface antigen, anti-HBs antibodies, total and IgM class antibodies against hepatitis B core antigen using enzyme immunoassay kits (Murex Diagnostics, Dartford, England). Anti-HCV antibodies were detected using Version V anti-HCV ELISA kit

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Badawy et al. (Murex Diagnostics, Dartford, England). Circulating HCV-RNA was performed to confirm the presence of HCV antigenemia by nested RT-PCR using a set of primers within the 5’ non-translated region according to Saber et al[27] 4. Nitric Oxide Level: Quantitative determination of nitric oxide concentration in serum was done using total NO/Nitrite/Nitrate assay (R&D systems, Inc., Code no. KGE001, Minneapolis, Minnesota, USA). Patients were included in this study if they had: (a) Clinical and laboratory evidence of CHC (b) Circulating anti-HCV antibodies by enzyme-linked immunosorbent assay (ELISA) (c) HCV-RNA viraemia (d) Histological evidence of chronic hepatitis consistent with HCV disease. The study also included 5 controls(needle liver biopsies taken from donors for liver transplantation). They had normal clinical, biochemical and ultrasonographic findings. Hepatitis B surface antigen and anti-HCV antibodies were negative. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Patients were informed and signed the study informed consent following instructions of TBRI ethical committee. Histological Assessment Liver biopsies were fixed in formalin and embedded in paraffin. The slides were stained by Hematoxylin-eosin and Masson trichrome for routine histopathological evaluation and assessment of fibrosis.Degree of CHC disease necroinflammation (activity) and stage of fibrosis were scored according to METAVIR scoring system using grade and stage system as follow:[28] Grade of hepatitis activity; based on amount of inflammation: A0 = no activity. A1 = mild activity. A2 = moderate activity. A3 = severe activity. OO Stage of fibrosis; representing amount of fibrosis or scarring: F0 = no fibrosis. F1 = portal fibrosis without septa. F2 = portal fibrosis with few septa. F3 = numerous septa without cirrhosis. F4 = cirrhosis. A1 included 34 cases; A2:26 cases and A3:12 cases. F0/F1 included 44 cases; F2:14 cases; F3:8 cases and F4:6 cases. OO

A-251 Immunohistochemical Technique The paraffin embedded liver biopsies were used by applying the Avidin-Biotin peroxidase complex (ABC) method. Sections were dewaxed and rehydrated. Endogenous peroxidase activity was quenched by incubation of the slides in a solution of 0.3 % hydrogen peroxide in methanol for 10 minutes. Antigen retrieval was performed to unmask the antigens by microwaving the slides in citrate buffer solution (pH 6.0) for 15 minutes at 700w. Duplicate liver sections were incubated overnight at 4oC withrabbit anti-human polyclonal antibodiesfor iNOS(Santa Cruz Biotechnology Inc., Catalog no. SC-65, Dallas, Texas, USA) and eNOS (NeoMarkersLab vision corp., Code no.RB-1711, Cheshire, UK). Both antibodies were used in the appropriate dilutions (1:50) using antibody diluent.Sections were incubated at room temperature for 10 minutes with biotinylated secondary anti-rabbit antibody (Universal detection kit, DAKO, Code no.K0673, Glostrup, Denmark). Then streptavidin horseradish peroxidase conjugate was applied on the slides for 10 minutes. The antigen was visualized by the addition of diaminobenzidine substrate solution for 10 minutes. Sections were counterstained with Mayers Hematoxylin then mounted. For each antibody tested, we performed a negativecontrol in which the primary antibody was replaced byphosphate buffer saline. Immunostaining Interpretation Immunstained sections were examined under light microscopy at 400X to assess type of cells expressing the studied antibodies, type of expression either nuclear or cytoplasmic as brown deposits in labeled structures, and the extent of expression by semi-quantitative evaluation of the mean percentage of positive cells in 10 consecutive microscopic fields. Statistical Analysis All data were analyzed by SPSS software version 18 (IBM corporation, Armonk, New York,USA) and expressed as mean ± SD. Comparisons were performed by using ANOVA test. Spearman correlation coefficient served to clarify the relationship between variables. A “p” value less than 0.05 was considered statistically significant.

Results

The study included 72 patients (50 men and 22 women) with a mean age of 38.67± 8.38 (age range from 20-50). All patients were categorized as Child-Pugh class A of chronic liver disease.

Schistosomal granulomatous lesions and steatosis were also assessed.

All cases were positive for HCV-RNA with a viral load ranged from low (<200 000 genome/ml) to high (500 000-

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Nitric Oxide Isoforms in Chronic Hepatitis C

1000 000 genome/ml). No significant difference was found in viraemia among grades of hepatitis activity (p>0.05). AST, ALT, as well as bilirubin levels were steadily elevated along grades of hepatitis activity (Table 1). Table 2 showed that most cases were in METAVIR grades A1 and A2 of hepatitis activity (47.2% and 36.1% respectively) and F0/F1 METAVIR stage of fibrosis (61.2%). Schistosomiasis was diagnosed as co-infection with CHC in 33.3% of the examined cases (24/72). Associated schistosomal affection was diagnosed upon presence of hepatic schistosomal granulomatous lesion or serological positivity for anti-schistosomal antibodies ± circulating schistosomal antigen. All cirrhotic cases (6 cases) were found to be associated with schistosomiasis (Table 2, Fig.1). Steatosis was present in 41.7% of examined cases (30/72) (Table 2), without significant difference between male and female cases. Steatosis correlated significantly with grades of hepatitis activity (r=0.446, p<0.001), stages of fibrosis (r= 0.256, p<0.05), bilirubin (r=0.541, p<0.001) and ALT levels (r=0.290, p<0.001). In control hepatic tissue, the mean immunoexpression value of iNOS was 17.5±4.63 appeared as faint cytoplasmic staining localized in periportal hepatocytes (zone 1) with a sharp transition between iNOS-rich and iNOS-poor regions of the acinus. Endothelial NOS was expressed as widespread cytoplasmic staining in hepatocytes, sinusoidal and vascular endothelium (throughout all zones) with a mean value of 56.25±20.48 (Table 3). In CHC, extent of iNOS expression was expanded to involve zones 2 and 3 of hepatic acini, inflammatory cells and endothelial lining of bile ducts and sinusoids with a mean value of 43.19 ± 18.21. The immunoexpression of iNOS was significantly up-regulated in higher grades of activity (p<0.05) compared to either lower grades or controls (Table 3). Liver biopsies ofF0/F1 and F2 patients revealed

a discrete and isolated iNOS positive immunoreactions in the cytoplasm of hepatocytes, mainly surrounding areas of portal fibrosis (Fig.2A).while in advanced stages of liver fibrosis and cirrhotic patients (F4), there was a uniform distribution of iNOS in the hepatocytes of cirrhotic regions with a significant increase of iNOS immnoexpression compared to other stages(p<0.05) (Table4&Fig.2B). No difference was found in iNOS expression in liver biopsies with or without hepatic schistosomal infection (Table 5). The pattern of eNOS immunoreactivity in CHC patients was similar to that of iNOS with additional translocation to hepatocytic nuclei. Extent of tissue expression of eNOS showed significant down-regulation in A3 grade compared to other ones as well as over-expression in F4stage compared to other stages (Tables3&4). In addition, it was found that eNOS was significantly over-expressed in cases associated with schistosomal infection compared to negative ones (p<0.01) (Table 5; Fig.3). Measurement of serum nitrite/nitrate levels (serum NO) showed a significant increase in CHC patients group (8.86±7.42) compared to controls (6.95±0.33) (Tables 3,4) without significant difference between schistosomal and non schistosomal patients (Table 5). Serum NO level significantly correlated to hepatitis activity, fibrotic stage, steatosis extent, and serum bilirubin level (Table 6). Statistical evaluation of data showed that extent of iNOS expression correlated significantly with hepatic activity, fibrosis, steatosis extent, bilirubin, ALT and nitrite/nitrate levels (Table 6). Spearmen correlation test proved that eNOS extent of expression inversely correlated with upgrading of hepatitis activity (r=-0.301, p<0.01) and positively correlated with presence of schistosomal affection (r=0.253, p<0.01) (Table 6).

Discussion

Nitric oxide (NO), an important nitrogen reactive species, is a free radical with controversial roles which can be

Table 1: Serological parameters in different grades of hepatitis activity.

Items

Normal value

AST (IU/L) ALT (IU/L) Bilirubin (mg/dL)

≤12 ≤12 0-1

METAVIR grades of activity A1

Mean ± SD

41.82±26.46 49.52±41.92 0.68±0.23

59.30±34.48 65.30±33.41 0.9±0.32

93.0±61.3*# 117.0±97.59*# 1.23±0.36*#

* p<0.01 versus grade A2 activity. # p<0.001 versus grade A1 activity. AST: Asparate aminotransferase, ALT: Alanine aminotransferase SD: Standard Deviation

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Table 2: Incidence of schistosomiasis, steatosis, and fibrosis in different grades of hepatitis activity METAVIR grades of activity ItemsN of cases (%) Schistosomiasis Positive cases, N: 24 (33.3%) Negative cases, N: 48 (66.7%) Steatosis Positive cases, N: 30 (41.7%) Negative cases, N: 42 (58.3%) METAVIR stages of fibrosis F0/ F1 N: 44 (61.2%) F2 N: 14 (19.4%) F3 N: 8 (11.1%) F4 N: 6 (8.3%) Total N of cases: 72 (100%)

N (%)

16 (47.1) 18 (52.9)

6 (23.1) 20 (76.9)

2 (16.7) 10 (83.3)

6 (17.6) 28 (82.4)

16 (61.5) 10 (35.5)

8 (66.7) 4 (33.3)

30 (68.2) 4 (28.6) -

12 (27.3) 8 (57.1) 3 (37.5) 3 (50.0)

2 (4.5) 2 (14.3) 5 (62.5) 3 (50.0)

34 (47.2)

26 (36.1)

12 (16.7)

Table 3: Immunoexpressions of iNOS and eNOS ,and serum level of nitrite/nitrate in different grades of hepatitis activity

Items iNOS eNOS Nitrite/ nitrate

METAVIR grades of activity

Control (N=5)

CHC (N=72)

Mean ± SD

17.5±4.63 56.25±20.48 6.95±0.33

43.19±18.21 58.53 ±18.91 8.86±7.42*

A1 (N=34)

A2 (N=26)

Mean ± SD 33.17±15.18 64.41±21.80 6.91±7.62

A3 (N=12)

Mean ± SD

50.15±13.59 57.69±15.95 8.21±4.49

65.5±11.65*# 50.83±14.89# 15.82±8.36*#

p<0.05 compared to control.# p<0.05compared to other grades iNOS: indicible nitric oxide synthase; eNOS: endothelial nitric oxide synthase. SD: Standard Deviation; CHC: chronic hepatitis C *

Table 4: Immunoexpressions of iNOS and eNOS, and serum level of nitrite/nitrate in different stages of hepatic fibrosis Control (N=5)

Items

iNOS eNOS Nitrite/nitrate

CHC (N=72)

METAVIR stages of fibrosis F0/F1 (N=44)

F2 (N=14)

F3 (N=8)

F4 (N=6)

Mean ±SD

Mean ± SD

Mean±SD

17.5±4.63 56.25±20.48 6.95±0.33

43.19±18.21* 58.53 ±18.91 8.86±7.42

35.20±14.20* 60.65±20.56 5.82±5.13#

57.14±12.09*∆ 50.71±14.26∆ 9.94±3.74*

58.33±16.93*∆ 56.67±13.66 12.18±6.23*

80.1±0.20*# 70.2±0.30# 20.45±10.86*ο

p<0.01 compared to control. # p<0.05 compared to other stages. p<0.05 compared to F2 stage. ∆ p<0.05 compared to F0/F1 stage iNOS: indicible nitric oxide synthase; eNOS: endothelial nitric oxide synthase. SD: Standard Deviation; CHC: chronic hepatitis C * ο

Table 5: Immunoexpressions of iNOS and eNOS, and serum level of nitrite/nitrate in relation to schistosomiasis Schistosomiasis Positive (N=24) Negative (N= 48)

iNOS

eNOS

Nitrite/nitrate

Mean ± SD

40.58±21.21 46.25±16.77

66.67±14.50* 56.25±20.49

8.53±8.49 9.03±6.92

* p<0.01 compared to negative group. iNOS: indicible nitric oxide synthase; eNOS: endothelial nitric oxide synthase.

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Table 6: Statistical correlation for studied parameters. Studied parameters iNOS immunoexpression VS grades of hepatitis activity iNOS immunoexpression VS stages of hepatic fibrosis. iNOS immunoexpression VS steatosis. iNOS immunoexpression VS bilirubin level. iNOSimmunoexpression VS ALT level. iNOS immunoexpression VS nitrite/nitrate level. eNOS immunoexpression VS grades of hepatitis activity eNOS immunoexpression VS Schistosomal affection. Nitrite/nitrate level VS grades of hepatitis activity. Nitrite/nitrate level VS stages of hepatic fibrosis. Nitrite/nitrate level VS extent of steatosis. Nitrite/nitrate level VS bilirubin level

Correlation co efficient (r)

p value

0. 648 0.625 0.337 0.405 0.294 0.755 -0.301 0.253 0.484 0.596 0.300 0.306

p<0.001 p<0.001 p<0.01 p<0.01 p<0.001 p<0.001 p<0.01 p<0.01 p<0.001 p<0.001 p<0.05 p<0.01

VS = versus. iNOS: indicible nitric oxide synthase; eNOS: endothelial nitric oxide synthase.

Fig. 1: Granulomatous lesion developed encircling schistosomal ova in a case of HCV infected liver with evident steatosis (Hx and Eosin stain X400).

Fig. 2: Immunohistochemistry for iNOS in liver sections expressed as brown cytoplasmic staining in hepatocytes, (A) a case of CHC with mild activity and fibrosis (A1F0/F1) showing zonal distribution of staining mainly periportal; (B) wide marked iNOS expression in hepatocytes and portal inflammatory cells in a cirrhotic liver on top of CHC . iNOS: inducible nitric oxide synthase; CHC: chronic hepatitis C (X400).

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Fig. 3: Immunohistochemistry for eNOS in liver sections expressed as brown cytoplasmic and additional nuclear staining in hepatocytes (A) wide cytoplasmic expression of eNOS in hepatocytes in a case of CHC with mild activity and fibrosis (A1F0/F1). (B) Marked cytoplasmic expression of eNOS in regenerating nodule of cirrhotic liver as well as in endothelium of portal vasculature (X400).

generated in almost all hepatic cells (hepatocytes, kupffer, endothelial and hepatic stellate cells).[8]NO is an important mediator of liver physiology and pathophysiology.[29]It is conceivable that, in the intra-hepatic microenvironment of CHC, an important source of NO exists. Since a marked induction of iNOS expression is observed in the majority of hepatocytes from patients with CHC, it is likely that these liver cells are a powerful cellular source of large quantities of NO.[30] In CHC, it has been suggested that the viral infection itself, by mechanisms not yet fully understood, may be a triggering factor. It could be suggested that this nitration process represents a non-specific pathogenic mechanism, common to different chronic inflammatory diseases and secondary to the elicitation of inflammation rather than its cause.[15] Machida et al. concluded that HCV infection can stimulate production of NO through activation of the gene for iNOS by the viral core.[31]

cirrhosis (Child class B&C),[23, 33-35]while McNaughton et al were unable to detect significant changes in serum NO of their patients with viral hepatitis, cirrhosis, and cholestasis when compared with controls.[36]In our study, serum nitrite/nitrate level variably increased in diseased cases with definite correlation to grade of hepatitis activity, stage of fibrosis, and extent of tissue expression of iNOS. No significant difference was recorded in NO serum level or hepatic expression of iNOS between our schistosomal and non schistosomal patients. Although Hassan et al estimated enhanced NO levels in cirrhotic patients with HCV infection and schistosomiasis compared to nonschistosomal HCV infected patients.[23]

Serum NO level in CHC patient was studied through estimation of nitrate or nitrite/nitrate level in multiple researches. In chronic active hepatitis patients, Parvu et al did not found difference in nitrite/nitrate levels of the patients and healthy control,[32]multiple studies recorded increased nitric oxide serum level in advanced stages of

We found that iNOS was constitutively expressed in a zonal pattern in the normal hepatic acinus. The distribution of iNOS showed the strongest expression in the periportal region, with diminution of intensity toward the perivenous regions of the hepatic acinus. These results agree with those of McNaughton et al.[36]Our study also provides the evidence that there was increased production of NO in CHC patients, appeared as over-expression of iNOS with an almost diffuse distribution pattern throughout the hepatic lobules mainly in hepatocytic cytoplasm but also in nuclei. These results were in contention with several studies conducted before.[8,10,11,15,18]Furthermore iNOS consistently labeled mononuclear cells infiltrating portal tracts in all our hepatitis samples. Also,this over-expression of iNOS correlated with disease severity in the form of hepatitis

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Hepatitis C virus infection induces the production of total nitric oxide (NO), i.e., NOX which includes both nitrites (NO2-) and nitrates (NO3-).[31]

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activity and fibrosis which is consistent with the results of Atik et al.[15] In this study, eNOS immunostaining was uniformly distributed in hepatocytes, present in the endothelium of hepatic arteries, terminal hepatic venules and sinusoids. Interestingly, the epithelium of biliary ducts showed strong expression of eNOS. Thus, in addition to endothelial cells, both hepatocytes and biliary epithelium express eNOS. There are very few studies that have examined the role of NO in biliary epithelial cell function. The functional significance of eNOS expression in the endothelium as a regulator of blood flow and cell–cell interactions has been proposed.[36]

patients. So, our findings triggered us to hypothesize the possible use of NO markers as an independent predictor of virological response in HCV genotype 4 infected patients. Further studies are required to determine whether iNOS inhibitors could be useful in reducing liver disease severity and improve the benefits of antiviral therapies.

Abbreviations

World Health Organization, WHO; hepatitis C virus, HCV; nitric oxide, NO; nitric oxide synthase, NOS; endothelial NOS, eNOS; inducible NOS, iNOS; chronic hepatitis C, CHC; Asparate aminotransferase, AST; Alanine aminotransferase, ALT; enzyme-linked immunosorbent assay, ELISA.

The immunohistochemical analysis in our study showed profound changes in the cellular distribution of eNOS in CHC tissue samples, leading to its translocation to hepatocytic nuclei. Interestingly, growth factors such as vascular-endothelial growth factor are known to cause nuclear translocation of eNOS in vascular endothelium. [37] The significance of this observation is unclear. Again, nuclear translocation of eNOS may merely reflect “growth factor storm” that is characteristic of chronic liver inflammation and cirrhosis.[38]Alternatively, the eNOS translocation may be a part of a liver defense mechanism aimed at limiting the effects of growth factors by decreasing the rate of cellular proliferation or apoptosis that is known to be regulated by NO.[36]

Funding

Schistosomiasis is an intravascular disease associated with inflammation, fibrosis and venous intrahepatic portal lesions which are the main pathogenic factors in production of portal hypertension leading to perisinusoidal block and increased resistance to portal blood flow.[39] Increased NO metabolism is associated with the hemodynamic alteration induced by portal hypertension,[33, 35] as endothelial cells control vascular tonus and permeability [21] via NO generation by eNOS.[13] Chang et al suggested that eNOS rather than iNOS involved in vascular response to vasoconstrictors in portal-systemic collaterals of portal hypertension.[22]This may explain the significant over-expression of eNOS in our cases associated with Schistosomal affection compared to negative ones.

3. Ibrahim M, Gomaa W, Ibrahim Y, El Hadad H, Shatat M, Abdel Aleem A, et al. Nitric oxide levels and sustained virological response to pegylated-interferon alpha2a plus ribavirin in chronic HCV genotype 4 hepatitis: a prospective study. J Gastrointesin LiverDis 2010; 19(4):387-392.

Conclusion

Both iNOS and eNOS proteins are differentially expressed in healthy human liver, and this expression is altered in chronic hepatitis C with or without schistosomiasis. Similarly, serum nitrite/nitrate level is elevated in these disease entities. Schistosomal coinfection can be considered as a risk factor for haemodynamic disturbance in CHC

This work was financed by TBRI internal project No78T. Principal investigator: Prof. Dr. Afkar A. Badawy.

Competing Interests None Declared

References

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4. Barakat SMR. Epidemiology of Schistosomiasis in Egypt: Travel through time:review. J Advanced Research 2013; 4(5):425-432. 5. World Health Organization (WHO). Schistosomiasis. h t t p : / / w w w. w h o . i n t / s c h i s t o s o m i a s i s / e n / . Accessed2011; 23 Feb 2012. 6. Strickland GT. Liver disease in Egypt: hepatitis C superseded schistosomiasis as a result of iatrogenic and biological factors. Hepatol. 2006; 34(5):915-937. 7. Diesen DL, Kuo PC.Nitric oxide and redox regulation in the liver: Part I. General considerations and redox biology in hepatitis. J SurgRes 2010;162(1): 95-109 8. Tache DE, Stanciulescu CE, Banit IM, Purcaru SO, Andrei AM, Comanescu V,et al. Inducible nitric oxide synthase expression (iNOS) in chronic viral

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29. Iwakiri Y, Kim MY. Nitric oxide in liver diseases.Trends in Pharmacological sciences 2015;36(8):524-536. 30. Sanz-Cameno P, Medina J, Garcia-Buey L, GarciaSanchez A, Borque MJ, Martin-Vilchez S, et al. Enhanced intrahepatic inducible nitric oxide synthase expression and nitrotyrosine accumulation in primary biliary cirrhosis and autoimmune hepatitis. J Hepatol 2002; 37: 723-9. 31. Machida k, Cheng k, Sung V, Lee kj, Levine A, Lai M. Hepatitis C virus infection activates the immunologic (Type II) isoform of nitric oxide synthase and thereby enhances DNA damage and mutations of cellular genes. J Virol 2004; 78: 8835–8843. 32. Parvu AE, Negrean V, Manea LP, Cosma A, Draghici A, Uifalean A, et al.Nitric oxide in patients with

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Original Article Evaluation of RAGE (Receptor for Advanced Glycation End-products) Expression in Gastric Carcinoma of Egyptian patients in Relation to Helicobacter pylori Infection Tarek Aboushousha1*, Afkar Badawy1, Mona Moussa1, Zeinab Omran1, Ahmed-Hazem Helmy2 and MagdyYoussef3 Pathology1, Surgery2 and Gastrointestinal and Hepatology Medicine3 departments, Theodor Bilharz Research Institute, Cairo, Egypt. Keywords: Gastric Carcinoma, Helicobacter Pylori, Rage.

ABSTRACT Background: Gastric cancer is one of the most common malignancies and is the second most common cause of death from cancer worldwide. Gastric cancer is a multistep process that is regulated by intrinsic and extrinsic cellular signals. Extrinsic factors include molecular patterns that are derived from either pathogens or cellular damage, which can promote tumourigenesis. Helicobacter pylori plays an important role in the pathogenesis of chronic gastritis and gastric adenocarcinoma. Receptor for Advanced Glycation End products (RAGE) is a pattern recognition receptor that binds multiple ligands derived from a damaged cell environment, and plays a critical role in promoting the intestinal tumorigenesis. The over-expression of RAGE has been associated with increased invasiveness and metastasis generation in different types of cancer, including gastric cancer. Therefore, the aim of this study was to evaluate the expression of RAGE protein in gastric carcinomas either in cases associated with Helicobacter pylori (Hp) infection or not, so as to predict its value as a target for therapy. Methods: 51 endoscopic and 19 surgical gastric biopsies including cases of gastric carcinoma, intestinal metaplasia and chronic gastritis were histopathologically and immunohistochemically studied for RAGE expression and were statistically discussed. Result: RAGE was not expressed in any case of gastritis or signet-ring gastric carcinoma. The RAGE cellular expression parameters were correlated significantly with the stages of gastric adenocarcinoma and lymph node metastasis and nonsignificantly with the grade of neoplasia. Our results showed no significant correlation between RAGE expression and Hp infection, either in chronic gastritis or malignant cases. Conclusion: RAGE expression could be identified as a possible marker for target therapy in some types of gastric carcinoma, possibly to control its invasive and metastatic potential, however, its relation to Hp infection was not quite evident in our current study.

*Corresponding author: Dr. Tarek Aboushousha, M.D., Professor of Pathology, Theodor Bilharz Research Institute, El-Nile Street, Warrak El-Hadar, Imbaba, P O Box: 30, Giza 12411,Cairo, Egypt Phone: 00201222186036 Email: taboushousha@gmail.com

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

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Introduction

Gastric cancer is one of the most common malignancies worldwide, with an estimated 934,000 cases reported globally in 2011, and is the second most common cause of death from cancer.[1] Gastric cancer is a multistep process that is regulated by intrinsic and extrinsic cellular signals. Extrinsic factors include molecular patterns that are derived from either pathogens or cellular damage, which can promote tumorigenesis.[2] Helicobacter pylori (Hp) is a gram-negative, spiral bacterium that colonizes gastric mucosa and plays an important role in the pathogenesis of chronic gastritis, peptic ulcer, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma.[3] Although the vast majority of Hp in colonized hosts are free-living, ~20% bind to gastric epithelial cells and adherence is required for prolonged persistence in the stomach and for induction of injury.[4] RAGE is a membrane receptor, belonging to the immunoglobulin family, and the overexpression of RAGE has been associated with increased invasiveness and metastasis generation in different types of cancer, including gastric cancer.[3] RAGE is a pattern recognition receptor that binds multiple ligands derived from a damaged cell environment, and plays a critical role in promoting the intestinal tumorigenesis.[2] RAGE is also an important inflammatory mediator that modulates crosstalk between survival pathways and autophagy in tumor cells. It sustains autophagy and limits apoptosis promoting tumor survival.[5] Although expressed at very low levels in normal tissues, chronic inflammatory conditions increase not only RAGE expression, but also the formation and release of some RAGE ligands.[6,7,8,9] Therefore, the aim of this study was to evaluate the expression of RAGE protein in gastric carcinomas either associated with Hp infestation or not, in order to estimate the value of it as a possible target for therapy.

Methods

Paraffin blocks of gastroscopic biopsies (51) and gasterectomy specimens (19) of 70 cases diagnosed as ; gastric carcinoma (48 cases) , intestinal metaplasia associated with gastritis (16 cases) and chronic gastritis without intestinal metaplasia (6 cases) received in the pathology department of Theodor Bilharz Research institute, Cairo, Egypt in the interval from Jun. 2012 to Apr. 2014 were included in this study. Hematoxylin and eosin staining was done for routine diagnosis, grading and staging. Giemsa stain was used to help detection of Hp in examined gastric mucosal sections, applying the Sydney updated scoring system.[10] Immunohistochemical staining for RAGE was done, and its expression was evaluated and discussed.

Immunohistochemical Method Anti-RAGE antibody (Santa Cruz Biotechnology) was used for immunohistochemical (IHC) detection of the expression of RAGE protein in tissue. Tissue sections were processed for IHC analysis of RAGE protein as follows. IHC examinations were carried out on 3 µm thick sections. For anti-RAGE IHC, unmasking was performed with 10 mM sodium citrate buffer, pH 6.0, at 90°C for 30 min. Sections were incubated in 0.03% hydrogen peroxide for 10 min at room temperature, to remove endogenous peroxidase activity, and then in blocking serum (0.04% bovine serum albumin, A2153, Sigma-Aldrich, Shanghai, China, and 0.5% normal goat serum X0907, Dako Corporation, Carpinteria, CA, USA, in PBS) for 30 min at room temperature. Anti-RAGE antibody (A11): sc80652 RAGE Antibody (A11) is a mouse monoclonal IgG2a provided at 200 µg/ml, raised against a truncated extracellular domain of RAGE of human origin (Santa Cruz Biotechnology , USA) The antibody was used at a dilution of 1:100. The antibody was incubated overnight at 4°C. Sections were then washed three times for 5 min in PBS. Non-specific staining was blocked 5% normal serum for 30 min at room temperature. Finally, staining was developed with diaminobenzidine substrate and sections were counterstained with hematoxylin. PBS replaced RAGE antibody in negative controls.[11] Quantification of protein expression: The expression of RAGE was semiquantitatively estimated as the total membrano-cytoplasmic immunostaining scores, which were calculated as the product of a proportion score and an intensity score. The proportion and intensity of staining was evaluated independently. The proportion score reflected the fraction of positive staining cells (score 0: <5%, score 1: 5%-10%, score 2: 10%-50%, score 3: 50%-75%, score 4: >75%), and the intensity score represented the staining intensity (score 0: no staining, score 1: weak positive, score 2: moderate positive, score 3: strong positive). Finally, a total expression score was given ranging from 0 to 12. Based on the analysis in advance, RAGE was regarded as negative expression in gastric cancer tissues if the score <2, and positive expression if the score ≥2.[11] It is to be noted that expression of RAGE in cases of malignancy was estimated only in malignant cells, also, in cases of intestinal metaplasia estimation of RAGE expression was estimated in metaplastic cells, not including non-metaplastic gastric epithelial cells. Statistical analysis: Statistical analysis was performed using SPSS.19 software program. For RAGE monoclonal

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Aboushousha et al. antibody, data were summarized as means and percentage. Means of groups were compared using unpaired t-test. For RAGE monoclonal antibody, data were summarized using cross tabulation. correlation tests served in correlating extent, intensity, and pattern of expression of the different RAGE parameters with other pathological features (grade, stage, inflammation, Hp,...etc). The interpretation of p value: p value of <0.05 was considered statistically significance. p value of <0.01 was considered of high statistical significance.

Results

Seventy cases were examined in this study; of them 57 biopsies were from male patients and 13 were from female patients, with the mean age of 52 years for males (range 34 -72) and 56 years for females (range: 38-84), with no statistically significant difference (p>0.05). As regard malignant cases females represent 17 cases, while males represent 31 cases (p<0.01). For malignant cases, the mean age for female patients was 58 years while it was 55 years for male patients, with no significant difference (p>0.05). (Table 1) Most of the studied cases were positive for Hp (70%). Signet-ring carcinoma cases showed the highest percentage of Hp positivity (80%), followed by cases of intestinal metaplasia (68.75%), adenocarcinoma (68.42%) and gastritis (66.67%) without significant differences (p>0.05). (Table 2). On the other hand a significant positive correlation was obtained between Hp score and the score of gastritis activity, and significantly inverse correlation with intensity of gastric inflammation (p<0.05 and p<0.01 respectively). In all examined cases, the intensity of RAGE expression mostly in the cytoplasm of positive cells was inversely correlated with inflammatory activity (p< 0.05), while percentage of positive cells expressing RAGE was

A-261 positively correlated with intensity of inflammatory reaction (p< 0.01). The overall RAGE score showed positive correlation with the inflammatory intensity (p< 0.05) and inverse correlation with the inflammatory activity (p< 0.01). (Table 3) In cases of gastric adenocarcinoma, all RAGE expression parameters were non-significantly correlated with tumor grade and Hp score ( p>0.05), while they were correlated positively with tumor stage and inflammatory intensity (p<0.01). On the other hand, Hp score was correlated positively with the inflammatory activity (p<0.05) and correlated inversely with the inflammatory intensity (p<0.01) (Table 4) As regard RAGE expression, our study showed that it was absent in non-malignant gastric glandular epithelial cells and in all cases of signet-ring carcinoma. On the contrary, in cases of intestinal metaplasia, RAGE was expressed with significant intensity (p<0.05) and highly significant percentage (p<0.01) and consequently showed higher RAGE score compared to cases of adenocarcinoma (p<0.05). (Table 5) In cases of adenocarcinoma, there were non-significantly higher values of RAGE intensity and scores in high grade tumors compared to low grade ones, also, there was a nonsignificant difference between the mean percentage of RAGE expression in low and high grade tumors. (Table 6) RAGE expression parameters (intensity, percentage and score) were non-significantly higher in Hp negative cases of adenocarcinoma compared to Hp positive cases (Table 7) All RAGE expression parameters were significantly higher in high stages of gastric carcinoma compared to low ones (p<0.01). Also, cases with lymph node metastases (LN+ve) showed significantly higher values of RAGE intensity and overall score (p<0.01) but a non-significantly higher value of RAGE percentage compared to cases without lymph node metastases (LN-ve) (p>0.05). (Table 8).

Table 1: Demographic data for studied cases: Sex

Diagnosis

Total

Adenocarcinoma

Signet-ring carcinoma Int. Metaplasia*

3 1

7 15

10 16

Gastritis

Total

*Intestinal metaplasia of gastric mucosa

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Table 2: Histopathological Diagnosis versus Helicobacter pylori infection: Hp

Histopathology

Diagnosis

Negative 12(31.58%) 2(20%) 5(31.25%) 2(33.33%) 21(30%)

Adeno Ca Signet-ring Ca Metaplasia Gastritis Total

Total

Positive 26(68.42%) 8(80%) 11(68.75%) 4(66.67%) 49(70%)

38(100%) 10(100%) 16(100%) 6(100%) 70(100%)

Non-significant differences (p> 0.05)

Table 3: Spearman’s correlation (rho) in the all-studied cases: Hp (score) -0.068 0.067 -0.064 0.526* -0.428**

RAGE score RAGE % RAGE Intensity Inflammatory Activity Inflammatory Intensity

Inflammatory Intensity 0.301* 0.336** 0.175 0.077

Inflammatory Activity -0.392** -0.105 -0.408**

*p<0.05 significant correlation ; **p<0.01 highly significant correlation

Table 4: Spearman’s correlation (rho) in adenocarcinoma cases: Tumor Grade

Tumor Stage

Hp (score)

0.188 0.030 0.285 -0.192 -0.231 0.110

0.840** 0.687** 0.765** -0.454 0.313 -0.099

-0.206 0.004 -0.171 0.351* -0.419**

RAGE score RAGE % RAGE Int. Inf. Act. Inf. Int. Hp (score)

Inflammatory Intensity 0.535** 0.587** 0.331* 0.023

Inflammatory Activity -0.314 0.148 -0.316

*p<0.05 significant correlation ; **p<0.01 highly significant correlation

Table 5: Differences in means of RAGE parameters between Adenocarcinoma and Intestinal metaplasia: RAGE Intensity Mean±SE 1.76±0.71 1.95±0.36 NS

Diagnosis (Number) Adenocarcinoma (38) Metaplasia (16) P value

RAGE (%) Mean±SE 73.95±20.47 91.52±8.26 p< 0.01

RAGE Score Mean±SE 6.42±3.24 10.25±2.11 p< 0.05

NS: nonsignificant difference (p>0.05)

Table 6: Difference in RAGE parameters in relation to grades of adenocarcinoma: RAGE Intensity Mean±SEM 1.59 ± 0.14 2.03 ± 0.18

Grade of AdenoCa (N) Low Grdae (22) High Grade (16)

RAGE % Mean±SEM 74.09 ± 4.25 73.75 ± 5.47

RAGE Score Mean±SEM 5.91 ± 0.69 7.13 ± 0.81

No significant difference between groups (p>0.05)

Table 7: RAGE expression in relation to Hp infection in cases of gastric adenocarcinoma: Hp detection (N)

RAGE Intensity Mean±SEM

RAGE % Mean±SEM

RAGE Score Mean±SEM

Hp -ve (12) Hp +ve (26)

2.03 ± 0.25 1.65 ± 0.12

76.67 ± 3.55 72.69 ± 4.59

7.50 ± 1.06 5.92 ± 0.58

No significant difference between groups (p>0.05)

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Table 8: RAGE parameters in studied adenocarcinoma cases as regards histopathological stage and lymph nodes metastasis: RAGE Parameter

Stage of Malignancy Mean±SEM Low (7)

High(12)

LN metastasis Mean±SEM -ve(4)

+ve(6)

RAGE Intensity

1.29±0.18

2.50**±0.15

1.00±0 .00

2.50**±0.22

RAGE %

67.14±5.22

89.17**±0.29

85.00± 2.89

93.33± 3.33

RAGE Score

3.71±0.47

10.00**±0.55

3.75±0.25

10.33**± 0.95

* Significant difference between both groups (p< 0.05). * High significant difference between both groups (p< 0.01).

Fig.1: Sections in gastritis cases showing low(1A) and high (1B) scores of Hp infection within superficial mucosal glands. (Giemsa stain, X400).

Fig. 2: Sections in case of gastritis showing (2A) moderate inflammatory intensity and activity (H&E stain, X200) and (2B) negative expression of RAGE (Immunohistochemical stain for RAGE, X200).

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Fig. 2C: Sections in case of gastric intestinal metaplasia. (H&E stain, X200). Fig. (2D): High scores of RAGE expression in case of intestinal metaplasia. (Immunohistochemical stain for RAGE, X200).

Fig. 3: Sections in cases of low grade gastric superficial adenocarcinoma (3A) exhibiting acinar pattern (H&E stain,X200) showing mild RAGE expression (3B). (immunohistochemical stain, X200)

Fig. 3C: Invasive papillary carcinoma (H&Estain, X100) showing high expression of RAGE (3D) by immunohistochemistry (X100).

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Fig. 4: Sections in signet-ring gastric carcinoma (H&E stain,X200) (4A), immunohistochemistry X200(4B).

showing negative expression for RAGE by

Fig. 5: Sections in grade 3 invasive gastric adenocarcinoma, H&E stain, X200(5A), showing high RAGE expression stained by immunohistochemistry, X200 (5B). Graph. (1): Comparison between RAGE scores in different grades, stages, LN metastasis and Hp detection status in cases of gastric adenocarcinoma.

**: high significant difference between relevant groups (p<0.01

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Discussion

RAGE Expression in Gastric Cancer

Worldwide, gastric cancer (GC) is the 5 most common malignancy in both sexes.[11] In Egypt, GC is the 12th most common cancer in both sexes. The median age of GC in the Egyptians is 56 years.[12] This was in accordance with our results in which there was a significantly higher percentage of male patients with GC than females. Within this group, the difference in the mean age for female and male patients, was non-significant. Hp causes gastritis and peptic ulceration and it is an important risk factor for gastric adenocarcinoma, the second highest cause of cancer deaths worldwide. The disease process is thought to have a multifactorial etiology; bacterial strain type, pattern of gastritis, and environmental conditions, are all thought to contribute.[13] Seventy percent of our all studied cases were positive for Hp. Signet-ring carcinoma cases showed the highest percentage of Hp positivity (80%). It was found by some authors that the best established risk factors for GC were Hp infection, male sex, a family history of GC, and smoking. Dietary risk factors are related to diet type and food preservation.[14] th

All strains of Hp induce a marked inflammation in the gastric mucosa which is characterized by neutrophil, lymphocyte and other inflammatory cell infiltration. While antral-predominant gastritis leads to increased acid production from the uninflamed corpus and predisposes to duodenal ulceration, corpus-predominant gastritis leads to hypochlorhydria and predisposes to gastric ulceration and adenocarcinoma.[15] Our study showed a significant positive correlation between Hp score and the scores of gastritis activity, and significantly inverse correlation with intensity of gastric inflammation. Fundamental characteristic of infection with Hp is chronic inflammation of the gastric mucosa, being the role of inflammation as a factor favoring tumor growth of widely recognized neoplastic lesions [16,17,18] and in recent years associated with tumorigenesis related to multiligand / RAGE axis. [19,20] Activation of RAGE axis also plays a particular role in the malignant transformation of gastric glandular epithelium. [3] In our study RAGE expression was absent in cases of gastritis without intestinal metaplasia, but was upregulated in association with intestinal metaplasia as well as cases of gastric adenocarcinoma. Xu et al. (2013) found that the positive expression of RAGE protein was detected in the cytoplasm of gastric cancer cells and was increased in gastric cancer tissues compared with the adjacent non-cancerous tissues (ANCT).[11] Immunoexpression observing a nuclear level RAGE is a rare event that needs to be corroborated by other studies to confirm their presence at this level and assess the functional significance of this finding.[3]

We found that the overall RAGE score showed significantly positive correlation with the inflammatory intensity and inverse correlation with the inflammatory activity. Aso, we found that all RAGE expression parameters (intensity, percentage and score) were non-significantly higher in Hp negative cases of adenocarcinoma compared to Hp positive cases. Studies in which gastritis has been followed up over a lengthy period by endoscopy and biopsy after eradication of Hp infection have been able to show that the neutrophil infiltrate disappeared completely, while infiltration of the mucosa with lymphocytes and plasma cells persisted, albeit only to a very slight degree. However, other parameters such as intestinal metaplasia or lymphoid follicles, which are often formed in association with Hp infection, may still be found in the mucosa several years after eradication of Hp.[21,22] These, together with othe factors, occasionally make the differential diagnosis vis-a-vis chemically induced/reactive gastritis somewhat difficult.[10] These observations could explain -at least partially- why we could not found a valuable correlation between Hp infection with other parameters of inflammatory mucosal reaction and RAGE expression. Another important point is that Hp is not the sole factor exciting gastric mucosal inflammatory reaction and consequently carcinoma. Other factors may be of equal or more importance in Egyptian patients, like diet, toxins, or even therapeutic agents, that should be studied intensively. Kuniyasu et al.(2002) have reported that, RAGE expression is closely associated with the invasion and metastasis in GC patients, which provides us a basis for the immunohistopathological study of RAGE in gastric cancer.[23] In our work, we found positive correlation of RAGE expression parameters with grades of gastric adenocarcinoma, although these correlations were nonsignificant. However, there was non-significantly higher values of RAGE intensity and scores in high grade tumors compared to low grade ones, also, there was a nonsignificant difference between the mean percentage of RAGE expression in low and high grade tumors. Zhang et al. (2015), confirmed in his study that RAGE receptor activation is required for gastric cancer cell proliferation.[24] RAGE expression parameters were correlating positively and significantly with ascending stages of malignancy. All RAGE expression parameters were significantly higher in high stages of gastric carcinoma compared to low ones. Also, cases with lymph node metastases (LN+ve) showed significantly higher values of RAGE intensity and score (p<0.01) and non-significant higher value of RAGE

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Aboushousha et al. percentage (p>0.05) compared to cases without lymph node metastases (LN-ve). We found also that RAGE intensity and score of expression were significantly higher in lymph node positive cases than in lymph nodes negative ones . A finding that was similar to the results achieved also by Xu et al. (2013).[11] Previous study found that upregulation of RAGE expression was significantly associated with poor clinicopathological characteristics and poor overall survival, suggesting that it may contribute to the malignant potential of GC. Therefore, RAGE could therefore serve as a valuable novel biomarker for predicting prognosis and a potential therapeutic target for patients with GC. However, further studies are warranted to clarify the underlying mechanisms of RAGE overexpression, thereby contributing to better understanding and further developing of its potential use.[21]

Conclusion

The present study indicated that RAGE was evidently upregulated in gastric cancer and pre-cancerous lesions namely intestinal metaplasia, but the correlation of RAGE expression with Hp infection was indefinite. There may be unexplained variation in the distribution of virulence factors and gastritis patterns that fail to explain the discordance between Hp infection rates and the variations in RAGE expression in gastric cancer in Egyptian patients. Other carcinogenic dietary, genetic and therapeutic factors should be intensively studied in these patients. However, we have reported that RAGE expression is closely associated with increased tumor grade, stage and lymph node metastasis, so, it could be used as a predictor prognostic factor. However, further work is needed to estimate the reliability of RAGE expression in relation to variable etiological factors and for being a target for possible therapy in prophylaxis as well as early intervention in high risk patients.

Acknowledgement

We acknowledge Tech. Nadia Abdullah for her kind help in preparing histopathological and immunohistochemical sections.

Ethical Statement

All our biopsy specimens were archival un-identified paraffin preserved materials from the Pathology Department of Theodor Bilharz Research Institute.

References

1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61:69-90 2. Heijmans J, Büller NV, Hoff E, Dihal AA, van der Poll T, van Zoelen MA, et al. Rage signalling promotes intestinal tumourigenesis. Oncogene 2013;32:1202-6 www.pacificejournals.com/apalm

A-267 3. Morales M E, Rojas R A, Monasterio A V, González B I, Figueroa C I, Manques M B, Romero E J, Llanos L J, Valdés M E, Cofré L C. Expression of RAGE in Helicobacter pylori infested gastric biopsies. Rev Med Chil. 2013 Oct;141(10):1240-8. 4. O’Brien DP, Romero-Gallo J, Schneider BG, et al. Regulation of the Helicobacter pylori Cellular Receptor Decay-accelerating Factor. J Biol Chem 2008; v.283(35) 5. Kang R, Tang D, Schapiro NE, Livesey KM, Farkas A, Loughran P, et al. The receptor for advanced glycation end products (RAGE) sustains autophagy and limits apoptosis, promoting pancreatic tumor cell survival. Cell Death Differ. 2010;17:666-76 6. Sparvero LJ, Asafu-Adjei D, Kang R, et al., RAGE (Receptor for Advanced Glycation Endproducts), RAGE Ligands, and their role in Cancer and Inflammation J Transl Med. 2009; 7: 17. 7. Odenbreit S. Adherence properties of Helicobacter pylori: impact on pathogenesis and adaptation to the host, Int. J. Med. Microbiol. 2005; 295317-324. 8. Schmidt AM, Yan SD, Yan SF, Stern MD. The multiligand receptor RAGE is a progression factor amplifying immune and inflammatory responses, J. Clin. Invest. 2001; 108 949e955 9. Donato R. RAGE: a single receptor for several ligands and different cellular responses: the case of certain S100 proteins, Curr. Mol. Med. 7(2007) 711e724 10. Stolte M, Meining A. The updated Sydney system: classification and grading of gastritis as the basis of diagnosis and treatment. Can J Gastroenterol. 2001; 15 No 9, 11. Xu XC, Abuduhadeer X, Zhang WB, T. Li, Gao H, Wang YH. Knockdown of RAGE Inhibits Growth and Invasion of Gastric Cancer Cells. Published online (2013) Nov 18. 12. Gharbiah Population Based Cancer Registry, Egypt. Ministry of Health and Population. Cancer in Egypt, triennial report of 2000–2002. (2007). MOH: Tanta, Egypt. 13. Peek RM Jr, Blaser MJ. Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nat Rev Cancer. 2002;2:28–37. 14. Brenner H, Rothenbacher D, Arndt V. Epidemiology of stomach cancer. Methods Mol Biol Clifton NJ, 2009;472:467–477 15. Asaka M, Kato M, Kudo M, et al. Atrophic changes of gastric mucosa are caused by Helicobacter pylori eISSN: 2349-6983; pISSN: 2394-6466

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infection rather than aging: studies in asymptomatic Japanese adults. Helicobacter. 1996 ;1:52–56. 16. Bornschein J, Kandulski A, Selgrad M, Malfertheiner P. From gastric inflammation to gastric cancer. Dig Dis. 2010; 28 (4-5): 609-14. 17. Blakwill F, Montovani A. Inflammation and cancer: back to Virchow? Lancet. 2001; 237: 539-45. 18. Mantovani A, Allavena P, Sica A, Balkwill F. Cancerrelated inflammation. Nature. 2008, 454, 436-44. 19. Rojas A, Figueroa H, Morales E. Fueling inflammation at tumor microenvironment: the role of multiligand / rage axis. Carcinogenesis .2010; 31: 334-41. 20. Riehl A, J Nemeth, Angel P, Hess J. The RAGE receptor: Bridging inflammation and cancer. Cell Communication and Signaling . 2009, 7: 12. Available at: www.biosignaling.com.

21. Wang D, Li T, YeG, Shen Z, Hu Y, Mou T, et al. Overexpression of the Receptor for Advanced Glycation Endproducts (RAGE) Is Associated with Poor Prognosis in Gastric Cancer. PLoSONE10. 2015 ; 4. 22. Stolte, M, Eidt, S. Lymphoid follicles in antral mucosa: immune response to Campylobacter pylori J. Clin. Pathol. 1991: 42, 1269-1271 23. Kuniyasu H, Oue N, Wakikawa A, et al. Expression of receptors for advanced glycation end-products (RAGE) is closely associated with the invasive and metastatic activity of gastric cancer. The Journal of pathology. 2002;196(2):163–70. 24. QIu-Yu ZHANG, LIN-QING Wu, TAO ZHANG, YANFEI HAN, Xu LIN. Autophagy-mediated HMGB1 release promotes gastric cancercell survival via RAGE activation of extracellular signal-regulated kinases 1/2. ONCOLOGY REPORTS 2015; 33: 1630-1638.

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Original Article Evaluation of Latex Agglutination Test Efficacy in Diagnosis of Acute Pyogenic Meningitis Shilpa Dayanand Putta1*, Smita Deshpande2 and Renu Bharadwaj2 Department of Microbiology, Rajarshi Chhatrapati Shahu Maharaj Govt. Medical College, & CPR General Hospital campus, Dassara Chowk, Kolhapur â&#x20AC;&#x201C; 416002, Maharashtra, INDIA. 2 Department of microbiology, B. J. Govt. Medical College and Sassoon General Hospital, Pune- 411001, Maharashtra, INDIA

Keywords: Acute Pyogenic Meningitis, Culture, LAT, CSF, Sensitivity, Specificity

ABSTRACT Introduction: Acute pyogenic meningitis is one of the most common and devastating disease of Central Nervous System. Difficulty and delay in diagnosis adds to problem in early management of patients. Rapid tests need to be invented so as to curbing the acute pyogenic meningitis cases. Aims: To identify the bacterial pathogens causing acute pyogenic meningitis and to evaluate the role of microscopy, Latex agglutination vis a vis culture in the diagnosis of pyogenic meningitis Methods: Clinically diagnosed cases of Acute pyogenic meningitis were included in the study. Cerebrospinal fluid (CSF) was the specimen of choice. CSF was processed in the microbiology laboratory for bacteriological aerobic culture, microscopy and Latex Agglutionation test (LAT). All the results analyzed statistically. Results: A total of 321 CSF specimens were screened. Among these 50 were from exclusive acute cases . On analysis gram stain positivity was 52%. Sensitivity of the gram staining was 64.29% and specificity was 52.78% while Culture positivity was 28%. Though LAT positivity (27.28%) was less as compared to microscopy and culture, LAT was found to be having higher sensitivity of 71.43% and specificity 80.55% . Conclusion: LAT can be a valuable tool in early diagnosis of acute pyogenic meningitis cases even after administration of antibiotics. It can be used as an adjunctive to culture and microscopy. In country like India, use of LAT should be concentrated on highly suspicious cases to make it cost-effective.

*Corresponding author: Dr. Shilpa Dayanand Putta, Assistant Professor, Department of Microbiology, Rajarshi Chhatrapati Shahu Maharaj Govt. Medical College, & CPR General Hospital campus, Dassara Chowk, Kolhapur â&#x20AC;&#x201C; 416002, Maharashtra, INDIA. Phone: +91 9049687272, 9860193300 Email: shilpa.putta21@gmail.com

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

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Latex Agglutination Test in Acute Pyogenic Meningitis

Introduction

Central nervous system (CNS) infections have been recognized as one of the most devastating of diseases. Among these CNS infections, bacterial meningitis found to be most common disease worldwide.[1] Approximately one million of pyogenic meningitis cases reported globally and around 2,00,000 of these die annually excluding epidemics. [2] In developed countries, mortality due to pyogenic meningitis is < 10% while in developing countries it may be 30% or more.[3] Clinically there is always difficulty in diagnosis of bacterial meningitis, this may be due to frequent non-specific symptoms and signs. Clinical diagnosis is often difficult in case of young children as compared to older. As acute bacterial meningitis has very high morbidity and mortality, its early diagnosis and treatment is vital.[4] Unfortunately in developing countries like India, there is delay in specimen transport and in addition recommended conditions are often not maintained during storage or transport. Also administration of antibiotics before collection of sample can clear the organisms from CSF leading to low number of pathogens from CSF. So, culture can yield false negative results. On the other hand gram stain is skill based; results can vary according to observer. Latex agglutination test can yield rapid results and is not affected by antibiotics or delay in transport, also is not a skill based technique.[5] The present study was conducted to evaluate usefulness of rapid diagnostic tests in acute pyogenic meningitis.

Material and Methods

The current study was conducted in the Department of Microbiology, in a tertiary care hospital. An ethical committee approval was obtained prior to commencing study. A total of 321 cases of acute pyogenic meningitis admitted in pediatric and medicine wards were included in the above study. Inclusion Criteria 1. The presence of clinical signs compatible with diagnosis of bacterial meningitis 2. CSF analysis with typical abnormalities suggestive of pyogenic meningitis 

WBC counts >10 cells/mm3



Protein >40mg/dl and/or



Sugar <40mg/dl

Specimen were collected by clinician as per standard guidelines. Standard 3 tubes of CSF were collected for chemistry, microbiology, and cytology. If possible 3-4 ml (1 ml minimum) CSF were collected into sterile, screwcap tubes. These all tubes were labeled and transported

to laboratory as soon as possible. If there was inevitable delay, then samples were kept at room temperature. CSF biochemical analysis was done by protein and sugar estimation. Simultaneously cytological examination was also performed and differential count estimated. Microbiological Processing On receipt of CSF into laboratory we looked for macroscopic examination. Specimen was preheated in boiling water bath for 3 min. After cooling, CSF was centrifuged for 5 min at 3000g. Supernatant separated and stored in a sterile tube. Sediment used for Gram staining and culture inoculation. 0.15 ml of deposit was inoculated on each of 5% sheep blood agar , chocolate agar and MacConkey Agar. 1 ml of deposit was deposited on brain heart infusion agar. [2,6] All plates were incubated at 37ᵒC in 5-10% CO2 for 4872 hours while brain heart infusion agar were incubated for 7 days and examined for growth daily. Identification of different isolates were done by standard microbiological techniques. [2,6] Latex agglutination test [2] was performed on CSF supernatant. CSF sample were tested for bacterial antigen detection using PASTOREXTM MENINGITIS (BIORAD), a latex antigen detection kit that included reagents: H.influenzae b, S.pneumoniae, Gp B Streptococcus, N. meningitides A, C, Y/W 135, N. meningitides B/E.coli, polyvalent negative Control and polyvalent positive control. A drop (40-50 µl) of the pre-treated sample supernatant placed in each of the agglutination card and one drop of latex regent delivered on the card following indicated distribution pattern. Sample and latex reagent mixed using a rod. The Card was rotated ̴ 120 rpm gently for 10 minutes and looked for agglutination visible to the naked eye within 10 minutes. All the observations and results were analyzed further using In Silico statistical software. Chi-square and P-value was calculated by using this software for comparison of difference to know the statistical significance.

Results

CSF specimens were collected from 321 patients for analysis which were screened for presence of leucocytes. 50 of these screened specimens included in the study and were subjected to different laboratory tests. Maximum number of cases were in the age group 12-50(48%) years followed by 1-6(26%) years age group. Males(64%) were more commonly affected than females(36%). Cytological analysis of CSF in patients of pyogenic meningitis was done . In 24 (48%) CSF samples (majority

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A-271 negative, 2 Enterococci, 1 S. aureus and 1 Citrobacter spp.) organisms isolated by culture, so those were excluded from analysis. One isolate was Citrobacter spp. which gave false positive reaction for N.meningitidis by latex agglutination. Among these 12 samples maximum identified organism was S.pneumoniae - 8(18.60%) followed by N.meningitidis â&#x20AC;&#x201C; 2(4.65%) and S.agalactiae- 2(4.65%). In this test, no cases showed LAT positive for H.influenzae and E.coli. When we further analysed LAT was found to be having sensitivity 71.43% and specificity 80.55% while positive predictive value was 41.67%.(Table 5)

of patient) cell count noticed were >1000 cells/cmm . In 14(28%) samples cell count were between 101-500 cells/cmm while Protein values were significantly higher ( >200 mg/dL) in 23(46%) patients. In 25(50%) sugar concentration was between 11-30 mg/dL; in 17(34%) it was between 1-10 mg/dL (Table 1). Out of 50 samples, total yield of gram stain positivity was 26(52%). Cocci (both gram positive and gram negative cocci) were common finding in microscopy. Sensitivity of the gram staining was 64.29% and specificity was 52.78% (Table 2). Out of 50 cases of acute pyogenic meningitis, Culture positivity was 28% in the present study. S. pneumoniae (10%) were the commonest isolates followed by K. pneumoniae (6%). (Table 3) . LAT was applicable to 43 samples. As LAT was not available for 7 (3 gram

On further statistical analysis using In Silico statistical software, gram stain was found to be stastically insignificant while LAT was found statistically significant in rapid diagnosis of pyogenic meningitis. (Table 6)

Table 1: CSF Cytology 1nd Biochemical Parameters Cytological parameters

Range <100 101-500 501-1000 >1000 50-100 101-200 >200 1-10 11-30 31-50 Total

Cells/cmm

Proteins (mg/dL)

Sugar (mg/dL)

No. of cases (Percentage) 6(12%) 14(28%) 6(12%) 24(48%) 15(30%) 12(24%) 23(46%) 17(34%) 25(50%) 8(16%) 50(%)

Table 2: Gram Stain Observations Morphology

Number

Gram positive diplococci

7(14%)

Gram negative diplococci

2(4%)

Gram positive cocci in short chains

4(8%)

Gram positive cocci in cluster

4(8%)

Gram negative bacilli

7(14%)

Budding yeast cells

2(4%)

Total

26(52%)

Table 3: Pathogens Isolated from CSFSamples By Culture Organisms

No of isolates

Percentage

Streptococcus pneumoniae

10%

Klebsiella pneumoniae

E.coli

Enterococci

S.aureus

Citrobacter spp.

Total

28%

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Table 4: Observations by Latex Agglutination Test Organism

Number of LAT positive

Culture positive

Percentage of LAT positive

S.pneumoniae

18.60%

N.meningitidis

4.65%

H.influenzae

S.agalctiae

4.65%

E.coli

Total

27.28%

Table 5: Gram Stain Versus Lat Sensitivity

Specificity

PPV

NPV

Gram stain

64.29%

52.78%

42.86%

82.76%

LAT

71.43%

80.55%

41.67%

93.54%

Table 6: Stastical Analysis Culture No growth

9(18%)

17(34%)

Negative

5(10%)

19(38%)

24(48%)

Total

14(28%)

36(72%)

50(100%)

Positive Gram stain

LAT

Total

Growth

Chi-square value

P value

1.1758

0.2782

7.8717

0.005

26(52%)

Positive

7(%)

12(%)

Negative

29(%)

32(%)

Total

7(%)

37(%)

43(100%)

*P value < 0.05 , taken as statistically highly significant

Fig. 1: Negative Latex agglutination test with positive and negative controls

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Fig. 2: LAT showing Positive agglutination with N.menigitidis A (Left) and Group B Streptococci (Right) antigen

Discussion

An accurate laboratory confirmation is essential in proper management of case of acute bacterial meningitis. Nowadays there has been increase interest in early and rapid diagnosis of etiological agent of acute pyogenic meningitis. There has been much techniques evolved to fulfill above needs; one of the commonly used technique is latex agglutination test.[2] The present study was undertaken to study the bacteriological profile of meningitis and also to do a comparative evaluation of Gram stain, culture and LAT, in clinically suspected cases of acute bacterial meningitis. Maximum samples in the present study, (table 1) showed cell count > 1000 cells/cmm which was 48%, followed by in 28% between 101-500 cells/cmm. Total 40% were showed CSF cytology between 101-1000 cells/cmm. In all the cases majority of cells were neutrophils. These findings were comparable to study conducted by Adhikary et al(7) in which maximum cell count was about, was >1000 cells/ cmm in 60.53% . Total 46% of cases showed protein concentration in the cerebrospinal fluid >200 mg/dL while in 30% cases, it was between 50-100 mg/dL while in remaining 24% of cases it was between 101-200 mg/dL. So majority of cases were above 50 mg/dL of protein concentration.It was seen that there was associated decrease in sugar levels in cerebrospinal fluids of patients. This typical picture of high protein and low sugar levels were found in all the cases. Typical high protein count and low sugar levels similar to the present study was also found in a study done by Adhikary et al(7), Gurley et al(8), Domingo et al(9) and AlMarzoqi et al. (10) www.pacificejournals.com/apalm

In our study when the sensitivity of gram stain (table 2,5,6) was evaluated, it was 64.29% and specificity was 52.78%, positive predictive value 42.86% and negative productive value 82.76%. In the present study, gram stain found to be statistically non significant by applying chi-square test as P value was more than 0.05. Mohammadi et al[11], had reported gram stain sensitivity 53.33%, specificity 83.52%,positive predictive value 36.36% and negative predictive value of 91.02%. in another study by Awari et al[12], had reported sensitivity 97.22%, but much lesser specificity of 14.28%, positive predictive value 74.47% and negative predictive value of 66.67%. While Vishwanath et al[13] in their study found gram sensitivity of 90%. Higher sensitivity in other studies correlates with the concentration of bacteria in cerebrospinal fluid of the patients of pyogenic meningitis. The likelihood of increased positivity or sensitivity is also depends on specific pathogen leading to meningitis. Gram stain results were observed to be ~20% lower in patients of pyogenic meningitis who received prior antimicrobial therapy.[14] Bacterial culture(table 3 and 4) detected 28% of etiological agents causing acute pyogenic meningitis. In the majority of patients, 16% isolates were showed gram positive organisms. Among gram positive bacteria, S.pneumoniae (10%) was the commonest organism isolated. Gram negative bacteria isolated in culture were 12%. Among gram negative bacteria, K.pneumoniae (6%) was the major cause of meningitis followed by E.coli (4%) and Citrobacter spp.(2%). These findings correlates with results reported by Adhikary et al[7], Mohammadi et al[11] and Bajaj et al[15]. eISSN: 2349-6983; pISSN: 2394-6466

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Reasons for low culture yield may be: False negative results can be seen in case of if specimen been transported or stored under inappropriate conditions[16] OO If an antibiotic therapy started before collection of CSF specimen[16] OO Low bacterial load[11] Detection of bacterial antigen in the present study was done in 43 patients by using latex agglutination and the test was positive in 12(27.90%) samples. Among these 12 latex positive samples 8(18.60%) were S.pneumoniae and 2(4.65%) were N.meningitidis, S.agalctiae each. Similar results were observed by Shivaprakash et al[17], Ceyhan et al[18], Mishra et al[19], which had 28.94%, 23% and 19.3% latex agglutination positivity respectively. OO

In the present study, no N.meningitidis, H.influenzae b and E.coli isolated. Detection of N.meningitidis especially group b antigen poses a problem by immunological techniques as it has been suggested that it had poor immunogenicity of this antigen. Also it is possible other organisms including N.meningitidis which are not detected in LAT or produced false negative results, reason for this might be that the antiserum included in latex agglutination kit does not detect all capsular serotypes prevalent in geographical area.[11] Also Enterobacteriaceae other than E.coli, that were culture isolated not included in the antigen kit. In the present study (table 5 and 6) on culture, 7 were grown the organisms that are not included in latex agglutination ; so latex agglutination test was applicable to 43 cases. Also LAT was observed to be statistically significant, as its P value was < 0.05. LAT also found statistically significant in Bajaj et al[15] study in diagnosis of bacterial meningitis. Similar accuracy indices results correlating with our studies were reported by Mohammadi et al[11] study, in which sensitivity of latex agglutination was 66.66%, specificity 87.91%, positive predictive value 35.29% and negative predictive value was 96.38%. Statistical analysis such as sensitivity, specificity, positive predictive value and negative predictive value considering CSF culture as gold standard has its own limitations which could affected sensitivity, specificity and positive predictive value negatively. In the present study, LAT had less sensitivity as it cannot detect all bacteria which are included in panel. And from results which showed low culture yield of fastidious organisms, it is clear that LAT is superior in detecting fastidious organisms such as S.pneumoniae, H.influenzae and S.agalctiae. Though it had low positive predictive value(41.67%) high negative predictive value(93.54%) rules out acute bacterial meningitis. [11]

Though the culture is considered as gold standard for diagnosis bacterial meningitis, it has a certain limitations in diagnosis. It is time consuming, give false negative results due to improper storage and transport, use of poor growth media or antibiotic therapy before specimen collection. So, a culture is less sensitive in diagnosis of bacterial menigitis.[16, 15] Latex agglutination test also has certain limitations. Cross reactions are common with bacteria which share common antigen which can be nullified or minimized by heating the CSF before testing. It cannot detect other bacteria that are also important cause of acute pyogenic meningitis such as K.pneumoniae, L.monocyotogenes and other gram negative bacilli. [20]

Conclusion

In spite of drawbacks of LAT, When culture is compared with LAT in various previous studies it is observed that it is useful rapid diagnostic test for acute pyogenic meningitis. Also it had been an important diagnostic tool in bacterial meningitis cases where poor diagnostic facilities or resources available and also important in identifying pathogen in whom preadmission antibiotics administered. Thus, LAT provides rapid microbiological diagnosis of acute bacterial meningitis earlier than culture so as to guide the clinicians for administration of appropriate antibiotics. Considering its high cost we advice its use based on the individual case history appears to be rational.

Funding None

Competing Interests None Declared

References

1. Somand D, Meurer W. Central Nervous System Infections. Emerg Med Clin N Am 2009; 27:89-100. 2. World Health Organization, Centers for Disease Control and Prevention. Laboratory methods for the diagnosis of meningitis caused by neisseria meningitidis, streptococcus pneumoniae, and haemophilus influenzae: WHO manual. 3. Cartwright KAV. Bacterial meningitis. In: Borriello P, Murray PR, Funke G. ed. Topley and Wilsonâ&#x20AC;&#x2122;s Microbiology and Microbial Infections. 10th edition. London: Edward Arnold, 2006. 4. Bashir H El, Laundy M, Booy R. Diagnosis and treatment of bacterial meningitis. Arch Dis Child 2003;88:615â&#x20AC;&#x201C;620.

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5. Surinder K, Bineeta K, Megha M. Latex Particle Agglutination Test as an adjunct to the diagnosis of bacterial meningitis. Ind J Med Microb 2007; 25 (4): 395-397. 6. Forbes BA, Sahm DF, Weissfeld AS. Meningitis and Other Infections of the Central Nervous System. In: Bailey and Scott’s Diagnostic Microbiology. 12th edition. China: Mosby Elsevier Co.; 2007. 7. Adhikary M, Chatterjee RN. Laboratory evaluation of meningitis attending a tertiary care hospital in India: an observational study. Int J Nutr Pharm Neuro Dis. 2013; 3(3): 282- 88. 8. Gurley ES, Hossain MJ, Montgomery SP, Petersen LR, Sejvar JJ, Mayer LW,et.al. Etiologies of bacterial meningitis in Bangladesh: results from a hospitalbased study. Am J Trop Med Hyg. 2009; 81(3):475-83. 9. Domingo P, Pomar V, Benito ND, Coll P. The spectrum of acute bacterial meningitis in elderly patients. BMC Infect Dis. 2013; 13: 108. 10. Al-Marzoqi AH, Al-Janabi DK, Al Taee ZM, Hussain HJ. Prevalence and Physiological analysis of Acute Bacterial Meningitis infections at care center in Babylon province. J Natur Scien Resear. 2012; 2(9): 75-81. 11. Mohammadi SF, Patil AB, Nadagir SD, Nandihal N, Lakshminarayana SA. Diagnostic value of latex agglutination test in diagnosis of acute bacterial meningitis. Ann Indian Acad Neurol. 2013; 16(4): 645-9. 12. Awari A, Nighute S. Incidence of bacterial meningitis with special reference to latex agglutination test. J Rec Adv App Sci. 2012; 27: 65-68.

13. Viswanath G, Praveen, Hanumanthappa AR, Chandrappa NR, Mahesh CB. Bacteriological study of pyogenic meningitis with special reference to latex agglutination. Indian J Pathol Microbiol. 2007 ;50(1):97-100. 14. Pomar V,Benito N,López-Contreras J,Coll P, Gurgui M,Domingo P. Spontaneous gram negative bacillary meningitis in an adults: as characteristics and outcome. BMC Infect Dis. 2013 ;13: 451. 15. Bajaj S, Garg D, Bajaj M, Agrawal N, Mandowara SL. Value of rapid antigen assay in diagnosis of pyogenic meningitis and partially treated meningitis. Int J Cur Rev. 2013; 5(16): 47-53. 16. Surinder K, Bineeta K, Megha M. Latex Particle agglutination Test as an adjunct to the diagnosis of bacterial meningitis; Ind J Med Microbio. 2007; 25 (4) :395-7. 17. Shivaprakash MR, Rajagopal V, Nagarathna S. Latex agglutination test in the diagnosis of pyogenic meningitis. J Commun Dis. 2004; 36(2): 127-31. 18. Ceyhan M, Yildirim I, Balmer P, Borrow R, Dikici B, Turget M. et. al. A prospective study of etiology of childhood acute bacterial meningitis, Turkey. Emerg Infect Dis. 2008; 14(7): 1089-94. 19. Mishra B, Mahaseth C, Rayamajhi A. Latex agglutination test for early detection of causative organism in acute bacterial meningitis. J Nepal Pediatr Soc. 2013; 33(1): 34-38. 20. Kalpana L. Diagnostic evaluation of latex agglutination test and study of spectrum of bacterial pathogenesis of neonatal meningitis. IOSR J Dent Med Sci. 2013; 7(4): 57-62.

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Original Article Platelet Volume Indices: Silver Linings for Vascular Complications in Diabetes Mellitus

Mitakshara Sharma1*, Sanjeev Narang2 and Maj. (Gen.) S.K. Nema2 Dept. of Pathology, All India Institute of Medical Sciences, Rishikesh, India Dept. of Pathology, Index Medical College & Research Centre, Indore, India

1 2

Keywords: Platelet Volume Indices, Mean Platelet Volume, Platelet Distribution Width, IFG, HBA1c, Diabetes

ABSTRACT Background: Morbidity and mortality in diabetes owing to micro and macro angiopathic complications is well known. Platelet indices are potentially useful surrogate markers for early diagnosis of diabetic complications attributed to platelet activation and recognized by increase in Platelet Volume Indices (PVI) including Mean Platelet Volume (MPV) and Platelet Distribution Width (PDW). The aim is to determine and compare MPV and PDW in known cases of diabetics with and without diabetes related complications. To document changes in platelet indices with duration of diabetes and to assess utility of platelet indices in early identification of vascular complications especially in developing countries like India. Methods: A two year cross sectional study with total 330 individuals segregated into two groups:- (a) Diabetic subjects with diabetes related complications (b) Diabetic subjects without diabetes related complications. Samples for HbA1c and platelet indices were obtained and processed on SYSMEX-X-800i autoanalyser. Result: The study revealed significant positive correlation between PVI and duration of diabetes across the groups (MPV-HbA1c r = 0.951; PDW-HbA1c r = 0.875). MPV & PDW of subjects with and without diabetes related complications were (15.14 ± 1.04) fl & (17.51±0.39) fl and (18.96 ± 0.83) fl & (20.09 ± 0.98) fl respectively with a significant p value 0.00. Conclusion: The current study demonstrates raised platelet indices in association with rising glycaemic levels and diabetes related vascular complications. This is the first study of its kind in India which is comprehensive with an adequately powered study design. PVI should be researched and explored further as surrogate marker to develop a clinical tool for early recognition of diabetic vascular changes.

*Corresponding author: Dr. Mitakshara Sharma, AIIMS, Rishikesh (UK)- 249203 Phone: +91 9993795966 Email: mitakshara.sharma@yahoo.com

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

Sharma et al.

Introduction

Diabetes mellitus (DM) is a metabolic disorder prevalent in pandemic proportions, incurring significant morbidity and mortality due to associated complications chiefly arising out of micro and macro angiopathic changes. Platelet related thrombogenesis plays a key role in the pathogenesis of these complications. These complications are attributed to platelet activation which can be recognized by an increase in Platelet Volume Indices (PVI) which includes Mean Platelet Volume (MPV) and Platelet Distribution Width (PDW). Therefore, it is postulated that Platelet Indices can potentially be useful surrogate markers for the early detection of thromboembolic and cardiovascular complications in diabetes. Patients with Diabetes Mellitus have a higher risk of vascular complications and burden of atheromatous plaque and recurrent thrombotic events which worsens the prognosis of vascular angiopathies as compared to non diabetic patients. [1] The vascular complications caused in diabetes include Coronary Artery Disease (CAD), atherosclerosis, medial calcification, retinopathy, nephropathy, neuropathy and peripheral arterial disease (PAD). [2] Data suggests that there is a 2-4 fold increase in CAD and PAD and also a 3 fold increased risk for stroke as compared to non diabetics. Deaths due to diabetes are increasing and there is a need to prevent these deaths by early diagnosis of impending complications. A large proportion of patients with Type 2 DM suffer from preventable vascular angiopathies and so there is a need to develop risk factor modifications and interventions to reduce the impact of such complications. Thrombogenic platelets are large, active and dysfunctional and these patients of DM with larger platelets can easily be identified during routine haematological analysis and could possibly benefit by early preventive measures. The introduction of automated cell counters has facilitated the availability of PVI (MPV and PDW) as routine parameters. Platelet Volume Indices as a group constitute an important, simple, effortless and cost effective tool that should be extensively explored and used as a marker for early diagnosis of diabetic complications, especially in developing countries with limited financial resources for predicting the possibility of impending vascular events in patients with Diabetes mellitus.

Materials and Methods

A-277 of the institute.The current study is a prospective analytical case control study conducted over 2 years. The cases and controls were those attending the Out Patient Department (OPD) and admitted in the hospital. As per the American Diabetes Association (ADA) criteria, [3] the subjects were segregated into 3 groups. The patients with HbA1c < 5.7% were considered as controls and put into non-diabetic (ND) group, those with HbA1c between 5.7% and 6.4% were considered as pre-diabetics and put into IFG (I) group and patients with HbA1c â&#x2030;Ľ 6.5% were considered as diabetics and put into the diabetic (D) group. Further, the diabetic group was divided into two groups on the basis of history of diabetes related vascular complications Patients suffering from idiopathic thrombocytopenic purpura, acute post streptococcal glomerulonephritis, renal failure, iron deficiency anaemia, cyanotic congenital heart diseases, hypertension, aplastic anaemia and patients on antiplatelet drug therapy were excluded. Pregnant women were also excluded from the study. Informed prior consent was taken from the patients and clinical details were recorded in a predesigned proforma. All the investigations were done in the central laboratory (pathology and clinical biochemistry) of INDEX Hospital, Indore. Since platelets are extremely easily activated; therefore, in order to avoid artefactual results, the sampling procedure was standardized and clean venipunctures were performed. All subjects were made to abstain from tobacco and caffeinecontaining beverages on the day of sampling. In every case 2.0-5.0 ml blood was collected under aseptic precautions in the respective blood collection tube. All the samples were processed within 2 hours of collection. Samples for Platelet Indices and Platelet Count Sample of the blood was drawn from the antecubital veins of the patients that allowed technically good sampling for platelet function testing and the blood sample was, with few exceptions, collected by a single laboratory technician/nurse in each study. Venipunctures were always performed without stasis with the subjects in a semi-recumbent position. They were collected using K3 EDTA (ethylene diamine tetra acetic acid) as an anticoagulant and were processed on SYSMEX-XS-800i autoanalyser.

The present study was conducted in the Department of Pathology, Index Medical College and associated Hospital, Nemawar Road, Indore between June 2012 and May 2014. The study protocol was implemented after obtaining approval and due clearances from the Ethical Committee

Samples for FPG and HbA1c Blood sampling was performed after an overnight fast. The samples for blood glucose and HbA1c were drawn from the antecubital vein. The samples for FPG were collected in plain tube and for HbA1c were collected in the EDTA tube and were processed on ERBA EM 360 autoanalyser.

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Statistical Analysis The p value was calculated for each parameter and p value <0.05 was considered to be significant. The power of study was kept at 99% and level of significance (α) at 5%. “Correlation Coefficient (r)” was also done to find the correlation between glycaemic levels (HbA1c & FPG) and PVI & PVI and duration of diabetes. Statistical analysis was done by the Statistical Package for Social Sciences (SPSS) version 16 for windows.

Result

A total of 1100 individuals were selected based on the laboratory investigations and all cases were subjected to clinical examination and diagnoses were reviewed, out of which 930 individuals fulfilled the selection criteria. A total of 330 individuals were enrolled in the diabetic group (D) of which 194 (58 %) were male and 136 (42 %) were females. A total of 300 individuals were enrolled in impaired fasting glucose (I) group of which 176 (58.6 %) were males and 124 (41.4 %) were females. Non diabetic (ND) group also had a total of 300 patients of which 134 were males (44.6%) and rest were females (43.4 %). The mean age in the diabetic group was 64.82 with a SD of 8.16. The youngest patient in this group was 45 years and the eldest patient was 99 years. The mean age in the IFG group was 60.50 with a SD of 7.34. The youngest patient in this group was 46 years and the eldest patient was 75 years. The mean age in the non diabetic group was 59.80 with a SD of 10.09. The youngest patient in this group was 37 years and the eldest patient was 82 years. The mean fasting plasma glucose (FPG) was highest in the diabetic group (128.12 mg/dl) with a SD of 37.26, followed by the IFG group (109.36mg/dl) with a SD of 7.81 and lowest in the non diabetic group (98.90 mg/dl) with a SD of 4.26. The difference of mean of FPG between all the three groups was analyzed and it was statistically significant (p value <0.001). The mean HbA1c was highest in the diabetic group (9.55%) with a SD of 1.80 followed by the IFG group (6.01%) with a SD of 0.21 and the non diabetic group (4.97%) with a SD of 0.32. The difference of mean for HbA1c between all the three groups was analyzed and it was statistically significant (p <0.001). The mean platelet count was recorded for various study groups. The mean platelet count was highest in the non diabetic group (2.97 lacs) with a SD of 0.82 followed by IFG group (2.55 lacs) with a SD of 0.71 and lowest in the diabetic group (2.51 lacs) with a SD of 0.69. The difference of mean of platelet count between all the three groups was analyzed and it was statistically significant (p value <0.001). The mean platelet volume (MPV) was highest in

the diabetic group (17.60 fl) with a SD of 2.04 followed by IFG group (11.76 fl) with a SD of 0.73 and lowest in the non diabetic group 9.93 with a SD of 0.64. The difference of means of MPV between all the three groups was analyzed and it was statistically significant (p value <0.001). The platelet distribution width (PDW) was highest in the diabetic group (19.17µm) with a SD of 1.48 followed by IFG group (15.49 µm) with a SD of 0.67 and lowest in the non diabetic groups was analyzed and it was statistically significant (p value <0.001). (Table 1, Figure 1) On correlation coefficient r, a positive correlation was found between PVI (MPV & PDW) and FPG, HbA1c & duration of diabetes. (Table 2, Figure 2a, 2b, 2c, 2d, 2e, 2f). In the diabetic group, based on the presence or absence of diabetes related vascular complications two groups were formed. Out of 330 diabetic patients, 213 patients (64.55%) suffered from diabetic complications and 117 patients (35.45%) were diabetic patients without diabetes related complications. There were 67 males & 50 females in the diabetic group without complications and 127 males & 86 females in the diabetic group with complications. The sex distribution was more equitable in the group of diabetics without complications. The mean age in the diabetic group with complication was (65.93) with a SD of 8.22 and the mean age in the diabetic group without complications was (62.80) with a SD of 7.71. The mean duration of diabetes among those with diabetic complications was found to be 9.82 years with a SD of 3.86 and only 0.85 years with a SD of 0.93 in those without complications. (p value <0.001) The mean FPG was higher in the diabetic group with complications (129.64 mg/dL) with a SD of 42.84 as compared to the group without diabetic complications (125.37mg/dL) with a SD of 23.95. (p value 0.246) HbA1c was also higher (10.70%) with the SD of 1.04 in the group with diabetic complications as compared to the group without complications (7.46%) with a SD of 0.65. (p value 0.001) In diabetic patients with complications MPV was higher (18.96fl) with a SD of 0.83 as compared to the other group with MPV (15.14fl) with a SD of 1.04. (p value 0.000) Also, in diabetic patients with complications PDW was higher (20.09 fl) with a SD of 0.98 as compared to the diabetic group without complications PDW (17.51 fl) with a SD of 0.39. (p value 0.000) The platelet count was slightly decreased in diabetic group with complications (2.46 lacs) with a SD of 0.77 as compared to those without complications (2.54 lacs) with a SD of 0.53. (p value 2.340) (Table 3, Figure 3)

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Table 1: Comparison of the demographics, glycaemic characteristics (FPG & HbA1c), platelet counts and platelet volume indices (MPV & PDW) among different groups using ANOVA (analysis of variance). Parameter Total No. Male (No.) Female (No.) FPG (mean ± SD) (fl) HbA1c (mean ± SD) (%) TotalPlatelet count (mean ± SD) (lacs) MPV (mean ± SD) (fl) PDW (mean ± SD) (µm)

F value

P value

Remark

300 176 124 109.36 ± 7.81 6.01 ± 0.21

Non-Diabetic Group (N) (N=300) 300 134 166 98.09 ± 4.26 4.97 ± 0.32

139.23 1521.09

<0.001 <0.001

S S

2.51 ± 0.69

2.55 ± 0.71

2.97 ± 0.82

34.56

<0.001

17.60 ± 2.04 19.17 ± 1.48

11.76 ± 0.73 15.49 ± 0.67

9.93 ± 0.64 10.59 ± 0.67

2832.89 5345.21

<0.001 <0.001

S S

Diabetic Group (D) (N=330)

IFG Group (I) (N=300)

330 194 136 128.12 ± 37.26 9.55 ± 1.80

Table 2: Correlation coefficient (r) between PVI and Various Parameters Parameter FPG HbA1c Duration of diabetes

PDW r value 0.462 (positive correlation) 0.875 (positive correlation) 0.630 (positive correlation)

MPV r value 0.460 (positive correlation) 0.951 (positive correlation) 0.714 (positive correlation)

p value 0.000 0.000 0.000

Remark S S S

Table 3: Comparison of demographics, glycaemic characteristics (FPG & HbA1c), platelet counts and platelet volume indices (MPV & PDW) between diabetic patients with complications and diabetic patients without complications.

Total (No.) Male (No.) Female (No.) Age (mean ± SD)

With diabetic complications 117 (35.45%) 67 50 62.80 ± 7.71

Without diabetic complications 213 (64.55%) 127 86 65.93 ± 8.22

Duration of diabetes (mean ± SD)

0.85 ± 0.93

FPG (mean ± SD) ( mg/dL) HbA1c (mean ± SD) (%) MPV (mean ± SD) (fl) PDW (mean ± SD) (fl) Total Platelet count (mean ± SD) (lacs)

Parameters

z value

p value

Remark

9.82 ± 3.86

-32.25

<0.001

129.64 ±42.84

125.37 ± 23.95

-1.16

0.246

10.70 ± 1.04 15.14 ± 1.04 17.51 ± 0.39

7.46 ± 0.65 18.96 ± 0.83 20.09 ± 0.98

-34.76 -34.20 -33.85

0.001 0.000 0.000

S S S

2.54 ± 0.53

2.46 ± 0.77

-1.11

0.267

Fig. 1: Comparison of the platelet counts and platelet volume indices (MPV & PDW) of study participants in diabetics, impaired fasting group (I) and the non diabetic (D).

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Fig. 2a: Scatter plot showing a positive correlation between MPV and HbA1c.

Fig. 2b: Scatter plot showing a positive correlation between MPV and duration of diabetes.

Fig. 2c: Scatter plot showing a positive correlation between PDW and HbA1c.

Fig. 2d: Scatter plot showing a positive correlation between PDW and duration of diabetes.

Fig. 2e: Scatter plot showing a positive correlation between Total Platelet Count and HbA1c0.

FiG. 2f: Scatter plot showing a positive correlation between Total Platelet Count and duration of diabetes.

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Fig. 3: Comparison of platelet counts and platelet volume indices (MPV & PDW) of study participants in diabetics with complications and diabetics without complications.

Discussion

Diabetes is increasing in leaps and bounds and so are its complications. This pandemic disorder is the cause for large scale morbidity and mortality owing to micro and macro angiopathic complications and is proving to be a big economic burden especially in poor countries with scarcity of resources. Any marker that can be useful in predicting the onset of complications in DM will be a boon for the patient in particular and country in general. In keeping with this line of thinking, this study was undertaken Studies have shown that there is a constant association of increased MPV with atherosclerotic diseases like coronary ischemic disease, myocardial infarction and cerebral infarct as well as with diabetes and hypertension.[4] MPV is a new emerging risk factor for atherothrombosis. In our study, MPV as a parameter was studied and evaluated across different glycaemic groups. A direct relationship was found between MPV and glycaemic status. The poorer the glycaemic control the higher was the MPV. It was found to be significantly higher in the diabetic group, followed by the IFG group. Normal MPV was found in all the non diabetic individuals. It was very conspicuously obvious that MPV increased with the duration of diabetes and it was found to be significantly higher in those with longer duration and high glucose level as compared to patients with short duration of diabetes (Figure 2 a & b). Zuberi et al (2008) [5] evaluated MPV in three different glycaemic categories (diabetics, IFG and non diabetics). The results of the present study are in agreement with this study which www.pacificejournals.com/apalm

also reported a stepwise increase in MPV from non diabetic group to IFG group to diabetic group. Authors viz. Papanas et al (2004),[6] Demirtunc et al (2009),[7] Jindal et al (2011) [8] have compared MPV values only in diabetics and non diabetics and have found an increased MPV in diabetics as compared to the non diabetics. The present study was compared with that of Bhayana et al (2013).[9] They documented no significant changes in MPV found in diabetic and non diabetic group (MPV in nondiabetics and diabetics was 8.04 fl). This was in contrast to our study where significantly higher MPV was found in diabetics (17.60Âą2.04)fl as compared to the non diabetics (9.93Âą0.64)fl with a significant p value. All other workers have reported results similar to the present study. In this study, PDW as an isolated parameter was also studied and evaluated in different glycaemic groups. PDW was corroborated as being directly proportional to the glycaemic status and duration of diabetes. The patients with poor glycaemic control revealed higher values of PDW. It was found to be significantly higher in the diabetic group, followed by the IFG group whereas normal PDW was found in the non diabetic group. It was also seen that values of PDW were also affected by the duration of diabetes and were significantly higher in those with longer duration of diabetes (Figure 3 a & b). Farah Jabeen et al (2013) [10] and Kir Young Kim et al (1986) [11] evaluated and compared PDW in diabetics and non diabetics and showed an increase in PDW in diabetics as compared to the non diabetics. Hence it can be stated that Platelet Volume Indices (PVI) increase during platelet activation. In the present eISSN: 2349-6983; pISSN: 2394-6466

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study, PVI were evaluated and compared across different groups based on glycaemic status. Stepwise increase in the PVI from non-diabetics to IFG to diabetics was observed and MPV & PDW were found in increasing order from: - Non-diabetics IFG Diabetics. They were found to have a significant linear positive correlation and a direct relationship with glycaemic control and duration of diabetes. With rising glycaemic levels, it was found that PVI levels also increased correspondingly. Similar relation was found with duration of diabetes. The longer the duration, the higher are the PVI. The present study also evaluated and compared PVI (MPV and PDW) and platelet count amongst diabetic cases with and without vascular complications such as acute and after recovery Coronary artery disease (CAD), Diabetic retinopathy (DR), Peripheral neuropathy (PN), Peripheral vascular disease (PVD) and Diabetic nephropathy (DN). In our analysis it was found that PVI (MPV & PDW) were significantly higher in diabetic cases with vascular complications as compared to diabetic cases without complications. Kir Young Kim et al (1986) [11] compared MPV and PDW in patients with known platelet activation with the controls and have concluded that PVI (MPV in particular) are increased in patients with known platelet activation as seen in the vascular diseases like myocardial infarction (MI), Coronary artery disease (CAD) and Ischaemic heart disease (IHD) as compared to the healthy individuals. The findings of our study were in accordance with the results of these studies and the PVI were found to be significantly increased in patients with vascular complications of diabetes. The present study also reported significantly higher PDW in patients suffering from Ischaemic heart disease (IHD). This was similar to the findings of Khanderkar et al (2006). [12]

Conclusion

This is the first study of its kind in India representing a pioneering effort including a large number of subjects and adequately powered to evaluate both platelet indices viz. MPV and PDW in diabetics, non diabetics and patients with IFG. PVI are a useful means for identifying larger & active platelets which play an important role in the development of micro and macro angiopathic complications of diabetes leading to mortality and morbidity. Simultaneous and serial measurements of platelet indices is easy and therefore

might serve as a valuable predictor of impending vascular complications, predict a poor outcome in patients with vascular disease and also prevent the worse outcome of these angiopathies. Due to the introduction of automated cell counters, PVI (MPV & PDW) are routinely available in almost all clinical laboratories with ease, speed and accuracy. The prevalence of DM and its vascular burdens are increasing day by day and itâ&#x20AC;&#x2122;s the need of the hour to prevent and monitor them. Thus, PVI should be researched and explored further as surrogate markers to develop a clinical tool for early recognition of diabetic vascular changes and thereby help prevent them. They can prove to be more useful in developing countries with limited financial resources. Patients with larger platelets can easily be identified during routine haematological analysis because PVI are generated as a by product of automated blood counts.[13]

Acknowledgements Nil

Funding Nil

Competing Interests Nil

Reference

1. Mahdavi et al. The effect of seeing a family physician on the level of glycosylated hemoglobin (HbA1c) in type 2 Diabetes Mellitus patients. Journal of Diabetes & Metabolic Disorders 2013;12:2. 2. Ferroni P, Basili S. Platelet activation in type 2 diabetes mellitus. J Thromb Haemost 2004;2:1282â&#x20AC;&#x201C;1291. 3. American Diabetes Association. Standards of Medical Care in Diabetes. Diabetes care 2013;36(Supplement 1):S11-S66. 4. Khemka, R., Kulkarni, K. Study of relationship between Platelet Volume Indices and Hyperlipidemia. Annals Of Pathology And Laboratory Medicine, 2014;1(1), 8-14. 5. Zuberi BF. Comparison of mean platelet volume in patients with diabetes mellitus, impaired fasting glucose and non-diabetic subjects. Singapore Medical Journal 2008;49(2):114-6. 6. Papanas N, Symeonidis G, Maltezos E. Mean platelet volume in patients with type 2 diabetes mellitus. Platelets 2004;15:475-8.

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Sharma et al. 7. Demirtunc R. The relationship between glycemic control and platelet activity in type 2 diabetes mellitus. J Diabetes Complications 2009;23(2):89-94.

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10. Farah Jabeen. Role of platelet indices, glycemic control and hs-CRP in pathogenesis of vascular

complications in type-2 diabetic patients. Pak J Med Sci 2013;29(1):152-156. 11. Kim KY. Mean platelet volume in the normal state and in various clinical disorders. Yonsei Medical Journal 1986;27(3). 12. Khandekar MM, Khurana AS. Platelet volume indices in patients with coronary artery disease and acute myocardial infarction: an Indian scenario. J Clin Pathol 2006;59:146â&#x20AC;&#x201C;149. 13. Manchanda, J, Potekar R, Badiger S, Tiwari, A. The study of platelet indices in acute coronary syndromes. Annals Of Pathology And Laboratory Medicine, 2015;2(1), A30-A35.

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8. Jindal S. Platelet indices in diabetes mellitus: indicators of diabetic microvascular complications. Hematology 2011;16(2):86-9. 9. Bhayana A. Is large platelet size a risk factor for acute coronary syndrome: A retrospective case-control study. J MGIMS, 2009 Sep;14(ii):52-55.

Original Article Study of Serum Magnesium Levels in Type 2 Diabetes Mellitus at Tertiary Medical Centre, Karnataka, India Vidya .B1*, Kishan Prasad HL2, U Sanjana Rao1, Chandrika Rao2 and Suchetha Kumari3 Intern, K.S. Hegde Medical Academy, Mangalore, India Department of Pathology, K.S. Hegde Medical Academy, Mangalore, India 3 Department Of Biochemistry, K.S.Hegde Medical Academy, Mangalore, India 1

Keywords: Correlations, Fasting Blood Sugar, Serum Magnesium, Type 2 Diabetes, Post Prandial Blood Sugar

ABSTRACT Background: Diabetes and poor glycaemic control alters the metabolism of magnesium (Mg) by increasing their urinary excretion and lowering serum Mg levels. Low serum Mg levels will contribute to the evolution of diabetic complications such as retinopathy, abnormal platelet function, cardiovascular disease and hypertension via reduction in the rate of inositol transport and subsequent intracellular depletion. This study aimed at evaluation of serum Mg levels with Type 2 diabetics and control non diabetic individuals. Methods: After the Ethical approval, blood samples from recruited subjects (50 diabetics and 50 non diabetics) were collected and estimation of Mg in serum by xylidyl blue method, Fasting blood sugar (FBS) and post prandial blood sugar (PPBS) by Glucose oxidase peroxidase (GOD-PAP) method was done. Descriptive statistics mean and standard deviation, independent sample â&#x20AC;&#x2122;tâ&#x20AC;&#x2122; test, level of significance 5% were used for analysis. Result: Mean value of FBS in control group of 100.98mg/dL and in case group with 134.80mg/dL. Mean value of PPBS in control group of 108.76mg/dL and in case group with 236.6mg/dL. Mean value of serum Mg in control group was 1.64mmol/L while in case group=1.20mmol/L. Serum magnesium was significantly decreased (p<0.001) in diabetics than controls. A negative linear relationship (p<0.05) between, Mg and FBS (r=-0.198 and p=0.048), Mg and PPBS (r=-0.206 and p=0.040) was found. Conclusion: The present findings demonstrate the imbalance in levels of serum Mg among the patients of type 2 DM in comparison to controls. Since serum magnesium is easily and inexpensively measured, and as oral magnesium replacement is cheap and safe, there is an argument for screening of diabetic patients for hypomagnesaemia and institution of supplementation if it is detected.

*Corresponding author: Dr. Vidya. B; D.No.3-27-2234/37 Gokulam house, adri Rocks, Kadri Kambla Road, Mangalore-575004, Karnataka, India Phone: +91 9964144055 Email: vidyabnayak1993@gmail.com

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Vidya .B et al.

Introduction

Type 2 diabetes mellitus (DM) is a major growing public health problem that affects over 200 million individuals worldwide. It is on track to become one of the major global public health challenges of the 21st century. India has become the “Diabetic Capital of the World”. Thus, there is an urgent need to develop primary prevention strategies aimed at controlling this epidemic.[1,2] Homeostasis of the trace elements such as zinc, copper, iron and magnesium (Mg) has been found to play an important role in the pathogenesis of diabetes and its complications. [1] Mg, one of the important components of many foods such as grains, nuts and green leafy vegetables is an essential cofactor for enzymes involved in glucose metabolism. Deficiency of Mg has been associated with wide variety of clinical conditions, including Type-2 DM. Hence it’s expected to have a negative impact on essential biochemical processes. [3, 4] Mg has received considerable attention for improving insulin sensitivity and preventing diabetes and its complications. [2] Diabetes and poor glycaemic control alters the metabolism of Mg by increasing their urinary excretion and lowering serum Mg levels. This association may reflect a ‘vicious cycle’ with hyperinsulinemia associated with insulin resistance contributing to extracellular Mg depletion and, in turn, further augmentation of insulin resistance by hypomagnesaemia. Low serum Mg levels may contribute to the evolution of diabetic complications such as retinopathy, abnormal platelet function, cardiovascular disease and hypertension via reduction in the rate of inositol transport and subsequent intracellular depletion. Further, Mg deficiency has been proposed as a novel factor implicated in the pathogenesis of late diabetic complications. [3-8] The primary prevention of type 2 DM and its complication through diet and lifestyle modifications is of paramount public health importance. In Indian scenario, very few studies are conducted regarding its association between type 2 diabetes and serum magnesium level. Hence this study was taken up to assess the correlation of serum Mg level with fasting and post prandial blood sugar levels in diabetics and non diabetics.

Materials And Methods

Study conducted by obtaining informed consent from the subjects after the institutional ethical approval. It is a prospective study conducted in tertiary hospital of south canara district between May to July 2014. The study group comprised 50 cases of Type 2 DM of either sex and control group of 50 non diabetic subjects of either sex. www.pacificejournals.com/apalm

A-285 Inclusion Criteria 50 cases of type-2 DM, confirmed by biochemical investigations as per WHO criteria. The following table summarizes the 2006 WHO recommendations for the diagnostic criteria for diabetes: (Table-1) Exclusion Criteria Patients with Type 1 DM, acute complications such as severe infection, major operations, severe cardiovascular or respiratory diseases. Under aseptic precautions, 2ml of plain blood is drawn by venepuncture, for estimation of Serum Mg levels. The serum is separated by centrifugation at 1500rpm for 15 minutes and stored at 4° Celsius. Estimation of serum Mg by xylidyl blue method is performed. 2ml of fluoride anticoagulated blood in appropriate vacutainer by venepuncture after 8 hours of fasting, for estimating FBS levels and 2 hours after a standard meal for estimating PPBS levels. Its estimation is done by Glucose oxidase peroxidase (GOD-PAP) method. Instrument used for measuring the absorbance: Semi auto analyzer. Statistical Analysis: - Descriptive statistics mean and standard deviation. -

Independent sample‘t’ test.

Level of significance 5%

Result

It is a prospective study in which 100 subjects were included. Out of which 50 were Type 2 DM patients confirmed by biochemical investigations as per WHO criteria and 50 were non-diabetic apparently healthy control subjects. The age group of cases and controls were between 20-80 years. (Table 2) Serum magnesium, FBS and post prandial blood sugar levels was measured in these subjects. FBS, PPBS and Mg levels are as shown in Table 3. They were significantly higher in cases than in controls. Independent Sample T-Test The result was significant showing that, there is difference in mean Mg, FBS, and PPBS between control and diabetic. Serum magnesium was significantly decreased (p<0.001) in diabetics than controls. (Table 3) Correlations Pearson’s correlation coefficient was used to find out the association between magnesium, FBS and PPBS. A significant negative correlation between serum magnesium, FBS and PPBS was observed. (Table 4). eISSN: 2349-6983; pISSN: 2394-6466

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Table 1: 2006 WHO recommendations for the diagnostic criteria for diabetes Fasting Blood Glucose (FBS) (mg/dL) Normal <110 DM ≥126 Table 2: Comparison of 2 parameters in two groups (control and case): Parameters Groups Control(n=50)

Gender

Case(n=50)

Age (Mean)

Control Case

Table 3: COMPARISON OF 3 PARAMETERS IN TWO GROUPS (CONTROL AND CASE): Parameters Mean Control 100.98 Fasting Blood Glucose (FBS) (mg/dL) Case 134.80 Control 108.76 Post Prandial Blood Glucose (PPBS) (mg/dL) Case 236.6 Control 1.64 Serum magnesium (mmol/L) Case 1.20 Table 4: Pearson’s Correlation Pearson Correlation Serum Mg v/s FBS Serum Mg v/s PPBS

Discussion

Diabetes accounts for a significant part of the morbidity and mortality. Diabetes is estimated to affect 25.6 million American adults and 366 million people worldwide, and the numbers will continue to increase to 552 million by 2030 globally. Therefore, primary prevention of type 2 diabetes through diet and lifestyle modifications is of paramount public health importance. Research over the past two decades has provided evidence of a clinical correlation between diabetes and low magnesium intake which may be a contributing factor in the progression of DM and its complications, [9] Comparing our study to the study done by Shrabani Mohanty, et al (2013) which showed that FBS in their control group was 81.96mg/dL and 218.62mg/dL in case group. This comparison shows that the FBS value in our case group (134.80mg/dL) was in discordance when compared to theirs (218.62mg/dL). The reason for this might be that most of the diabetics in our study group were probably under strict diabetic diet and hence under control.[10] PPBS value in control group of our study was found to be 108.76mg/dL and in case group it was 236.6mg/dL. This was in concordance with their study which showed 113.56mg/dL for control group and 285.04mg/dL for case group. [10]

r Value -0.198 -0.206

2 hour glucose (mg/dL) <140 ≥200

Male = 29 Female = 21 Male = 25 Female = 25 46 53 Std Deviation 11.52 45.96 25.27 88.10 0.778 0.345

p Value <0.001 <0.001 <0.001

p Value 0.048 0.040

The mean value of serum Mg in our control group was found to be 1.64mmol/L and in case group it was 1.20mmol/L. It is observed that serum Mg was significantly decreased (p<0.001) in diabetics than controls which is in concordance with the previous study. [10] Similar such decrease in serum magnesium level in diabetic’s patients as compared to controls has been reported by some authors. [2, 10-17] (Table 5). Correlation of serum Mg level with FBS and PPBS levels in type 2 DM patients and control group was assessed using Pearson’s correlation coefficient. A significant negative correlation between serum Mg v/s FBS and serum Mg v/s PPBS was observed in our study. Mg depletion has a negative impact on glucose homeostasis and insulin sensitivity in diabetic patients as well as on the evolution of complications such as retinopathy, thrombosis and hypertension. Preventing low Mg status in diabetics may therefore be beneficial in the management of the disease. The reasons for the high prevalence of Mg deficiency in diabetes are not clear, but may include increased urinary loss, lower dietary intake, or impaired absorption of Mg compared to healthy individuals. Several studies have reported increased urinary Mg excretion in type 1 and 2 diabetes. [10] Patients with DM had altered

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Table 5: Comparison between FBS, PPBS and Mg in various studies with our study: Studies Group FBS PPBS Mg Control 83.13 1.37 Salem M,et al.[2] Case 264.07 0.89 Control 86.8 3.36 Mahendra B G, et al.[11] Case 107.8 1.60 Control 81.96 113.56 1.91 Shrabani Mohanty,et al. [10] Case 218.62 285.04 1.58 Control 94.52 140 2.62 Dipankar Kundu,et al. [12] Case 165 201 2.02 Control 92.8 128.72 2.40 [13] M Prasad Naidu, et al. Case 170.23 265.70 1.80 Control 85.57 2.26 [14] Mahadeo Mane,et al. Case 112.77 1.98 Control 93.80 132.20 2.47 Without Without Without Asha S Khubchandani,et al. [15] Case complications:172.00 complications:204.80 complications:1.94 With complications: 235.44 With complications: 268.48 With complications:1.32 Control 88.13 127.03 2.17 Without Without Without Mirza Sharif Ahmed Baig,et al. [16] complications:230.70 Case complications:142.97 complications:1.61 With complications: With complications:187.83 With complications:1.29 317.00 Control 93.6 136.32 2.375 A.G. Kulkarni,et al. [17] Case 124.1 210.4 1.96 Control 100.98 108.76 1.64 Our Study Case 134.80 236.6 1.20

metabolism of Mg, probably due to hyperglycemia. Previous studies showed the impaired metabolism of the elements may contribute to the progression of DM and its complications. [2, 4-7]

Conclusion

The present findings demonstrate the imbalance in levels of serum Mg among the patients of type 2 DM in comparison to controls. Since serum magnesium is easily and inexpensively measured, and as oral magnesium replacement is cheap and safe, there is an argument for screening of diabetic patients for hypomagnesaemia and institution of supplementation if it is detected.

Acknowledgements Nil

Conflicts of Interest Nil

Funding

ICMR- Indian Council Of Medical Research

Competing Interests None Declared

www.pacificejournals.com/apalm

Reference

1. Jiancheng Xu, Wei Xu, Hanxin Yao, Weixia Sun, Qi Zhou, Lu Cai. Associations of serum and urinary magnesium with the pre-diabetes, Diabetes and diabetic complications in the chinese northeast population. PLoS ONEÂ 2013; 8(2):e56750. 2. Salem M, Kholoussi S, Î&#x161;holoussi N, Fawzy R. Malondialdehyde and trace element levels in patients with type 2 diabetes mellitus. Arch Hellen Med 2011; 28(1):83-8. 3. Cavalot F, Petrelli A, Traversa M, Bonomo K, Fiora E, Conti M, et al. Postprandial blood glucose is a stronger predictor of cardiovascular events than fasting blood glucose in type 2 diabetes mellitus, particularly in women: Lessons from the San Luigi Gonzaga Diabetes Study. J Clin Endocrinol Metab 2006; 91:813-9. 4. Ankush RD, Suryakar AN, Ankush NR. Hypomagnesaemia in Type-2 Diabetes Mellitus Patients: A study on the status of oxidative and nitrosative stress. Indian J Clin Biochem 2009; 24(2):184-9. 5. Dong JY, Xun P, He K, Qin LQ. Magnesium intake and risk of Type 2 diabetes: Meta-analysis of prospective cohort studies. Diabetes Care 2011; 34(9):2116-22. eISSN: 2349-6983; pISSN: 2394-6466

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6. Meenakshi Puri, Uma Gujral, Nayyar SB. Comparative study of serum Zinc, Magnesium and Copper levels among patients of type 2 Diabetes mellitus with and without Microangiopathic complications. Innovative Journal of Medical and Health Science 2013; 3(6):274 -8. 7. Song Y, He K, Levitan EB, Manson JE, Liu S. Effects of oral magnesium supplementation on glycaemic control in Type 2 diabetes: a meta-analysis of randomized double-blind controlled trials. Diabetic Med 2006; 23(10):1050-6. 8. Peters KE, Chubb SA, Davis WA, Davis TM. The relationship between hypomagnesaemia, metformin therapy and cardiovascular disease complicating type 2 diabetes: The Fremantle Diabetes Study. PLoS ONE 2013; 8(9):e74355. 9. Kao WH, Folsom AR, Nieto FJ, Mo JP, Watson RL, Brancati FL. Serum and dietary magnesium and the risk for type 2 diabetes mellitus: the Atherosclerosis Risk in Communities Study. Arch 1999; 159(18):2151-9. 10. Supriya, Mohanty S, Pinnelli VB, Murgod R, Raghavendra DS. Evaluation of serum copper, magnesium and glycated haemoglobin in Type 2 Diabetes mellitus. Asian J Pharm Clin Res 2013; 6(2):188-90.

11. Gandhe MB, Jain K, Gandhe SM. Evaluation of 25(OH) Vitamin D3 with reference to magnesium status and insulin resistance in T2DM. J Clin Diagn Res 2013; 7(11):2438-41. 12. Kundu D, Osta M, Mandal T, Bandyopadhyay U, Ray D, Gautam D. Serum magnesium levels in patients with diabetic retinopathy. Nat Sci Biol Med 2013; 4(1):113-6. 13. Naidu MP, Shiva Kumar, Vali SM, Madhav D, Subrahmanyam G. Study of the role of copper, zinc and magnesium in diabetic nephropathy. RJPBCS 2013; 4(4):71. 14. Mane M, Gunwant RC, Reddy EP. Hypomagnesaemia in diabetic patients and biochemical action on the cardiovascular system. Int J Biol Med Res 2012; 3(1): 1273-6. 15. Khubchandani AS, Sanghani H. Study of serum magnesium and HbA1C in diabetic patients along with changes in their lipid profiles. Indian Journal of Clinical Practice 2013; 23:11. 16. Sharif M, Baig A, Sugoor M, Sarwari KN. Serum HSCRP and magnesium in type 2 diabetic patients without and with complications. International Journal of Basic and Applied Medical Sciences 2013; 3(3):218-28. 17. Kulkarni AG, Shendge SK, Shinde V. Study of serum magnesium levels in Type 2 Diabetes mellitus. IOSR Journal of Dental and Medical Sciences (IOSR-JDMS) 2014; 13(4):115-9.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Original Article An Insight Into Pituitary Fossa Lesions: A Single Institutional Experience

Purwa Rangrao Patil, Bhavana Madhukar Bharambe* and Divyaja Sondankar Dept. of Pathology, Grant Government Medical College & Sir J J Group of Hospitals, Byculla, Mumbai, India Keywords: Sellar, Pituitary, Adenoma, Craniopharyngioma, Squash Cytology

ABSTRACT Introduction: The complex placement of the sellar region and the various native tissues provide a fertile ground for the development of spectrum of non-neoplastic and neoplastic lesions in this area. Methods: The prospective study of two years in a tertiary care hospital was conducted. A total of 67 specimens of sellar and parasellar region were received from the neurosurgery department for intraoperative as well as histopathological diagnosis. Squash cytology was utilised for rapid intraoperative consultation. Results: Pituitary adenomas were the most common tumours followed by craniopharyngiomas. Conclusion: Multidisciplinary approach of radiology, neurosurgery and pathology helps in arriving at the right conclusion and diagnosis in central nervous system lesions.

*Corresponding author: Dr. Bhavana Madhukar Bharambe, (Assistant Professor) Pathology in Grant Government Medical College & Sir J J Group of Hospitals, Byculla, Mumbai-400008, INDIA Phone: +91 9892906747 Email: bhavanab.136@gmail.com

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Lesions of Sellar/Parasellar Region

Introduction

The anatomical placement of the sellar region is an important factor for varied differential diagnosis of the pathological lesions of this area. The complex localisations and vicinity of important anatomical landmarks like optic chiasma is primarily an important cause of disability in patients. The classification of tumours of sellar region is enumerated in Table 1. Pituitary adenomas (PA) constitute 10% to 15% of all intracranial neoplasms. The genesis is not known exactly. Most PAs are sporadic but association with multiple endocrine neoplasia (MEN) type 1 is identified in 2.7% of individuals.[1]The family history is encountered in 5% cases. Clinically, PA can be functional or non-functional depending on their behaviour. Radiologically, PAs are classified by their size based into microadenoma (less than <10 mm) or macroadenoma (equal or greater than ≥10 mm). Histologically, they used to be classified as either basophilic, acidophilic or chromophobic on basis of Hematoxylin and Eosin (H&E) staining, which is not used in this era. Functionally, PAs can be classified depending on the hormones elaborated by them, detected immunohistochemically and serologically (Table 2). WHO classifies pituitary tumours as typical and atypical adenomas and pituitary carcinoma. In addition to symptoms produced by hormone secretion, the localisation and size of the tumour in pituitary fossa also leads to pressure symptom like diminuition of vision, vision loss, bitemporal hemianopia, headache, loss of visual acuity and pituitary insufficiency (hypopituitarism)[2] Radiology is an important tool in determining the location, size and extent of the tumour. Magnetic resonance imaging (MRI), on T1 weighted imaging, shows hypointense tumour whereas on T2 imaging, isointense to grey matter and diffusely hyperintense and show moderate to strong post contrast enhancement. PA is classified as adenomas into 0 of 4 grades (0–IV) based on radioanatomical findings.[3] The tan brown colour of PA is characteristic. Being soft in consistency, they are spread easily during squash cytology done for intraoperative diagnosis. The squash cytology shows monolayer sheets of uniform cells with moderate cytoplasm, round nuclei, delicate stippled chromatin and inconspicuous nucleoli. The histological examination shows a tumour arranged in sheets or papillary or trabecular pattern. The cells may be acidophilic, basophilic or chromophobic. The typical PA is generally mitotically inactive. The PA showing increased mitosis and Ki67 index with p53 overexpression can be labelled as atypical adenomas. The tumours infiltrating the bone, the cavernous sinus, diaphragm sellae can be labelled as infiltrative PAs. Pituitary carcinomas are very rare tumours elaborating prolactin as well as ACTH commonly.[4] Mere presence of

local invasion, cellular atypia or increased mitosis cannot differentiate infiltrative PAs from pituitary carcinoma.[4] The sole presence of metastasis is only confirmatory of carcinoma. The incidentally detected PAs during imaging performed for some unrelated cause or at autopsy are called as incidentalomas. Pituitary apoplexy is a condition that occurs when pituitary adenomas suddenly haemorrhage internally, causing a rapid increase in size or when the tumour outgrows its blood supply which causes tissue necrosis and subsequent swelling of the dead tissue causing visual loss and sudden onset headache. Spindle cell oncocytoma is a very rare subtype of primary tumour of adenohypophysis, the diagnosis of which is largely based on the pathologic characteristics of the tumour. Craniopharyngiomas (CP) represent 1–2% of all intracranial neoplasms and about 10% of the tumours of the sellar region.[5,6] They show bimodal sex distribution and also present with pressure symptoms or endocrinologic abnormalities. On neuroimaging, CPs are typically calcified, solid or cystic (or mixed solid-cystic) lesions that have a complex lobular appearance. On gross pathological examination, they exhibit mixed solid-cystic appearance. The cut surface shows cysts (containing dark greenishbrown liquid resembling machinery oil) and secondary changes such as fibrosis, calcification, ossification and the presence of cholesterol-rich deposits. CP is difficult to spread on squash smears. The cytology shows sheets of closely packed epithelial cells with clear spaces, resembling transitional or stratified squamous epithelium. The histological examination of CP is characterised by islands of densely packed squamous epithelium in cords, lobules and irregular trabeculae bordered by palisaded columnar epithelium along with intermingled stellate reticulum. Nodules of “wet keratin” are present. Cystic cavities containing squamous debris are lined by flattened epithelium. The surrounding gliosis may be evident in case of infiltration. The sella turcica can harbour other neoplasms derived from bone, meninges, neural tissue and nasal sinuses like plasmacytoma, giant cell tumour of bone, chordoma, meningioma, schwannoma, haemangiopericytoma and salivary gland tumours.[7]The metastatic tumours to pituitary gland account for only 1% of cases. Metastases of breast, lung or gastrointestinal carcinoma are often encountered at autopsy. With thourough understanding of features of all these lesions, histopathologist plays an important role in the definitive intraoperative or post operative diagnosis of lesions in this complex area.

Materials and Methods

This was a prospective study of sixty seven patients with sellar and suprasellar lesions operated at a tertiary care

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hospital in Mumbai, India. The clinically and radiologically suspected and histologically confirmed cases of sellar and parasellar tumours were enrolled in the study. The study was approved by the institutional ethics committee of the instituition. The details of the each patient were taken from medical records i.e. age, gender; clinical presentation, radiological evaluation (MRI and/or CT scan), location, brain infiltration and recurrence were noted. Intra-operative consultation was done by squash smears for which sample was received in isotonic normal saline or wet guaze as early as possible from the operating room to avoid artifacts. Squash diagnosis was noted and was correlated with final histology. Both squash cytology and histological sections were viewed by experienced pathologists. In all cases, specimen if remaining after squash cytology as well as those received following surgery were fixed in 10% buffered neutral formalin for 24 hours. The received specimen, fragmented or in toto, whole tissue was submitted for processing. The paraffin embedded blocks were cut into 4-5 micron sections and stained with routine Haematoxylin and Eosin stain (H&E). The reticulin stain was done wherever needed to differentiate between hyperplasia and adenoma.

Results

The prospective study over a period of two years conducted in a tertiary care hospital. A total of sixty seven cases were studied. The lesions of sellar, suprasellar and parasellar lesions were as shown in Table 3. The commonest age group affected was 31-40 years (34.15%) followed by 41-50 years group (26.83%). Headache was the commonest symptom seen in 75.60% cases followed in close frequency by visual disturbances seen in 68.30% cases. Symptom related to hormonal disturbances (amenorrhoea, galactorrhoea, menstrual irregularity) was seen in about 21.96% cases (9.76, 2.44, 9.76% respectively). Majority were nonfunctioning adenomas (63.41%) (Table 4). Prolactin was secreted by most functioning PAs. 8 cases out of 41 were

recurrent contributing to 19.51%. The radiology, cytology and histology of the PA was characteristic (Figure 1,2 & 3). CP contributed 4.35% of all intracranial neoplasms and 5.48% of all extra-axial lesions. The younger age group was commonly affected from 11-30 years (28.57%). 12 out of 21 cases were males. 18 out of 21 presented with headache (85.71%), 12 out of 21 presented with visual disturbances (57.14%). 3 cases out of 21were recurrent contributing to 14.28%. The radiology showed a solid cystic lesion (Figure 4). The squash smear cytology showed squamous epithelium with keratin (Figure 5) while histopathology confirmed the diagnosis (Figure 6). Four cases of meningiomas were diagnosed with varied histological (meningothelial, papillary, psammomatous and metaplastic) pattern in our study (Figure 7). The squash cytology was 100% sensitive and specific in such cases. A single case of lymphocytic hypophysitis was also seen (Figure 8).

Discussion

The PAs are the most common tumours of the sellar region which was also seen in our study. They constituted 61.19% of all tumours followed by CP (31.35%). The findings were in accordance with Saegeret et al[9] reported who found 86.64% of PA in sellar neoplasms and Valassi et al[10] who stated it as 15% of all intracranial neoplasms and Cho et al.[11] In present study, most common affected age group was 31-40 years constituting 34.15% cases. Cases in 3th and 5th decade contributed 73.17% (30 out of 41) while Arvind Rishi et al [12] who found 50.6% cases in 3rd to 5th decade. The mean age in present study was 40.26 years which was in concordance with findings of John et al [13], Rishi A et al [12] and Alma Ortiz plata et al [14] who found it as 44.9 years, 46.2 years and 41.4 years respectively. In present study 7.31% cases were less than 20 years of age. In a study done by Mindermann and Wilson et al [15] less than 6% cases were below 20 years of age, while in a study

Table 1: Tumour and tumour like lesions in pituitary gland and sellar region7 Tumours of anterior pituitary

Pituitary adenoma, Atypical pituitary adenoma Pituitary carcinoma, Spindle cell oncocytoma

Tumours and tumour like lesions of non-pituitary origin

Craniopharyngioma , Meningioma, Langerhans cell histiocytosis, Metastasis, Chordoma.

Inflammatory lesions

Lymphocytic hypophysitis, Granulomatous hypophysitis, Sarcoidosis.

Cystic lesions

Rathkeâ&#x20AC;&#x2122;s cleft cyst, Arachnoid cyst Epidermoid / dermoid cyst

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Table 2: Classification of pituitary adenoma8 Adenoma type Lactotrophic adenoma Somatotrophic adenoma Mixed lactotrophic&somatotrophic Corticotrophic Gonadotropic adenoma Thyrotropic adenoma Pluerihormonal adenoma Silent subtype Null cell adenoma

Hormone Prolactin (PRL) Growth hormone(GH) PRL/GH Adrenocorticotrophic Hormone (ACTH), Endorphins Follicle Stimulating Hormone (FSH), Leutinising Hormone (LH) Thyrotropin Stimulating Hormone (TSH) GH, PRL, TSH -

Table 3: Lesions of sellar, suprasellar and parasellar regions (n=67) Lesions No. Pituitary adenoma 41 Craniopharyngioma Meningioma Lymphocytic hypophysitis

% 61.19

21 04 01

31.35 5.97 1.49

Table 4: Incidence of functioning and non-functioning pituitary adenoma Normal (Non-functioning)

No. of cases

Total %

26 63.41

Fig. 1: T2 weighted coronal MRI image shows a well defined, solid intensely enhancing sellar mass (pituitary adenoma).

Deranged (Functioning) Growth hormone Prolactin TSH ACTH (Cortisol )

02 09 02 02 15 36.59

Fig. 2: Pituitary adenoma (Squash smear) - Shows monolayer sheets of monotonous cells having round nuclei with speckled chromatin (H&E, X 400).

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Fig. 3: Pituitary adenoma – Shows sheets & nests of tumour cells having round nuclei with speckled chromatin (H&E, X 400).

Fig. 4: T2 weighted coronal MRI image shows a solid-cystic intensely enhancing sellar mass (craniopharyngioma).

Fig. 5: Craniopharyngioma (Squash smear) - Shows sheets of epithelial cells against keratinous background (H&E, X100).

Fig. 6: Craniopharyngioma - Shows cystic spaces along with sheets of squamous epithelium bordered by palisaded columnar epithelium (H&E, X100).

Fig. 7: Meningioma – Shows characteristic whirling pattern of arrangement of cells. (H&E, X100).

Fig. 8: Lymphocytic Hypophysitis – Dense infiltration of lymphocytes and plasma cells in the pituitary gland. (H&E, X100).

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done by Plata et al [14] 9.8% cases were less than 20 years of age. In a present study female cases contributed 41.46% while studies done by John et al [13] showed 55% and Daly AF et al [16] which showed 67.6% female cases. In present study, most common presenting symptom was visual disturbances in 80.49% of cases, followed by headache in 68.29% cases. In a study done by John et al [13] visual disturbances were seen in 94.4% cases while headache was present in 75.3% cases. Study done by Paulo Andrade de Mello et al [17] showed visual disturbances in 87.3% of cases and headache in 70% cases. In present study about 63.41% pituitary adenomas were non-functioning. This correlated with the studies done by John et al [13] in which 77.3% were non-functioning, Rishi et al [12] (51% nonfunctioning) and Paulo Andrade de Mello et al [17] (60.6% non-functioning). In the present study, out of 36.59% of functioning adenoma, prolactin secreting adenoma were the most common contributing to 21.95% cases. This finding was correlated with Matshana Kennedy John et al13 in which it was 14.7%. In present study 90.24% were macroadenoma while Alma Ortiz plataet al [14] showed 80% macroadenomas. In general tumour size correlated with functional activity. Non-functioning are usually diagnosed as macroadenomas due to absence of clinical manifestations which allow tumour growth over a period of time. The squash cytology correlated with histology in all cases except that of a case of pituitary apoplexy. The case of lymphocytic hypophysitis was diagnosed on intraoperative squash cytology which was overdiagnosed as pituitary adenoma on radioimaging. Frequency of recurrence varies between 19 to 34% in different studies.18In our study recurrent cases contributed 19.51% while in Alma Ortiz plata et al [14] 29.4% cases were recurrent. CPs represent 1–2% of all intracranial neoplasms and about 10% of the tumours of the sellar region.[5] In present study CP was second most common neoplasm of sellar region contributed to 31.35% of sellar neoplasms and 4.35% (21 out of 383) of all intracranial neoplasms. In the present study headache was seen in 85.71% and visual disturbances in 61.90% of cases of CP. In a study done by Jennifer L Shin et al [19] 67% of the cases were having visual disturbances. In our study most of the cases are seen in 2nd -3rd decade with slight male preponderance while Jennifer L Shin et al [19] found most cases in later half of 3rd decade with no gender bias. The squash smears were diagnostic in all but one case where the diagnosis of epidermoid cyst was offered. 3 out of 21 cases were recurrent contributing 9.52%. Role of Pathology The pathologist has a prime role in the intraoperative diagnosis of the lesion when the surgeon has to decide the further plan of surgery. The squash smears have

become a useful tool in giving prompt opinion about the nature of the lesion. Pituitary adenomas can be reliably differentiated from inflammatory and cystic lesions, like lymphocytic hypophysitis and Rathke’s cleft cysts in our study. However, the pathologist needs to be trained with pathological aspects of various central nervous system tumours. These smears had a fairly acceptable correlation with histology in our cases.

Conclusion

PA constituted 8.49% of all intracranial neoplasms and 61.19% cases of sellar tumours in the study over a period of two years. Majority were non- functioning adenomas and seen in young adults. CP was second commonest (31.35%) tumour in the region, also found in young adults. The squash smear cytology correlated well with histology in 97.56% cases in cases of PA and 95.24% cases of CP. Four cases of meningiomas and a single case of lymphocytic hypophysitis were diagnosed.

Acknowledgment Nil

Source of Funding Nil

Competing Interest Nil

LIST OF ABBREVIATIONS USED

PA - Pituitary Adenoma CP - Craniopharyngioma H&E - Haematoxylin and eosin CNS - Central nervous system CT - Computerised tomography MRI - Magnetic resonance imaging WHO - World Health Organisation PAS - Periodic acid Schiff GH - Growth hormone ACTH - Adrenocorticotrophic hormone PRL - Prolactin FSH - Follicular stimulating hormone LH - Luteinizing hormone TSH - Thyroid stimulating hormone hpf - High power field N/C - nuclear to cytoplasmic ratio

References

1. Scheithauer BW, Laws ER, Kovacs K, et al. Pituitary adenoma of the multiple endocrine neoplasia type I syndrome. SeminDiagnPathol. 1987;4:205-211. 2. Kleinschmidt BK, De Masters. Pituitary gland. In: Juan Rosai, editor. Rosai and Ackerman’s Surgical

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Pathology. 10th ed. Edinburgh. Mosby Elsevier; 2012. 2441-2461. Lloyd RV, Kovacs K, Young Jr WF, et al. Tumours of the Pituitary gland. In: De Lellis RA, Lloyd RV, Heitz PU, Eng C, editors. World Health Organization Classification of Tumours. Pathology and genetics of Tumours of Endocrine Organs. Lyon, France. IARC; 2004. 10-13. Scheithauer BW, Kovacs K, Horvath E, et al. Pituitary carcinoma. In: DeLellis RA, Lloyd RV, Heitz PU, Eng C, eds. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Endocrine Organs. Lyon, France. IARC Press; 2004. 36-39. Rushing EJ , Giangaspero F, Paulus W, et al. Craniopharyngioma. In: Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, eds. WHO Classification of Tumours of the Central Nervous System. Lyon, France. IARC Press; 2007. 238-240. Adamson TE, Wiestler OD, Kleihues P, Yasargil MG. Correlation of clinical and pathological features in surgically treated craniopharyngiomas. J Neurosurg. 1990;73:12-17. Burger PC, Scheithauer BW, Vogel FS. Region of the sella turcica. In: Surgical pathology of the Nervous system and its coverings. 4th ed. New York. Churchill Livingstone; 2001. 437-490.

8. Scheithauer BW. The Pituitary and Sellar Region. In: Mills SE, Carter D, Greenson JK, Reuter VE, Stoler MH, editors. Sternberg’s Diagnostic Surgical Pathology. 5th ed. Philadelphia. Lippincott Williams & Wilkins; 2010. 460-488. 9. Saeger W, Lüdecke DK, Buchfelder M, Fahlbusch R, Quabbe HJ, Petersenn S. Pathohistological classification of pituitary tumours: 10 years of experience with the German pituitary tumour registry. European J Endocrinol. 2007;156: 203-216.

11. Cho HJ, Kim H, Kwak YJ, et al. Clinicopathologic Analysis of Pituitary Adenoma - A Single Institute Experience. J Korean Med Sci 2014;29:405-410. 12. Rishi A, Sharma MC, Sarkar C, Jain D, Singh M, Mahapatra AK, Mehta VS, Das TK. A clinicopathological and immunohistochemical study of clinically non-functioning pituitary adenomas: A single institutional experience. Neurology India. 2010;58:418-423. 13. Matshana, John K. Analysis of pituitary tumours: retrospective study at Chris Hani Baragwanath and Charlotte Maxeke Johannesburg academic hospitals, 1999-2008. [Available from: http://hdl.handle. net/10539/8798] 14. Plata AO, Tena-Suck ML, Neri IP, Bojórquez DR, Fernández A. Pituitary Adenomas – Clinico-Pathological, Immunohistochemical and Ultrastructural Study, Pituitary Adenomas, VafaRahimi-Movaghar (Ed.). 2012. ISBN: 978-95351-0041-6, InTech, Available from: http://www. intechopen.com/books/pituitary-adenomas/pituitaryadenomas-clinico-pathologicalimmunohistochemicaland-ultrastructural-study 15. Mindermann T, Wilson CB. Age related and gender related occurrence of pituitary adenomas. ClinEndocrinol (Oxf). 1994;41:359-64. 16. Daly AF, Jaffrain-Rea ML, Ciccarelli A, Socin HV et al. Clinical Characterization of Familial Isolated Pituitary Adenomas. J Clin Endocrinol Metab. 2006;91:3316-3323. 17. De Mello PA, Naves LA, Neto AP, et al. Clinical and laboratorial characterization and post-surgical follow-up of 87 patients with non-functioning pituitary macroadenomas. ArqNeuropsiquiatr. 2013;71:307-312.

10. Valassi E, Biller BM, Klibanski A, Swearingen B. Clinical features of nonpituitarysellar lesions in a large surgical series. ClinEndocrinol (Oxf). 2010;73:798-807.

18. Reddy R, Cudlip S, Byrne JV, Karavitaki N, Wass JA. Can we ever stop imaging in surgically treated and radiotherapy–naïve patients with nonfunctioning pituitary adenoma? Eur J Endocrinol. 2011; 165:739-44.

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Original Article Clinicopathological Study of Primary Focal Segmental Glomerulosclerosis: A New Vision of all Variants Sakhi Anand1, Aminder Singh1* and Mary Mathew2 Department of Pathology, Dayanand Medical College & Hospital, Ludhiana, India 2 Department of Pathology, Kasturba Medical College, Manipal, Karnataka, India

Keywords: Primary Focal Segmental Glomerulosclerosis, Immunofluorescence, Nephrotic Syndrome, Variant.

ABSTRACT Background: This study is a detailed clinical and histomorphological analysis of Primary Focal Segmental Glomerulosclerosis diagnosed by combined approach of histopathology and direct immunofluorescence. The aim was to identify the morphologic variants with histopathological prognostic features of primary focal segmental glomerulosclerosis and to establish the clinical, laboratory & pathologic findings in nephrotic syndrome & renal insufficiency associated with primary focal segmental glomerulosclerosis. Methods: It was a prospective & retrospective analysis of 41 cases of primary Focal Segmental Glomerulosclerosis. Routine & special stains were done all renal biopsies. Immunofluorescence studies were performed. Multiple comparisons among the groups were performed using ANOVA. Mean comparison of two groups was performed using independent sample t-test. A categorical variable was tested using Chi-square test and fishers exact test. Results: Out of 718, all the 41 renal biopsies of primary FSGS classified into morphologic variants. Primary Focal segmental glomerulosclerosis constituted 5.7% of total kidney biopsies. Mean age was 35.93 years having male preponderance. Proteinuria was highest in Perihilar variant while hematuria was more in the cellular variant. Nephrotic syndrome was most commonly associated with the cellular and perihilar variant. It was only histological parameter whose distribution among the different variants was statistically significant. A statistically significant correlation (p<0.05) was noted between the percentage of globally sclerosed glomeruli with hypertension & serum creatinine. A significant correlation was found between serum creatinine and mesangial hypercellularity, serum albumin, podocyte hyperplasia, arteriolar hyalinosis, intimal sclerosis and medial hypertrophy. Conclusions: This comprehensive study of primary FSGS reiterates that different histological variants of FSGS have substantial differences in clinical and histological features.

*Corresponding author: Dr. Aminder Singh, Assistant Professor, Department of Pathology, Dayanand Medical College & Hospital, Tagore Nagar, Ludhiana Phone: +91 8968966550 Email: dramisingh@gmail.com

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

Anand et al.

Introduction

Focal and segmental glomerulosclerosis (FSGS) is a clinicopathological entity that affects both adults and children and manifests clinically by persistent nephrotic syndrome, non-selective proteinuria, microscopic haematuria, hypertension and commonly, renal insufficiency at presentation. The clinical course of patients with FSGS is one of progressive deterioration of renal function leading to end-stage renal disease (ESRD) over 5-10 years. Some patients with FSGS respond to steroids, others do not. FSGS can be primary or secondary to other primary processes.[1,2] At present, there is no agreement on histological feature or constellation of features that separates patients with primary FSGS into prognostically distinct groups. A new classification scheme has been proposed by D’ Agati et al in 2004[3] which subcategorizes FSGS into distinct histomorphologic categories, which have distinct aetiopathogenetic factors. The morphologic variants based on specific diagnostic inclusion/exclusion criteria are an attempt to separate lesions with different clinical outcomes. This study was planned to identify morphologic variants and other histo-morphologic prognostic features of FSGS and correlate with clinical parameters at the time of renal biopsy. In addition, this study also correlates the clinical, laboratory and pathologic findings in renal insufficiency associated with primary FSGS.

Material and Methods

This is a prospective and retrospective study of 4 years duration. A total of forty-one patients diagnosed with primary Focal Segmental Glomerulosclerosis (FSGS) were included in this study. Diseases associated with secondary FSGS: morbid obesity, chronic hypertension, renal dysplasia, solitary kidney, reflux nephropathy, infections (HIV, parvovirus B19), medication (pamidronate, lithium, interferon-α) or intravenous drug abuse, family history of renal disease and sickle cell anaemia, history of genetic diseases with predisposition to FSGS, history of previous dialysis/transplant or other glomerular disease with sclerotic lesions were excluded. The sections were stained with Haematoxylin and eosin (H&E), Periodic acid-Schiff (PAS), Periodic acid methenamine silver (PASM) or Jones methenamine silver (JSM) and Masson trichrome (MT). Immunofluorescence studies were performed on biopsies. The sections were studied using the direct immunofluorescence technique with commercially prepared fluorescence isothiocyanate-conjugated antisera to IgG, IgM, IgA and C3. The histopathological lesions on the renal biopsy were classified in accordance with the Columbia classification system described by D’Agati et al.[3] Clinical and laboratory data were obtained in each patient at the time of biopsy. The data included age, sex, www.pacificejournals.com/apalm

A-297 duration of onset of illness, hypertension, and the degree of proteinuria, presence or absence of haematuria, serum albumin levels and creatinine levels at the time of renal biopsy. Glomerular filtration rate was calculated as GFR (ml/min) = 186 x serum creatinine (mg/dl)-1.154 x age0.203 x 0.742 (for female). Morphologic variants, various histomorphologic parameters and immunofluorescence were correlated with clinical and laboratory parameters at the time of presentation. Statistical comparisons were carried out with the aid of SPSS statistical software. Multiple comparisons among the groups were performed using ANOVA. Mean comparison of two groups was performed using independent sample t-test. Categorical variable was tested using Chi-square test and fishers exact test. Pearsons correlation was performed among continuous variables. A p-value of <0.05 was set to be statistically significant.

Results

A total of 718 kidney biopsies were examined. Presence of minimum 10 glomeruli taken as adequacy criteria for a renal biopsy to be included in the study. Out of these 106 cases (14.76%) were diagnosed as Focal Segmental Glomerulosclerosis. Only 41 cases (5.7%) were diagnosed as primary FSGS and selected for further histomorphological analysis. The mean age of presentation in adult patients with primary FSGS was 35.93±15.53 years whereas in children it was 10.8±3.25 years. A male preponderance was seen in the adult population with M: F ratio of 1.28:1 whereas in children female predilection was noted with M: F ratio of 0.8:1. Clinical parameters at the time of biopsy i.e. duration of onset of illness, presence or absence of hematuria, hypertension, nephrotic syndrome and renal insufficiency were studied. The mean duration of onset of illness in months in primary FSGS was 13.44±28.48 months. The most common presenting symptom was nephrotic syndrome followed by hypertension. More than half of the total patients presented with hypertension. Haematuria and renal insufficiency were less frequently observed. The mean serum creatinine, serum albumin, urine protein and GFR values 1.32±0.70 mg%, 2.18±0.99 gm, 4.16±2.12 g/day, 7.59±41.95 ml/min/m2 respectively. No significant difference was noted in the mean age of presentation of patients with and without renal insufficiency (p=0.848). Significantly higher urea levels and serum creatinine levels in patients with renal insufficiency. (p=0.03, 0.00 respectively). Mean GFR values were significantly lower in patients with renal insufficiency (p=0.00). Patients manifesting with nephrotic syndrome had a higher mean urinary protein excretion (5.12±1.76 g/day) compared to cases with non-nephrotic proteinuria (1.79±1.59 g/day). This difference was highly statistically significant. eISSN: 2349-6983; pISSN: 2394-6466

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Immunofluorescence was done in 35 cases, of which 4 biopsies were inadequate. In 7 cases (22.58%) both IgM & C3 were positive. Six cases showed IgM and 2 with C3 positivity (19.35% and 6.45% respectively). Immunofluorescence was negative in 16 cases. The frequency of the morphologic variants in the present study was, Not otherwise specified (NOS) 51.2%, perihilar 24.4%, tip 19.55 and cellular 4.9%. NOS was the commonest variant and cellular the least common. Collapsing variant of FSGS was not identified in this study. The majority of the cases of primary FSGS (31.7%) were between 17-25 years at the time of biopsy. 60% of the cases of perihilar variant were less than 25 years, whereas the majority of the cases of the tip variant were older and ranged between 26-50 years. There was no statistical significance between the age group of different morphologic variants [Table 1].

The cellular variant presented the earliest (0.83Âą0.24 months). The perihilar variant had a longer duration of onset of illness (20.10Âą52.07 months). This difference was however statistically not significant. (p=0.078). On an average 36.6% cases of FSGS presented with hematuria. The cellular variant was most often associated with haematuria (50%) and the lowest association was seen with the perihilar variant and the difference was statistically not significant (p value=0.947). The majority of the cases were hypertensive. Among the hypertensive cases, the maximum patients had mild hypertension (26.8%) followed by moderate hypertension (22%). All the cases of cellular variant and 80% of cases of perihilar variant presented with nephrotic syndrome. The NOS and tip variant were less frequently associated with nephrotic syndrome [Figure 1-3].

Table 1: Age distribution among the morphologic variants. Variant

Age group < 17 Years

17-25 Years

26-50 Years

> 50 years

Total

NOS

5(23.8%)

7(33.3%)

5(23.8%)

4(19%)

Tip

1(12.5%)

2(25%)

4(50%)

1(12.5%)

Cellular

0(0%)

1(50%)

0(0%)

1(50%)

Perihilar

3(30%)

2(20%)

Total

9(22%)

13(31.7%)

11(26.8%)

8(19.5%)

p-value

0.836

Fig. 1: A) NOS Variant (H & E x 200) B) NOS variant (Jones Methenamine silver x200) C) Tip variant (H & E X200) D) Tip variant (PASM X200) E) Perihilar variant (H & E x200) F) Perihilar variant (Jones Methenamine silver x200) G) Cellular variant with intraglomerular foam cells (H & E X200) H) Cellular variant with podocyte hyperplasia (Jones Methenamine silver x200).

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Fig. 2: I) Cellular variant (PAS X400) J) Glomerular hylinosis (H & E X200) K) Glomerular hylinosis (PAS X200) L) Medial hypertrophy (H & E X400) M) Intimal sclerosis (PASM X200) N) Mesangial hypercellularity (H & E X200) O) Global sclerosis (Jones Methenamine silver x200) P) Tubular atrophy (Jones Methenamine silver x200).

Fig. 3: Q) Interstitial fibrosis (Masson Trichrome x100) R) Segmental C3 deposits Direct Immunofluorescence.

Overall renal insufficiency was seen in 36.5% of the cases. In the tip and the cellular variants about half of the cases presented with renal insufficiency whereas 30% of cases of the perihilar variant were associated with renal insufficiency, The difference was however statistically not significant.(p=0.787). The average serum creatinine level in 41 cases of primary FSGS was 1.32± 0.70mg/dL. Different morphologic variants presented with different creatinine levels, which were highest in the cellular variant (1.80±0.84) and lowest in perihilar variant (1.13±0.419), but the comparison was statistically not significant.

(p=0.394). The average urine protein excretion rate was 4.16±2.12g/day. The degree of proteinuria did not vary significantly (p=0.916) among the different variants and ranged from 4.59±2.5g/day in the perihilar variant to 4.03± 2.26 g/day in the tip variant. The mean serum albumin levels were 2.18±0.944gm. Serum albumin levels were significantly higher in the tip (2.31 ±1.29) and the perihilar variants (2.72±0.88) than NOS (1.92±0.8) and cellular variants (1.8±0.28).However, this difference was statistically significant between NOS and perihilar variant. (p =0.025).

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Correlation Between Percentage of Glomerular Sclerosis with Clinical & Histological Parameters [Table 2]: Hypertensive patients have a higher degree of glomerular sclerosis (total), global and segmental sclerosis. A statistically significant difference was observed between globally sclerosed glomeruli with presence or absence of hypertension at the time of biopsy. (p=0.03). No significant association was observed between the percentage of totally glomeruli sclerosed, globally sclerosed and segmentally sclerosed with presence of hematuria at the time of biopsy. (p>0.05). The interstitial fibrosis became more severe with an increase in the percentage of glomerular sclerosis. There is statistically significant difference in the degree of interstitial fibrosis in the biopsies with different percentages of segmentally sclerosed glomeruli. (p=0.02). There was statistically significant difference in the degree of tubular atrophy in the biopsies with different percentages of glomeruli sclerosed (total), global and segmental sclerosis (p=0.002, 0.002, 0.038 respectively). Tubular atrophy was absent when the percentages of totally sclerosed, globally and segmentally sclerosed glomeruli were 22.66±10.17%, 3.0±6.7% and 19.66±9.67% respectively and were severe when the same were, 69.09±37.55%,23.08±18.92%, 46.01±25.47% respectively. Thus, the tubular atrophy became more severe with an increase in the percentage of glomerular sclerosis. The histological parameters (intraglomerular hyalinosis, glomerulomegaly, podocyte hyperplasia, mesangial hypercellularity, intraglomerular foam cells, adhesion, tubular atrophy, interstitial fibrosis and inflammation, arteriolar hyalinosis, intimal sclerosis, medial hypertrophy) were correlated with clinical and laboratory parameters at presentation i.e. blood pressure, hematuria, serum creatinine, serum albumin, proteinuria, GFR and

urea in forty-one cases of primary FSGS. A statistically significant correlation was found between serum creatinine and mesangial hypercellularity, serum albumin and podocyte hyperplasia, arteriolar hyalinosis, intimal sclerosis and medial hypertrophy of vessels. Similarly, there was statistically significant correlation between urine protein, adhesion and intimal sclerosis, between haematuria and intraglomerular foam cells and interstitial fibrosis and between blood pressure, chronic interstitial inflammation and medial hypertrophy of vessels. A higher percentage of segmentally sclerosed and globally sclerosed glomeruli were involved in patients with renal insufficiency. This difference was statistically significant (p=0.004, 0.05 respectively). The association of podocyte hyperplasia, adhesion, mesangial hypercellularity and interstitial fibrosis with renal insufficiency was not statistically significant. Chronic interstitial inflammation was observed in 86.7% cases with renal insufficiency and in only 53.8% of cases without renal insufficiency. This difference was again statistically significant (p=0.04) . The percentage of segmental sclerosis and global sclerosis was higher in patients not presenting with nephrotic syndrome, 37.43±24.84% and 11.11±13.33% respectively. However, the difference was not statistically significant. (p=0.296& 0.587).

Discussion

It is important to realize that FSGS is a histological diagnosis and not a single disease entity.[3-6] FSGS is responsible for 2% to 41% of primary glomerulopathies as reported from different countries.[7-13] This study was undertaken with the main aim of identifying the various morphologic variants and other histo-morphologic prognostic features of FSGS and their correlation with clinical parameters at the time of renal biopsy. Banfi et al[14] and Shiki et al[15] suggesting that hypertension at presentation is induced

Table 2: Correlation of morphologic variants with glomerular histological parameters: Glomerular Histological Parameters Variant

Foam cells

Adhesion

Podocyte hyperplasia

8 (38.1%)

20 (95.2%)

5 (23.8%)

17 (81%)

Tip

4 (50%)

7 (87.5%)

0 (0%)

Cellular

1 (50%)

2 (100%)

Perihilar

4 (40%)

All Cases

NOS

P value

Mesangial Glomerulomegaly hypercellularity

Hyalinosis

Number of cases

20 (95.2%)

10 (47.6%)

3 (37.5%)

8 (100%)

5 (62.5%)

0 (0%)

2 (100%)

1 (50%)

10 (100%)

2 (20%)

10 (100%)

7 (70%)

17 (41.5%)

39 (95.1%)

7 (17.1%)

32 (78%)

40 (97.6%)

23 (56.1%)

0.939

0.655

0.425

0.011

0.807

0.668

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by hemodynamic changes, not by permanent histological changes. Comparisons of histopathological features were performed with different studies.[16-20,23] [Table 3]. Association of Nephrotic Syndrome with The Morphologic Variants: In our study, all cases of cellular variant and 80% of cases of perihilar variant presented with nephrotic syndrome. Least association of nephrotic syndrome was seen in the tip variant. This finding is in discordance with the studies of Thomas et al[20], Stokes et al[21], Chun et al[22] and Deegens et al[23] where the tip variant was associated with highest percentage of nephrotic cases (97%, 94.8%, 88% and 91% respectively). In a study by Alexopoulous et al[24] patients manifesting with nephrotic syndrome had a significantly higher mean urinary protein excretion as seen in our study. Association of Renal Insufficiency with The Morphologic Variants: The incidence of renal insufficiency among the morphologic variants as reported in two North American and one west European studies[20,21,23] has shown that renal function is usually preserved in patients with the tip variant at presentation and collapsing and cellular FSGS present with maximum renal insufficiency. In our study, no

association was found between interstitial fibrosis and renal insufficiency as opposed to the findings of Taheri et al[25] where a statistically significant association was established between interstitial fibrosis and renal insufficiency. Another study in Iranian adults[26] with primary FSGS was unable to establish a significant association between interstitial fibrosis and renal insufficiency. Association Between Sclerosed Glomeruli with The Morphologic Variants: Laura et al & Chun et al[27,22] demonstrated the percentage of glomeruli involved was maximum in the classic lesion (43±23%) and least in tip lesion (24±17%) (p<0.05). Similarly, the percentage of glomeruli sclerosed (total) was also maximum in perihilar variant (39.55±18.82%) and least in tip variant (17.87±12.26%) (p=0.015). However Few studies, showed that the number of glomeruli involved was maximum in perihilar variant and least in the tip variant whereas segmentally sclerosed glomeruli was maximum in perihilar but lowest in the cellular variant. [27,18] Thomas et al[20] found maximum glomerular sclerosis/ consolidation in collapsing and least in tip variant.

Table 3: Comparative clinical and laboratory parameters of variants of FSGS in different series: Variant

Collapsing

Various studies

Age

Duration (months)

Thomas et al [20]

38±12

3.1±3.8

Nada et al

46±10

10.75±16.88

2.75±1.3

63±18

1.9(1.5- 2.2)

2.3±1.6

[18]

Deegens et al [23] Present study

Cellular

Tip

Haematuria %

6.1±4.6

33.3

10.4±6.7

10.0 ±5.3

45±13

2.5±1.7

16±15

Nada et al [18]

30±13

4.38±5.57

1.49±0.7

4.6±2.8

61.5

Present study

Deegens et al

36± 22.62

0.83±0.24

1.8±0.84

4.3±2.1

Thomas et al

54±13

1.5±0.9

9.7±7.0

30±14

20.39±41.07

1.84±1.02

3.3±1.3

20.83

44±16

2.3(2-10.6)

1.3±0.9

10.0±5.7

33.8± 19.79

14.69±13.5

1.6±0.9

2.7±2.8

37.5

Nada et al

[23]

[20]

[18]

Deegens et al [23] Thomas et al

[20]

Nada et al [18] Deegens et al

[23]

Present study Thomas et al NOS

Hypertension %

Thomas et al [20]

Present study

Perihilar

Serum creatinine Proteinuria (mg/dL) gm/24 hrs

Nada et al

[20]

[18]

Deegens et al [23] Present study

50±16

2.0±1.4

4.4±3.3

27±17

6 5.33± 99.3

0.93±1.53

1.9±.9

100

50±12

49 (0.7-303)

1.6±0.9

5.2±2.6

1.13±0.41

4.59±2.5

2.1±1.8

5.5±4.6

32±14

20.25±36.09

2.29±2.49

3.3±1.8

24.8

51±17

4.3 (0.15-10)

2.0±1.3

6.9±4.9

29.6±17.6

10.39±16.02

1.243±0.72

4.1±2.05

47.6

38.1

28.5± 16.26 20.10±52.07) 50±15

*NA= Not available, NF= Not found, NOS= Not otherwise specified

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The Frequency of Tubulointerstitial Changes in The Morphologic Variants: Nada et al[18] found mild inflammatory infiltrate in all the cases of perihilar variant and severe inflammation was seen in only NOS variant. Thomas et al[20] showed that interstitial inflammation was maximum in collapsing and least in tip variant (p<0.001). However, cases of collapsing variant were not identified in our study. Our findings are similar to the two studies by where the difference in the degree of interstitial fibrosis between different variants was statistically not significant. [22,18] They also showed that interstitial fibrosis was absent or of a mild degree in the majority of cases (41% and 38.5% respectively) and was severe in only 5% of total FSGS cases. Severe degree of interstitial fibrosis was observed only in NOS variant (6.6%). Association of Vascular Changes with the Morphologic Variants: In the present study, arteriolar hyalinosis and intimal sclerosis were absent in the cellular variant. Arteriolar hyalinosis was maximum in the tip variant (60%) and intimal sclerosis was maximum in the perihilar variant. Varying degree of medial hypertrophy was seen among the variants (37.5% to 50%). No statistical significance could be established. Many studies[28-32] particularly Nada et al[18] showed the absence of arteriolar hyalinosis and intimal sclerosis in all of the cases of collapsing variant. Frequency of The Morphologic Variants: Overall, cellular variant was less frequent in most series, comprising only 3–4.5% of cases in multiethnic cohorts[20,21] except the Chinese series in which only one-quarter showed the cellular variant, and the Indian cohort in which one-tenth of the cases belonged to the cellular variant.[33,18] Regarding tip lesions, these are just morphologic abnormalities in glomeruli that otherwise appear normal on light microscopy and it would considered as a part of minimal change disease which clinically like only. The prognosis in tip lesion may be more benign than other morphologic variants so that’s the reason some studies[27] thought that tip lesion is an intermediate form between Minimal change disease and FSGS but according to latest study done by Luis F et al[34] it should not to be considered as benign disease. They found that tip lesions presents with less chronic histologic alterations, but the prognostic implications were not favourable as chronic kidney disease developed in 30.8% and end stage in 19.2% of patients. So their study suggested that the tip variant should not be considered a prognostically favourable disease rather it could be a more early stage of a severe glomerular disease. Another study with predominant African Americans documents 32% of cases as cellular variant in the paediatric age group.[35]

Limitations of The Study: The study included only 41 cases so the sample size is a limiting factor in analyzing all the clinicopathological variables. Few morphological variants like cellular variant was seen only in a couple of biopsies thus, might not represent the actual prevalence. The study comprised of all age groups and not specifically related to children. Electron microscopy & genetic testing were not done because of financial constrains.

Conclusion

To conclude, this comprehensive study of primary FSGS reiterates that different histological variants of FSGS have substantial differences in clinical and histological features. Although histological appearance does not permit to know the cause of FSGS and it is not a perfect indicator of outcome but a statistically significant correlation (p<0.05) was noted between the percentage of globally sclerosed glomeruli with hypertension & serum creatinine. A significant correlation was found between serum creatinine and mesangial hypercellularity, serum albumin, podocyte hyperplasia, arteriolar hyalinosis, intimal sclerosis and medial hypertrophy.

Acknowledgements

No Financial and material support and conflict of interests

Funding None

Competing Interests None Declared

References

1. Alpers CE: The Kidney. In: V. Kumar, Abbas AK, Nelson Fausto, editors. Robbins and Cotran: Pathologic Basis of Disease. 7th edition: Saunders; 2004. 955-1021. 2. Nadasay T, Silva FG. Adult Renal Diseases. In: Mills SE, Carter D, Reuter VE, Greenson JK, Stoler MH, Oberman HA. editors. Sternberg’s Diagnostic Surgical Pathology. 4th edition, Philadelphia: Lippincott -Williams & Wilkins; 2004. 1863-1954. 3. D’Agati V, Fogo AB, Bruijn JA, Jennette JC. Pathologic classification of Focal segmental Glomerulosclerosis: A working Proposal. American Journal of Kidney Diseases. 2004;43: 368-82. 4. Cameron JS. The enigma of focal segmental glomerulosclerosis. Kidney Int. 1996; 57: S-119-31. 5. D’Agati V. Pathologic classification of focal segmental glomerulosclerosis. Semin Nephrol. 2003; 23:117 –134.

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Anand et al. 6. Rao TKS, Soman Anjana S. Focal segmental glomerulosclerosis. e-medicine Nephrology, http:// emedicine.medscape.com/article/245915-overview. 7. Newman WJ, Tisher CC, McCoy RC, Krueger RP, Clapp JR, et al. Focal glomerular sclerosis:Contrasting clinical patterns in children and adults. Medicine. 1976;55:67. 8. Cameron JS, Turner DR, Ogg CS, Chantler C, Williams DG, et al. The long-term prognosis of patients with focal segmental glomerulosclerosis. Clin Nephrol. 1978;10:213-218. 9. Ingulli E, Tejani A. Racial differences in the incidence and renal outcome of idiopathic focal segmental glomerulosclerosis in children. Pediatr Nephrol. 1991;5:393. 10. Korbet SM, Schwartz MM. The prognosis of focal segmental glomerular sclerosis of adulthood. Medicine (Baltimore). 1986; 65: 304-11. 11. Rydell JJ, Korbet SM, Borok RZ, Schwartz MM, et al. Focal segmental glomerular sclerosis in adults:Presentation, course and response to treatment. Am J Kidney Dis. 1995; 25: 534. 12. Habib R. Focal glomerular sclerosis [Editorial]. Kid Int. 1973; 4: 355-61. 13. Kitiyakara DC, Kopp J B, Eggers DP. Trends in the epidemiology of focal segmental glomerulosclerosis. Semin Nephrol. 2003;23:172. 14. Banfi G, Moriggi M, Sabadini E, Fellin G, D’Amico G, Ponticelli C. The impact of prolonged immunosuppression on the outcome of idiopathic focal-segmental glomerulosclerosis with nephrotic syndrome in adults. Clin Nephrol. 1991;36: 53-59. 15. Shiiki H, Nishino T, Uyama H, Kimura T, Nishimoto K, Iwano M, et al. Clinical and morphological predictors of renal outcome in adult patients with focal and segmental glomerulosclerosis (FSGS). Clin Nephrol. 1996; 46: 362-368. 16. D’Agati Vivette D. Spectrum of FSGS new insight. Current Opinion in Nephrology and Hypertension. 2008;17:271-281.

A-303 different? Nephrol Dial Transplant. 2009; 24 (12): 3701-3707. 19. Laurinavicius A, Rennke HG: Collapsing glomerulopathy: A new pattern of renal injury. Semin Diagn Pathol. 2002; 19: 106 –15. 20. Thomas DB, Franceschini N, Hogan SL, Holder S, Jennette CE, Falk RJ and Jennette JC. Clinical and pathologic characteristics of focal segmental glomerulosclerosis pathologic variants. Kidney In. 2006; 69: 920-2. 21. Stokes MB, Valeri AM, Markowitz GS and D’Agati VD. Cellular focal segmental glomerulosclerosis: Clinical and pathologic features. Kidney Int. 2006; 70: 1783-92. 22. Chun MJ, Korbet SM, Schwartz MM, Lewis EJ. Focal Segmental Glomerulosclerosis in Nephrotic Adults: Presentation, Prognosis, and Response to Therapy of the Histologic Variants. J Am Soc Nephrol. 2004;15: 2169-77. 23. Deegens JK, Steenbergen EJ, Borm GF, Wetzels JF. Pathologic variants of focal segmental glomerulosclerosis in an adult Dutch population – epidemiology and outcome. Nephrol Dial Transplant. 2008; 23:186–192. 24. Alexopoulous Efstathios, Stagnou Maria,Papagianni Aikaterini,Pantzaki Aphroditi Pantazaki, Papadimitriou. Factors influencing the course and the response to treatment in primary focal segmental glomerulosclerosis. Nephrol Dial Transplant. 2000;15:13348-1356. 25. Diana T, Ali C, Pargol S, Amar H, Shohreh S, Shiva S. Correlation of kidney biopsy findings and clinical manifestations of primary focal and segmental glomerulosclerosis. SJKDT. 2009; 20(3):417-42. 26. Diana T, Ali C, Pargol S, Amar H, Shohreh S, Shiva S. The predictive role of histopathological findings in renal insufficiency and complete remission in a sample of Iranian adults with primary focal segmental glomerulosclerosis. JRMS. 2010;15(1):14-19.

17. Stokes MB, Glen SM, Julie L, Anthony MV, D’Agati V. Glomerular tip lesion: A distinct entity within the minimal change disease/focal segmental glomerulosclerosis spectrum. Kidney Int. 2004; 65: 1690-02.

27. Barisoni L, Schnaper HW, Kopp JB. A Proposed Taxonomy for the Podocytopathies: A Reassessment of the Primary Nephrotic Diseases. Clinical Journal Of The American Society Of Nephrology. 2007; 2: 529-542.

18. Ritambhara N, Kaur KJ, Amulyajit B, Walker MR, Sakhuja V, Joshi K. Primary focal segmental glomerulosclerosis in adults: is the Indian cohort

28. Gubler MC, Waldherrs R, Levy M, Habib R. Idiopathic nephrotic syndrome with focal and segmental sclerosis and/or hyalinosis: clinical course, response

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to therapy, and long- term outcome. In: Strauss J, ed. Nephrotic syndrome: current concepts in diagnosis and management. New York: Garland, 1979: 193-212. Velosa JA, Donadio JV Jr, Holley KE. Focal sclerosing glomerulonephropathy: a clinicopathologic study. Mayo Clin Proc. 1975; 50: 121-33. Brown CB, Cameron JS. Focal segmental glomerulosclerosis with rapid decline in renal function (“malignant FSGS”). Clin Nephrol. 1978; 10: 51-61. Lee HS, Spargo BH. Significance of renal hyaline arteriolosclerosis in focal segmental glomerulosclerosis. Nephron.1985; 41: 86-93. Schoeneman MJ, Bennett B, Greifer I. The natural history of focal segmental glomerulosclerosis with

and without mesangial hypercellularity in children. Clin Nephro. 1978; 9: 45-54. 33. Shi SF, Wang SX, Zhang YK, Zhao MH, Zou WZ. Clinicopathologic study of different variants of focal segmental glomerulosclerosis. Zhonghua Bing Li Xue Za Zhi. 2007;36 (1):11-4. 34. Luis F AriasI, Carlos A, JiménezII, Mariam J ArroyaveI. Histologic variants of primary focal segmental glomerulosclerosis: presentation and outcome. J Bras Nefrol. 2013;35(2):112-119. 35. Silverstein DM, Craver R. Presenting features and short-term outcome according to pathologic variant in childhood primary focal segmental glomerulosclerosis. Clin J Am Soc Nephrol. 2007; 2:700–707.

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Original Article Correlation Of Histopathological, Biochemical & Clinical Spectrum Of Chronic Hepatitis B A New Look In To Old System Aminder Singh1*, Bhavna Garg1, Neena Sood1 and Ajit Sood2 Department of Pathology, Dayanand Medical College & Hospital, Ludhiana, India 2 Gastroenterology, Dayanand Medical College & Hospital, Ludhiana, India

Keywords: Hepatitis B Virus; HAI Score; HBeAg; Anti HBe Antibody; Fibrosis

ABSTRACT Background: Hepatitis B virus infection is a global health problem. Histopathological examination is the mainstay in evaluating the disease progression and designing treatment protocols in these patients. The aim of this study is a correlation of histological features with clinical and biochemical parameters and what new could be proposed for the future by analyzing the limitations of scoring system used. Methods: We evaluated 195 patients of hepatitis B virus infection over a period of 9 years & analysed their clinical & histological features and correlated them. Histological evaluation was done according to modified Ishakâ&#x20AC;&#x2122;s grading and Scheuer scoring system. Results: Patients were divided into age groups of < 40 & > 40 years. The majority of the patients were asymptomatic, male sex , younger age & had lower grade and stage. The mean values of total bilirubin and Prothrombin time of patient groups were within the normal ranges. A significant correlation was seen with stage III & IV and presence of HBe antigen & Anti HBe antibody cases [P-value < 0.05]. Conclusions: We concluded that liver biopsy remains the only diagnostic test in evaluating liver pathology in hepatitis B infection. With certain modifications in the current existing scoring systems, histopathology along with clinical & biochemical correlation is essential to plan the proper management of patients.

*Corresponding author: Dr. Aminder Singh, Assistant Professor, Department of Pathology, Dayanand Medical College & Hospital, Tagore Nagar, Ludhiana, INDIA Phone: +91 8968966550 Email: dramisingh@gmail.com

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

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Introduction

Hepatitis B virus [HBV] infection is an emerging worldwide health problem among all viral hepatitis. According to Lavanchy D[1] there are about 2 billion people who are infected with HBV and 350 million among them are suffering from chronic HBV infection. The culprit of 10th leading cause of death worldwide in 2004 is HBV.[2] But in the present scenario; it is the 3rd most common cause of death.[3] The histopathological changes of hepatitis B virus can reflect the clinical course of the disease.

four separate parameters were considered and scores for individual parameters were added to calculate the total score.[9, 10] Statistical Analysis: All the clinical, biochemical and histological findings were then analysed statistically using Chi-square test, Fisher Exact test, Unpaired t test.

Results

The salient histopathological features in viral hepatitis B infection are different in various clinical disease scenarios. Few studies noted that seroconversion of HBe Ag to antiHBe is followed by a significant improvement of disease activity.[4-7] Fong et al[8] documented that persistence of HBsAg, HBeAg and high titre of HBV DNA for more than 6 months implies progression to chronic HBV infection.

The age varied from seven to seventy years with a maximum number of patients being between 20 - 40 years. Male to female ratio was 5.9:1. The majority of the patients 164 [>80%] were asymptomatic. Others presented with jaundice, pain, discomfort in right hypochondrium and constitutional symptoms like weakness, lethargy, anorexia & easy fatigability. On physical examination, 59 [30.25%] cases had mild hepatomegaly. Some had mild icterus, splenomegaly and ascites. No clinical evidence of portal hypertension seen.

The prognosis of chronic HBV infection is determined predominantly by the presence or absence of active viral replication, the degree of histological liver damage by assessing scoring systems (Modified Ishak activity grade & Scheuer system staging) for the degree of liver fibrosis & that of necroinflammatory activity so this retrospective study was planned to see these effects.

On investigations; 28 patients had anaemia <10 gm/dl. Thrombocytopenia noted in 2 patients. Mild leukocytosis noted in 2 and leucopenia seen in one patient only. Raised serum bilirubin levels in 21 patients with level ranging from 2.6 to 7.3 mg/dl. Patients had AST and ALT levels were more >1.5 times of normal. 110 patients were HBeAg positive while 85 patients were anti-HBe antibody positive.

Material and Methods

Histological Findings: All the 195 liver biopsies were graded according to Ishakâ&#x20AC;&#x2122;s Modified grading system & Scheuer Staging system [Figure1, 2].

Study Design: There were 195 patients who underwent liver biopsy as per Standard Treatment Protocols following the inclusion & exclusion criteria as described: Inclusion Criteria: HBs Ag positive for more than 6 months, HBV DNA viral load >2000 copies for HBe Ag negative patients, HBV DNA >20000 copies for HBe Ag Positive patients, No definite evidence of Cirrhosis clinically, Alanine transaminase [ALT]/Aspartate transaminase [AST] Levels >1.5 times of upper limit. Exclusion Criteria: Co-infection with HCV & HIV infection, significant alcohol intake >40 gm/day, coexisting metabolic diseases like Wilson disease, alpha 1 antitrypsin deficiency, autoimmune hepatitis etc, failure to give consent, coagulopathy with International Normalized Ratio >1.5, Platelet count < 80 x 109/l, inadequate liver biopsy. The study was a hospital based retrospective study & protocol was approved by the institutional ethics committee and written consent for liver biopsy was already obtained from the patients. Detailed Clinical & Biochemical data were analysed. Liver biopsies were graded according to Ishak Modified Histological Activity Index [HAI] grading and staged according to Scheuer staging system. For this,

The first parameter considered was Piecemeal Necrosis. The scores ranged from 0 to 4. A maximum number of patients had 0 score i.e. 115 cases [58.97 %], followed by score 1 in 48 [24.61 %] patients. 16 patients had a score 2 & 10 with 3 while 6 had a score of 4. The second parameter considered was Confluent Necrosis and scores for this ranged from 0 to 6. Maximum cases i.e. 169 [86.66 %] had a score of 0. 8.2 % cases had confluent necrosis of score 1 while the rest few cases had a score of 2 and 3. Score 4-6 was not seen in any of the cases. The next parameter analysed was spotty Necrosis of hepatocytes which were again scored from 0 to 4 depending on the number of these foci seen per 10 x magnification. A maximum number of cases i.e. 110 [56.41 %] had a score of 1. Sixty-five patients had a score of 2; 16 had 3 while 4 with no focus of spotty necrosis. Portal inflammation score ranged from 0 to 4. A maximum number of cases i.e. 101 [51.79%] had a score of 1. Sixty patients had a score of 2, followed by 15 patients of score 3, 9 with a score of 4 and 10 with 0. Scores for individual grade were added and an overall histological activity index was calculated. A maximum number of patients [62%] were with HAI score of 1-3 [minimal activity] followed by

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Fig. 1: HAI Score 5, Grade 2, Stage 1, [a & b] A solitary focus of spotty necrosis- arrow [H & E, x 100 & x 400]. [c] Moderate to marked degree of portal inflammation [H & E, x 400]. [d] Portal inflammation with a focus of piecemeal necrosis [H & E, x 400]. HAI Score 3, Grade 1, Stage 2, [e & f] Mild degree of portal inflammation [H & E, x 100 & x 400]. [g & h] Periportal septal fibrosis [Masson Trichrome x 400 & x100].

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Spectrum of Chronic Hepatitis B

Fig. 2: HAI Score 2, Grade 1, Stage 3, [a & b] Bridging fibrosis look like incomplete nodule formation [H & E, x 100 & Masson Trichrome x100] [c] C shaped fibrosis [Reticulin x 100 [d] Thick fibrous septae indicating old lesion[Orcein x 400]. HAI Score 6, Grade 2, Stage 4, [e] Nodule formation [H & E, x 100].] [f] Collagen bundles [Masson Trichrome x 400].

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29.7% with HAI score of 4-8 [mild activity]. HAI score of 9-12 [moderate activity] was seen in 7.2% of cases while least HAI score 13-18 [Severe activity] was observed in only 1% of patients. It means maximum patients were of grade I & II. Fatty change which was noted in 52.8%, ballooning degeneration in 24.6%, bile ductular proliferation in 2.05% and ground glass appearance of hepatocytes in 17.94% of cases. A maximum number of patients [76 cases] had no increased fibrosis i.e stage 0. Seventy-one case had stage I. Stage II was noted in 25 cases while stage III in 10 patients. 13 cases had Stage IV. HBs Ag was positive in 28 of our cases. Correlation of Histological Features with Clinical and Biochemical Parameters Correlation of Grade and Stage with Age [Table 1]: Minimal, mild and severe activity was more common in the age group <40 years and moderate activity in >40 years. Though, statistically, it was not significant. There was no certain trend of age with worsening activity; the observation was not significant on the overall basis. Stage 0, I and II were more common in age group <40 years than in >40 years with a P-value of >0.05 [Not significant]. Advanced fibrosis and cirrhosis were

observed more in the age group >40 years than in < 40 years with a P value of >0.25. Correlation of Grade and stage with Sex & Clinical Features: There were 162 males and 33 females. No significant difference was seen in the distribution of activity and stage with sex [P >0.01]. Clinical features [all together] were correlated with the various grades. It was seen that severity of symptoms did correlate with histological grading & staging [Table 2]. Symptoms could predict the histological outcome. Most of the patients were having a necroinflammatory grade in the form of minimal or mild activity [HAI score 0-8] of which 151 patients with had no symptoms while 17 had jaundice, 9 had constitutional symptoms and 2 had discomfort in right hypochondrium. In moderate to severe activity [HAI score 9-18] category, 13 patients with had no symptoms while 1 each had both jaundice and constitutional symptoms. In stage III & IV of advanced fibrosis and cirrhosis, 14 patients were asymptomatic, 7 had a history of jaundice while 2 had constitutional symptoms. None of the patients included in the study had clinical, radiological & endoscopic evidence of portal hypertension however 23 patients had histopathological features of advanced cirrhosis.

Table 1: Correlation of histological grade and stage with age groups [<40 & >40 years] Grade / Stages No activity [Grade 0] Minimal activity [Grade I] Mild activity [Grade II] Moderate activity [Grade III] Severe activity [Grade IV] Stage 0,I,II [Mild fibrosis] Advanced Fibrosis and cirrhosis [Stage III, IV]

Group I [< 40 years] [n = 126] 0 82 40 02 02 116 10

Group II > 40 years [n = 69] 0 39 18 12 00 77 12

P value / Significance Chi Square Test P > 0.05 Non-Significant Fisher Exact Test [2 tailed Test] 0.2530

Table 2: Correlation of histological grade and stage with presenting symptoms With Symptoms [n=31] 0 12 14 3 2 With Symptoms [n=31]

No symptoms [n=164] 0 109 42 11 02 No symptoms [n=164]

Stage 0,I,II

150

Advanced Fibrosis and cirrhosis [Stage III, IV]

Grade No activity Minimal activity Mild activity Moderate activity Severe activity Stage

Statistical Analysis

P value or Significant

Chi= 10.55 d.f.=3

Significant [P < 0.05]

Fisher Exact Test [2 tailed]

P = 0.0035 Highly Significant

[d.f. = Degree of freedom]

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Correlation of Grade and Stage with AST and ALT Levels: The majority of the patients having minimal activity had these enzyme levels >1.5 to 2 times of normal. However, as there was no certain trend in these enzyme levels and activity, so this observation was not significant on an overall basis. There was also no significant association between the severity of fibrosis and AST & ALT levels [P >0.01]. Correlation of Age, Sex, Symptoms, Grade & Stage with HBe Ag Status: The mean age of HBe Ag-positive patients was 33.56 + 4 and anti-HBe antibody positive patients 43.3 + 7 [p-value <0.05]. Males were more in both HBeAg-positive and anti HBe antibody positive chronic hepatitis. The majority of the patients were asymptomatic in HBeAg-positive i.e. 89 patients and 75 of anti HBe positive patients. There was no difference between the two groups with respect to ALT/AST levels. Minimal to mild activity was observed in 104 patients of HBeAg-positive as compared to 75 patients of anti-HBe positive chronic hepatitis. Moderate to severe activity was more common in anti-HBe positive patients i.e. 10 patients as compared to 6 patients with HBe Ag positive patients. Advanced fibrosis and cirrhosis were observed in 18 patients of HBeAg-positive chronic hepatitis as compared to 5 patients of anti-HBe positive chronic hepatitis which were statistically significant [Table 3]. Few of the parameters were not considered in any of the existing scoring systems [Figure 3].

Discussion

No certain trend was found between the severity of activity and age group. However, cirrhosis was commoner in

age group >40 years. Similar, observations were also recorded by DiMarco et al[11] & Fattovich et al[12] who in their study encountered age group of 9 years to 60 years with male preponderance. Campbell et al[13] stated that old age is an independent risk factor for progression to cirrhosis. Brunelto et al[14] also emphasized that old age is a well-established predictor for cirrhosis development. Ott JJ et al[15] stated that the pattern of age-specific HBs Ag prevalence varied greatly by region and the trend of a decreasing prevalence with age was more evident in 1990 as compared to 2005, where some regions, e.g. South East Asia showed an exceptional increase with age. Yang CX et al[16] documented the percentages of the chronic HBV carriers with liver histopathology inflammatory grading & fibrosis staging. He observed Significant difference existed among groups in general [P value <0.01]. YunFan Liaw & Maurizia R Brunetto et al[17] documented the differences about geographical variations. In our study majority of the patients were completely asymptomatic. In a similar study conducted by Fattovich et al[18] stated that out of 105 patients of chronic hepatitis B infection; 56 were asymptomatic. However Hoofnager et al[19] pointed out that, symptoms of chronic hepatitis B infection correlate poorly with the activity of liver disease. In the studies performed by Razario et al[20] and Fattovich et al[18] most of the cases had mild and minimal activity. Fibrosis was assessed for each case in our study. The results were similar with Tor Borg et al[21] who found that on the evaluation of 174 liver biopsies; maximum patients were in stage 0 and I and cirrhosis identified in only 9 [5.2%] cases. Similarly, Huo et al[22] in a study of 1355 patients only found 6 patients of cirrhosis. Sigal SH et al[23] concluded

Table 3: Status of HBeAg-positive and HBe antigen negative [Anti HBe antibody positive] and its comparison with age, sex, symptoms, grade & stage HBeAg positive [n = 110]

HBe antigen negative [Anti HBe positive] [n = 85]

Statistical Analysis

P value / Significant

33.56 + 4

43.3 + 7

t = 12.23 d.f. = 193 [unpaired â&#x20AC;&#x2DC;tâ&#x20AC;&#x2122; test]

0.0001 H.S.

Male

Female

Fisher Exact Test [2 tailed]

0.70 N.S.

Symptomatic

Asymptomatic

Fisher Exact Test [2 tailed]

0.235 N.S.

Minimal to mild activity

104

Moderate to severe activity

Fisher Exact Test [2 tailed]

0.122 N.S.

Stage 0,I,II

Advanced fibrosis and cirrhosis [Stage III,IV]

Fisher Exact Test [2 tailed]

0.026 Significant

Mean age

[d.f.= Degree of freedom]

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Fig. 3: Ignored areaâ&#x20AC;&#x2122;s in the current scoring system [a] Macrovesicular Steatosis with confluent & spotty necrosis [H & E, x 100]. [b & c] Ground glass hepatocytes in a patient with hepatitis B virus infection [H & E, x 400 & Orcein x 400]. [d] Increased copper seen as red granules in the hepatocytes [Rhodanine x 400].

that there was no correlation between inflammatory activity and age, ethnicity, enzyme aminotransferase, bilirubin & HBV DNA levels, HBeAg seropositivity. They stated that patients with high-grade inflammation had greater degrees of hepatic decompensation and high-grade inflammation is common in end-stage HBV cirrhosis, but it is not readily detected by virologic and biochemical parameters. Highgrade inflammation is associated with a greater degree of liver decompensation. Our study showed significant differences between age, symptoms and advanced fibrosis in HBeAg positive and HBe antigen negative while grading matches poorly. Another histological feature frequently encountered in many of our cases was a fatty change. According to Peng et al[24] & Tor Borg F et al[21] steatosis can be due to hepatitis B virus which facilitates development of steatosis and fatty change seen in 54% of cases of hepatitis B. Other histological features which were identified were ductular

proliferation, ground glass appearance etc. Yang JD et al[25] & Yang LM et al[26] also concluded in their study that elevated enzyme levels do not mean more disease activity as patients with normal AST and ALT levels were found to have prominent inflammation and fibrosis. We also performed HBeAg and anti-HBe in all the patients and analysed the results. 110 patients were HBeAg positive while 85 patients were positive for anti-HBe antibody. There was no significant difference in AST and ALT levels in both these groups. HBeAg positive chronic hepatitis patients had minimal to mild activity while anti-HBe antibody positive ones had moderate to severe activity and this finding was statistically significant. Similar results were also presented by Zarski et al[27] who also inferred that anti HBe antibody positive patients had more active, advanced and progressive liver disease as compared to patients having HBeAg. However, no difference in enzyme levels was noted. Mc Mohan et al[28] also quoted that in

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Spectrum of Chronic Hepatitis B

Alaskan natives with chronic hepatitis B infection, loss of HBeAg was more likely to occur in older patients and presence of anti-HBe antibody is associated with severe necroinflammation and more progression to cirrhosis. Difficulty were noted in stage 3 and 4 biopsies because by definition Fibrosis with architectural distortion, but no obvious cirrhosis was stage 3 and Probable or definite cirrhosis came under stage 4. But these terms had not been discussed and explained in a meaningful sense. If incomplete septae were found with but with thick septae, what category would it be! Also the HBs Ag positivity, copper binding protein load, Steatosis had not been given any importance in the existing scoring systems. The histological scores are not representing the measurement of continuous variable but different categories of severity. The inter-observer variation cannot be avoided. Sampling variation may exist on needle biopsy at different parts of liver, in term of both necro-inflammation and fibrosis. Limitation of this study was related to factors that were not considered in our analysis such as genotype information & data pertaining to secondary causes of steatosis in HBV infection.

Conclusion

Liver biopsy remains the main diagnostic standard test in evaluating the hepatic pathology in hepatitis B infection. Modified HAI scoring system by Ishak and staging system by Scheuer are excellent for evaluating liver biopsy. By grading & staging with this system the physician can plan their management well. Age & enzyme levels had no correlation with disease activity and fibrosis. However, clinical features had a significant correlation with disease activity, particularly advanced fibrosis. Amount of fibrous tissue can be given in reports with Progressive & Regressive fibrotic changes. Cirrhosis should not be considered as an end point rather clinical correlation could be done to document compensated or decompensated cirrhotic changes. Size of nodules, thickness of septa and amount of fibrosis should be included in the revised classification which is the need of future. Thus sub classification is the necessity for clinical usefulness. It does not matter which system we follow for grading & staging. Pathologist & physicians can determine what is most comfortable and what type of reporting makes sense. Give graphic representation and write the system clearly in diagnosis.

Acknowledgements

Authors thank Dr. Vikram Gupta, Assistant Professor, SPM, DMCH for his assistance and comments of this manuscript

Funding None

Competing Interests None Declared

References

1. Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat. 2004; 11: 97-107. 2. Bréchot C. Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology. 2004; 127: 56–61. 3. Singh S, Singh PP, Lewis R. Roberts & William Sanchez, Chemopreventive strategies in hepatocellular carcinoma. Nature Reviews Gastroenterology & Hepatology. 2014; 11: 45–54. 4. Ruiz-Moreno M, Otero M, Millan A. Clinical and histological outcome after hepatitis B e antigen to antibody seroconversion in children with chronic hepatitis B. Hepatology. 1999; 29: 572–5. 5. Fattovich G, Bortolotti F, Donato F. Natural history of chronic hepatitis B: special emphasis on disease progression and prognostic factors. J Hepatol. 2008; 48: 335–52. 6. Yun-Fan Liaw. HBeAg seroconversion as an important end point in the treatment of chronic hepatitis B. Hepatol Int. 2009; 3: 425–433. 7. ter Borg F, ten Kate FJ, Cuypers HT, LeentvaarKuijpers A, Oosting J, Wertheim-van Dillen PM, Honkoop P, Rasch MC, de Man RA, van Hattum J, et al. Relation between laboratory test results and histological hepatitis activity in individuals positive for hepatitis B surface antigen and antibodies to hepatitis B e antigen. Lancet. 1998; 351: 1914–1918. 8. Fong T L, Di Bisceglie A M, Gerber M A. Persistence of hepatitis B virus DNA in the liver after the loss of HBsAg in chronic hepatitis B. Hepatology. 1993; 18: 1313-8. 9. Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, Denk H, Desmet V, Korb G, MacSween RN, et al. Histological grading and staging of chronic hepatitis. J Hepatol. 1995; 22: 696–699. 10. Schener PJ. Scoring of liver biopsies: are we doing it right? Europ J Gastroenterol Hepatol. 1996; 8: 1141-43. 11. Di Marco V, Lo Iacono O, Cammà C, et al. The longterm course of chronic hepatitis B. Hepatology. 1999; 30: 257-64.

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12. Fattovich G, Bortolotti F, Donato F. Natural history of chronic hepatitis B: special emphasis on disease progression and prognostic factors. J. Hepatol. 2008; 48: 335–352. 13. Campbell MS, Reddy KR. The evolving role of liver biopsy. Alimentary Pharmacology & Therapeutics. 2004; 20: 249-259. 14. Brunetto MR, Oliveri F, Coco B, Leandro G. Outcome of anti-HBe positive chronic hepatitis B in alphainterferon treated and untreated patients: a long-term cohort study. J Hepatol. 2002; 36: 263-70. 15. Ott JJ, Stevens GA, Groeger J, Wiersma ST. Global epidemiology of hepatitis B virus infection: new estimates of age-specific HBsAg seroprevalence and endemicity. Vaccine. 2012; 30: 2212-9. 16. Yang CX, Yang WB, Fan JH, et al. Pathological grading analysis of liver biopsies in chronic hepatitis B virus carriers of different age ranges. Zhonghua Gan Zang Bing Za Zhi. 2011; 19: 881-3. 17. Yun-Fan Liaw, Brunetto MR, Hadziyannis S. The natural history of HBV infection and geographical differences. Antiviral therapy. 2010; 15: 25-33. 18. Fattovich G. Natural history and prognosis of hepatitis B. Semin Liver Dis 2003; 23: 47-58. 19. Hoofnagle JH, Shafritz DA, Popper H. Chronic type B hepatitis and the “healthy” HBsAg carrier state. Hepatology. 1987; 7: 758-63. 20. Rozario R, Ramakrishna B. Histopathological study of chronic hepatitis B and C. A comparison of two scoring system. J Hepatol. 2003; 38: 223-29.

21. Tor Borg F, Ten Kate FJ. A survey of liver pathology in needle biopsies from HBsAg and anti-HBe positive individuals. J Clin Pathol. 2000; 53: 541-8. 22. Huo TI, Wu JC, Lee PC, et al. Sero-clearance of hepatitis B surface antigen in chronic carriers does not necessarily imply a good prognosis. Hepatology. 1998; 28: 231-6. 23. Sigal SH, Ala A, Ivanov K, et al. Histopathology and clinical correlates of end-stage hepatitis B cirrhosis: a possible mechanism to explain the response to antiviral therapy. Liver Transpl. 2005; 11: 82-8. 24. Peng D, Han Y, Ding H, Wei L. Hepatic steatosis in chronic hepatitis B patients is associated with metabolic factors more than viral factors. J Gastroenterol Hepatol. 2008; 23: 1082-8. 25. Yang JD, Kim WR, Coelho R, et al. Cirrhosis is present in most patients with hepatitis B and hepatocellular carcinoma. Clin. Gastroenterol. Hepatol. 2011; 9: 64–70. 26. Yang LM, Chang K, Zhao YL, Whu ZR. Clinical significance of liver biopsy in chronic hepatitis B patients with persistently normal transaminase. Clin J Diag Dis. 2002; 3: 150-53. 27. Zarski JP, Marcellin P, Cohard M, Lutz JM, Bouche C, Rais A, Group FM. Comparison of anti-HBepositive and HBe-antigen-positive chronic hepatitis B in France. Journal of hepatology. 1994; 20: 636-640. 28. McMahon BJ, Heyward WL, Templin DW, Clement D, Lanier AP. Hepatitis B-associated clinical and epidemiologic features and long-term follow-up. Hepatology. 1989; 9: 97-101.

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Original Article Emergence of Chikungunya infection in North India Malik Shruti1, Mehta Krunal D2*, Singh K.P.3 and Dhole T.N.1 Department of Microbiology, SGPGIMS, Lucknow, U.P., India Department Of Microbiology, Shri M.P.Shah Govt. Medical College, Jamnagar, Gujarat, India 3 Department of Microbiology, KGMU, Lucknow, U.P. India 1

Keywords: Age; Chikungunya; Clinical Features; Seasonal Variation; Sex Correlation.

ABSTRACT Background: Since re-emergence of chikungunya virus (CHIKV) infection in Indian subcontinent in 2005, it has become a major public health threat. Because of paucity of literature from north India, we undertook this study to determine incidence of Chikungunya infection and to assess clinical spectrum of disease. Methods: 248 clinically suspected cases of CHIKV were enrolled. IgM ELISA for CHIKV was done on all samples. RT- PCR for CHIKV was performed on 53 selected samples. Result: Thirty samples were positive for CHIKV IgM Ab of total 248 suspected cases. Of 53 samples tested by RTPCR, 17 were found to be positive. Majority of the confirmed cases were in age group 21-40 yrs. Male: female ratio was 1.38:1. The most common clinical features were fever with joint pain and rash. Apthous ulcers were seen in 36% and arthritis was seen in 25% of the confirmed cases. Lymph nodes were enlarged in 16% and hemorrhagic manifestations were seen in 15.9% of the positive cases. Neurological involvement was present in 7 of the CHIKV infected cases and was more common in young children. Case fatality rate amongst CHIKV infected was 4.5% (2/248, both children). Conclusion: CHIKV IgM positivity of 12% was seen in the present study. Largest proportion of confirmed cases was in the age group 21-40 years. Neurological manifestations were present in 7 of CHIKV confirmed cases, five being children. Mortality in confirmed cases was 4.5%. The increased severity of illnesss & high case fatality rate among children demands for a more detailed understanding of the neurotropism & needs to be analysed in detail.

*Corresponding author: Dr Krunal D. Mehta; Assistant professor, Department of Microbiology, Shri M.P.Shah Govt. Medical College, Jamnagar-361008, Gujarat, India Phone: +91 9979890765 Email: krunaldmehta@yahoo.co.in

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

Shruti et al.

Introduction

Chikungunya (CHIKV) fever is a re-emerging viral disease characterized by abrupt onset of fever, skin rash and incapacitating arthralgia. [1] Chikungunya virus (CHIKV) is a RNA virus belonging to genus Alphavirus and family Togaviridae. Chikungunya fever outbreaks have been affecting many countries since January 2005. The outbreak which occurred in 2006 appeared to be more severe and one of the biggest outbreaks caused by CHIKV in India affecting over 13 lakh people.[2][3] This disease was first described in 1955, following an outbreak on the Makonde Plateau along the border of Tanganyika and Mozambique.[4] Chikungunya virus is no stranger to the Indian subcontinent. Since its first isolation in Kolkata in 1963, [5][6] Recently CHIKV resurfaced in India affecting several South Indian states.Â [7][8] The outbreak started in 2005 from the coastal regions of Andhra Pradesh and Karnataka. With more than 1.3 million people estimated to be affected CHIKV prevailed across 150 districts of 8 states in India.Â [9] Despite the number estimated, the actual disease burden was thought to be much higher due to potential underestimation from lack of accurate reporting.[10] Due to paucity of literature about incidence, clinical profile, atypical manifestations and complications of the Chikungunya from Northern India we carried out a study on diagnosing and analyzing various manifestations of Chikungunya cases at KGMU & SGPGIMS, two main tertiary care hospitals in Lucknow, Uttar Pradesh State from September 2010- August 2011.

Materials And Methods

In this prospective study, the study population comprised of all suspected cases (as per NICD, New Delhi) which included patients with an acute illness characterized by sudden onset of fever with one or more of the following symptoms: joint pain, headache, backache photophobia, arthralgia, rash visiting out/inpatient department of Paediatrics, Medicine and Rheumatology at our institute during the period of September 2010-August 2011. Detailed history was taken and clinical examination was carried out. After obtaining informed consent, blood (3-5 ml) was collected in plain serum vials. Routine investigations (total leucocytes count, differential leucocytes count, platelets count and haemoparasites in peripheral smear) were done. Confirmation of cases was carried out by detection of CHIKV IgM antibodies in serum using IgM Antibody capture ELISA Kit (NIV, Pune, India). Reverse Transcriptase PCR was also performed on 53 selected samples as per Hasebe et al(2002). Chikungunya strain TND/SGPGI/2007/01 from Department of Microbiology, SGPGIMS, Lucknow served as the positive control. www.pacificejournals.com/apalm

A-315 Special investigations like IgM ELISA for Dengue (IVD Research Inc. Quality Diagnostics, USA) IgM ELISA for JE (Xcyton Diagnostics, Bangalore, India), smears and Optimal test (Diamed, USA) for malarial parasite, Widal test (In- house) and Typhidot test (SD Biodiagnostics, Korea) for typhoid fever were carried out as requested by the concerning clinician. Statistical Analysis: Data were entered in an excel file and analyzed using Stata 9.2 (College Station Tx, USA). Clinical and epidemiological features were compared between Chikungunya positives and negatives using bivariate analysis. p<0.05 was taken as significant.

Result

A total of 248 patients presented with clinical suspicion of Chikungunya from September 2010 to August 2011. IgM ELISA (NIV, Pune) for Chikungunya was done on all the 248 serum samples of which 30 were IgM positive. RTPCR was done on 53 selected samples (45 IgM negative and 8 IgM positive) of which 17 were positive. Three of these were IgM positive as well & so a total of 44 cases were considered as Chikungunya infected. Majority of Chikungunya cases (suspected as well as positive) occurred in the months of September-December (71.8%) (Table 1). Largest proportion of suspects was in the age group 21-40 years (Fig 1). Amongst Chikungunya confirmed cases, majority (40%) hailed to the age group 21-40 years. Male: female ratio was 1.38:1 in CHIKV confirmed cases and majority of the positive cases (61.3%) were from urban areas, maximum (47.7%) being reported from Lucknow district. The most common clinical features seen in CHIKV infected patients were fever with joint pain and rash. (Table2) Rashes were generalized, erythematous and maculopapular. Aphthous ulcers present on oral mucosa and tongue were seen in 36% of CHIKV confirmed cases. Arthritis mainly involving joints of lower limbs was seen in 25% of the confirmed cases. Lymph nodes were enlarged in 16% and hemorrhagic manifestations were seen in 15.9% of the positive cases. Neurological involvement with encephalitis and seizures was present in 7 of the CHIKV infected cases. CSF analysis in these cases revealed predominantly lymphocytes. Proportion of neurological involvement was more common in young children as of these 7 cases, five were children. On bivariate analysis, involvement of elbow, wrist and hip joint, rash, conjunctival congestion and photophobia were found to have significant association with CHIKV infection positivity as shown in Table 2. Hematological investigations revealed lymphocytosis in 52% of confirmed cases. Total leukocyte count did not show any significant abnormality. Thrombocytopenia was eISSN: 2349-6983; pISSN: 2394-6466

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seen in 7(16%) cases. Platelet count was in the range of 25,000 -80000/μl in these cases.

etiologies including J.E., Dengue and Malaria and turned out to be negative for them.

Case fatality rate amongst CHIKV infected was found to be 4.5% (2/44), which was slightly higher as compared to Chikungunya negative patients. Both the cases belonged to paediatric age group were 3 & 7 years of age respectively. They had neurological manifestations comprising of encephalitis and seizures & were CHIKV RT-PCR positive. They were tested for other possible

All the 44 CHIKV infected cases were tested for DENV by IgM ELISA. Out of total 44 confirmed Chikungunya cases 16 (36.4%) turned out to be positive for Dengue IgM. JE IgM was done in 20 of the 44 CHIKV infected cases and 4 (20%) were positive. Smear & Optimal test for malaria was done in 41 cases and 3 (7.3%) were positive for Plasmodium falciparum.

Table 1: Month wise Distribution of clinically suspected and positive cases of Chikugunya. Month & Year

Clinically suspected cases (n=248)

Positive cases (n=44)

% positivity

September 2010

100

October 2010

November 2010

December 2010

10.8

January 2011

February 2011

March 2011

April 2011

May 2011

June 2011

7.1

July 2011

8.3

August 2011

7.1

Total

248

Table 2: Comparison of clinical and demographic features at initial presentation of Positive and Negative cases (n=248). Negative (n=204) Positive (n=44) Variable OR (95% CI) p-value N Mean±SD or n (%) N Mean±SD or n (%) History Seasonal variability 1.241 204 138:66 44 41:3 0.001 (Sep-Nov vs Dec-August) (1.129-1.364) 1.050 Sex ratio: Female:Male 204 73:131 44 31/13 0.430 (0.934-1.179) 1.039 Fever 204 204 (100) 44 44 (100) 0.562 (0.917-1.177) 1.121 Joint Pain/Arthralgia 197 175 (88.8) 41 39 (95.1) 0.224 (0.978-1.285) 1.171 Joint swelling 202 28 (13.9) 44 11 (25.0) 0.067 (0.953-1.438) 0.834 Headache 197 183 (92.9) 41 35 (85.4) 0.114 (0.662-1.117) 1.116 Rash 204 85 (41.7) 44 25 (56.8) 0.046 (0.988-1.260) 1.071 Lymphadenopathy 204 24 (11.8) 44 7 (15.9) 0.451 (0.878-1.308) 1.130 Aphthous ulcers 204 48 (23.5) 44 16 (36.5) 0.078 (0.969-1.319) 1.190 Pruritis 204 19 (9.3) 44 8 (18.2) 0.087 (0.925-1.530)

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Variable

Negative (n=204) MeanÂąSD or n (%)

Positive (n=44) MeanÂąSD or n (%)

Conjunctival congestion

204

56 (27.5)

21 (47.7)

Photophobia

202

13 (6.4)

7 (16.3)

Haemorrhagic manifestation

204

35 (17.2)

7 (15.9)

Altered sensorium

184

58 (31.5)

10 (24.4)

Seizures

188

22 (11.7)

7 (16.7)

Encephalitis

188

18 (9.6)

7 (16.7)

JOINTS INVOLVED Elbow

170

42 (24.7)

22 (55.0)

Knee

170

165 (97.1)

37 (92.5)

Shoulder

170

15 (8.8)

3 (7.5)

Wrist

170

20 (11.8)

19 (47.5)

Ankle

170

110 (64.7)

28 (70)

Hip

170

35 (20.6)

2 (5)

Metacarpophalangeal

170

2 (1.2)

2 (5)

OR (95% CI)

p-value

1.190 (1.025-1.381) 1.292 (0.932-1.791) 0.984 (0.848-1.143) 0.941 (0.830-1.067) 1.089 (0.878-1.350) 1.152 (0.895-1.482) 1.336 (1.108-1.611) 0.765 (0.466-1.314) 0.969 (0.779-1.205) 1.711 (1.253-2.335) 1.045 (0.915-1.194) 0.825 (0.739-0.921) 1.631 (0.611-4.355)

0.008 0.032 0.841 0.369 0.381 0.182 <0.001 0.175 0.788 <0.001

Fig:1 Age wise Distribution of clinically suspected and positive cases of Chikungunya.

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0.526 0.020 0.111

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Chikungunya in North India

Discussion

Since the last outbreak of chikungunya fever there had been hardly any active or passive surveillance carried out in our country suggesting disappearance of the virus from the subcontinent. However, large scale outbreaks of fever caused by this virus in several States of India including Andhra Pradesh, Kerela, Tamilnadu, and Maharashtra have confirmed its re-emergence. [11][13] [14] At present, Chikungunya has become an important health concern in India. For treatment and prevention, a detailed understanding of epidemiology and pathogenesis is required. Furthermore, it is important to have the information regarding the prevalence rate of chikungunya infection and course of the disease. In the present study, Chikungunya IgM positivity was found to be 12%. 17 of the 53 tested samples were positive by RT-PCR. . Largest proportion of suspects and confirmed cases were in the age group of 21-40 years. Movement of people outdoors during daytime when the activity of mosquito vector is at its peak, lesser personal protection (towards mosquitoes) and individual’s different immune response to disease are some of the speculative reasons for increased CHIKV positivity seen in this age group.[15] In the present study majority of Chikungunya suspected and positive cases occurred in the months of SeptemberDecember (71.8%) which can be explained by the high vector density in the post monsoon period. Majority of the positive cases (61.3%) were from urban areas. Most of the previous outbreaks in India were also found to be confined mainly to urban areas and large cities. This can be attributed to A aegypti being the dominant CHIKV vector in India which has a strong predilection for urban and semi-urban environments. The main clinical features in the present study were fever with joint pain, rash and apthous ulcers. Lymphadenopathy and hemorrhagic manifestation were also seen in 16% of the cases. In our study neurological involvement with encephalitis and seizures was present in 7 of CHIKV infected cases, of which 5 were children. Also the severity of infection in CHIKV infected children was quite high, terminating in death in 2 of the positive cases. Our study strongly supports CHIKV to be an important cause of neurological disorders in children and that clinicians should be aware of the fact that CHIKV may be a cause of CNS infections in children. No State or central government has officially declared any deaths caused by chikungunya except for Gujarat, where 11(4.8%) deaths out of 225 laboratory confirmed cases of the virus had been reported. Although the Kerala State government reported 74 deaths, the central government

team investigated 56 of these and concluded that they were not caused by chikungunya virus. [16] In the present study, case fatality rate amongst CHIKV infected was found to be 4.5% (2/44), which is slightly higher as compared to Chikungunya negative patients. Both the cases had neurological involvement and were 3 &7 years of age. The high case fatality rate among children is a serious and unusual finding and demands for a more detailed understanding of the neurotropism and virulence of CHIKV. In our study, 36.4% of the CHIKV positive patients tested positive for Dengue IgM. This can be attributed to either coinfection or dual infection. In Asia, the CHIKV-affected areas overlap with DENV-epidemic areas and provide opportunities for mosquitoes to become coinfected with both viruses resulting in significant coinfection.

Conclusion

CHIKV IgM positivity of 12% was seen in the present study. 17 of the 53 samples tested positive by RT-PCR. Largest proportions of confirmed cases were in the age group 2140 years. Neurological manifestations were present in 7 of CHIKV confirmed cases, five being children. Mortality in confirmed cases was 4.5%. The increased severity of illness & high case fatality rate among children demands for a more detailed understanding of the neurotropism & needs to be analyzed in detail.

Acknowledgements

Authors thank all the Clinic staff for providing support for recruitment of patients and collection of samples, and also acknowledge the Department of Microbiology for extending support for the study.

Funding None

Competing Interests None Declared

Reference:

1. Pialoux G, Gaüzère B, Jauréguiberry S, Strobel M. Chikungunya, an epidemic arbovirus. Lancet Infect Dis. 2007;7:319–27. 2. Directorate of National Vector Borne Disease Control

Programme (NVBDCP), Directorate General of Health Services, Government of India. Chikungunya Fever situation in the country during 2006. Available at: http://www.namp. gov.in/chikun-cases.html, accessed on October 28, 2006.

3. Government of India, Ministry of Health & Family Welfare. Update on Chikungunya. Available at: http://www. nvpdep:gov.in/doc/chikungunyaupdate.pdf, accessed on October 28, 2006.

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5. Chatterjee SN, Chakravarti SK, Mitra AC, Sarkar JK.

Virological investigation of cases with neurological complications during the outbreak of haemorrhagic fever in Calcutta. J Indian Med Assoc. 1965; 45: 314-6.

6. Shah KV, Gibbs CJ Jr, Banerjee G. Virological investigation

of the epidemic of haemorrhagic fever in Calcutta: isolation of three strains of Chikungunya virus. Indian J Med Res. 1964; 52: 676-83.

7. Arankalle VA et al. Genetic divergence of Chikungunya viruses in India (1963–2006) with special reference to the 2005–2006 explosive epidemics. J Gen Virol. 2007; 88: 1967–76. 8. Yergolkar PN et al. Chikungunya outbreaks caused by African genotype, India. Emerg Infect Dis. 2006; 12: 1580–3. 9. Lahariya C, Pradhan SK. Emergence of chikungunya virus in Indian subcontinent after 32 years: A review. J Vector Borne Dis. 2006; 43: 151–60. 10. Kumar NP, Joseph R, Kamaraj T, Jambulingam P A226V mutation in virus during the 2007 chikungunya outbreak in Kerala, India. J Gen Virol. 2008; 89: 1945–8.

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A-319 11. Ravi V. Re-emergence of chikungunya virus in India. Indian J Med Microbiol. 2006; 24: 83-4.

12. National Institute of Communicable Disease, New Delhi. Chikungunya fever. Commun Dis Alert. 2006; 10: 6-8.

13. NVBDCP: Epidemiological profile of chikungunya fever in the country. (prov.). 2009 [http://nvbdcp.gov. in/Doc/chikun-update07.pdf]. New Delhi: National Vector Borne Disease Control Programme. 14. NVBDCP: Status report on Dengue and Chikungunya as on 31.12.09(Dengue and chikungunya update). 2010 [http://nvbdcp.gov.in/Doc/Den_Chik_Dec09. pdf]. New Delhi: National Vector Borne Disease Control Programme. 15. Moro ML et al. Chikungunya virus in North-Eastern Italy: A Seroprevalence study. Am J Trop Med Hyg. 2010;82:508-511. 16. Mavalankar D, Shastri P, Raman P. Chikungunya epidemic in India: a major public-health disaster. Lancet Infect Dis. 2007; 7: 306-7.

17. Suryawanshi et al. Clinical profile of chikungunya fever in patients in a tertiary care centre in Maharashtra, India. Indian J Med Res. 2009; 129:438-441.

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Original Article Apoptosis and Micronucleus in Cervical Pap Smears:

Promising Assays to Increase the Diagnostic Value of The Test Amoolya Bhat1*, Vijaya C1 and Padmasri R2 Department of Pathology, Sapthagiri Institute of Medical Sciences and Research Centre , Bangalore, India. Department of Obstetrics & Gynaecology, Sapthagiri Institute of Medical Sciences and Research Centre, Bangalore, India 1

Keywords: Apoptosis, Cervical, Correlation, Micronucleus, Pap Smear

ABSTRACT Background: The micronucleus (MN) test on exfoliated cells has been successfully used to screen population groups at risk for cancers of cervix, oral cavity, esophagus and urinary bladder. MN originates from chromatin fragments or whole chromosomes; their presence in cells is a reflection of chromosomal aberration. Their frequency rises in carcinogenexposed tissues much earlier than the symptoms. Apoptosis is easily discernible in cervical Pap smears in the form of karyorrhexis, karyolysis and condensation of chromatin. Very few studies are available in literature have studied the significance of MN scoring and apoptosis in cervical nonneoplastic, pre-neoplastic and neoplastic conditions. We compared the MN score and apoptosis in the whole spectrum of cervical lesions and also evaluated the role of MN score and apoptosis as biomarkers in different non-neoplastic, pre-neoplastic and neoplastic lesions. Aim: This is a retrospective study to evaluate the significance of apoptosis and micronucleus counts in the spectrum of lesions in cervical pap smears. Methods: It was a retrospective study conducted using Papanicolau stained cervical smears archived in the department of pathology. We evaluated a total of 230 smears, which included 63 normal smears, 106 inflammatory smears, 15 smears of Atypical squamous cells of undetermined significance, 20 smears of Low grade squamous intraepithelial neoplasm, 12 smears of high grade squamous intraepithelial neoplasm, and 14 smears of invasive carcinoma. Two pathologists separately and independently counted the number of cells with micronucleus and the number of apoptotic cells per 1,000 squamous epithelial cells. Results: Micronucleus score was higher in invasive carcinoma and preneoplastic conditions of cervix than the normal and inflammatory conditions. Apoptotic cells were more in preneoplastic lesions and invasive carcinomas as compared normal and inflammatory conditions except in case of inflammatory atrophic smears and erosions of cervix. There was a positive correlation between micronucleus score and apoptosis in the study groups. Conclusions: Micronucleus score and apoptotic cells increases with increasing grade of dysplasia. It is maximum in malignancy and minimal in normal and inflammatory smears. There is a positive correlation between increased apoptosis and high micronucleus score. These two additional features increase the accuracy of reporting.

*Corresponding author: Dr. Amoolya Bhat, MBBS, MD. Assistant Professor, Department of Pathology, Sapthagiri Institute of Medical Sciences and Research Centre, #15, Chikkasandra, Hesarghatta Main Road, Bangalore â&#x20AC;&#x201C;5600 90, INDIA. Phone: +91 9480315066 Email: simpathologist@gmail.com

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

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Introduction

Approximately 20 per 100,000 Indian women are likely to suffer from carcinoma of cervix. [1] The common age group is 35-65 years. [2] Cervical Pap smears are the most commonly used screening tests in detecting cancers of cervix. The micronucleus (MN) test on exfoliated cells has been used to screen population at risk for cancers of cervix, oral cavity, esophagus and urinary bladder in the past. MN originates from DNA fragments or whole chromosomes; their presence is a reflection of chromosomal aberration. They occur in high numbers in carcinogen-exposed tissues much earlier than the symptoms. [1] Apoptosis is easily discernible in cervical Pap smears in the form of karyorrhexis, karyolysis and pyknosis. Their frequency of occurence increases with increase in the severity of the premalignant and malignant lesions of cervix. [3,4] These features are not routinely included in cervical Pap smear reports. Very few studies are available in literature which have studied the association between MN scoring and apoptosis in cervical non-neoplastic, pre-neoplastic and neoplastic conditions. We compared the MN score and apoptosis in the whole spectrum of cervical lesions and also evaluated the role of MN score and apoptosis as biomarkers in different nonneoplastic, pre-neoplastic and neoplastic lesions Aim This is a retrospective study to evaluate the significance of apoptosis and micronucleus counts in the spectrum of lesions in cervical pap smears.

Materials and Methods

Two thousand cells were included in each slide following criteria[5-8]: (a) Intact cytoplasm and relatively flat cell position on the slide with little or no overlap with adjacent cells (b) Nucleus normal and intact, with smooth and distinct nuclear perimeter and (c) Little or no debris Criteria used for identification of micronucleus were, (a) Regular rounded structure with perimeter suggestive of a membrane located within inner half of the cytoplasm. (b) Diameter variable from 1/16 to 1/3 the diameter of the main nucleus without any overlap or a bridge to the nucleus. (c) Staining intensity and texture similar to that of the nucleus (d) Same focal plane as nucleus Cells with single or multiple MN were given a score of one. All slides are screened by two pathologists. Thus for each smear a total of 2,000 cells were counted and the numbers of MNC in each case were expressed per 1,000 cells (MN score) (Figure 1a and b). The frequency of MN was expressed as mean count among the study groups and comparison was done statistically. Criteria for Apoptosis: Presence of karyorrhexis, karyolysis and pyknosis in the squamous cells are considered as features of apoptosis [4] and the count is expressed in terms of number of apoptotic cells per 1000 squamous epithelial cells after screening 2,000 squamous cells (Figure 1c,d,e).

We retrospectively evaluated 230 cervical smears stained with Papanicolau stain received during the period of 3 years from January 2012 to December 2015 that fulfilled the inclusion and exclusion criteria mentioned below. The study material consisted of 15 smears of ASCUS, 20 smears of LSIL, 12 smears of HSIL, 14 smears of squamous cell carcinoma, 106 smears of specific and nonspecific inflammatory lesions and 63 normal smears.

Inclusion Criteria

Cytological smears were obtained on microscopic slides using Auer spatula from the uterine cervix. These smears were fixed with commercially available spray fixative (available with the RAPID PAP TM KIT) for 15 min and stained by PAP technique using a commercially available staining kit RAPID PAP.

MN scoring: Slides were examined under optical microscope at 400Ă&#x2014;, magnification in a zigâ&#x20AC;&#x2018;zag method. www.pacificejournals.com/apalm

Squamous cells lying singly will be preferred for counting of MN and apoptotic cells. Squamous cells not obscured by blood, inflammatory ] cells, debris and organisms.

Exclusion Criteria

Clumps of cells with obscured nuclear or cytoplasmic boundaries and overlapping cells were avoided. Degenerated cells, cell covered with debris, mucus, WBC, bacteria and RBC were exempted from counting and scoring. Anucleate squames, endocervical and endometrial cells were avoided. Inadequate and faded slides were excluded. eISSN: 2349-6983; pISSN: 2394-6466

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Fig.1: Photomicrographs showing a) and b) squamous cells with micronucleus (black arrow) (Papanicolau stain; x1000); c) Squamous cell with pyknotic nucleus (black arrow); d) and e) squamous cells with karyorrhectic nuclei (black arrow) (Papanicolau stain; x400).

Statistical Analysis: The data was analysed using SPSS software version 19.0. MeanÂąSD values of micronucleus scores and apoptotic counts were calculated. The micronucleus scores and apoptotic counts of various study groups were compared using Chi square test. This test was applied after combining the smear type, micronucleated and apoptotic cell counts until not less than 20% of expected values were less than 5. Pearsonsâ&#x20AC;&#x2122; correlation test was performed to discern the relationship between micronucleus and apoptosis in various smear types.

Results

In this study we found that majority of the normal and inflammatory smears along with those associated with atrophic changes, reactive atypia, erosions and polyps were showing low scores for micronucleus, LSIL and HSIL and invasive carcinomas showed higher micronucleus scores.

This was statistically significant. Chi-square = 126. 77 Degree of freedom [ DF ]= 4, p < 0.001(Table 1&2). Also apoptotic cells were found to be more in invasive carcinomas, LSIL and HSIL than in normal non specific inflammatory smears and inflammatory smears with reactive atypia. However inflammatory smears associated with atrophic changes, polyps and erosisons of cervix showed more number of apoptotic cells in the smears. This was statistically significant. Chi-square = 74.2 Degree of freedom [ DF ]= 4, p < 0.001.(Table 3&4). Mean micronucleus scores and mean number of apoptotic cells [Mean+/-SD] in case of normal smear, inflammatory smear, ASCUS, LSIL, HSIL and invasive carcinoma are shown in table 5 along with Pearson correlation values. There was gradual increase in micronucleus scores and apoptosis in the study groups from normal to the invasive carcinoma group. There was a positive correlation between micronucleus score and apoptosis in the study groups.

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Table 1: Distribution of micronucleus in various study groups. Micronucleated cells /1000squamous cells

Smear Type

<1 1-2 2–3 52 11 0 82.5% 17.5% 0.0% 61 31 13 57.5% 29.2% 12.3% 6 8 1 40.0% 53.3% 6.7% 0 7 8 0.0% 35.0% 40.0% 0 2 3 0.0% 16.7% 25.0% 0 1 4 0.0% 7.1% 28.6% 119 60 29 51.7% 26.0% 12.6% Chi-square = 126. 77 DF = 4, p < 0.001

Normal smear Inflammatory smear ASCUS LSIL HSIL Invasive carcinoma Total

3 & above 0 0.0% 1 .9% 0 0.0% 5 25.0% 7 58.3% 9 64.3% 22 9.5%

Total 63 100.0% 106 100.0% 15 100.0% 20 100.0% 12 100.0% 14 100.0% 230 100.0%

Table 2: Distribution of micronucleus in various types of inflammatory smears. Micronucleated cells /1000squamous cells in various subtypes of inflammatory smear

Inflammatory Atypia Erosions

1-2

2–3

3 & above

Total

72.2%

27.8%

0.0%

100.0%

54.5%

45.5%

0.0%

100.0%

40%

60%

100%

33.3%

66.7%

100%

Atrophic smear Polyps

Table 3: Distribution of apoptotic cell count in various study groups. Smear Type Normal smear Inflammatory smear ASCUS LSIL HSIL Invasive carcinoma Total

<1 43 68.3% 65 61.3% 4 26.7% 0 0.0% 1 8.3% 0 0.0% 113 49.2%

Apoptotic cells/1000squamous cells 1-2 2–3 3-4 10 10 0 15.9% 15.9% 0.0% 14 5 13 13.2% 4.7% 12.3% 9 2 0 60.0% 13.3% 0.0% 2 5 12 10.0% 25.0% 60.0% 3 4 3 25.0% 33.3% 25.0% 0 2 10 0.0% 14.3% 71.4% 38 28 38 16.5% 12.2% 16.5% Chi-square = 74.2 DF = 4, p < 0.001

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4 & above 0 0.0% 9 8.5% 0 0.0% 1 5.0% 1 8.3% 2 14.3% 13 5.6%

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Total 63 100.0% 106 100.0% 15 100.0% 20 100.0% 12 100.0% 14 100.0% 230 100.0%

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Table 4: distribution of apoptosis in various types of inflammatory smears. Apoptotic cells /1000squamous cells in various types of inflammatory smears

Inflammatory Atypia Erosions Atrophic smear Polyps

1-2

2–3

3 -4

4 & above

Total

61.1%

16.7%

0.0%

16.7%

5.6%

100.0%

0.0%

27.3%

72.7%

100.0%

20%

80%

100%

66.7%

33.3%

100%

Table 5: Mean micronucleus score and apoptotic cell count with Pearson correlation in various study groups. Type of smear

Micronucleus score [Mean±SD]

Apoptotic cells [Mean±SD]

Pearson Correlation

Normal smear

0.17±0.38

0.47±0.75

0.0024

Inflammatory smear

0.56±0.74

1.10±1.79

0.0818

ASCUS

0.76±0.59

0.9±0.38

0.8690

LSIL

2.02±0.73

2.25±1.01

0.7048

HSIL

2.79±1.01

2.95±1.45

0.0479

Invasive carcinoma

3.14±0.94

3.57±1.10

0.0200

Discussion

Cytogenetic markers like chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), and micronuclei (MN) are sensitive indicators of genetic damage. [9] The mean prevalence of cells with micronucleus in the general populations is 0.0 to 0.9%. Higher range of micronucleus counts can be the result of chromosomal alterations. [10] The analysis of MN in the epithelial cells has been shown to be a sensitive assay for monitoring chromosomal damage in human populations. [9,10] Three most commonly hypothesized mechanisms responsible for the formation of MN are metabolic stress, clastogenic substances released from neoplastic cells and the oncogenic viruses like HPV. [11,12] Chromosomal instability, involving chromosomes 1,3,5,11 and 17 is associated with the development of carcinoma of uterine cervix. The presence of MN correlates with malignancy. The MN are indicative of numerical and/or structural chromosome aberrations during cell division. [13] MN monitoring must be considered as an additional criterion for the early detection of cytogenetic damage in routine gynaecological examinations. The genetic instability caused by human papilloma virus involves the expression of viral oncogenes. E6 expression may result in failed cytokinesis, E7 expression may uncouple centrosome duplication from cell division. Also the absence of a functional TP53 gene, makes the cells aneuploid. False

negatives account for 10-50% of Pap test results as a result of limited sensitivity in detecting precancerous lesions of cervix, coupled with the subjective interpretation of results. Complementary methods that increase the sensitivity of screening for cervical cancer include high-risk HPV test and micronucleus (MN) counts. The people from low socioeconomic strata cannot afford HPV testing in many developing and poor countries. [8] MN is a biomarker of chromosomal aberration which has increased risk of cancer.[14] The combination of cytology and Mn Ag immunostaining may be helpful to decrease the false negative cases. Differentiation between cellular atypia due to benign reactive changes versus cellular atypia due to dysplasia in the category of ASC-US and AGUS is also possible. [15] The MN test is a simple, rapid, inexpensive, practical, and noninvasive screening technique that is well accepted by the patients for management of subjects under carcinogenic risks after exposure to genotoxic agents like tobacco, ionizing radiation and oncogenic viruses. Increased micronuclei frequency in the grossly normal appearing buccal mucosa of the high risk individuals is associated with greater risk of carcinogenesis. [16] Micronuclei (MN) are small chromatin bodies that appear in the cytoplasm by the condensation of acrocentric chromosomal fragments (clastogenesis) or by whole

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chromosomes(aneugenesis), lagging behind due to mitotic malfunction. Their number increases with increasing chromosomal alterations. The damaged chromosomes, in the form of acentric chromosome fragments, lag behind in anaphase when centric elements move towards the spindle poles. After telophase, the lagging elements are included in the daughter cells in the form of one or several secondary nuclei, which are considerably smaller than the principal nucleus, situated around the main nucleus within the inner half of the cytoplasm and are therefore called the micro nuclei. This takes place in the basal layer of the epithelial tissue, where cells undergo mitosis. This rapid turnover of epithelial tissue brings the cells to the surface, where they exfoliate. [7] Micronucleus scoring or assay can also be done in human erythrocytes, and lymphocytes. [7] It has been shown to have a sensitivity of 94%, specificity of 100%, and an accuracy of 95%. [6,7] Micronuclei scoring can be interfered by small dye granules, bacteria and keratohyaline granules. Bacteria can be differentiated by their shape, smaller size and their location either over or in between the squamous cells. Dye granules have a slightly different refractility and color intensity. Keratohyaline granules are numerous and are dispersed throughout the cytoplasm. Misinterpretation of nuclear anomalies like karyorrhexis, karyolysis, condensed chromatin, and binucleates as MN sometimes may occur. [7] Chemopreventive agents including beta‑carotene and other vitamins, have been shown to significantly decrease MN levels in healthy tobacco users, as well as in individuals with precancerous lesions. [7] DNA specific stains are preferred over Papanicolau stain for evaluating MN, and other nuclear anomalies in exfoliated cells. Many studies have utilized Feulgen‑Fast Green as it is DNA specific and enables easy identification of MN in clear transparent cytoplasm [4,16-19]; however method is relatively time consuming and may lead to the underscoring of MN.[16] The assessment of degenerative abnormalities suggestive of apoptosis increases the sensitivity of micronucleus test. [3,4] and therefore can be employed while assessing micronucleus in smears stained with non DNA specific stains. Other stains include fluorescent dyes, such as diamidino2-phenylindole (DAPI), acridine orange, Hoechst, and propidium iodide and nonspecific stains like May‑Grunwald Giemsa (Giemsa), PAP, hematoxylin and eosin, and Orcien. [6,7] www.pacificejournals.com/apalm

In our study we used 230 cervical pap smears stained with Papanicolau stain consisting of 63 normal,106 inflammatory smears, 15 ASCUS, 20 LSIL,12HSIL and14 invasive carcinomas. We found that micronucleus scores were higher in invasive carcinomas ,LSIL and HSIL as compared to ASCUS, normal and inflammatory smears(including those with reactive atypia, erosions, polyps and atrophic changes). The numbers of apoptotic cells were higher in invasive carcinomas, LSIL, HSIL, atrophic inflammatory smears, cervical erosions and polyps as compared to ASCUS, normal and non specific inflammatory lesions. The mean micronucleus score and mean apoptotic cell counts were higher in ASCUS, LSIL and HSIL groups as compared to inflammatory and normal smears. The smears showing reactive atypia exhibit lower micronucleus scores and apoptotic cells except in cases of atrophic changes, erosions or polyps. Thus micronucleus score and apoptotic cell count helps to distinguish reactive atypia from LSIL and HSIL once the postmenopausal status, erosions and polyps are ruled out. Micronucleus and apoptotic cells can be counted even in unsatisfactory smears due to low cellularity and those exhibiting micronucleus scores and apoptotic cells in high numbers should be carefully reevaluated to rule out the presence of LSIL, HSIL and invasive neoplasms. Many authors have reported spuriously higher micronucleus scores while using Papanicolau smears. The accuracy of micronucleus score can be improved in Papanicolau stained smears by combining it with the apoptotic cell count as the LSIL, HSIL and invasive carcinomas exhibit high micronucleus and apoptotic cell counts as compared to benign non-neoplastic conditions as shown in this study. Thus Bueno C T et al evaluated the association between the frequency of micronuclei (MN) and the cellular changes in 174 Papanicolaou test smears. MN frequencies were significantly higher in the group with pre neoplastic and neoplastic lesions compared to the control group (p < 0.001). The mean MN frequencies were 0.95 +/-1.12 (mean +/-SD) in the control group (n = 223), 2.98 +/- 1.20 in individuals with atypical squamous cells of undetermined significance (ASC-US) (n = 50), 4.04 +/- 1.45 in cervical intraepithelial neoplasia (CIN) I (n = 52), 5.97 +/-1.83 in CIN II (n = 30), 7.29 +/- 1.55 in CIN III (n = 17) and 8.64 +/- 1.55 in invasive cancers(n = 25). [8] Patel BP et al conducted a study on 47Healthy tobacco chewers and 48controls using the peripheral blood eISSN: 2349-6983; pISSN: 2394-6466

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lymphocyte and exfoliated buccal mucosa cells for Chromosomal Aberrations (CA) and micro nucleated cell count (MNC) respectively. MNC was significantly higher (p=0.001) in tobacco chewers than controls. Chromosomal aberration was higher in chewers than controls. However MN test is better indicator for genotoxicity damage than CA. The study concluded that MNC is a better surrogate biomarker to predict genotoxicity than chromosomal aberration for tobacco exposure and DNA damage index in tobacco chewers. [16] Naderi NJ et al compared micronucleus count in buccal mucosa of smokers to non smokers. Fourteen samples from individuals with a smoking history less than 10 years and 26 samples from individuals with the smoking history of more than 10 years were analysed. The control group consisted of 23 samples from nonsmokers. The mean number of micronucleus of buccal mucosa cells in nonsmokers, smokers with a smoking history less than 10 years and second group smokers with smoking history of more than 10 years was 0.94 ± 0.94, 1.89 ± 0.62 and 2.01 ± 0.93 respectively. The differences between these groups were statistically significant (P < 0.002). The mean number of micronuclei in buccal mucosa cells of the nonsmokers was significantly lower than that of the smokers. The mean number of micronucleus of buccal mucosa cells in smokers who smoked more than 10 years was higher than smokers who smoked less than 10 years. [10] Fareed M et al investigated the frequency of micronucleus in oral mucosa of pan masala chewers and healthy controls and showed that MN frequency was greater in exposed cases (3.56±0.719) as compared to the controls (0.75±0.171). [9] Ambroise MM et al studied the routine Papanicolaoustained cervical smears in a total of 132 cases consisting of 42 pre-neoplastic and neoplastic cases and 90 non-neoplastic cases. In each smear, the number of micronucleated cells and binucleated cells were counted under oil immersion. They concluded that the micronucleus count and the binucleated cell count were significantly higher (p value<0.001) in the high-grade squamous intraepithelial lesion (HSIL) and invasive carcinoma patients compared to low–grade squamous intraepithelial lesion (LSIL) and non-neoplastic cases. Expression of HPV 16 E6/E7 inhibits p 53 and RB and facilitates transformation of the binucleated cells which would otherwise undergo apoptotic cell death. Binucleated cells contain double the quantity of chromosomal material. Propagation of these cells can promote genomic instability resulting in development of cervical cancer. Thus binucleated cell count augments the predictive value of MN count. These biomarkers are useful in identifying the borderline cases on cytology. [19]

In a study conducted by Gayathri et al, the mean MN scores ± SD in normal, inflammatory, atypical squamous cells of undetermined significance, LSIL, HSIL and Infiltrative carcinoma cases were 0.84±0.68, 1.06±0.84, 3±0.73, 4.06±1.13, 8.03±1.64 and 10.5±2.01, respectively. MN scores of Infiltrating carcinoma and HSIL were significantly high compared to normal (P<0.000), inflammatory (P<0.000), ASC-US (P<0.000), ASC-H (P<0.000) and LSIL (P<0.000) cases.[20] Aires GMA et al evaluated distribution of micronucleus and apoptosis among women with normal smears and women with cervical abnormalities, i.e., 12 inflammatory lesions and 10 low- grade and 27 high-grade squamous intraepithelial lesions(HSIL). The frequency of end points of apoptosis was similar in women without cervical abnormalities and women showing HSIL (P > 0.50), and significantly lower in women without cervical abnormalities and in women showing HSIL than in women showing inflammatory lesions or low grade squamous intraepithelial lesions(LSIL)(P < 0.0001). Micronucleus counts were significantly greater in women with HSIL than in the women without cervical abnormalities or inflammatory processes (P < 0.001) or in the women with LSIL(P < 0.005). [3] Shoji Y et al examined forty six cases of CIN I/I, 75 of CIN III, 16 of microinvasive carcinoma of cervix, and 44 of invasive carcinoma of cervix using formalin fixed and paraffin wax embedded samples. The apoptotic cells were detected using TdT mediated dUTP-biotin nick end labeling (TUNEL) method. Apoptotic labelling indices were calculated after counting positive nuclei among at least 2000 nuclei. Significant positive correlation with histological malignant grading in CIN and tumour cell invasion into stroma was noted. They concluded that apoptosis in cervical neoplasias may be closely related to tumour progression. [4] Mergener M et al studied nasal swab samples collected from 40 infants under 12 months of age to evaluate DNA damage related to air pollution in infants. They recorded 0.13% of cells with micronuclei; 1.20% karyorrhexis; 0.03% pyknosis; 10.85% karyolysis; 1.11% condensed chromatin; 0.54 binucleated cells; and 0.02% nuclear bud. [21]

Conclusions

MN and apoptosis counts helps in mass screening for cervical cancer. The study of micronuclei and apoptosis in Pap smears will increase the sensitivity and specificity of cervical cytology as screening test for cancer.

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There was a positive correlation between increased apoptosis and high micronucleus score in our study. These features can be used as biomarkers of cervical intraepithelial lesion and carcinoma in cervical Pap smear even while interpreting Papanicolau stained smears. Also presence of these two markers in high numbers in inadequate cervical smears due to low cellularity indicates the presence of underlying LSIL or HSIL or carcinoma cervix. Hence these patients must undergo repeat Pap test without fail.

Acknowledgements

We thank the Mr.Shivraj Somanna N.S(Statistician , M.S. Ramaiah Institute of medical sciences and research centre, Bangalore) and Mrs. Lavanya (Statistician, SIMS&RC) for their valuable work in statistical analysis of the study data. We also thank Mr.Chinswamy, technician for his assistance in staining the samples.

Funding None.

Competing Interest

The authors declare that there is no competing of interest.

References

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Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Original Article Utility of Modified Bleach Method Technique for the Demonstration of Acid Fast Bacilli in the Diagnosis of Tuberculous Lymphadenopathy in Comparison to Routine Ziehl-Neelsen Staining Manika Khare* and Manju Kaushal PGIMER, Dr Ram Manohar Lohia Hospital, New Delhi, India Keywords: Acid Fast Bacilli, Ziehlâ&#x20AC;&#x201C;Neelsen stain, Modified Bleach Method, Fine Needle Aspiration

ABSTRACT Background: There is major role of fine needle aspiration in diagnosis of tubercular lymphadenitis as it is a less invasive procedure and Ziehl-Neelsen (ZN) method for acid-fast bacilli (AFB) plays a key role in diagnosis and also for the monitoring of treatment in tuberculosis. The bleach concentration method is a technique with many benefits over routine ZN staining as it improves the sensitivity of direct microscopy for the detection of AFB. Methods: A total of 129 cases of tuberculous lymphadenitis diagnosed by fine needle aspiration cytology (FNAC) were categorized into six cytomorphological patterns. The acid-fast bacilli positivity by routine staining was correlated with modified bleach methods of ZN staining. Sensitivity of routine ZN and modified bleach concentration was compared. Grading of acid fast bacilli and time taken to detect bacilli was also compared. Result: The classic cytomorphological pattern of tuberculosis of epithelioid granulomas and caseous necrosis was seen in 21.1% of cases. Routine ZN staining detected AFB in 58.9% of cases and the modified bleach method in 89.1%. The modified bleach method showed AFB positivity in all cases where routine ZN staining was positive. The mean time taken for detection of AFB by modified bleach method was 8.91 minutes and by routine ZN staining method was 23.36 minutes. Conclusion: The modified bleach method was more sensitive and safer than routine ZN staining. As the background was clear, the bacilli were easily visible and the screening time was shorter.

*Corresponding author: Dr Manika Khare, PGIMER, Dr Ram Manohar Lohia Hospital, New Delhi, India Phone: +91 8527266802 Email: drmanika09@gmail.com

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Modified Bleach Method Technique for AFB

Introduction

India accounts for nearly one third of the global burden of TB and it is the most important public health problem. India alone accounts for 26% (2.0-2.4 million) of global cases. [1] Every day more than 20,000 people become infected with tubercle bacillus, more than 5000 develop the disease and more than 1000 die from Mycobacterium tuberculosis infection.[ 2] Lymphadenopathy is the most common form of extra pulmonary TB.[3,4,5] FNAC provides an alternative to excision biopsy for lymph nodes and is an easy procedure for collection of material for cytomorphological and bacteriological examination. There is major role of fine needle aspiration in diagnosis of tubercular lymphadenitis as it is a less invasive procedure and Ziehl-Neelsen (ZN) method for acid-fast bacilli (AFB) plays a key role in diagnosis and also for the monitoring of treatment in tuberculosis. Inspite of great utility, the major disadvantage of ZN staining is low sensitivity, ranging from 9 to 46%.[6,7] The bleach concentration method is a technique with many benefits over routine ZN staining as it improves the sensitivity of direct microscopy for the detection of AFB, can effectively kill mycobacterium tuberculosis which makes the specimen safe to handle and is inexpensive & easy to perform and requires no additional equipment.

Materials and Methods

In a time course of 1 year, total of 129 cases suspected of tuberculosis were taken. In each case smears were stained by Giemsa stain, pap stain and routine ZN staining. In rest of the left out material, modified bleach technique was applied. In this method the aspirated material was subjected to liquefaction with 5% sodium hypochlorite (NaOCl, bleach) solution at room temperature for 30 minutes, followed by centrifugation at 3000 rpm for 15 mins. Smears were made from the deposit and were stained by routine ZN staining. The spectrum of cytomorphological features observed on Papanicolaou and MGG stained smears was broadly categorized into six patterns â&#x20AC;&#x201C; 1) Epithelioid granuloma with langhans giant cells and caseous necrosis (pattern 1); 2)Epithelioid cells in a reactive background (pattern 2); 3) Caseous necrotic material with epithelioid cells (pattern 3); 4) Caseous necrotic material with a few lymphocytes and histiocytes, with no epithelioid cells (patter 4); 5)Smears with predominantly neutrophils, degenerating epithelioid cells and necrotic material (pattern 5). Smears stained by both the methods were thoroughly screened for presence of AFB under 100X oil immersion.

After identifying AFB in the routine ZN slide and modified bleach technique grading of the AFB was done on the slide and comparison between the both staining system was done. (table 1) This grading system was created on the basis of pilot study done in our department with the help of grading system done on the sputum smear.

Result

All 129 cases of suspected tubercular lymphadenitis were screened for AFB by routine ZN stain and modified bleach method. The age range of the cases varied from 1 to 85 years. There was female preponderance accounting 55.9% of total cases. Most patients were in the age group of 16 â&#x20AC;&#x201C; 30 years constituting 48.8%, followed by age group of 31-45 years constituting 23.7%. The youngest patient was 1 year old. The oldest patient was 81 years old. Among the 129 lymphnodes studied, maximum cases involved cervical lymphnodes (71.3%), followed by axillary region (7.7 %). Least number of patients presented with preauricular and posterior occipital region lymphadenopathy. The smear positivity for AFB on conventional ZN method was 58.9% (76/129) while the positivity increased to 89.1% (115/129) by bleach method. The comparison between conventional ZN method and bleach method showed statistical significance. (table 2) The mean time taken for detection of AFB by modified bleach method was 8.91 minutes and by routine ZN staining method was 23.36 minutes. The time difference between two methods was statistically significant. (table 3) There was a significant difference between the grading of AFB positivity by modified bleach method and routine Z-N staining (p value <0.001) with modified bleach method showing higher grades of positivity.(table 4)

Discussion

Tuberculosis (TB) is an ancient infection that has plagued humans since times immemorial and still continues to remain a major public health problem especially in developing countries like India. Hence, early diagnosis of tuberculosis and initiating optimal treatment would not only enable cure of an individual patient but will significantly reduce the transmission of infection and disease to others in the community. Diagnostic modalities must also be tailored according to the needs of the population and epidemiology of TB in that region. These include improved microscopy, usage of liquid culture for childhood and extrapulmonary TB, chemical and physical detection of mycobacterial antigens in paucibacillary condition, antigen capture,

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antibody detection, cellular immune recognition, nucleic acid amplification and phage assay. However all these investigations are time consuming and expensive.

cases which was inconsistent with the distribution pattern of the present study.[9] Majority of the AFB positive cases by modified bleach method showed more number of bacteria present in clean thin background making them easily visible.

In the present study smears suggestive of tubercular pathology were divided in to six patterns depending upon cytomorphological findings. The most common pattern (n=35, 27.1%) was pattern 3 and least common (n=16, 12.4%) was pattern 5. However in a study by chandrashekhar et al in the year 2012, pattern 1 was seen in 42 (38%) cases, pattern 2 in 27 (24%), pattern 3 in 8 (7%), pattern 4 in 17 (15%), and pattern 5 in 16 (14%)

The present study validates the literature on the utility of concentration of AFB by modified bleach method for detection of tuberculous bacilli in lymph node aspirates. The comparison of percentage of smears positive with routine ZN staining and modified bleach method in several other studies is shown in the table no 5.

Table 1: Grading of AFB on FNAC smears. NUMBER OF AFB

GRADE

NUMBER OF FIELDS EXAMINED

>10 per oil immersion field

1-10 per oil immersion field

10-99 per 100 oil immersion fields

100

Scanty

200

0-9 per 100 oil immersion fields

Table 2: Comparison of routine ZN staining and modified bleach technique in detection of AFB AFB – BLEACH TECH

Positive

AFB-ZN STAINING

Negative

Total

Positive

Negative

115

129

Chi-square value

p-value

16.33

<0.001

TABLE 3: Comparison of time taken for detection of AFB by routine ZN staining and modified bleach technique by applying paired t test. TIME ZN TIME Bleach

Pair 1

Paired Samples Statistics Mean N 23.36 129 8.91 129

Std. Deviation 6.26 6.94

p-value <0.001

TABLE 4: Comparison of grading of AFB positivity in routine ZN staining and modified bleach technique. GRADING BY ZN STAINING

Scanty Grade 1 Grade 2 Grade 3

Total

GRADING BY BLEACH METHOD Scanty Grade 1 Grade 2 Grade 3 7 20 10 2 0 16 12 2 0 0 4 1 0 0 0 2 7 36 26 7

Total

Chi-square value

p-value

39 30 5 2 76

43.13

<0.001

TABLE 5: Comparison of AFB positivity in different studies by ZN & modified bleach technique. AUTHORS

ROUTINE-ZN STAINING

MODIFIED BLEACH TECHNIQUE

Khubani et al[9] (2005)

21.8%

71.9%

Gangane et al

51.27%

72.0%

Annam et al [11] (2009)

33.33%

63.44%

Chandrashekhar et al [9] (2012)

12.5%

60.7%

Patel M M (2013) [12]

21.3%

61.74%

Present study

58.9%

89.1%

[10]

(2008)

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Modified Bleach Method Technique for AFB

In the current study, mean time taken by the routine ZN staining in detecting AFB was 23.36 minutes and by modified bleach method it was 8.91 minutes. Paired T test was applied and significant difference was noted in time taken by modified bleach method (mean time 8.91 min) and routine ZN staining (mean time 23.36 min), resulting in the p value of <0.001which was significant. Chandrashekhar et al in the year 2012, in his study also suggested reduction in time taken for detection of AFB by modified bleach method, although they did not quantify the difference of time in their study.[9] In the present study the number of bacilli per oil immersion field in routine ZN staining and modified bleach method was graded. Numbers of bacilli were found to be more per oil immersion field in modified bleach method. There was significant difference in positivity grade of bacilli in two methods with higher grade in bleach method (p value < 0.001). The difference in time for detection of AFB and grading of bacilli on smear by two methods is compared in this study. However this comparison was not done in earlier studies.

Conclusion

The bleach method for AFB is simple, safe and costeffective. The results would be more accurate if the concentration by bleach solution and incubation time is strictly followed. The implementation of the bleach method clearly improves microscopic detection and can be a useful contribution to routine cytology. This would be of benefit to the patients to receive an early and effective treatment.

Funding None

Competing Interests None

Reference

1. World Health Organization (WHO). Global tuberculosis report 2013. Geneva: WHO; 23 Oct 2013. Available from: http://apps.who.int/iris/ bitstream/10665/91355/1/9789241564656_eng.pdf

2. Bhatia AS, Kumar S, Harinath BC. Immunodiagnosis of tuberculosis: An update. Ind J Clin Biochem 2003;18(2): 1-5. 3. Dandapat MC, Mishra BM, Dash SP, Kar PK. Peripheral lymph node tuberculosis: a review of 80 cases. Br J Surg 1990; 77: 911-2. 4. Lau SK, Kwan S, Lee J, Wei WI. Source of tubercle bacilli in cervical lymph nodes: A prospective study. J Laryngol Otol 1991;105:558-61. 5. Shahid M, Sirwar SM, Indupalli AS, Bhatt S . In Extrapulmonary Tuberculosis in sputum negative cases. International J of Bioassays; 2013. p. 971-74. 6. Dandapat MC, Mishra BM, Dash SP, Kar PK. Peripheral lymph node tuberculosis: a review of 80 cases. Br J Surg1990;77 : 911-2. 7. Lau SK, Kwan S, Lee J, Wei WI. Source of tubercle bacilli in cervical lymph nodes: A prospective study. J Laryngol Otol 1991;105:558-61. 8. Chandrashekhar B, Prayaga AK. Utility of concentration method by modified bleach technique for the demonstration of acid-fast bacilli in the diagnosis of tuberculous lymphadenopathy. J Cytol 2012 July; 29:165-8 9. Khubnani H, Munjal K. Application of bleach method in diagnosis of extrapulmonary tuberculosis. Indian J Pathol Microbiol 2005; 49(4): 546-50. 10. Gangane N, Anshu, Singh R. Role of modified bleach method in staining of acid-fast bacilli in lymph node aspiration. Acta Cytol 2008;52:325-28. 11. Annam V, Mohan HK, Rekha BP. Improved microsopical detection of acid-fast bacilli by the modified bleach method in lymph node aspirates. Indian J Pathol Microbiol 2009;52:349-52 12. Patel MM, Patel KK, Italiya SL, Kaptan KR. Improved diagnosis of tuberculosis in lymph node cytology by bleach method for detection of acid fast bacilli in comparison to conventional Ziehl Neelsen staining method. Int J Med Sci Public Health. 2013; 2(4): 935-939.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Original Article Seroprevalence of Transfusion Transmitted Infections by Using 4th Generation Enzyme- Linked Immunosorbent Assay kit: A 3 Year Study in a Tertiary Health Care Centre of Delhi Mohammad Jaseem Hassan, Sabina Khan, Zeeba S Jairajpuri, Safia Rana, Salamah Parveen Imteyaz, Sujata Jetley* Department of Pathology, Hamdard Institute of Medical Sciences and Research (HIMSR), Jamia Hamdard, New Delhi, India. Keywords: Transfusion transmitted infections, Blood donors. HIV, Hepatitis B, Hepatitis C, Syphilis, Malaria

ABSTRACT Background: One of the greatest challenges of the blood transfusion services is to prevent the transmission of Transfusion transmitted infections (TTI), because these unsafe blood transfusions leads to increase morbidity and mortality and eventually leads to economic burden on the society. The objective of our study was to find out the seroprevalence of TTI among blood donors at our blood bank by using 4th generation ELISA kit and to compare our study with other studies conducted at national and state level. Method: 2401 units of blood collected during 3 years period were screened for 5 infections. HIV, Hepatitis B and Hepatitis C infections were screened by using 4th generation ELISA kit. Test for syphilis was done by Rapid plasma reagin card test and test for malaria parasite was done by Advantage Mal card test. Results: A total of 2401 units were collected during the period of three years. 98.25% donors were male. 91.09% donors were replacement donors. A total of 71 blood donors (2.95%) were tested positive for any one of the TTI. Out of these 71 cases, 69 were males and only 2 were females. The overall prevalence of HIV, HBV, HCV and syphilis in our study were 0.33%, 1.7%, 0.74% and 0.16% respectively. None of our donors was tested positive for malaria. Conclusion: In order to minimize the risk of TTI, voluntary donors should be encouraged by means of educating general people about benefits of blood donation and motivating them by conducting regular blood camps.

*Corresponding author: Dr. Sujata Jetley, Department of Pathology, Hamdard Institute of Medical Sciences and Research (HIMSR), Jamia Hamdard, New Delhi, 110062 INDIA Email: sujatajetley@gmail.com

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TTI Among Blood Donors

Introduction

Although blood transfusion (BT) is an integral part of medical and surgical therapy which can saves millions of lives worldwide each year and also reduces morbidity, however it has a life threatening hazards as well, which may vary from only trivial to potentially life threatening complications. Thus proper selection of donors and meticulous testing of all donated blood is essential to reduce the morbidity and mortality associated with blood transfusion.[1,2,3] The infectious agents must have the following characteristics in order to be transmitted by blood. These include “presence in the blood for long periods, stability in blood stored at 4̊ C or lower temperature, long incubation period before the appearance of clinical signs and asymptomatic phase or only mild symptoms in the blood donors hence not identifiable during the blood donor selection process”.[4] Human immunodeficiency virus (HIV), Hepatitis B virus (Hep B), Hepatitis C virus (Hep C), syphilis and malaria is responsible for majority of transfusion transmitted infections (TTI), however infrequently other infections like Cytomegalovirus (CMV), Epstein bar Virus (EBV), Herpes virus, Human T-cell leukotropic virus (HTLV), Toxoplasmosis, Brucellosis and Chagas disease etc. may also be transmitted through BT.[1] The World Health Organization (WHO) recommended mandatory screening of all donated bloods for HIV-1 & HIV-2, Hep B, Hep C and syphilis. [5] They also recommended screening of other infections like malaria, chagas disease or HTLV based on local epidemiological evidence. [5] In India, according to guidelines of National AIDS Control Organization (NACO), all donated blood must be tested for HIV-1 & HIV-2, Hep B, Hep C, syphilis and malaria.[6] There is always 1% chance of transfusion associated problems including TTI with every unit of blood transfused.[7] Thus one of the greatest challenge of the transfusion services is to prevent the transmission of these TTI, because these unsafe blood transfusions leads to increase morbidity and mortality and eventually leads to economic burden on the society.[8] According to WHO “the minimum evaluated sensitivity and specificity level of all assay used for blood screening should be as high as possible and preferably not less than 99.5%’’. The NACO recommended use of 3rd or 4th generation ELISA kit, which is 100% sensitive for testing donated blood for HIV-1 & HIV-2.[9] The present study was carried out to find out the seroprevalence of TTI among blood donors at our blood bank attached to newly establish medical college by using 4th generation ELISA kit, since inception of our blood bank in March, 2012. This is essential for monitoring the safety of blood supply. We also compare our study with other

studies conducted at national level as well as with studies conducted at Delhi and adjoining states.

Material and Method

The present study was conducted at our newly established blood bank attached to our Medical college. The 3 years data was collected between the periods 20th March, 2012 to 20th March, 2015. The blood was collected in our blood bank both from replacement donors and voluntary donors. The first step of blood donation starts with filling of a registration form by the donors which include age, sex, address, contact number, occupation, history of previous donation along with medical history like history of previous major or minor surgery, blood transfusion, hospitalization, history of any febrile illness in the recent past, weight loss, uncontrolled diarrhea, recent jaundice, liver disease, lung disease, cardiovascular disease, malignancy, epilepsy, malaria, dog bite and intake of alcohol or any contraindicated drugs etc. Pre-donation counseling of the donor was the next step which includes explanation of the procedure of the blood donation, post-donation care and the final outcome of the donation in the form of result of TTI screening test. Blood grouping and hemoglobin (Hb) estimation was done. All the donors were then screened by blood transfusion officer (BTO) as per the strict donor selection criteria led down by NACO. Height, weight, pulse, blood pressure and temperature were recorded. Thorough inspection was done for any marks of drug abuse, infection or skin lesion at the site of venepuncture. This screening procedure was helpful in excluding professional donors. All the donors between the age group of 18 to 60 years, weight more than 45 kg, Hb level of more than 12gm%, without any history of hepatitis, jaundice or any major surgery in past one year and with normal pulse and blood pressure were considered fit for blood donation. Blood was then collected by taking all aseptic precautions as per surgical operative procedure (SOP) of our blood bank after obtaining a written informed consent from the donor. As per NACO guidelines, all 2401 units of blood collected during 3 years period were screened for 5 infections, i.e HIV, Hep B, Hep C, Syphilis and malaria. HIV, Hep B and Hep C infections were screened by using 4th generation ELISA kit. HIV screening was done by using Erba sure HIV Gen4 ELISA kit (Transasia, India) with reported sensitivity and specificity of 99.8% and 99.7% respectively. The screening of Hep B was done by Hepalisa ultra kit (J.Mitra, India) with reported sensitivity of 100% and specificity of 99.92%. The Hep C was screened by using Monolisa HCV

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Jaseem Hassan et al.

A-335 donors (VD). (Table-1) A total of 71 blood donors (2.95%) were tested positive for any one of the TTI. Out of these 71 cases, 69 were males and only 2 were females. Out of these 71 positive cases, 67 cases (3.06%) were positive in RD and only 4 cases were positive in VD (1.86%). The overall prevalence of HIV, HBV, HCV and syphilis in our study were 0.33%, 1.7%, 0.74% and 0.16% respectively. None of our donor was tested positive for malaria.(Table-2) Out of 71 positive cases, 41 cases were positive for HBV, 18 cases for HCV, 8 cases for HIV and 4 cases for syphilis. (Table-3) The age wise distribution of positive cases showed that majority of positive cases (63.28%) were seen in age group of 18-30 years, followed by 30.98% cases in age group of 31-40 years.(Table-3) Blood group distribution of positive cases showed that majority of positive cases (32 cases) belongs to blood group B, followed by 21 cases of blood group O. (Table-4)

Ag-Ab ultra kit (Bio-Rad, France) with reported sensitivity and specificity of 100% and 99.83% respectively. Test for syphilis was done by Rapid plasma reagin (RPR) card test (Tulip, India) and the test for malaria was done by Advantage Mal card test (J. Mitra, India). Manufacturerâ&#x20AC;&#x2122;s instructions were strictly followed for performing all these tests. Before labeling a test as seropositive, the samples were repeated in duplicate. The donated blood found positive for any TTI were discarded according to SOP of our blood bank.

Results

A total of 2401 units were collected during the period of three years. The majority of the donors were male (98.25%) with female donors constituting only 1.75% of total donation. Out of 2401 donors, 2187 (91.09%) were replacement donors (RD) and 214 (8.91%) were voluntary Table 1: Sex distribution and Type of donors. Voluntary Donors

Replacement Donors

Total Donors

192

2167

2359 (98.25%)

Male Females

42 (1.75%)

Total

214 (8.91%)

2187 (91.09%)

2401 (100%)

Table 2: Sex distribution of seropositive markers. TTI

Male

Females

No. of cases

HIV

08 (0.33%)

HBsAg

41 (1.7%)

HCV

18 (0.74%)

Syphilis

04 (0.16%)

Malaria

00 (0.00%)

Total

71 (2.95%)

Table 3: Age wise distribution of transfusion transmitted infections. Age (Years)

HIV

HBsAg

HCV

Syphilis

Malaria

Total

18-30

45 (63.38%)

31-40

22 (30.98%)

41-50

4 (5.63%)

51-60

00 (0.00%)

Total

8 (11.26%)

41 (57.74%)

18 (25.35%)

4 (5.63%)

00 (0.00%)

71 (100%)

Table 4: Blood group distribution of seropositive markers. TTI HIV HBsAg

A 02 07

B 02 19

AB 01 04

O 03 11

Total 08 41

HCV

Syphilis

Malaria

Total

13 (18.30%)

32 (45.07%)

05 (7.04%)

21 (29.57%)

71 (100%)

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TTI Among Blood Donors

Table-5: comparison of prevalence rate of TTI from various parts of India. Duration of study

Place

Patel PA et al.8

2005-2011

Ahmadabad, Gujrat

Ahmed Z et al.

2008-2011

Mangalore, Karnataka

Sastry JM et al.12

2008-2013

Pune, Maharashtra

2005-2011

Akola, Maharashtra

2008-2011

Kolkata, WB

2012-2015

Delhi

Chandra T et al.

2001-2007

Lucknow, UP

Pahuja S et al.14

2002-2005

Delhi

Kaur G et al.

2001-2005

Chandigarh

2002-2006

Hisar, Haryana

0.3

1.7

1.0

0.9

4.0

2005-2009

Bellary, Karnataka

0.91

3.28

0.35

0.04

4.58

Gupta R et al.18

2003-2008

Delhi

0.35

1.66

0.65

2.8

5.46

Sinha SK et al.

2007-2008

Kolkata, WB

0.64

2.27

1.62

1.31

5.8

Author

Raut MM et al

Karmakar PR et al. Our study 24

Arora D et al.

Kulkarni N13 20

Discussion

The main objective of blood transfusion services is not only to provide safe and adequate blood supply at all levels but also to eliminate or reduce the risk of TTI associated with BT, because BT is a significant route of transmission of these infections. HIV and hepatitis are two deadly infections which are transmitted by BT. It is reported that on an average one HIV positive transfusion leads to death after 2 years in children and after 3-5 years in adults.[1] With prevalence of 0.3%, an estimated 2.4 million people are living with HIV in our country.[10] Despite the availability of safe and effective vaccine against hepatitis B since 1982, India is still placed in the Intermediate zone of prevalence of Hepatitis B by WHO (prevalence rate of 2-7%) with an estimated 40 million HBsAg carriers.[11] In our study out of 2401 blood donors, 98.25% were males and only 1.75% female donors. Our findings were comparable to the study done by various authors from different parts of India, who found more than 95% male donors in their study. These include Arora et al, Haryana (96.2%), Mangalore (97.5%) , Raut MM et al, Wardha (97.51%) , Patel PA et al, Ahmadabad (95.48%) , Pallavi P et al, Mysore (97.84%), Sastry JM et al, Pune (96.6%), Kulkarni N, Bellary (98%) and Pahuja S et al, Delhi (97.24%). [1,2,3,8,11-14] The less number of female donors in our study may partly be due to social and cultural habit in our country but mainly due to the fact that majority of female donors are declared unfit during pre donation screening because of very high incidence of anemia during child bearing age in India. [2]

HIV (%)

HBV (%)

HCV (%)

Syphilis (%)

Overall TTIs (%)

0.08

0.30

0.09

0.06

0.53

0.1

0.5

0.08

0.07

0.82

0.28

1.23

0.41

0.008

1.56

0.53

1.6

0.14

0.03

2.30

0.6

1.41

0.59

0.23

2.79

0.33

1.7

0.74

0.16

2.95

0.23

1.96

0.85

0.01

3.05

0.56

2.23

0.66

3.45

0.6

1.7

0.8

0.7

3.8

The majority of our donors were RD (91.09%) as compared to VD (8.91%). The predominance of RD was also reported by Pahuja S et al. (99.48%), Kakkar N et al (94.7%) and Singh B et al (82.4%). [14-16] Contrary to our study, the majority of VD was reported by Raut MM et al (87.13%), Patel PA et al. (95.58%) and Karmakar PR et al (100%). [3,8,17] In this study out of 2401 blood donations, 71 blood was found to be positive for any one TTI. Thus the overall prevalence of TTI in our study was 2.95%. This is much less than that reported by Arora D et al, Haryana (4%), Pahuja S et al, Delhi (3.45%), Kulkarni N, Bellary (4.58%),Gupta R et al, Delhi (5.46%), Singh B et al, Delhi (5.61%), Sinha SK et al, Burdwan, WB (5.8%) and Kaur G et al, Chandigarh (3.8%), [1,14,15,18-21] but overall prevalence of TTI in our study was higher than that reported by Fernandez H et al, Mangalore (0.6%) Raut MM et al , wardha (2.30%), Patel PA et al, Ahmadabad (0.53%), Pallavi P et al, Mysore (2.22%), Fernandez H et al, Sastry JM et al, Pune (1.56%), Karmakar PR et al, Kolkata (2.79%) and Adhikari L et al, Sikkim (1.63%). [2,3,8,11,12,17,22] This may be because majority of donors in their study were VD. Out of 71 positive cases, HIV was positive in 8 cases, HBsAg was positive in 41 cases, HCV in 18 cases and syphilis in 4 cases. Thus the overall prevalence of HIV, HBsAg, HCV and syphilis in our study was 0.33%, 1.74%, 0.74% and 0.16% respectively. None of our case was positive for malaria parasite. The prevalence of HIV was 0.33%, which was similar to the prevalence of HIV (0.3%) in our country. [10] The prevalence of HIV in this study was much less than that reported by Pallavi P et al (0.44%), Pahuja S et al (0.56%), Kulkarni N (0.9%), Singh B et al

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Jaseem Hassan et al. (0.54%), Karmakar PA et al (0.6%), Singh B et al (0.8%), Sinha SK et al (0.64%) and Kaur G et al (0.6%), [11,14-17,1921] but it was much higher than Fernandes H et al (0.06%), Patel PA et al (0.08%), Ahmed Z et al (0.1%) and Chandra T et al (0.23%). [2,8,23,24] The prevalence of HIV close to 0.33% were reported by Arora D et al (0.3%), Sastry JM et al (0.28%), Gupta R et al (0.35%)and Adhikari L et al (0.32%). [1,12,18,22] The prevalence rate of HBsAg in present study was 1.7%, which was less than the lower limit of prevalence rate of 2-7% as laid down by WHO for India. Our finding was less than that reported by Kulkarni N (3.28%), Pahuja S et al (2.23%) and Sinha SK et al (2.27%), [13.14,20] but it was more than that reported by Fernandes H et al (0.34%), Patel PA et al (0.30%), Sastry JM et al (0.28%) and Adhikari L et al (0.78%). [2,8,12,22] The prevalence rate of HBsAg close to our finding of 1.7% was also reported by Arora D et al (1.7%), Raut MM et al (1.6%), Gupta R et al (1.68%), Singh B et al (1.8%), Kaur G et al (1.7%) and Chandra T et al (1.96%).[1,3,18,19,21,24] The 0.74% prevalence of HCV in our study was comparable to study by Pahuja S et al (0.66%), Gupta R et al (0.65%), Kaur G et al (0.8%) and Chandra T et al (0.85%).[14,18,21,24] Arora D et al (1%) and Sinha SK et al (1.62%) reported higher prevalence of HCV as compared to our study, [1,20] while Fernandes H et al(0.06%), Patel PA et al (0.09%), Pallavi P et al (0.23%), Sastry JM et al (0.41%),Kulkarni N (0.35%)and Adhikari L et al (0.27%) reported lower prevalence of HCV than our study. [2,8,11-13,22] The overall prevalence of syphilis in our study was 0.16%, which was comparable to study done by Fernandes H et al (0.11%), Pallavi P et al (0.28%), KarmakarPR et al (0.23%) and Adhikari L et al (0.27%). [2,11,17,22] Arora D et al (0.9%) , Singh B et al (2.6%), Gupta R et al (2.8%), Sinha SK et al (1.3%) and Kaur G et al (0.7%) reported higher prevalence of syphilis than our study, [1,16,18,20,21] while Raut MM et al (0.03%), Patel PA et al (0.06%), Sastry JM et al (0.008%), Kulkarni N (0.04%), Chandra T et al (0.01%) and Khageshan AP et al (0.04%) reported lesser prevalence of syphilis as compared to our study. [3,8,12,13,24,25] None of our donors were found positive for malaria parasite, which was comparable to the study done by Patel PA et al, Pallavi P et al, Sastry JM et al, Kulkarni N, and Ahmed Z et al. [8,11,12,21,23] This may be due to the presence of typical fever with rigor in patients of malaria, therefore these patients were usually eliminated during pre donation screening. Fernandes H reported one positive case of malaria in their study.[2]

A-337 TTI in RD was also reported by Arora D et al, Pahuja S et al and Singh B et al .[1,14,16] RD are usually one time donor who donate blood in emergency for their family members, close relatives or friends, thus because of compulsion to donate blood in these situations, information regarding past illnesses or high risk behavior may be concealed by the donors.[17] In our country due to lack of awareness and motivation, RD still constitute the largest group of blood donors.[11] VD donates blood at regular intervals and they are usually young motivated college students and employee of various institutions. Thus in order to reduce the risk of TTI, there is need to promote VD by proper education, motivation and providing accurate information about the advantages of blood donation by conducting regular blood donation camps. .[11,13] Age wise distribution of positive cases in this study showed that 63.38% of positive cases fall in the age group between 18-30 years, which was similar to the study conducted by Arora D et al (69.95% between age group 18-31 years), Patel PA et al (42.79% between age group18-30 years) and Ahmed Z et al (59.55 between age group 18-35%).[1,8,23] The younger age group (18-40 years) is most sexually active group of the community, therefore they are more prone for developing TTIs, however this is of much concern as well because this age group is also the most productive and economically viable group of the society.[1,8,23] Blood group profile of positive cases showed that 32 cases (45.07%) were of blood group ‘B’, followed by 21 cases (29.57%) of blood group ‘O’, 13 cases (18.3%) of blood group ‘A’ and 5 cases of blood group ‘AB’. This may be explained by the fact that the most common blood group of the donors in our study was blood group B followed by blood group O. When we compare our study with previous studies from Delhi and adjoining states, there is definite decrease in overall prevalence of TTIs over last one and half decade, with decrease in prevalence of HIV and syphilis. The prevalence of hepatitis is almost similar as compared to previous studies.[1,14,16,18,19]

Out of 71 positive cases of TTI, 67 cases were found in RD (3.06%, 67/2187), while only 4 cases were positive in VD (1.86%, 4/214). Similar finding of high prevalence of

The increasing seropositivity of TTIs among RD is of major concern for BTS. By practicing the deferral of high risk donors by donor self exclusion and use of nucleic acid amplification test (NAT) for screening the blood, the developed countries more or less achieve the decreased risk of TTI. .[11,21] However in underdeveloped countries like ours, because of inability to use NAT for blood screening due to its high cost and lack of awareness about voluntary blood donation, TTI is still a major concern for BTS. Thus in order to minimize the risk of TTI, VD should be encouraged by means of educating general people

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about benefits of blood donation and motivating them by conducting regular blood camps along with use of NAT for blood screening, which can detect very low levels of viral DNA or RNA in the donated blood.

Conclusion

During three years period, 2401 donors were tested for various TTIs, comprising of 98.25% male donors and 1.75% female donors. Most of the donors were RD (91.09%) with only 8.91% VD. The seropositivity was highest for HBsAg (1.7%), followed by HCV (0.74%), HIV (0.33%) and syphilis (0.16%). None of our donors were tested positive for MP. Out of 71 positive cases, 67 cases were seen on RD with only 4 positive cases among VD. One of positive outcome of our study was definite decrease in prevalence of TTI in Delhi over the past one and half decade. The TTI can be minimized by encouraging voluntary nonrenumerated blood donors by proper education, motivation and counseling of general population along with judicious use of blood only in cases of absolute emergency and use of advanced technique like NAT for pre transfusion screening of blood.

References

1. Arora D, Arora B, Khetarpal A. seroprevalence of HIV, HBV, HCV and syphilis in blood donors in southern Haryana. Indian J Pathol Microbiol 2010;53:308-309. 2. Fernandes H, D’souza PF, D’souzaPM. Prevalence of transfusion transmitted infection in voluntary and replacement donors. Indian J Hamatol Blood Transfus 2010;26:89-91. 3. Raut MM, Joge US, Choudhary SG, Malkar VR, Ughade HM. Seroprevalence of transfusion transmitted infections among healthy blood donors at blood bank attached to a tertiary care hospital in Maharashtra state of India. International Journal of Health Sciences & Research 2012;2:18-24. 4. Contreras M (ed). ABC of transfusion (3rd edn). London BMJ Books, 1998. 5. Screening donated blood for transfusion-transmissible infections: recommendations. 2009, Geneva, World Health Organization. 6. National AIDS control organization. Standards for blood banks and blood transfusion services. New Delhi: Ministry of Health and family welfare Government of India; 2007 7. Widmann FK, editor. Technical manual American association of blood banks. Aglington USA. 1985:325-344.

8. Patel PA, Patel SP, Oza HV. Seroprevalence of transfusion transmitted infections (TTIs) in blood donors at western Ahmedabad- A secondary care hospital based study. Int J Biol Med Res. 2012;3:1806-1810. 9. “Manual on quality standards for HIV testing laboratories” produced and published by national AIDS control organization, Ministry of health and family welfare, Government of India, New Delhi published in 2007. 10. Global report: UNAIDS report on the global AIDS epidemic. 2010:6. http://www.unaids.org/globalreport/ global_report.htm. 11. Pallavi P, Ganesh CK, Jayashree K, Manjunath GV. Seroprevalence and trends in transfusion transmitted infections among blood donors in a university hospital blood bank: a five year study. Indian J Hematol Blood 2011;27:1-6. 12. Sastry JM, agarwane SU, harke VA. Retrospective study of the five-year prevalence and trends of transfusion transmitted infections (TTIs) among blood donors at a charitable hospital blood bank in Pune, India. International J. of Healthcare and Biomedical Research 2014;2:193-200. 13. Kulkarni N. Analysis of the seroprevalence of HIV, HBsAg, HCV and syphilitic infections detected in the pretransfusion blood: A short report. International Journal of Blood Transfusion and Immunohematology 2012;2:1-3. 14. Pahuja S, Sharma M, Baitha B, Jain M. Prevalence and trends of markers of Hepatitis C virus, Hepatitis B virus, Human Immunodeficiency virus in Delhi blood donors: a hospital based study. Jpn J Infect. Dis. 2007;60:389-391. 15. Kakkar N, Kaur R, Dhanoa J. Voluntary donorsneed for a second look. Indian J Pathol Microbiol 2004;47:381-383 16. Singh B, Verma M, Kotru M, Verma K and Batra M. Prevalence of HIV & VDRL seropositivity in blood donors of Delhi. Indian J Med Res 2005;122:234-236. 17. Karmakar PR, Shrivastava P, Ray TG. Seroprevalence of transfusion transmissible infections among blood donors at the blood bank of a Medical College of Kolkata. Indian J Public Health 2014;58:61-64. 18. Gupta R, Singh B, Singh DK, Chugh M. Prevalence and trends of transfusion transmitted infections in a regional blood transfusion centre. Asian J Transfus Sci. 2011;5:177-178.

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Jaseem Hassan et al. 19. Singh B, Kataria SP, Gupta R. Infectious markers in blood donors of East Delhi: prevalence and trends. Indian J pathol Microbiol 2004;47:477-479.

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21. Kaur G, Basu S, Kaur R, Kaur P, Garg S. patterns of infections among blood donors in a tertiary care centre: A retrospective study. The National Medical Journal of India 2010;23(3): 147-149.

22. Adhikari L, Bhatta D, Tsering DC, Sharma DK, Pal R, Gupta A. Infectious disease markers in blood donors at central referral hospital, Gangtok, Sikkim. Asian J Transfus Sci 2010;4:412. 23. Ahmed Z, Umaru N, Shreesha K. Seroprevalence of transfusion transmitted infections among blood donors in Mangalore. Medica Innovatica 2012;1:24-26. 24. Chandra T, Kumar A, Gupta A. Prevalence of transfusion transmitted infections in blood donors: an Indian experience. Trop Doct 2009;39:152-154. 25. Khageshan AP, Kulkarni KR, baragundi MC. Seroreactivity of syphilis among blood donors of a blood bank. Annals of pathology and laboratory medicine 2016;3:A41-44.

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20. Sinha SK, Roychoudhary S, Biswas K, Biswas P, Bandopadhya R. prevalence of HIV, Hepatitis B, Hepatitis C and syphilis in donor’s blood: A study from eastern part of India. Open Journal of Hematology 2012;3

Original Article Association of Thyroid Disorders with the Hormones of Anterior Pituitary in Female Infertiltiy Rakhee Yadav*, Sanjiv Kumar Bansal, BusiKarunanand, Eram Hussain Pasha, Birendra Kumar and Smita Prasad Shastry Dept. of Biochemistry, Faculty of Medicine and Health Sciences, SGT University, Gurgaon, India Keywords: Infertility, Females, Hypothyroidism, Prolactin, LH, FSH

ABSTRACT Background: Fertility in females is centred on the functional hypothalamo-pituitary-ovarian (HPO) axis. Thyroid dysfunctions are also known to interfere with the physiology of reproduction and pregnancy. This study was conducted with an objective to evaluate the relationship between thyroid and pituitary hormones in female infertility because abnormalities of HPO axis and thyroid dysfunction are one of the common and often treatable causes of infertility in females. Methods: Hundred female patients of infertility who visited the Department of Biochemistry, SGT Medical College, Hospital and Research Institute, Gurgaon for hormonal evaluation were recruited for the study. After excluding other causes of infertility including tubal factors, genetic or other anatomical factors; blood samples for TSH, LH, FSH and Prolactin were collected. Hundredage matched healthy fertile females were recruited as controls and their blood samples were collected for similar hormone assay. These parameters were estimated by the ELISA technique. Results were statistically analysed. Result: Out of the patients in our study group, 78% had primary infertility while secondary infertility was seen in 22% of the patients. Majority of the patients came out to be euthyroid (79%). There was a significant (p value<0.05) high serum level of Prolactin in patients of infertility as compared to controls. LH and FSH were found to be lower in infertile patients as compared to healthy controls. Moreover there was a significant positive correlation between the levels of TSH and Prolactin in infertile patients thus it might be imperative to say that hypothyroidism was strongly associated with hyperprolactinemia. Conclusion: For the normal physiology of reproduction, the functions of pituitary as well as thyroid should be normal. From our study we have elucidated that abnormalities of the thyroid gland and pituitary gland are interrelated. Thus their evaluation becomes necessary in order to elucidate the etiopathogenesis of female infertility which will further enhance the dimension towards designing effective treatment protocols.

*Corresponding author: Dr. Rakhee Yadav, H.No. 323, Sector 10-A, Gurgaon-122001(Haryana), INDIA Phone: +91 8826640014 Email: y.rakhee@yahoo.com

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

Yadav et al.

Introduction

Infertility is the inability of a couple to achieve pregnancy over an average period of one year despite of having regular unprotected sexual intercourse.[1] Infertility can be primary or secondary. In primary infertility, the couples have never been able to conceive; while in secondary infertility the couple has conceived previously but have a difficulty currently with conception.[2] There are no reliable estimates for global prevalence of infertility.[3]The incidence of female infertility is rising and varies from 10 to 20%.[4]Infertilities, either primary or secondary occurs for almost 15% of all women worldwide.[5] Infertility may be caused by an underlying medical condition that may damage the fallopian tubes, interferes with ovulation or causes hormonal complications. To find out the etiology and to treat it successfully; has always been the matter of concern for clinicians and is a potential area of research by researchers. The physiology of reproduction in females depends upon a complex interplay of the Hypothalamo-Pituitary-Ovarian (HPO) axis.[6] The hypothalamus through the release of gonadotropin releasing hormones (GnRH), controls the pituitary gland which directly or indirectly controls most other hormonal glands in the human body. Thus, alterations in the chemical signals from the hypothalamus can affect the pituitary gland, ovaries, thyroid, mammary gland and hence is a cause of hormonal abnormalities. Hormonal imbalance is an important cause of anovulation. Women with hormonal imbalance will not produce enough follicles to ensure the development of ovum.[2] Clinical and experimental studies have suggested a close relationship between Hypothalamo-Pituitary-Ovarian (HPO) axis and Hypothalamo-Pituitary-Thyroid (HPT) axis. The specific thyroid hormone receptors at the ovarian level may regulate the reproductive function.Moreover the influence of estrogens at higher levels of the HPT axis seem to integrate the reciprocal relationship between these two major endocrine axis.[7] Hormones from pituitary gland like TSH, prolactin or growth hormone may act synergistically with FSH and LH to enhance the entry of non-growing follicles into growth phase. In addition to this, thyroid hormones may be necessary for maximum production of both estradiol and progesterone.[8] It is well known that the function of the thyroid hormones includes modulation of carbohydrates, proteins and fat metabolism, gene expression and also sexual and reproductive function and when the thyroid hormone gets out of balance, many body functions are affected.[9]Thyroid www.pacificejournals.com/apalm

A-341 dysfunction in females have been found to be associated with anovulatory cycles, decreased fecundity and increased morbidity during pregnancy.[7,10] Hyperprolactinemia also adversely affects the fertility potential by impairing the pulsatile secretion of GnRH and hence interfering with ovulation.[11]Moreover it is associated with menstrual as well as ovulatory dysfunctions like amenorrohea, oligomenorrhoea, anovulation, inadequate corpus leuteal phase and galactorrhoea.[12] Thus we conducted our study with an objective to elucidate a relationship between thyroid disorders andpituitary hormones i.e. LH, FSH and prolactin in female infertility, thereby further consolidating our knowledge about the complex interplay of the Hypothalamo-Pituitary-Ovarian axis and HypothalamoPituitary-Thyroid axis in female reproduction.

Materials and Methods

Our study was the hospital based case control study where hundred female patients who were suffering from infertility formed our study group. They were the patients who visited the Department of Biochemistry, SGT Medical College, Hospital and Research Institute, Gurgaon for hormonal evaluation. Careful history was taken and the patients of both primary as well as secondary infertility were recruited in the study group. They were subjected to the exclusion criteria where causes of infertility like tubal blockage, cervical factors, pelvic inflammatory diseases or any other infective etiologies, other genetic factors including congenital anomalies of urogenital tract and male factor infertility were taken into consideration. Hundred healthy fertile females of the similar age groups were taken as controls. After taking the informed consent from both the groups; blood samples for thyroid profile (T3, T4 and TSH), LH, FSH and Prolactin were taken in plain vacutainers on second day of menstrual cycle following standard laboratory procedures. These parameters were assayed by competitive enzyme immunoassay; ELISA using ELISA kit method (Accubind ELISA Microwells). Results were analysed statistically. Continuous variables were expressed as mean and standard deviation and discrete variables were expressed as a percentage; Student t-test was applied to the values of TSH, T4, T3, LH, FSH and prolactin for the comparison of data in the two groups. Differences were considered as significant if p value was <0.05. Pearsonâ&#x20AC;&#x2122;s correlation coefficient was calculated to find out the correlation between different parameters in our study.

Results

Out of the hundred patients of our study group; majority were suffering from primary infertility (78%) and only eISSN: 2349-6983; pISSN: 2394-6466

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Association of Thyroid and Pituitary in Female Infertility

22% had secondary infertility. Their age wise distribution is given in Table 1.

to be significantly lower in infertile females as compared to the healthy controls.

Mean age of the female patients having primary infertility was significantly lower (23.6±4.78) as compared to the mean age of the female patients with secondary infertility (29.9±5.65)(p value<0.05).

So, it is evident that there is derangement of the normal hormonal milieu in infertile females.We further divided the patients of our study group (cases) into three sub-groups based upon their TSH values as: euthyroid, hypothyroid and hyperthyroid and compared the levels of different hormones in these sub-groups in order to elucidate the impact of thyroid dysfunction on the pituitary hormone status (Table 3).

On evaluation of the clinical history, menstrual irregularities were much more frequently present in infertile patients as compared to controls. Figure 1 shows the distribution of the menstrual cycle patterns in the infertile patients as well as the control group. It was found that oligomenorrhoea was predominant in infertile females of our study group (48%) followed by amenorrhoea (19%). However menstrual disturbances were also seen in healthy controls in the form of oligomenorrhoea, amenorrhoea or menorrhagia but only in 12% of them and majority had normal menstrual cycles. Measurement of the hormones has always been considered an important component of the infertility work-up in females; thus estimation of T3, T4, TSH, LH, FSH and Prolactin was done in both the groups (cases as well as controls) in our study. On comparing the levels of pituitary and thyroid hormones in the cases and control groups; the levels of Prolactin and TSH were significantly higher in patients of infertility as compared to controls (Table 2). LH and FSH were found

Out of all the parameters studied, hyperprolactinemia was a consistent finding in all the thyroid disorders. Also LH and FSH levels were lower in infertile females; much more significantly in hypothyroidism than any other thyroid illness. There was a highly significant positive correlation between prolactin and TSH levels in the infertile patients group (r=0.658; p value<0.001) as depicted in Figure 2. However a negative correlation was found between TSH and LH as well as TSH and FSH but was not significant. Overall hyperprolactinemia was seen in 56% of the patients of our study group and as far as thyroid dysfunctions was concerned; majority of the patients presented were euthyroid (Figure 3) Further, there was no significant difference between the distribution of the hormonal abnormalities between the patients of primary and secondary infertility in our study.

Table 1: Age-wise distribution of the study group patients and controls. Age groups (years)

Primary infertility (n=78)

Secondary infertility (n=22)

Controls

20-25

26-30

31-35

>36

Table 2: Comparison of the study parameters between the infertile females and healthy controls. Normal serum levels

Cases (infertile females) (n=100)

Controls (n=100)

p value

TSH (mIU/ml)

0.39 – 6.16

6.98±3.81

1.23±2.64

<0.05

T3 (ng/ml)

0.49 – 2.02

1.9±0.96

1.6±0.05

NS*

T4 (μg/ml)

4.4 – 11.6

6.6±2.51

7.8±2.96

NS*

LH (mIU/ml)

1.5 – 8.0

2.4±1.19

7.1±3.76

<0.01

FSH (mIU/ml)

2.0 – 10.0

3.1±2.37

8.6±3.01

<0.01

Prolactin (ng/ml)

2.0 – 20.0

34.8±8.43

11.4±5.11

<0.001

Study parameters

(NS* = non-significant p value)

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Table 3: Comparison of the study parameters in different disorders of thyroid in the infertile group with the healthy controls Study parameters

Controls (n=100)

TSH (mIU/ml)

Infertile females (n=100) (Cases) Euthyroid (n=79)

Hypothyroid (n=14)

Hyperthyroid (n=7)

1.23±2.64

4.96±3.04*

11.48±7.03***

0.12±0.02*

T3 (ng/ml)

1.6±0.05

1.8±1.26

0.3±0.61*

4.6±1.01**

T4 (μg/ml)

7.8±2.96

6.4±1.48

3.8±3.11

18.8±2.96***

LH (mIU/ml)

7.1±3.76

5.6±3.11

2.6±3.76**

3.1±1.72**

FSH (mIU/ml)

8.6±3.01

5.8±2.42

3.1±2.86

3.4±1.16**

Prolactin (ng/ml)

11.4±5.11

26.5±4.76***

40.4±4.71***

29.4±3.82***

p values on comparing with healthy controls: * p value <0.05 ** p value <0.01 *** p value <0.001

Fig. 1: Distribution of the menstrual irregularities in the infertile patient group and controls.

Fig. 2: Correlation between serum prolactin and TSH levels in infertile females.

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Fig. 3: Distribution of different hormonal profiles in the infertile patients.

Discussion

Infertility, recently has picked up a rising trend and has got not only medical but also psychological as well as economic implications in the human society.[13] Hormonal evaluation has been an important aspect of the infertility diagnostic work-up and is also an area of research which forms a basis for the designing of the effective treatment protocols. In our study we evaluated the serum levels of thyroid and pituitary hormones in infertile female patients in order to find out a possible relationship between thyroid disorders and LH, FSH and prolactin.Majority of our patients came out to be euthyroid and hyperprolactinemia emerged as the most frequent hormonal abnormality in the infertile patients of our study. Prolactin is secreted from anterior pituitary and is primarily associated with breast development during pregnancy and induces lactation. However, prolactin also binds to specific receptors in the gonads, lymphoid cells and liver.Hyperprolactinemia may occur primarily as a result of normal physiological changes during pregnancy, breastfeeding, mental stress, hypothyroidism or sleep. Pathologically it may be due to diseases affecting the hypothalamus and pituitary gland or secondary to disease of other organs such as liver, kidneys, ovaries and thyroid. [14] Hyperprolactinemia causes infertility by increasing the release of dopamine from hypothalamus which inhibits gonadotrophin releasing hormones and thus gonadal steroidogenesis and eventually infertility. In our study we found a significant positive correlation between the TSH and prolactin levels in the infertile females.

Thus hypothyroidism can be said to be associated with hyperprolactinemia. It is well documented that in primary hypothyroidism, the serum thyroxine levels are low and there is decreased negative feedback on the hypothalamopituitary axis. The resulting increased secretion of thyrotropin releasing hormone (TRH) stimulates the thyrotrophs and lactotrophs, thereby increasing the levels of both TSH and prolactin and thus ovulatory dysfunction due to hyperprolactinemia.[15] Thyroid hormones have profound effects on reproduction and pregnancy.[16,17]Although majority of the infertile patients in our study group were euthyroidfollowed by hypothyroid, but TSH was frequently present in the upper normal limit in majority of the euthyroid patients when compared with the healthy controls. These might have compensated thyroid functions or raised antithyroperoxidase antibody titres and must be considered for a thorough investigation. Moreover, despite of the normal TSH and thyroxine levels, some patients might exhibited the clinical picture of hypothyroidism. It has also been documented that even in the absence of hyperprolactinemia, hypothyroidism itself may contribute to infertility since thyroid hormones may be necessary for the maximum production of both estradioland progesterone.[8] The final common pathway for TSH and prolactin secretion is thyrotropin releasing hormone (TRH), which stimulates the secretion of both TSH and prolactin.[18]TRH is under negative feedback control of TSH through a short negative feedback loop and any increase in TSH will decrease the release of TRH which in turn will inhibit the secretion of prolactin and will also normalise the TSH levels. The short

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Yadav et al. feedback loop is exemplified by the pituitary hormone that feeds back negatively on the hypothalamus operating through the cognate receptor.[19]This explains the normal TSH levels in majority of the cases. From our study we have elucidated that most frequent hormonal abnormality in female infertility has been hyperprolactinemia, also seen associated with hypothyroidism. There is a complex interplay between HPT and HPO axis in female reproductive physiology and any derangement in it, leads to anovulatory cycles, luteal phase defects, high prolactin levels, estrogen and progesterone hormonal imbalances.[14] Prolactin inhibits the hormones necessary for ovulation i.e. FSH and GnRH. Thus, in the setting of hyperprolactinemia; ovulation is inhibited and contributes to infertility. Moreover when GnRH secretions become low, LH and FSH secretions also fall. So, gamete production is not stimulated and gonadal steroidogenesis is hampered.[20-22] In our study also we found that the levels of LH and FSH were low in the infertile patients group; FSH more than LH and that too in hypothyroid patients. Some of the studies earlier also demonstrated that hyperprolactinemia was associated with a marked reduction in both the frequency and the amplitude of the LH pulses, which indirectly suggested that both the brain and the pituitary gland might be targets for prolactin. The increase which was observed in prolactin may be the cause of the low estrogen and progesterone concentrations in the infertile subjects, which showed that higher serum prolactin concentrations resulted in decreased serum LH and FSH levels in infertile women. Further it was found that there was lower concentration of serum LH and FSH and higher concentrations of prolactin in primary infertile women than in the control group.[23,24] Researchers have also found that decreased levels of LH in the midcycle clearly indicate the possibility of anovulation, which could result in infertility. Elevated levels of prolactin in such infertile patients show that there is a mechanism which operates at the anterior pituitary level and leads to abnormal distribution of LH and FSH, thus further explains the abnormal or delayed ovum maturation.[25]These also corroborate with the findings of our study. The most frequent menstrual abnormality in our study among the infertile females has been oligomenorrhea. The reason for this might be attributed to the derangement in the normal hormonal milieu which has been seen in the form of hyperprolactinemia may be resulting from longstanding primary hypothyroidism leading on to ovulatory dysfunctions ranging from inadequate corpus www.pacificejournals.com/apalm

A-345 luteal progesterone secretion when mildly elevated to oligomenorrhea or amenorrhea when circulating prolactin levels are high.[26] In addition to this, amenorrhea occurs in hypothyroidism due to hyperprolactinemia resulting from a defect in the positive feedback of estrogen on LH, and because of LH and FSH suppression. Thus thyroid or pituitary dysfunction can affect fertility in various ways resulting in menstrual abnormalities, anovulation, luteal phase defects to post conception sustenance of the pregnancy.

Conclusion

From the present study it is concluded that there occurs a derangement in hypothalamic-pituitary-ovarian and hypothalamic-pituitary-thyroid axis in female infertility especially in primary infertility. Hyperprolactinemia is seen more frequently as compared to other pituitary and thyroid dysfunctions. Moreover, there is a positive correlation between the levels of TSH and prolactin; TSH and LH in female patients of infertility. Thus, it becomes not only inevitable but also fundamental to evaluate the hormonal status of the females with infertility as it not only gives an insight towards the understanding of its etiopathogenesisbutalso helps in the designing of the effective treatment protocols.

Funding None

Competing interests None Declared

References

1. Cooper TG, Noonan E, VonEckardstein S, Auger J, Baker HW, Haugen TB. World Health Organisation Reference Values for Human Semen Characteristics. Human Reprod Update 2010; 16(3): 231-45. 2. EniolaOW, Adetola AA,Abayomi BT. A Review of Female Infertility; Important Etiological Factors and Management. J Microbiol Biotech Res 2012; 2(3): 379-385. 3. Mascarenhas MN, Cheung H, Mathers CD, Stevens GA. Measuring Infertility in Populations: Constructing a Standard Definition for Use With Demographic and Reproductive Health Surveys. Popul Health Metr 2012; 10(1): 17. 4. Romero RR, Romero GG, Abortes MI, Medina SHG. Risk Factors Associated to Female Infertility. Gynecol Obstet Mex 2008; 76(12): 717-21. 5. Kumar D. Prevalence of Female Infertility and Its Socio-Economic Factors in Tribal Communities of Central India. Rural Remote Health 2007; 7(2): 456. eISSN: 2349-6983; pISSN: 2394-6466

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Association of Thyroid and Pituitary in Female Infertility

6. Fischer DA, Nelson JC, Carlton EI, et al. Maturation of Human Hypothalamic-Pituitary-Thyroid Function and Control. Thyroid 2000; 10:229-234. 7. Donfas AG, Mastorakos G. The HypothalamicPituitary-Thyroid Axis and the Female Reproductive System. Ann N Y AcadSci 2000; 900: 65-76. 8. Wakim AN, Polizotto SL, Burholt DR. Influence of Thyroxine on Human Granulosa Cell Steroidogenesis in vitro. J Assist Reprod Genet 1995; 12(4): 274-7. 9. Veeresh T, Moulali D, Sarma DVH. A Study on Serum FSH, LH and Prolactin Levels in Women with Thyroid Disorders. International Journal of Scientific and Research Publications 2015; 5(3): 250-4. 10. Poppe K, Velkeniers B, Glinoer D. Thyroid Disease and Female Reproduction. ClinEndocrinol 2007; 66(3): 309-21. 11. Zollner U, Lanig K, Steck T, Dietl J. Assessment of Endocrine Status in Patients Undergoing InVitro Fertilization Treatment. Is It Necessary? Arch GynecolObstet 2001; 265(1): 16-20. 12. Mishra R, Baveja R, Gupta V. Prolactin Levels in Infertility with Menstrual Irregularities. J Obstet. Gyynecol India 2002; 52: 40-3. 13. Frey KA, Patel KS. Initial Evaluation and Management of Infertility by The Primary Care Physicians. Mayo Clin Proc 2004; 79: 1439-43. 14. Mancini T, Casanueva FF,Giustina A. Endocrinology and Metabolism Clinics of North America 2008; 37(1): 67-99. 15. Shoupe D, Mishell DR. Hypoprolactinemia, Diagnosis and Treatment. In: Mishellâ&#x20AC;&#x2122;s textbook of Infertility, Contraception and Reproductive Endocrinology. 4th edition. Massachusets: Blackwell Science; 1997. pp.323-41.

16. Elahi S, Tasneem A, NazirI, Nagra SA, Hyder SW. Thyroid Dysfunction in Infertile Women. J Coll Physicians Surg Pak 2007; 17(4): 191-4. 17. Micinsk P, Weilgus E, Wojcieszyn M, Pawlicki K. Abnormal Ovarian Reserve Test Reflects Thyroid Dysfunction. Pol J Gyn Invest 2006; 9(1): 30-4. 18. Freeman ME, Kanyiscka B, Lerant A, Nagy G. Prolactin: Structure, Function and Regulation of Secretion. Physiol Rev 2000; 80: 1523-1631. 19. Litwack G, Schmidt TJ. Biochemistry of Hormones 1: Polypeptide Hormones. In Devlin TM, editor. Textbook of Biochemistry with Chemical Correlation. 5th edition. New York: Wiley-Liss; 2002 .pp. 906-57. 20. Koutras DA. Disturbances of Menstruation in Thyroid Diseases. Ann N Y AcadSci 1997; 816: 280-4. 21. Cramer DW, Sluss PM, Powers RD, et al. Serum Prolactin and TSH in an In Vitro Fertilization Population: Is There a Link Between Fertilization and Thyroid Function? J Assist Reprod 2003; 20: 210. 22. Poppe K, Velkeniers B. Thyroid and Infertility. Verh K AcadGeneeskdBelg 2002; 64(6):389-99. 23. Bohnet HG, Dahlen HG, Wuttke W, Schneider HP. Hyperprolactinemic Anovulatory Syndrome. J ClinEndocrinolMetab 1976; 42: 132-43. 24. Matsuzaki T, Azuma K, Irahara M, Yasui T, Aono T. Mechanism of Anovulation in Hyperprolactinemic Amenorrhoea Determined by Pulsatile Gonadotropin Releasing Hormone Injection Combined with Human Chorionic Gonadotropin. FertilSteril 1994; 62: 1143-49. 25. Mohan K, Sultana M. Follicle Stimulating Hormone, Leutinizing Hormone and Prolactin Levels in Infertile Women in North Chennai. J BiolSci Res 2010; 1(4): 279-84. 26. Krassas GE. Thyroid Disease and Female Reproduction. FertilSteril 2007; 74(6): 1063-70.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Original Article Thyroid Cytology Evaluation Based on the Bethesda System with Clinico-Morphological Correlation Dimple Mehrotra*, Anita A.M., Sainath K. Andola and Anuradha G. Patil Department of Pathology, M.R. Medical College, Kalaburagi, Karnataka, India Keywords: Thyroid, Cytology, TBSRTC, Histopathology, Accuracy

ABSTRACT Background: FNAC of Thyroid gland is a widely accepted and accurate method for evaluation of thyroid nodules. In the past, terminology of reporting of Thyroid FNACs varied markedly, which made it difficult for the clinicians to interpret the reports and decide the management protocol. To cater to this issue, The Bethesda System of Reporting Thyroid Cytology was proposed with six diagnostic categories and appropriate management protocol for each category. This study was undertaken to categorize FNAC of Thyroid lesions according to the Bethesda System of Reporting Thyroid Cytology (TBSRTC) and to correlate with Histopathology wherever feasible. Methods: The present study includes 175 Thyroid FNAC cases studied over a 3-year period (August 2012- July 2015). These cases were categorized according to TBSRTC and the cytological diagnosis was correlated with histopathology wherever it was available. The Sensitivity, Specificity, Positive Predictive value (PPV), Negative Predictive value (NPV) and Accuracy was also calculated. Result: A total of 175 Thyroid FNAC cases were collected over 3 years. The mean age was 36.18 years and the male to female ratio was 1:9.3. Percentage of cases in Category I to VI according to the Bethesda system of reporting Thyroid cytology were 4.57%, 68.58%, 5.72%, 17.14%, 1.14% and 2.85% respectively. Histopathological details were available in 19.42% of the cases. The sensitivity, specificity, PPV, NPV and accuracy were 69.23%, 89.47%, 81.81%, 80.95% and 81.25% respectively. Conclusion: The findings of the present study were consistent with other studies that used TBSRTC.

*Corresponding author: Dr. Dimple Mehrotra, A-32, Nanda Marg, Adarsh Nagar, Delhi 110033, INDIA Phone: +91 -9900415163, 9810139357 Email: drdimplemehrotra@gmail.com

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

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Thyroid Cytology and Bethesda System

Introduction

Thyroid cytology has been regarded as the first line diagnostic procedure for assessment of thyroid lesions. Cytological reporting formats used in the past ranged from histology-equivalent categories to categories like equivocal, inconclusive, indeterminate, atypical, suspicious, uncertain, malignancy suspicious, possibly neoplastic, possibly malignant and probably malignant to report thyroid aspirates that fell between benign and malignant diagnostic categories. These made it difficult for clinicians to interpret the reports. [1] To address terminology related issues, the National Cancer Institute (NCI) hosted the NCI Thyroid FNA State of the Science Conference and proposed the Bethesda System for Reporting Thyroid Cytopathology. This was a standardized system with six general diagnostic categories and clear categorical nomenclature including malignancy risks. [2] This study was undertaken to classify thyroid lesions based on the Bethesda system of reporting Thyroid Cytology, which would help in planning prognostic and therapeutic approaches. The cytological diagnosis was also correlated with histopathology wherever available.

Materials And Methods

The present study was conducted in the Department of Pathology, Basaveshwar Teaching and General Hospital attached to M.R. Medical College, Kalaburagi, Karnataka, after taking approval from the Institutional Ethics Committee. The study includes 1 year retrospective (August 2012 to July 2013) and 2 years prospective (August 2013 to July 2015). All the patients with clinically diagnosed Thyroid lesions referred for Thyroid FNAC were included in the study. Neck swellings other than thyroid swellings and patients having known Bleeding diathesis were excluded. Both non aspiration and aspiration techniques were followed depending on the nature of the thyroid swelling. In special cases such as when the lesions were difficult to localize, Ultrasonography-guided FNAC was done after taking informed consent from the patient for FNAC. Slides were prepared by smearing the aspirate and stained with May Grunwald Giemsa (MGG) and Haematoxylin and Eosin (H&E). In doubtful cases, such as those where malignancy was suspected clinically and studying the nuclear details was important, wet smears were prepared and stained with Papanicolaou stain (PAP). Slides for wet smears were fixed in 95% ethyl alcohol, while the others were air dried. Stained smears were studied under light microscopy. The cases in which surgical intervention was decided upon by the surgeon based on the thyroid cytology report

were closely followed. The post-operative specimen was received 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. Cytological diagnosis was correlated with the histopathology and the efficacy of FNAC was estimated by using the methodology of Galen and Gambino [3] as follows:Sensitivity =

TP X 100 Tp + FN

Specificity =

TN X 100 TN + FP

Positive Predictive Value (PPV) =

TP X 100 TP + FP

Negative Predictive Value (NPV) = Accuracy/ Efficacy =

TN X 100 TN + FN

TP + TN X 100 TP + FP + TN + FN

[TP = True Positive, FP = False Positive, TN = True Negative, FN = False Negative] The statistical values are interdependent statistical concepts indicating the accuracy of results.

Results

In the present study, 175 cases of thyroid FNAC were received. These were categorized according to the Bethesda system of reporting thyroid cytology into 6 categories. Majority of the cases were received in the period of August 2014 to July 2015 (38.28%). Age of the patients ranged from 5 to 85yrs with the mean age of 36.29years. Maximum number of cases was seen in the age group of 21-30years (29.14%). There was a female preponderance with male to female ratio being 1: 9.3. The most common complaint was painless swelling in front of neck which was seen in all the patients who presented with thyroid swellings and the other presenting complaints are shown in Table1. In majority of the cases the duration of the thyroid swelling was in the range of 0-3 months (38.8%). The right lobe of thyroid was involved in majority of the cases (34.8%). Size of the swelling: ranged from 2x1cms to 12x7cms. The nature of the swelling was firm in most of the cases (44%). In majority of the cases direct unguided FNAC was performed (95%). The ratio between Direct: USG guided

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Mehrotra et al. was 19:1. In majority of the cases the aspiration technique of FNAC was employed (71%). The ratio between Aspiration: Non Aspiration was 2.4: 1. Amount of aspirate ranged from 0.5ml to 10 ml. In majority of the cases the aspirate was hemorrhagic (62%). The ratio between hemorrhagic: dark brown was 1.6: 1.These 175 cases were reported according to The Bethesda System of Reporting Thyroid Cytology. Category I â&#x20AC;&#x201C;Non Diagnostic or Unsatisfactory included 8/175 cases (4.57%). Out of which in 5 cases only cyst fluid was aspirated and in 3 the aspirate was hemorrhagic and acellular. Category II â&#x20AC;&#x201C; Benign included 120/175 cases (68.57%). Majority of the cases in Category II were Benign Follicular Nodule (Colloid nodule/ Adenomatoid nodule) accounting for 91/120 cases (75.8%). This was followed by Hashimoto/ Lymphocytic thyroiditis with 26/120 (21.6%) and finally Granulomatous thyroiditis with 3/120 (2.6%). Histopathological details were available in 21/91 cases of Benign Follicular Nodule, out of which positive correlation was found in 18 cases. Category III- Atypia of Undetermined Significance (AUS) Or Follicular Lesion of Undetermined Significance (FLUS) included 10/175 cases (5.7%). Category IV- Follicular Neoplasm (FN) or Suspicious for a Follicular Neoplasm (SFN) included 30/175 cases (17.14%). In this category, 2 cases were reported as Hurthle cell neoplasm. Histopathological details were available in 11/30 cases, out of which positive correlation was found in 9 cases. Category V- Suspicious For Malignancy (SFM) included 2/175 cases (1.14%). Resected Surgical specimen was received in 1 case in which the histopathological findings favoured a diagnosis of Follicular Adenoma. Category VI- Malignant included 5/175 cases (2.86 %). Out of the 5 cases, 2 cases were diagnosed as Papillary carcinoma and the remaining 3 cases as Anaplastic carcinoma. Resected Surgical specimen was received in 1 case the cytological and histopathological findings of which correlated with a diagnosis of Papillary carcinoma. The final categorization of these 175 cases according to TBSRTC and the cytohistopathological correlation in 34 cases which underwent surgery is shown in Table 2.

A-349 Statistical Analysis of the above data reveals that the number of True Positive (TP), True Negative (TN), False Positive (FP) and False Negative (FN) was 11 (32%), 18 (53%), 2 (6%) and 3 (9%) respectively. On applying Chi-square Test, The two-sided P value was 0.0002, which is considered extremely significant. The row/column association was statistically significant. Chi-square statistic (with Yates correction) was 13.622 and Degree of freedom was 1. The Accuracy of Thyroid FNAC in correctly diagnosing malignancy was 85.29%. Hence, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of Thyroid FNAC in detecting malignancy were 78.57%, 90%, 84.62%, 85.71% and 85.29% respectively.

Discussion

FNAC has an extremely important role in the evaluation of thyroid nodules or swellings. The present study was undertaken to categorize Thyroid FNAC cases according to the Bethesda system of reporting Thyroid cytology so as to improve communication between the pathologist and surgeon in deciding the treatment modality for the patients and avoid unnecessary surgeries. The FNAC diagnosis of these cases was also correlated with Thyroid Function Tests and Histopathology wherever feasible. Thereby, providing data for the statistical analysis in these cases i.e., sensitivity, specificity, positive predictive value, negative predictive value and accuracy of Thyroid FNAC in detecting malignancy. The mean age in the present study was 36.29 years, which correlates well with the study conducted by Naz et al [4] where the mean age at presentation was 39.7 years as shown in Table 3. The Male: Female ratio in the present study was 1: 9.3, which correlates well with the study conducted by Richmond et al [5] who reported a Male: Female ratio of 1:6.1 as shown in Table 4. In the present study, all of the cases presented with a swelling in front of the neck followed by dysphagia, pain and tenderness over the swelling, whereas, in the study conducted by Richmond et al [5] the majority of cases presented with dysphagia followed by enlarging nodule and hoarseness. In the present study, majority of the cases were in Category II followed by Category IV which correlates well with the studies conducted by Yang et al [6], Yassa et al [7] and Joshi et al [8] as shown in Table 5.

Cytological features of each of these Bethesda categories are shown in Fig.1. The gross and microscopic features of some of the cases are shown in Fig.2.

The accuracy in the present study was 85.29% , which is comparable to the accuracy reported by Ko et al [9] and Kessler et al [10] as, 84.4% and 87% respectively as shown in Table 6.

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Table 1: Clinical Features Complaints Swelling infront of the Neck Dyspnoea Change of Voice Dysphagia / Difficulty in swallowing Weight Gain Weight Loss Palpitation and Anxiety Pain and Tenderness on palpation Weakness Tremors Fever Referred pain- Ear, Shoulder Exophthalmos Polymenorrhoea Cold intolerance Sweating Numbness in hands and feet

Number Of Cases 175 2 3 13 2 2 3 9 2 3 5 3 1 1 5 2 1

Table 2: TBSRTC Category wise distribution of cases and their Cytohistopathological correlation. Bethesda No. of cases in the No. of cases in which HP FNAC Histopathological diagnosis Category category available diagnosis I 8 0 13 CG II 120 21 BFN 5 MNG 3 FA III 10 0 9 FA 1 HT IV 30 11 FN 1 MNG V 2 1 SFM FA VI 5 1 PC PC Table 3: Comparison of the Mean age of presentation. Study

Year

Mean Age (In Years)

Singh et al [11]

2011

57.5

Muratli et al

2014

51.24

2014

39.7

2014

2015

36.29

[12]

Naz et al [4] Richmond et al

[5]

Present Study Table 4: Comparison of Male: Female ratio. Study

Year

Male:Female

Singh et al [11]

2011

1: 4.7

Muratli et al

2014

1: 4.8

Naz et al

[12]

2014

1: 3.6

Richmond et al [5]

2014

1: 6.1

Present study

2015

1: 9.3

[4]

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Fig. 1-A) Category I- Cyst macrophages only- [H&E; 400X], B) Category II- Cyst macrophages and hemosiderin laden macrophages in a colloid background-[MGG; 400X], C) Mirror cracking appearance of dense colloid- [MGG; 400X], D) Fire Flare appearance-[MGG; 400X] , E) Hashimoto/Lymphocytic thyroiditis- Lymphocytes impingeing onto follicular cells -[MGG; 100 X]. Inset-Askanazy cells -[MGG; 400X], F) Granulomatous Thyroiditis- Epithelioid cells- [MGG; 1000X], InsetGranuloma, G) Category III-Some follicular cells show nuclear enlargement, often with prominent nucleoli [MGG; 400X], H) to J)=Category IV-H)-Microfollicles with follicular cells showing small uniform nuclei- [MGG; 100X], I)Same case of Category IV showing Follicular cells with hyperchromasia and some enlarged nuclei-[MGG; 400X], J) Hurthle cells showing abundant eosinophilic granular cytoplasm-[MGG; 400X], K) Category V- Follicular cells showing nuclear enlargement, macronucleoli, anisonucleosis and nuclear pallor- Suspicious for Papillary carcinoma [MGG; 400X], L) to P)-Category VI- Papillary carcinoma- L)FNAC-Intranuclear cytoplasmic pseudoinclusions, [MGG;1000X], M)Cells arranged in papillary fronds [MGG; 400X], N) Arrow- Multinucleated giant cell [MGG; 400X], O) Arrow- Nuclear Clearing[MGG; 1000X], P) Chewing â&#x20AC;&#x201C;gum colloid [H&E;1000X], Q) to T) Category VI-Anaplastic carcinoma-Q)- Follicular cells showing anisonucleosis, pleomorphism and multinucleation [MGG; 400X], R) Nuclear enlargement, prominent nucleoli and spindle cells [MGG; 1000X], S) Intranuclear cytoplasmic pseudoinclusions [MGG; 1000X], T) Prominent nucleoli and spindle cells.

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Thyroid Cytology and Bethesda System

Fig.2- A) & B)-Colloid goitre-A) Hemithyroidectomy specimen- cut section reveals multiple honey colored colloid filled nodules B) Same case showing variable sized thyroid follicles showing marked hyperplasia of lining epithelium-[H&E; 400X], C)&D)- Follicular adenoma-C) Lobectomy specimen- Cut section shows a well circumscribed, capsulated, solitary nodule, D) Same case showing a well defined capsule [H&E; 100X]. InsetMicrofollicular(fetal) pattern [H&E; 400X], E)&F)- Papillary carcinoma- E) Branching papillae with central fibrovascular core and stratified lining of cuboidal cells, F) Optically clear Orphan Annie eye nuclei.

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Table 5: Comparison of the distribution of cases according to TBSRTC . S.No. STUDY

YEAR

CAT I

CAT II

CAT III

CAT IV

CAT V

CAT VI

2007

10.4

64.6

3.2

11.6

2.6

7.6

Yang et al

Yassa et al

2007

Theoharis et al [13]

2009

11.1

73.8

5.5

1.3

5.2

Nayar et al [14]

2009

5.3

64.2

17.8

5.9

1.9

4.9

Marchevsky et al [15]

2010

12.9

71.6

9.8

1.5

2.3

Jo et al [16]

2010

18.6

3.4

9.7

2.3

Renshaw et al

2010

23.6

7.7

8.6

1.8

4.2

VanderLaan et al

2011

12.5

62.7

10.9

4.2

4.5

5.2

Kim et al

[6] [7]

[17] [18]

2011

1.8

58.3

16.3

1.2

6.2

16.2

Krane et al

[20]

2011

13.9

66.9

3.2

3.9

Singh et al

[11]

2011

13.2

41.3

3.7

5.6

3.9

4.5

Mondal et al

2013

1.2

87.5

4.2

1.4

4.7

Muratli et al

2014

10.8

59.5

8.7

0.6

2.8

17.6

Naz et al

2014

4.7

76.3

12.7

2.1

3.4

0.8

Mehra et al

2015

7.2

4.9

2.2

3.6

2.2

Mamtha et al

2015

10.84

12.5

3.34

4.16

9.16

Joshi et al

2015

65.5

7.3

14.5

4.5

8.2

Melo- Uribe et al

2015

4.08

23.47

2.04

16.84

37.24

16.33

Present Study

2015

4.57

68.58

5.72

17.14

1.14

2.85

[19]

[21]

[12]

[4] [22] [23]

[8] [24]

Table 6: Comparison of the statistical analysis. Study

Year

No.of cases

Sensitivity

Specificity

PPV

NPV

Accuracy

Al Sayer et al [25]

1985

Cusiok et al

[26]

1990

283

Bouvet et al

[27]

1992

93.5

85.3

88.2

79.6

Afroze et al [28]

2002

170

61.9

99.31

92.86

94.74

94.58

Ko et al [9]

2003

207

78.4

98.2

66.3

84.4

Al Hureibi et al [29]

2003

196

89.9

66.7

73.2

Kessler et al

2005

170

98.5

98.7

76.6

Mahar et al

[10]

2006

125

Haberal et al [31]

2009

260

92.6

91.6

83.5

96.5

91.9

Muratli et al [12]

2014

126

87.1

64.6

76.1

79.5

77.3

Present study

2015

175

78.57

90.00

84.62

85.71

85.29

[30]

Conclusion

This study was undertaken to classify the thyroid lesions based on The Bethesda system of reporting Thyroid Cytology and provide data that would help in planning the therapeutic approach in patients with thyroid swellings. The cytomorphological analysis was done to assess the diagnostic accuracy of cytology in diagnosis of thyroid lesions. www.pacificejournals.com/apalm

Acknowledgements None

Funding None

Competing Interests None Declared

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References

Thyroid Cytology and Bethesda System

1. Cibas ES, Ali SZ. The Bethesda System for Reporting Thyroid Cytopathology. Am J Clin Pathol 2009; 132: 658-665. 2. Wang HH. Reporting thyroid fine-needle aspiration: Literature review and a proposal. Diagnostic Cytopathology 2006; 34(1): 67–76. 3. Parikh R, Mathai A, Parikh S, Chandra SG, Thomas R. Understanding and using sensitivity, specificity and predictive values. Indian Journal of Ophthalmology. 2008; 56(1):45-50. 4. Naz S, Hashmi AA, Khurshid A, Faridi N, Edhi MM, Kamal A, Khan M. Diagnostic accuracy of Bethesda system for reporting thyroid cytopathology: an institutional Perspective. International Archives of Medicine 2014; 7: 46-50. 5. Richmond BK, Judhan R, Chong B, Uber At, Aburahma Z, Mangano W, Stephanie Thompson. False-negative Results with the Bethesda System of Reporting Thyroid Cytopathology: Predictors of Malignancy in Thyroid Nodules Classified as Benign by Cytopathologic Evaluation. Am Surg. 2014; 80(8): 811–816. 6. Yang J, Schnadig V, Logrono R, et al. Fine-needle aspiration of thyroid nodules: a study of 4703 patients with histologic and clinical correlations. Cancer. 2007; 5: 306-315. 7. Yassa L, Cibas ES, Benson CB, et al. Long-term assessment of a multidisciplinary approach to thyroid nodule diagnostic evaluation. Cancer. 2007; 6: 508-516. 8. Joshi D, Jesalpura NS. Comparison between Bethesda System and Conventional System in Thyroid Cytopathology. IJSR. 2015; 4(9): 78-80. 9. Ko HM, Jhu IK, Yang SH, Lee JH, Nam JH, Juhng SW, et al. Clinicopathologic analysis of fine needle aspiration cytology of the thyroid. A review of 1,613 cases and correlation with histopathologic diagnoses. Acta Cytol. 2003; 47: 727–732. 10. Kessler A, Gavriel H, Zahav S, Vaiman M, Shlamkovitch N, Segal S, et al. Accuracy and consistency of fine-needle aspiration biopsy in the diagnosis and management of solitary thyroid nodules. Isr Med Assoc J. 2005; 7: 371–373. 11. Singh RS, Wang HH. Eliminating the “atypia of undetermined significance/follicular lesion of undetermined significance” category from the bethesda system for reporting thyroid cytopathology. Am J Clin Pathol. 2011; 136:896–902.

12. Muratli A, Erdogan N, Sevim S, Unal I, Akyuz S. Diagnostic efficacy and importance of fine needle aspiration cytology of thyroid nodules. Journal of Cytology. 2014; 31:73-77. 13. Theoharis CG, Schofield KM, Hammers L, et al. The Bethesda thyroid fine-needle aspiration classification system:year 1 at an academic institution. Thyroid. 2009; 19: 1215-1223. 14. Nayar R, Ivanovic M. The indeterminate thyroid fine needle aspiration: experience from an academic center using terminology similar to that proposed in the 2007 National Cancer Institute Thyroid Fine Needle Aspiration State of the Science Conference. Cancer Cytopathol. 2009; 11(7):195 -202. 15. Marchevsky AM, Walts AE, Bose S, et al. Evidencebased evaluation of the risks of malignancy predicted by thyroid fine needle aspiration biopsies. Diagn Cytopathol. 2010;38:252–259 16. Jo VY, Stelow EB, Dustin SM, Hanley KZ. Malignancy Risk for Fine Needle Aspiration of Thyroid Lesions According to The Bethesda System for Reporting Thyroid Cytopathology. Am J Clin Pathol. 2010; 134: 450- 456. 17. Renshaw AA. Should “atypical follicular cells” in thyroid fine-needle aspirates be subclassified? Cancer Cytopathol.2010; 118: 186-189. 18. VanderLaan PA, Marqusee E, Krane JF,. Clinical Outcome for Atypia of Undetermined Significance in Thyroid Fine-Needle Aspirations .Should Repeated FNA Be the Preferred Initial Approach?. Am J Clin Pathol 2011;135:770-775 19. Kim SK, Hwang TS, Yoo YB, et al. Surgical results of thyroid nodules according to a management guideline based on the BRAFV600E mutation status. J Clin Endocrinol Metab.2011; 96:658-664. 20. Krane JF, VanderLaan PA, Faquin WC, et al. The atypia of undetermined significance/follicular lesion of undetermined significance: malignant ratio: a proposed performance measure for reporting in The Bethesda System for Thyroid Cytopathology. Cancer Cytopathol. 2012; 120(2):111-116. 21. Mondal SK, Sinha S, Basak B, Roy DN, Sinha SK. The Bethesda system for reporting thyroid fine needle aspirates: A cytologic study with histologic follow up. Journal of Cytology.2013; 30(2): 94-99. 22. Mehra P, Verma AK. Thyroid Cytopathology Reporting by the Bethesda System:A Two-Year Prospective Study in an Academic Institution. Pathology Research International.2015, 1-11.

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23. Mamatha M, Sekhar SC, et al.. A comparative study between conventional system and the Bethesda system applied for reporting thyroid cytopathology. International Archives of Integrated Medicine. 2015; 2(3): 87-95. 24. Melo-Uribe MA, Sanabria A, Romero-Rojas A, et al. The Bethesda system for reporting thyroid cytopathology in Colombia: Correlation with histopathological diagnoses in oncology and nononcology institutions. Journal of Cytology. 2015; 32(1):12-16. 25. Al-Sayer HM, Krukowski ZH, Williams VM, Matheson NA. Fine needle aspiration cytology in isolated thyroid swellings: A prospective two year evaluation. Br Med J (Clin Res Ed) 1985; 290: 1490–1492. 26. Cusick EL, MacIntosh CA, Krukowski ZH, Williams VM, Ewen SW, Matheson NA. Management of isolated thyroid swellings: A prospective six year study of fine needle aspiration cytology in diagnosis. BMJ.1990; 301: 318–321.

27. Bouvet M, Feldman JI, Gill GN, Dillmann WH, Nahum AM, Russack V, et al. Surgical management of the thyroid nodule: Patient selection based on the results of fine-needle aspiration cytology. Laryngoscope.1992; 102:1353–1356. 28. Afroze N, Kayani N, Hasan SH. Role of fine needle aspiration cytology in the diagnosis of palpable thyroid lesions. Indian J Pathol Microbiol. 2002; 45: 241–246. 29. Al-Hureibi KA, Al-Hureibi AA, Abdulmughni YA, Aulaqi SM, Salman MS, Al-Zooba EM. The diagnostic value of fine needle aspiration cytology in thyroid swellings in a university hospital, Yemen. Saudi Med J. 2003; 24: 499–503. 30. Mahar SA, Husain A, Islam N. Fine needle aspiration cytology of thyroid nodule: Diagnostic accuracy and pitfalls. J Ayub Med Coll Abbottabad. 2006; 18: 26–29. 31. Haberal AN, Toru S, Ozen O, Arat Z, Bilezikçi B. Diagnostic pitfalls in the evaluation of fine needle aspiration cytology of the thyroid: Correlation with histopathology in 260 cases. Cytopathology.2009; 20: 103–108.

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Original Article Bacteriological Profile and Antimicrobial Sensitivity Pattern of Clinical Isolates from Patients Attending Tertiary Care Hospital Kotgire Santosh. A*, Sunil Hatkar. S, Sufia Siddique, A.B. Deshmukh, Uzma Afreen and Sayyed Mariya Department of Microbiology, Indian Institute of Medical Sciences, Jalna, Maharashtra, India Keywords: Bacterial Pathogens, Antimicrobial Susceptibility, Surveillance, Carbepenems.

ABSTRACT Background: Bacteriological infection plays vital role in determining the outcome as well as cost and duration of hospital stay for patients. Therefore a regular surveillance of important bacterial isolates and their susceptibility pattern is mandatory. So the present study was undertaken to find out bacterial pathogens causing infection in patients attending at our tertiary care hospital and to know drug sensitivity pattern of isolates. Methods: The study was carried out in the Department of Microbiology, Indian Institute of Medical Science and Research during the period from July 2015 to February 2016. A total 8189 clinical samples (urine, blood, sputum, pus etc.) were collected and processed for culture, identification as per standard recommended procedures and antibiotic susceptibility testing were carried out on isolates as per Clinical Laboratory Standard Institute (CLSI) guidelines. Result: 2976 different types of bacterial pathogens were isolated. The prevalence of gram negative bacilli were 70.83% and gram positive bacilli were 29.17%.The commonest pathogen isolated was Escherichia coli 33.09%, followed by Staphylococcus aureus 26.27%, Klebsiella spp 23.85% and nonfermenters 10.68 % (Pseudomonas aeruginosa and Acinetobacter Spp). Most of gram negative bacilli were resistant to commonly used drugs such as cotrimaxozole, ciprofloxacin and were sensitive to carbepenems. whereas Gram positive bacteria shown resistance to erythromycin, cotrimaxozole and to some extent cefoxitin. Conclusion: The present study reveals microbiological profile in patients attending our hospital. Regular surveillance help in implementing better therapeutic strategies to reduce morbidity and mortality associated in patients in health care facility.There is, in general resistance amongst gram negative bacilli to commonly used drugs and shown good sensitivity to carbepenems and aminoglycosides. Resistance among gram positive is not acute, although the Methiciilin resistance staphylococcus aureus (MRSA) incidence is increasing in our setup.

*Corresponding author: Dr. KotgireSantosh .A, Associate professor, Department of Microbiology, Indian Institute of Medical Sciences, Jalna, Maharashtra, India Phone: +91 9922867658 Email: santosh_kots2001@yahoo.com

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Introduction

Collection of Sample and Processing :The samples collected were mainly urine, blood, pus, sputum, CSF, throat swab, stool, tracheal aspirate and other body fluids such as pleural fluid and ascetic fluid. The samples were then sent immediately to the microbiology laboratory for culture and sensitivity. Standard operating procedures were used to collect samples.

The increased risk of bacterial infection is further compounded by rising trends of antibiotic resistance in commonly implicated organisms all over the world. [3] Antibiotic resistance among bacteria is becoming more and more serious problem throughout the world. This is particularly true in the case of members of Enterobactericae group like Escherichia coli and Klebsiella Spp and nonfermenter group of bacteria such as Pseudomonas Spp and Acinetobacter Spp. [3,4]

The samples were then inoculated on Nutrient agar, Blood agar and MacConkey’s agar plates and incubated aerobically at 37° C temperature for 24 hours. Growth was processed according to standard microbiological techniques which includes Gram staining, colony characteristics and biochemical properties. [7,8]

In spite of vast advances made by medical science, bacterial infection remains major cause of concern. Throughout the world bacterial infections are one of leading cause of morbidity, mortality, responsible for increased health care cost and accounts for major burden on patients and public health system of any country. [1,2]

Increasing resistance among gram positive organisms is also matter of concern and high rates of methicillin resistance Staphylococcus aureus (MRSA) in clinical samples have been noted. Similarly resistance to glycopepetides antibiotics such as vancomycin and teicoplanin among clinical isolates of Enterococci Sppis also increasing. [5,6] The pattern of bacteria causing infections and their antibiogram vary widely from one country to another,as well as from one hospital to other and even among ICUs with one hospital. [2,4,5] There also appears to be a significant lack of studies highlighting susceptibility patterns of locally prevalent organisms. Knowledge of predominantly isolated bacterial microorganisms and their sensitivity to available drugs is of immense value to the rational selection of antimicrobial agents and for development of appropriate antibiotic policies. Therefore, the present study was undertaken to identify prevalence of common bacterial isolates and their antimicrobial susceptibility pattern of various clinical samples from patients attending tertiary care hospital.

Materials and Methods

Study Design: The present study is prospective type of study and was carried out at Department of Microbiology, Indian Institute of Medical Sciences Badnapur , Jalna, Maharshtra a tertiary care hospital after approval from institutional ethics committee. The study was carried out during the period of July 2015-February 2016; a total 8189 clinical samples were evaluated. The clinical samples received from various departments of the hospital were included in the study. www.pacificejournals.com/apalm

Antimicrobial Sensitivity Testing: Criteria for antimicrobial sensitivity testing was carried out as per Clinical Laboratory standard institute (CLSI). [9] Antimicrobial sensitivity testing was done on Muller Hinton Agar (MHA) by Kirby Bauer’s disc diffusion method . Commercially available discs (Himedia) were used. Concentration of discs used were Erythromycin (15 mcg), Vancomycin (30mcg), Cotrimoxazole (25mcg), Ciprofloxacin (5mcg), Linezolid (30mcg), Ampicillin (30mcg), Piperacillin+Tazobactum (100/10mcg), Ceftazidime (30 mcg), Amikacin (30 mcg), Ofloxacin (5mcg), Gentamicin (10mcg) & high level (30mcg) ,Furazolidone (300mcg), Azetronam (30mcg), Chloramphenicol (30mcg),and Imipenem (10mcg). Nitrofurantoin (300mcg) was used in case of urine isolates. Methicillin resistance in Staphylococcus aureus (MRSA) was tested using Muller Hinton Agar with Cefoxitin disc (30mcg) by Kirby-bauer disc diffusion methods as per CLSI guidelines. [9] Suspected extended- spectrum beta lactamases (ESBLs) producing Enterobactericae were confirmed by double disk synergy test as per CLSI guidelines.[9] Staphylococcus aureus (ATCC 25923), E. coli (ATCC 25922) and P. aeruoginosa (ATCC 27853) were used as quality control throughout the study for culture and antimicrobial susceptibility testing. Statistical Analysis: The data was analysed and evaluated on the basis of percentage values and the result were presented in the form of tables and figures. Microsoft excel was used for the interpretation of these results.

Results

During 8 month study period, 8189 clinical samples were analysed. 2976 (36.37%) organisms were isolated. Of all positive samples, 1956 (65.7%) were the organisms eISSN: 2349-6983; pISSN: 2394-6466

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â&#x20AC;&#x153;Bacteriological Profile and Antimicrobial Sensitivity Pattern... followed by Staphylococcus aureus 782(26.27%) and non-fermenters 318 (10.68%) (Pseudomonas aeruginosa / Acinetobacter spp). Table 2 & 3

isolated from hospitalised patients while 1020 (34.3%) organisms were isolated from those who attended outpatients department.Total number of organisms isolated from various clinical samples are shown in Table.1

The detailed bacteriological profile and their antibiogram from various clinical samples can be seen from table 4,5,6,7&8. The prevalence of multidrug resistance organisms is shown in table 9

2108 (70.83%) were gram negative isolates and 868 (29.17%) were gram positive isolates. Most common bacteria isolated were E.coli/ Klebsiella spp 1687(56.68%)

Table 1: Number Organisms isolated from various clinical samples (n-2976). Sr. No. 1. 2. 3. 4. 5.

Clinical samples Urine Blood Pus swab Sputum Others Total

Number of organisms isolated 1370 369 876 181 180 2976

Percentage% 46.01 12.48 29.43 6.08 6.04 100

Others- CSF, throat swab, stool, pleural fluid, ascetic fluid, tracheal aspirate

Table 2 Gram Negative Isolates (n-2108). Sr.No. 1. 2. 3 4 5 6

Gram negative isolates E.coli KlebsiellaSpp Pseudomonas aeruginosa Acinetobacterspp Salmonella typhi/ S.paratyphi A Proteus Total

Total numbers 985 710 198 120 51 44 2108

Percentage% 46.72 33.68 9.39 5.69 2.41 2.08 100

Total numbers 782 69 12 5 2108

Percentage% 90.09 7.94 1.38 0.58 100

Table 3 Gram positive Isolates (n-868). Sr. No. 1. 2. 3 4

Gram positive isolates Staphylococcus aureus Enterococci spp Streptococcus pneumonia Streptococcus pyogens Total

Table 4.Urinary Tract Infections (UTI) â&#x20AC;&#x201C; Antibiogram Microbiology data (n=1370) Most common pathogens

Prevalence% Antibiotic sensitivity

Ecoli (n=822)

Klebsiellaspp (n=383)

Staphylococcus aureus (n=137) Pseudomonas aeruginosa (n=14)

10 1

Imipenem (100%), Ofloxacin/ Ceftazidime (83%), Gentamicin (74%) Amikacin/ Nitrofurantoin(60%) Ciprofloxacin (54%), Cotrimaxozole(40%) Imipenem (100%), Ceftazidime (92%),Ofloxacin(78%), Gentamicin (70%) Amikacin/Nitrofurantoin (64%) Ciprofloxacin(58%), Cotrimaxozole(50%) Linezolid (100%),Vancomycin (96%), Cefoxitin(64%), Gentamicin (60%), Erythromycin(58%), Cotrimaxozole/ Nitrofurantoin (40%) Imipenem (92%),Pipercillin-tazobactam(85%),Amikacin(78%) Ceftazidime/ciprofloxacin (64%),Azetronam/ Cotrimaxozole (50%)

Enterococci (n=08)

0.5

Linezolid/ Vancomycin (100%), Ampicillin(87%), Erythromycin (75%) Gentamicin (high level)(62%), Nitrofurantoin(50%)

Proteus/Acinetobacter (n=07)

0.5

Imipenem (100%), Ofloxacin(85%), Gentamicin(71%) Amikacin /Nitrofurantoin (57%),Ceftazidime/ciprofloxacin(42%) Cotrimaxozole(28%)

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Table 5: Blood Stream Infections (BSIs) â&#x20AC;&#x201C; Antibiogram Microbiology data (n=369). Most common pathogens

Prevalence%

Antibiotic sensitivity

Staphylococcus aureus (n=181)

Linezolid/ Vancomycin (100%), Cefoxitin(77%), Gentamicin(69%), Erythromycin(56%), Cotrimaxozole/ Ciprofloxacin(50%)

Klebsiellaspp (n=77)

Imipenem (100%), Ofloxacin(90%), Gentamicin(80%) Amikacin (62%) Ceftazidime /Ciprofloxacin(54%), Cotrimaxozole(45%)

Salmonella typhi/Para typhi A (n=51)

Ceftrioxone(100%),Ofloxacin(88%),Amoxycillin(82%), Cotrimoxazole(80%), chloramphenicol(76%), furazolidone(62%)

Pseudomonas aeruginosa / Acinetobacter (n=41)

Imipenem/ Pipercillin-tazobactam (100%), Amikacin(85%) Ceftazidime/ciprofloxacin(78%), Azetronam/ Cotrimaxozole (50%)

Enterococci (n=18)

Linezolid/ Vancomycin (100%), Ampicillin(88%), Gentamicin (high level)(83%), Erythromycin(66%) Nitrofurantoin(50%)

Table 6: Skin & soft tissue infection- Antibiogram Microbiology data (n=876). Most common pathogens

Prevalence %

Antibiotic sensitivity

Staphylococcus aureus(n=351)

Linezolid(100%), Vancomycin(92%), Gentamicin(72%), Cefoxitin(63%), Erythromycin (56% ) Cotrimaxozole/ Ciprofloxacin(50%)

Ecoli /Klebsiellaspp (n=245)

Imipenem (100%), Ofloxacin(88%), Gentamicin(78%) Amikacin (72%) Ceftazidime /Ciprofloxacin (64%), Cotrimaxozole(51%)

Pseudomonas aeruginosa (n=122)

Polymyxin B (100%)Imipenem(95%), Pipercillin-tazobactam (83%) , Amikacin(77%), Ceftazidime (73%),Ciprofloxacin(69%), Azetronam/ Cotrimaxozole (50%)

Proteus spp / Acinetobacter (n=113)

Imipenem (100%), Ofloxcin(92%), Gentamicin(85%) AmikaciN(82%),Ceftazidime/ciprofloxacin (71%) Cotrimaxozole(48%)

Enterococci (n=43)

Linezolid/ Vancomycin (100%), Ampicillin(93%), Gentamicin (high level)(90%), Erythromycin(50%)

Table 7: Lower respiratory tract infection (pneumonia ) â&#x20AC;&#x201C; Antibiogram Microbiology data (n=181). Most common pathogens

Prevalence%

Antibiotic sensitivity

Klebsiella pneumonia and other enterobacteriaceae (n=103)

Imipenem (98%), Oflaxcin(92%), Gentamicin (85%), Ceftazidime(82%) Amikacin /Ciprofloxacin(77%), Cotrimaxozole(49%)

Staphylococcus aureus(n=41)

Linezolid(100%), Vancomycin(90%), Gentamicin (78%), Cefoxitin(50%), Erythromycin(43%), Cotrimaxozole/ Ciprofloxacin(27%)

Pseudomonas aeruginosa/ Acinetobacter (n=24)

Polymyxin B (100%),Imipenem(95%), Pipercillin-tazobactam (83%) Amikacin(75%), Ceftazidime (70%), Ciprofloxacin(69%), Azetronam/ Cotrimaxozole (62%)

Streptococci pneumonia (n=12)

Linezolid /Cetriaxone (100%), Ofloxacin(83%), Amoxyclavulante(75%), Erythromycin(43%)

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Table8: Bacteriological profile of other potential infectious clinical samples- Antibiogram(Thorat swab, pleural fluid, ascetic fluid, CSF, stool, tracheal aspirate) Microbiology data (n=180). Most common pathogens

Prevalence%

Antibiotic sensitivity

Staphylococcus aureus (n=72)

Linezolid(100%), Vancomycin(97%), Gentamicin(90%), Cefoxitin(75%), Erythromycin(69%), Cotrimaxozole/ Ciprofloxacin(50%)

E.coli and other enterobacteriaceae (n=57)

Imipenem (98%), Oflaxcin(92%), Gentamicin (85%), Ceftazidime(82%) Amikacin /Ciprofloxacin(77%), Cotrimaxozole(49%)

Pseudomonas aeruginosa/ Acinetobacter (n=45)

Polymyxin B/ Imipenem (100%), Pipercillin-tazobactam (94%) Amikacin(83%), Ceftazidime(80%),Ciprofloxacin(75%), Azetronam/ Cotrimaxozole (50%)

Streptococcus pyogens(n=5)

Ceftriaxone/Ofloxacin/Vancomycin(100%), Erythromycin(80%), Penicillin(80%)

Table 9: MDRO pattern Sr.No.

Organisms

Total numbers

MDRO

Percentage%

E.Coli/ Klebsiellaspp

1687

310 (ESBL- producers)

18.37

Staphylococcus aureus

782

282 (MRSA)

36.06

MDRO- multi drug resistant organisms.

Discussion

The microbial pathogens, as well as their antibiotic sensitivity patterns may change from time to time and place to place. The overuse and misuse of antibiotic is leading to emergence of resistance. Hospital antibiogram are commonly used to help guide empiric antimicrobial treatment and are important component of detecting and monitoring trends in antimicrobial resistance. In the present study the most common microorganism isolated were Escherichia coli (33.09%), Staphylococcus aureus (26.27%), Klebsiella spp (23.85%) and nonfermenter (10.68%) (Pseudomonas aeruginosa and Acinetobacter Spp) similar findings were seen in studies carried out by many researchers.[10,11,12,13] Amongst gram negative bacilli Escherchia coli was dominant pathogen isolated from urine and skin & soft tissue infections whereas Klebsiella pneumonia was mostly isolated from lower respiratory tract infections and blood stream infection. While amongst gram positive bacteria Staphylococcus aureus was dominant pathogen isolated in blood stream infections, urinary tract infection and from other potentially infectious samples followed by Enterococci Spp, similar trends were seen in studies carried out by K Yadav et al.[11] The study showed a very high percentage of resistance among organisms to betalactam antibiotics, combination of betalactam/ betalactamase inhibitors. Most of gram negative bacteria shown resistance to cotrimaxozole, ciprofloxacin, and to less extent to amikacin, whereas most of the uropathogens shown 50% resistance to nitrofuantoin,

similar findings regarding drug resistance pattern were observed by other researchers . [5,12,13] The present study also highlights that gram negative bacilli were 95-98% sensitive to carbepenems, and incidence of carbepenems resistance is very low in our setup as oppose to increasing trend of carbepenems resistance shown by other researchers. [14,15] Most of the non fermenter (Pseudomonas aeruginosa and Acinetobacter Spp) shown 100% sensitivity to polymyxin B and showed excellent sensitivity to carbepenems and pipercillin-tazobactam. In case of gram positive bacteria most of isolates especially Staphylococcus aureus were sensitive to vancomycin & linezolid and 50% of resistance was shown to ciprofloxacin, cotrimaxozole and erythromycin. Most of Enterococci Spp were 100% sensitive to vancomycin/linezolid, followed by high level gentamicin and ampicillin and 50% resistant was seen with erythromycin. Our sensitivity pattern was in concordance with studies carried out by many other researchers though in their study resistant pattern to vancomycin was on higher side as compared to our study.[14,15] Our study also found out that around 18% Enterobactericae isolates were ESBL producers which is in concordance with other studies. [12,13] Around 36.06% were detected as methicillin resistant Staphylococcus aureus (MRSA) which is on slightly higher side, though many other studies shown that overall prevalence of MRSA ranges from 1445%.[15,16]

Conclusion

The most common microorganism isolated were Escherichia coli (33.09%), Staphylococcus aureus

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Santosh .A et al. (26.27%), Klebsiella spp (23.85%) and nonfermenters (10.68%) (Pseudomonas aeruginosa and Acinetobacter Spp).Antimicrobial resistance pathogens in any hospital settings is major deterrent to patient outcome, increasing duration of patient stay as well as expense. Strict infection control measures like universal precaution, simple hand washing, rational use of antibiotics and strictly adhering to antibiotic policies are necessary for decreasing drug resistance in hospital.

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6. Pawar M, Mehta Y, Purohit A, Trehan N, Rosenthal VD. Resistance in gram-negative bacilli in a cardiac intensive care unit in India: Risk factors and outcome. Ann Card Anaesth 2008;11:20-6

7. Koneman EW, Allen SD, Janda WM, Schreckember PC, Winn WC. Koneman’s Colour Atlas and text book of Diagnostic Microbiology. 6th edition. Newyork: Lippincott; 2006; 97-99. 8. Forbes BA, Sahm DF, Weissfeld AS. In: Bailey and Scott’s Diagnostic Microbiology. 12th ed. Missouri: Mosby Elsevier; 2007. p. 779. 9. CLSI – Clinical and Laboratory Standards Institute 2016. Performance standards for antimicrobial susceptibility testing. Twenty-second informational supplement. Wayne, PA, USA: CLSI:2016. 10. Rao SP, Rama PS, Gurushanthappa V, Manipura R, Srinivasan K. Extended-spectrum beta-lactamases producing Escherichia coli and Klebsiella pneumoniae: A multi-centric study across Karnataka. J Lab Physicians 2014;6:7-13. 11. Shashwati N, Kiran T, Dhanvijay AG. Study of extended spectrum β-lactamase producing Enterobacteriaceae and antibiotic co-resistance in a tertiary care teaching hospital. J Nat SciBiol Med 2014;5:30-5. 12. Manoharan A, Chatterjee S, Mathai D; SARI Study Group. Detection and characterization of metallo beta lactamases producing Pseudomonas aeruginosa. Indian J Med Microbiol 2010;28:241-4. 13. Kala Yadav ML, Ashmita Raja; bacteriological profile and antibiogram of gram negative clinical isolates from tertiary care hospital. International J res in health sci 2014;3:734-39 14. Dash M, Padhi S, Pattnaik S, Mohanty I, Misra P. Frequency, risk factors, and antibiogram of Acinetobacter species isolated from various clinical samples in a tertiary care hospital in Odisha, India. Avicenna J Med 2013;3: 97-102. 15. 17. Menezes GA, Harish BN, Sujatha S, Vinothini K, Parija SC. Emergence of vancomycin-intermediate Staphylococcus species in Southern India. J Med Microbiol 2008;57(Pt 7):911-2. 18 16. Eshwara VK, Munim F, Tellapragada C, Kamath A, Varma M, Lewis LE, et al. Staphylococcus aureus bacteremia in an Indian tertiary care hospital: Observational study on clinical epidemiology, resistance characteristics, and carriage of the Panton-Valentine leukocidin gene. Int J Infect Dis 2013;17:e1051-5.

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Fortunately, in our study almost all gram negative bacteria were sensitive to carbepenems and also retained useful susceptibility to third generation cephalosporins and aminoglycosides whereas in case of gram positive bacteria all were sensitive to vancomycin/ linezolid though 36.06% were MRSA.

Funding None

Competing Interests None Declared

References

1. Antimicrobial resistance in India: A review. J Nat SciBiol Med 2013;4:286-91 2. Choudhury R, Panda S, Singh DV. Emergence and dissemination of antibiotic resistance: A global problem. Indian J Med Microbiol 2012;30:384-90. 3. Ghafur A, Mathai D, Muruganathan A, Jayalal JA, Kant R, Chaudhary D, et al. The Chennai declaration: A roadmap to tackle the challenge of antimicrobial resistance. Indian J Cancer 2013;50:71-3. 4. Muto CA, Jernigan JA, Ostrowsky BE, Richet HM, Jarvis WR, Boyce JM, et al. SHEA guideline for preventing nosocomial transmission of multidrugresistant strains of Staphylococcus aureus and Enterococcus. Infect Control HospEpidemiol 2003;24:362-86. 5. Forbes BA, Sahm DF, Weissfeld AS. Bailey and Scott‘s Diagnostic Microbiology. 12th ed. Elsevier -Mosby, St. Louis, Missouri 63043, USA; 2007.

Case Report A Unique Case of Multiple Endocrine Neoplasm-1 (MEN-1) Syndrome satisfying all Six WHO Criteria Rushabh Jitendra Shah* and Samruddhi Dilip Rajpurkar Department of Pathology, Seth GS Medical College & KEM Hospital, Parel, Mumbai Keywords: Multiple Endocrine Neoplasia Type 1, Parathyroid Hyperplasia, Thymic Carcinoid, Pituitary Adenoma

ABSTRACT Multiple Endocrine Neoplasia (MEN) syndromes are a group of genetically inherited diseases resulting in proliferative lesions (hyperplasia, adenomas and carcinomas) of the endocrine organs. MEN 1 syndrome principally includes tumors of parathyroid, pancreas and pituitary. Our case is a 45 year old male, previously operated for thymic carcinoma, subsequently presented with radiologically and pathologically confirmed neuroendocrine lesions involving pituitary, parathyroid, adrenal, pancreas, stomach and duodenum. This patient had a positive family history for MEN 1 syndrome thus satisfying all six WHO criteria for MEN 1 syndrome which is a rare observation with no previous published reports. Tumors in this syndrome are more aggressive and recur in higher proportion of cases than do similar tumors occurring sporadically in non-syndromic patients.

*Corresponding author: Dr. Rushabh Jitendra Shah, 403, Jolly Apts, Cama Lane, Near Fatima High School, Ghatkopar-West, Mumbai-400086, INDIA. Phone: +91 25133167, (M): +91 9867712372 E-mail: mrushabh2387@hotmail.com

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Shah et al.

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Introduction

Multiple Endocrine Neoplasia (MEN) syndromes are a group of genetically inherited diseases resulting in proliferative lesions (hyperplasia, adenomas and carcinomas) of the endocrine organs. MEN1 syndrome (also known as Wermer Syndrome) is a rare inherited autosomal dominant disorder with an incidence of one in 30,000, seen in all age groups (ranging from 8 to 81 years) and an equal sex distribution. It is characterized by polyendocrinopathies usually presenting by the fifth decade. This syndrome exists in sporadic and familial forms. [1] To label a case as MEN1 syndrome, two out of six WHO criteria need to be fulfilled. [2] We report a unique case of 45 year old male satisfying all the criterias and with equally rare thymic involvement as the initial manifestation.

Case Report

A 45 year old male, operated for thymic carcinoma two years ago and treated with radiation, presented to our hospital in November 2013 with proximal muscle weakness, bone pains, weight loss, bilateral renal calculi and hydronephrosis. Patient was on proton pump inhibitors for peptic ulcer disease since nine years and oral hypoglycemics for type two diabetes mellitus since the past three years. On examination in the present admission, there were multiple whitish small nodular skin lesions like tags over neck, right elbow and popliteal fossa clinically suspected to be collagenomas (figure-fig. 1a) and a popliteal fossa lipoma. Vital parameters and systemic examination was unremarkable. Investigations showed raised serum glucose levels (fasting-214mg/dl), alkaline phosphatase (440U/L) and calcium (13.4mg %) and low serum phosphorus (1.6mg %). With the clinical suspicion of MEN 1 syndrome, he was further evaluated. Hormonal studies revealed elevated serum parathormone (319pg/ml) and prolactin (167ng/ml). Serum gastrin levels could not be carried out due to daily proton pump inhibitors therapy. Subsequent radiologic examination and octreoscan study showed multiple lesions - tumors in pituitary and parathyroid, nodules in stomach, duodenum, head of pancreas and adrenal, residual thymic lesion, sphenoid bone destruction and lytic, sclerotic foci in axial and appendicular skeleton (suspected as metastasis of thymic carcinoma). He underwent pituitary and parathyroid tumor resection. Pituitary gland showed histology of pituitary adenoma (fig. 1b) with atypical features like bone invasion and increased mitotic activity (fig.1c) and on immunohistochemistry (IHC) was positive for neuroendocrine markers (fig.1d). On studying the gross (fig.1e) and microscopy (fig.1f) of parathyroid resection specimens it revealed adenoma in one gland and hyperplasia in remaining three glands. www.pacificejournals.com/apalm

On reviewing the histology, previously operated thymic tumor was a neuroendocrine carcinoma (NEC) (fig.1g) confirmed immunohistochemically by chromogranin and synaptophysin positivity and negativity for TTF 1 and cytokeratin 5/6. Stomach biopsy was labelled as neuroendocrine tumor and was confirmed by IHC (fig.1h). On family screening (fig.2-family tree), patientâ&#x20AC;&#x2122;s younger son had high serum levels of prolactin (1250ng/ ml) and alkaline phosphatase (215 U/L) with pituitary macroadenoma and a thymic nodule thus suggesting presence of MEN 1 syndrome in him too. Based on these findings, we conclude our case as MEN 1 syndrome, with uniqueness of satisfying all the six WHO criteria. [2] Post operatively in this patient, serum levels of prolactin, calcium and phosphorus started normalizing but bone pain persisted. On follow up after two months, he had severe diffuse bone pains in the back and extremities. Radiological examinations showed multiple nodules involving the sphenoid sinus, anterior mediastinum and axial skeleton with markedly elevated serum chromogranin levels (4530 ng/ml). Diagnosed to have neuroendocrine metastatic disease, he was put on palliative treatment with analgesics, dopamine receptor agonists and calcium and vitamin D supplements. Meanwhile genetic screening of both father (index case) and his son was carried out which did not reveal mutations in MEN1 gene. At two and half year follow up, (April 2016) patient has improved symptomatically and is doing well till date.

Discussion

Usually MEN1 syndrome is characterized by three principle lesions viz. multiglandular parathyroid disease, enteropancreatic endocrine tumors, and anterior pituitary tumors. [3] Clinical manifestations are related to tumor localizations and their secretory products. Spectrum of the lesions (endocrine and non-endocrine) and their incidence is elucidated in Table 1. [1, 4] Hypercalcemia due to parathyroid hyperplasia (primary hyperparathyroidism) is the commonest and usually the first presenting manifestation of MEN 1. [4] However this may not be always true, as in our case patient presented with thymic NEC. Thymic involvement is usually in form of thymic carcinoids seen in about 0-8% of patients with MEN 1. They are insidious tumor but can present in an aggressive fashion with poor prognosis if detected late. [4, 5] As our case presented well in time, despite having NEC, he had a relatively better outcome. After treatment for NEC, patient subsequently developed multiple endocrine tumors. So a clinical diagnosis of MEN 1 syndrome was suspected and confirmed by laboratory evaluation and histopathological examination there by making histopathological examination a helpful modality in MEN1 eISSN: 2349-6983; pISSN: 2394-6466

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Unique Case of MEN-1 Syndrome

Fig. 1: a. Whitish small nodular skin lesions like tags â&#x20AC;&#x201C; collagenomas b. Pituitary gland - tumor cells forming trabeculae, rossetoid and pseudopapillary architecture and cells are small, polyhedral to columnar with moderate eosinophilic cytoplasm, round to oval nuclei with finely dispersed chromatin (HE x 400) c. Pituitary tumor in nests and sheets reaching the bony tissue (HE x 100) d. Immunohistochemistry (IHC) - pituitary tumor cells are positive for synaptophysin (x400) e. Parathyroid glands- on left (above and below) showing hyperplasia, and on right is adenoma (grey brown with a few cystic and hemorrhagic areas) f. Parathyroid adenoma - multiple nodules containing many micro follicles with compressed parathyroid tissue at periphery (HE x 100) g. Thymus - tumor cells arranged in nests and cords showing nuclear atypia with foci of lymphovascular invasion (HE x 100) h. Stomach - antral biopsy showing nodules of round to polygonal monomorphic cells arranged in trabecular and insular pattern (HE x 40) (inset-IHC positive for synaptophysin)

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Fig. 2: Father of the index case died of abdominal cancer at 35 yrs and son has a thymic nodule and prolactinoma.

diagnosis. On studying the WHO diagnostic criteria for MEN 1 syndrome [2] we noticed that this case fulfills all of them which is tabulated below (Table 2) thus making it a unique scenario. MEN1 syndrome is caused by inactivating mutations of the tumor suppressor gene, MEN1, located on chromosome 11q13. [6] It has been observed that 5% and 10% of MEN1 patients do not have mutations in the coding region but may involve the promoter or untranslated regions, which remains to be investigated. [7] This could be possible in our case too as this patient was not positive for commonly occuring MEN1 mutations. Treatment results in MEN 1 are less successful due to tumor multiplicity, higher metastatic rate, and more aggressive Table 1: Spectrum of lesions in MEN 1 Lesion Incidence Parathyroid lesions

95 %

Pancreatic islet cell/ 30-80% neuro endocrine tumors Anterior pituitary tumors 30-40% Collagenomas

Facial angiofibromas Lipomas

>70% 40-90%

20-30%

Adrenocortical tumors

Thyroid tumors

20-30% 25%

Carcinoids

10%

Meningiomas Pheochromocytomas

8% <1%

tumors than their sporadic non-syndromic counterparts. Untreated MEN 1 patients have decreased life expectancy and disease specific mortality is attributed to malignant pancreatic NET and thymic carcinoids. [8] Hence there is need for early diagnosis which is another key feature of the current case as this patient had dramatic clinical improvement and subsequently good prognosis as a result of timely diagnosis and aggressive treatment of different encountered lesions. The incidence of MEN1 in the Indian population is unknown and reports highlighting the same were not available after literature search. [9] To the best of our knowledge, this is the first documented report ofÂ MEN-1 syndrome which fulfills all the criterias with a relatively rare presenting feature in the form of thymic tumor.

Clinical relevance

Commonest Endocrine Lesions Commonest presenting manifestation Commonest functional tumors - gastrinomas and insulinomas, overall about one third tumors are non-functional

Prolactinomas - commonest

Common skin lesions

Represents genodermatosis, helps in presymptomatic diagnosis before hormone secreting tumors appear Less Common lesions Majority non-functional adenomas, indolent behaviour, occurs late in disease course Incidental association noted Usually silent, asymptomatic, common locations- bronchi, thymus, gastrointestinal tract, pancreas Rare association, presents late

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Unique Case of MEN-1 Syndrome

Table 2: WHO Diagnostic Criteria for MEN 1 syndrome Sr. No WHO criteria

Our Case

Primary or recurrent primary hyperparathyroidism with multiglandular hyperplasia and/or adenoma

Parathyroid adenoma and hyperplasia

Duodenal and/or pancreatic endocrine tumor*- IHC proven, gastric enterochromaffin-like tumors

Gastric and pancreatic NET

Anterior pituitary adenoma*-IHC proven

Atypical pituitary adenoma

Adrenocortical tumors*

Adrenal adenoma

Thymic and/or bronchial endocrine tumors

Thymic NEC

First degree relative (parent, sibling, offspring) fulfilling MEN 1 syndrome criteria

Son with pituitary and thymic endocrine tumour

* - functioning, non-functioning or multi-secreting; abbr: IHC-immunohistochemistry, NET- neuroendocrine tumors, NEC-neuroendocrine carcinoma

Conclusion

High index of suspicion is required for this syndrome when there are rarer presenting symptoms. Pathological examination of the lesions is beneficial for confirmation of MEN 1 cases. Routine surveillance of asymptomatic atrisk individuals by using all the available modalities for diagnosing multiple tumors occuring at a younger age, will help to reduce syndrome associated morbidity and mortality.

Acknowledgements Nil

Funding None

Competing Interests None Declared

Reference

1. Marini F, Falchetti A, Del Monte F, Carbonell Sala S, Gozzini A, Luzi E et al. Multiple endocrine neoplasia type 1. Orphanet J Rare Dis 2006;1:38. 2. Calender A, Scoazec JY, Morrison CD, Sweet KM, Komminoth P, Teh BT. Inherited tumor syndromesMultiple endocrine neoplasia type one. In: DeLellis RA, Lloyd RV, Heitz PU, Eng C, editors. World Health Organisation Classification of Tumors. Pathology and Genetics of Tumors of Endocrine Organs. 3rd ed. Lyon, France: IARC Press; 2004. pp. 218-27.

3. Teh BT, Grimmond S, Shepherd J, Larsson C, Hayward N. Multiple endocrine neoplasia type 1: clinical syndrome to molecular genetics. Aust NZ J Surg 1995;65:708-13. 4. Hoff AO, Hauache OM. Multiple endocrine neoplasia type 1 (MEN 1): clinical, biochemical and molecular diagnosis and treatment of the associated disturbances. Arq Bras Endocrinol Metabol 2005;49:735-46. 5. Teh BT, Zedenius J, Kytölä S, Skogseid B, Trotter J, Choplin H et al. Thymic carcinoids in multiple endocrine neoplasia type 1. Ann Surg 1998;228:99105. 6. Chandrasekharappa SC, Guru SC, Manickam P, Olufemi SE, Collins FS, Emmert-Buck MR et al. Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 1997;276:404-7 7. Thakker RV. Multiple endocrine neoplasia type 1. Indian J Endocrinol Metab 2012;16 Suppl 2:272-74. 8. Gaztambide S, Vazquez F, Castaño L. Diagnosis and treatment of multiple endocrine neoplasia type 1 (MEN1). Minerva Endocrinol 2013;38:17-28. 9. Shah SR, Raghavan R, Desai DC, Chauhan PH, Lala M, Dherai AJ, Ashavaid TF. An Indian family of multiple endocrine neoplasia type 1(MEN1): molecular diagnosis, treatment and follow up. Indian J Gastroenterol. 2008;27:242-44.

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Case Report A Rare Case of Leukemia Cutis in T-cell Acute Lymphoblastic Leukemia (T-ALL) in an Elderly Patient Anshu Gupta*, Sadhana and Tanima Dwivedi Department of Emergency Laboratory, Institute of Human Behavior and allied Sciences (IHBAS), Delhi, India Keywords: T cell, Acute lymphoblastic leukemia, Leukemia cutis, Hepato-splenomegaly

ABSTRACT Primary cutaneous involvement in T-cell acute lymphoblastic leukemia (ALL) is rare in elderly patients. Here we present a case of 65 years old male admitted to a metropolitan tertiary care hospital because of maculo-papular lesions on chest and back for last 3 months as chief complaint along with on and off history of fever, fatigability and anorexia. On general examination, there was no lymphadenopathy. Per-abdomen examination revealed hepatosplenomegaly. CT scan of chest and abdomen showed para-tracheal, pre-tracheal, carinal, hilar and abdominal lymphadenopathy. Peripheral blood smear and bone marrow aspiration showed more than 80% of lymphoblasts. Flow cytometry of the bone marrow aspirate showed CD7, CD5, CD45 and TdT positivity indicating Tâ&#x20AC;&#x201C;cell origin. Skin biopsy from maculo-papular lesion revealed secondary leukemic infiltration of the skin. Corroborating all the above findings, a final diagnosis of T-cell ALL with leukemia cutis was made. Thereafter patient was given chemotherapy and after one month, the skin lesions started resolving and total leukocyte count (TLC) decreased. One week later, skin lesions relapsed with fall in hemoglobin and rise in TLC. Repeat bone marrow aspirate showed 90% blasts cells. Then, patient was put on palliative therapy but his condition progressively deteriorated and ultimately died within four months of diagnosis.

*Corresponding author: Dr.AnshuGupta, Associate Professor, Emergency Laboratory, Institute of Human Behaviour and Allied Sciences, Dilshad Garden, Delhi-110095, India. Phone: +91 9868396812 E-mail: dransh2002@yahoo.co.in

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

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T- ALL with Leukemia Cutis in an Elderly Patient

Introduction

Acute lymphoblastic leukemia (ALL) is a malignancy of bone marrow where normal hematopoietic cells are replaced by proliferating lymphoid precursor cells. It is the most common type of leukemia seen in paediatric age group constituting about 75% of all cases of leukemia [1] .Incidence of ALL in elderly patients (> 65 years) is 1-1.6 per 100000 patients[2].Among all newly diagnosed cases of ALL, approximately 11.2% were from age group of >65 years. WHO classifies ALL as Pre (precursor) B-lymphoblastic ALL (most common ALL in adults), Mature B cell ALL (BurkittÂ ALL corresponds to ALL-L3) and Pre (precursor) T-cell ALL[3]. Leukemia Cutis (LC) is extra-medullary manifestation of leukemia caused by infiltration of skin by neoplastic leukemic cells and thus resulting in clinically identifiable skin lesions[4]. Incidence of LC is more common in AML constituting 10-15% of cases as compared to chronic myeloproliferative disorders[5]. However LC is rare in cases of precursor B- or T-cell lymphoblastic leukemia/ lymphomas (1%)[6]. Here, we are reporting a case of T-cell acute lymphoblastic leukemia with cutaneous infiltration in an elderly, posing as a diagnostic challenge because of its unusual presentation and rare immunophenotype.

Case Report

A previously healthy 65 years old male was admitted to a metropolitan tertiary care hospital with chief complaints of fever, fatigue, anorexia, and pruritic maculo-papular lesions over chest and back for past 3 months. On clinical examination, the patient was averagely built and poorly nourished. Skin lesions weremaculo-papular, erythematous, non-tender without ulceration or induration and blanching with pressure (Fig.1).There was no palpable lymphadenopathy in cervical, axillary or inguinal region. Per-abdomen examination showed enlarged liver, 3cms below costal margin and moderate splenomegaly. Biochemistry investigations showed ALT: 45 IU/L, AST: 48 IU/L, LDH: 392 IU/L, with normal kidney function tests. Ultrasonography revealed hepatosplenomegaly with liver measuring 18 cm and spleen measuring 19 cm. Chest roentgenogram and bone scan were normal. Routine hematological investigations revealed haemoglobin: 11.5g/ dl and total leucocyte count (TLC): 55,500/cmm. Differential Leukocyte count was blast cells 80%, neutrophils 10%, lymphocytes 6%, monocytes 2%, eosinophils 2%, basophils 0%, and platelet count: 1,20,000/cmm. Leishman stained peripheral smear showed 80% of blasts which were small to intermediate in size and round with scant light-blue cytoplasm, moderately condensed to dispersed

chromatin and inconspicuous nucleoli (Fig.2& 3). CT scan of the chest showed right para-tracheal, pre-tracheal, carinal and bilateral hilar lymphadenopathy.CT scan of the abdomen revealed hepatosplenomegaly and discrete pre and para-aortic and aorto-caval lymphadenopathy. Geimsa stained bone marrow aspirate smears showed more than 80% of blast cells that had characteristics of lymphoblasts. Special cytochemical stains i.e. Myeloperoxidase, Periodic acid schiff (PAS) and Sudan black stains were negative on bone marrow aspirate.Skin biopsy performed from one of the maculo-papular lesions showed thinned out epidermis. Thedermisshowed infiltration by atypical immature and blast like cells arranged in nodules, around blood vessels and adenexal structures (Fig.4& 5).These tumour cells were positive for CD45, suggestive of secondary leukemic infiltration of skin. On flowcytometry, CD7, CD5, CD45 and TdT were positive on bone marrow aspirate while CD10, HLADR, CD34 and B cell markers (CD19 and CD20) were negative. With these findings, a final diagnosis of immunologically T-cell type ALL with leukemia cutis was made. Patient was put on chemotherapy. Initially, skin lesions started resolving along with decrease in total leukocyte count (TLC) after one month of treatment but one week later, skin lesions relapsed started appearing on post-auricular region and then spreading over to rest of the body. His hemoglobin dropped to 6gm/dl, TLC increased and bone marrow examination showed 90% of blast cells. Patient was put on palliative therapy only because of poor response. His condition started deteriorating progressively, ultimately leading to death within four months of diagnosis.

Fig. 1: Photograph showing maculo-papular lesions on chest of the patient

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Fig. 2: Microphotograph of peripheral smear showing lymphoblasts (Leishman stain x 10x)

Fig. 4: Skin biopsy showing thinned out epidermis and blast like cells in nodules within the dermis (H&E stainx10x)

Fig. 3: Same microphotograph under higher magnification (Leishman stain x 100x).

Fig. 5: Same microphotograph under higher magnification showing blast like cells (H&E stain x40x)

Discussion

Clinical presentation of ALL is heterogeneous. Fatigue and lethargy are frequent manifestations because of anaemia. About 50% of patients present with fever.Dermal and mucus membranes petechiae and bone tenderness may occur due to leukemic infiltration. Leukemia cutis (LC)

is an extra-medullary manifestation of leukemia. It is generally seen in previously diagnosed cases of leukemia. However, rarely it may be the presenting manifestation of systemic disease[9]. Clinically, LC presents with papules, plaques, or nodules with colour ranging from violaceous to red-brown[9]. In mature T-cell leukemia it has been reported in 20-70% of cases[10.]. However, LC is rare in cases of precursor B- or T-cell lymphoblastic leukemia/ lymphomas (1%).[6,11,12]. LC is associated with poor prognosis as concluded by some studies including study by Su WP(1984)[13]. Marti RM et al (2003) reported that there was no significant difference between leukemic patients with and without LC in terms of sex, age, WBC count, haemoglobin, platelet count, and fibrinogen level, however serum LDH and Î˛2 -microglobulin had been observed to be higher in cases with LC[14].In our case, patient presented with fever, lethargy and maculo-papular lesions on skin

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ALL is most common leukemia diagnosed in younger patients and is a rare disease of elderly. There is a peak in its incidence between 2-5 years followed by falling rates during later childhood, adolescence and young adults[7]. Its incidence increases again in the sixth decade and peaks in elderly patients. Its incidence in elderly patients (> 65 years) is 1-1.6 per 100000 patients while in young adults aged 25-54 years, it is 0.6-0.7 per 100000. Male/Female ratio in elderly is 267/282 (0.95) while in young adults it is 1.43[8]. Our patient was 65 years old male.

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without any petechiae or bone tenderness. Serum LDH was within normal limits. Various studies reported varied clinical findings regarding hepatosplenomegaly, lymphadenopathy, CNS involvement and mediastinal mass in ALL. Hepatosplenomegaly and lymphadenopathy are common findings in children whereas half of the elderly patients do not have lymphadenopathy, hepatomegaly or splenomegaly as reported by Pui C-H et al (1999), Chessells et al (1998),Thomas et al (2001)etc. [6,15,16,17] Another study concluded that here was no significant difference in hepatomegaly and CNS involvement between elderly patients and adults[17].Various studies quoted 4-5% of CNS involvement in ALL patients [18].Higher incidence of CNS involvement is seen in B-cell ALL patients[19]. While Hillard M et al (2005) have found more frequent CNS involvement in T-cell immunophenotype[20].Our patient did not have CNS involvement. Few studies observed that about 8% of childhood cases and 15% of adult cases present with anterior mediastinal mass[7].Our case had abdominal and right para-tracheal, pretracheal, carinal and bilateral hilar lymphadenopathy along with hepatosplenomegaly without any mediastinal mass. Laboratory findings suggestive of anaemia, neutropenia and thrombocytopenia are frequently seen in ALL patients. Only 12% of adult patients have leukocyte count in the range of 50,000-99,000/cmm as in our case [7].About 46% of adult patients have haemoglobin >10g/dl. Maximum number of elderly patients (about 71%) has > 90% leukemic

blasts in the marrow. Morphologically, ALL is divided into three classes according to FAB (French-American and British) classification: L1 (70-75%). Blasts are smaller, uniform with scanty cytoplasm and inconspicuous nucleoli with coarse and homogenous chromatin. L2 (20-25%): Blasts are variable in size with greater amount of cytoplasm and prominent nucleoli, irregular nuclear membrane and finer chromatin. L3 (1-2%): Blasts are larger, have deeply basophilic cytoplasm with multiple vacuoles, stippled nuclear chromatin with prominent 1-2 nucleoli. In adults L2 morphology is seen more commonly[21].On special stains, B cell ALL shows dot and block PAS positivity while T cell ALL does not stain with it rather shows acid phosphatase positivity[7]. In our case,on peripheral smear, blasts constituted 80% of differential count and were small to intermediate in size and round with scant light-blue cytoplasm, moderately condensed to dispersed chromatin and inconspicuous nucleoli.Myeloperoxidase and PAS staining was negative. On flowcytometry ,blast cells showed CD7, CD5, CD45 and TdT positivity on bone marrow aspirate and were negative for CD10, HLADR, CD34 and B cell markers (CD19 and CD20), Therefore, categorized as ALL- L1 and T-cell type .Elderly patients have been observed less commonly to have T-cell lineage lymphoblast[22]. On immunochemistry, another classification based upon immunological expression of surface antigen has been given in Table I[7].

Table I: Immunological classification– Based upon expression of surface antigen S.No. Type 1.

Pro-B ALL

Early- pre B ALL (Common ALL)

Pre-B ALL

Mature B-cell ALL

Characteristics Accounts 10% cases of ALL,seen commonly in infants and children, morphologically correlates with FAB- L1 and L2 comprises 2/3rd of childhood ALL cases, most cases are CALLA (CD 10) positive 20% of childhood ALL cases, express cIg, which is associated with t(1;19) and poorer outcome

Immunocytochemistry

Rare (1-2%), morphology is FAB-L3, poor prognosis

SIg, λ +, κ +

15-20% of ALL cases, have morphology of L1 and L2, PAS T-ALL negative, Acid phosphatase positive, Higher incidence of CNS involvement, associated with mediastinal mass Comprises 1-2% of acute leukemia, two populations of morphologically and immunophenotypically distinct cell, Mixed lineage acute Larger cells with myeloblastdifferentiation, MPO +, SBB leukemia +, usually with Auer rods or withmonoblastic morphology. Smaller blasts with L1 morphology. Blast express myeloid and lymphoid antigens Undifferentiated Blast have L2 morphology but no lineage differentiation acute leukemia

HLA-DR+, TdT+ HLA-DR+, TdT+,CD10+ HLA-DR+,\ cIg +

CD3+, CD 2+ CD5+, CD7+, TdT+

CD3+,CD79a+, CD22+,CD10+, TdT+, MPO+, CD13+, CD11b+ HLA-DR+,CD-34+, TdT+,CD38+,CD-7+

cIg-cytoplasmic immunoglobulin; sIg-surface immunoglobulin; TdT-terminal deoxynecleotidyltransfarase.

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Gupta et al. Most of the studies have shown that old age is in itself an independent poor prognostic factor in ALL patients. The prognosis of older ALL patients (>60years) is not as good as of younger patients, probably due to presence of various co-morbidities in elderly patients, for example poor performance status, decreased bone marrow function[22]. Elderly ALL patients with high white blood cell count is found to have poor prognosis. According to a study done by Su WP, LC is associated with poor prognosis[13]. But now only immunological, cytogenetic and molecular factors are considered. Additionally, since there is inadequate data from clinical trials with respect to the ALL patients with old age, probably they are not being treated by the most effective agents. The incidence of Philadelphia (Ph) chromosome which is resistant to conventional chemotherapy increases with age and was observed in 24% of elderly patients and 19% of young patients and this Ph-positive status is found to be a poor prognostic factor[24,25]. Treatment of ALL varies from palliative care to intensive therapy. The palliative or minimally aggressive therapy (vincristine and steroids) leads to poor survival because these therapies were usually given to the patients with poor performance status[26]. It has been noticed that in elderly patients, intensive induction therapy, was frequently associated with a high mortality as compared with younger patients given similar regimen[27]. Our patient was elderly male with leukemia cutis, hepatosplenomegaly; hilar and abdominal lymphadenopathy diagnosed as T cell ALL and was put on intensive chemotherapy initially. Patient responded well in his first month course but rapidly deteriorated thereafter with abrupt fall in haemoglobin, rise in TLC and increased number of blasts in bone marrow and ultimately leading to death.

Conclusion

This is the one of the rare cases of T-ALLwith LC in elderly male patient. So one should always keep acute leukemia as a differential if an elderly patient presents primarily with cutaneous manifestations along with hepatosplenomegaly even in the absence of palpable lymphadenopathy. Prognosis is poor in such patients because of old age and presence of other co-morbidities.

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T- ALL with Leukemia Cutis in an Elderly Patient

descriptive and prognostic clinicopathologic study of 29 cases. Leuk Lymphoma 2003; 44(1):59-69. Reiter A, Schrappe M, Ludwig WD, Hiddemann W, Sauter S, Henze G,et al. Chemotherapy in 998 unselected childhood acute lymphoblastic leukemia patients. Results and conclusion of multicentre trial ALL-BFM 86. Blood 1994; 84(9):3122-33. Chessells JM, Hall E, Prentice HG, Durrant J, Bailey CC, Richards SM: The impact of age on outcome in lymphoblastic leukemia; MRC UKALL X and XA compared: A report from the MRC Paediatric and Adult Working Parties. Leukemia 1998; 12(4):463-73. Thomas X, Olteanu N, Charrin C, Lhéritier V, Magaud JP, Fiere D. Acute lymphoblastic leukemia in the elderly: The Edouard Herriot Hospital experience. Am J Hematol 2001; 67(2):73-83. Petersdorf SH, Kopecky KJ, Head DR, Boldt DH, Balcerzak SP, Wun T. Comparison of L10M consolidation regimen to an alternative regimen including escalating methotrexate/L-asperginase for adult acute lymphoblastic leukaemia : A southwest Oncology Group Study; Leukaemia 2001;15(2):208-16. Thomas DA, Cortes J, O Brien S, Pierce S, Faderl S, Albitar M, Hagemeister FB et al. Hyper-CVAD program in Burkitt’s-type adult acute lymphoblastic leukemia. J Clin Oncol 1999; 17 (8): 2461-70. Lazarus HM, Richards SM, Chopra R, Litzow MR,Burnett AK, Wiernik PH. Central nervous system involvement in adults in adult acute lymphoblastic leukemia at diagnosis: results from the international ALL trial MRC UKALL XII/ECOG E2993. Blood 2006; 108(2):465-72.

21. Arora RS, Eden TOB, Kapoor G. Epidemiology of childhood cancer in India. Indian J Cancer 2009; 46(4):264-73. 22. Delannoy A, Sebban C, Cony-MakhoulP,Cazin B,Cordonnier C,Bouabdallah R. French Group for Treatment of Adult Acute Lymphoblastic Leukemia. Age-adapted induction treatment of acute lymphoblastic leukemia in the elderly and assessment of maintenance with interferon combined with chemotherapy: a multicentric prospective study in forty patients. Leukemia 1997; 11(9):1429–34. 23. Annino L, Goekbuget N, Delannoy A. Acute lymphoblastic leukemia in the elderly. The Hematology Journal 2002; 3: 219-23. 24. Larson RA. Acute lymphoblastic leukemia: older patients and newer drugs.Hematology Am Soc Hematol Educ Program 2005:131–136. 25. Thomas X, Thiebaut A, Olteanu N, Danaïla C, Charrin C, Archimbaud E. Philadelphia chromosome positive adult acute lymphoblastic leukemia: characteristics, prognostic factors and treatment outcome. Hematol Cell Ther1998; 40(3):119–28. 26. Taylor PRA, Reid MM, Bown N, Hamilton PJ, Proctor SJ. Acute lymphoblastic leukemia in patients aged 60 years and over: a population-based study of incidence and outcome. Blood 1992; 80(7): 1813-17. 27. Kantarjian HM, O’Brien S, Smith TL, Cortes J, Giles FJ, Beran M, Pierce Set al. Results of treatment with hyper-CVAD, a dose-intensive regimen, in adult acute lymphocytic leukemia. J Clin Oncol 2000; 18 (3): 547-61.

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Case Report Autoimmunization in Thalassemia: A Case Report with Review of Literature

Sangeeta Pahuja, Geetika Sharma*, P. Lalita Jyotsna and Richa Chauhan Department of Regional Blood Transfusion Center, Department of Pathology, Lady Hardinge Medical College & Kalawati Saran Children’s Hospital, New Delhi , India Keywords: Thalassemia Major, Red Cell Autoimmunization, Autoantibodies.

ABSTRACT Alloimmunization has been variably reported worldwide in multi transfused thalassemic patients and leads to delay in issue of compatible blood. However, there is paucity of literature on the frequency of red cell auto-immunization in thalassemics, particularly of Indian origin. We present a case of 4 year old female child, known case of thalassemia major, who presented to the pediatric emergency with impending congestive heart failure. The EDTA sample showed auto-agglutination on naked eye at room temperature which persisted on incubating the sample at 37°C.There was discrepancy in forward and reverse blood grouping at room temperature which was resolved by extended blood grouping at 3 temperatures (4°C, 22°C & 37°C) and patient’s blood group was confirmed to be B positive. The polyspecific direct antiglobulin test (DAT) was strongly positive with 4+ reaction and DAT profiling revealed presence of IgG and C3d on red cells. The antibody screening and identification [Biorad ID Diacell I, II & III and Diapanel (11 cell panel)] respectively showed a panagglutinating reaction in Coomb’s phase at 37°C. The titers for IgM antibody in saline phase were found to be clinically non significant. Multiple units put up for the cross match were all incompatible. However, she was given profile matched (“c”neg, “E”neg and “Kell”neg) least incompatible blood slowly, under strict clinical supervision after the written consent from the clinician. Thus, guidelines for transfusion policy for thalassemic child need to be formulated to minimize the risk of immunization. Rh and Kell matched blood for thalassemic children (better match) has been shown to reduce the rate of immunization in thalassemic children.

*Corresponding author: Dr. Geetika Sharma, 362, Sector 8, Panchkula, Haryana, Pincode - 134109, (India). Phone: +91 9560835225, 9013952791 E-mail: aqua.gs2009@gmail.com

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Introduction

Thalassemia is genetically inherited group of disorders that result from reduced rate of production of one or more of globin chains.[1] Survival of thalassemic child is dependent upon regular blood transfusion therapy. While blood transfusion therapy, on one hand improves longevity; on other hand causes complications like red cell immunization. Alloimmunization is a known complication in multitransfused thalassemic patients and leads to delay in issue of compatible blood. Autoimmunization i.e. formation of antibodies against self antigens is less commonly reported in thalassemics. Hemolysis of patients own red cells as well as transfused red cells occur due to presence of autoantibodies in these patients. As auto-agglutinins are commonly pan-agglutinins in nature, finding compatible blood units for the patients becomes very cumbersome.

Case Report

We present a case of 4 year old Sindhi, female child, resident of Gwalior, who presented in the Pediatric emergency of Kalawati Saran Children’s Hospital with complaints of high grade fever with chills and rigor, on and off along with pain abdomen and vomiting since last 1 month. On general physical examination, patient had severe pallor, icterus, tachycardia and tachypnea (heart rate – 150/ min and respiratory rate – 52/min). On per abdomen examination, child had hepatosplenomegaly (5.5cm & 4cm respectively). Past clinical history revealed that child was a known case of thalassemia major, diagnosed at the age of 2½ years [High performance liquid chromatograph (HPLC) of the child was suggestive of beta thalassemia major (HbF 98.2%, HbA 1.7% and HbA20.5%) while HPLC findings of the mother and father were consistent with beta thalassemia trait with HbA2 values of 5.3 and 6.1, respectively]. Since then child was on regular blood transfusion at interval of 4-5 weeks from Gwalior and was clinically stable. For the last 1 month child also had history of increased requirement of blood transfusion. Complete haematological and biochemical investigations were done. The hematological findings revealed severe anaemia with macrocytic normochromic red cell indices and increased reticulocyte count (Hb 2.5 g/dl, reticulocyte count 7%). Biochemical investigations revealed elevated levels of total and indirect bilirubin, 3.8 gm/dl and 2.26 gm/ dl, respectively. Thus, both hematological and biochemical investigations were suggestive of hemolytic anaemia. Viral markers for HCV, HBV & HBsAg, Rapid malarial antigen test were nonreactive and urine examination was within normal limits.

EDTA sample was received at Regional Blood Transfusion Center, Lady Hardinge Medical College, for cross match to issue packed red cell. Sample showed auto-agglutination on naked eye which persisted on incubating the sample at 37 °C. Also, there was evidence of hemolysis in the sample at 37° C. Thus, a repeat fresh sample was taken under strict warm conditions and the plasma and the red cells were immediately separated , which also showed auto-agglutination. There was discrepancy in forward and reverse blood grouping at room temperature following which an extended blood grouping was repeated at 3 temperatures (4°C, 22°C, 37 °C) after multiple washing of red cells. This showed the presence of saline reacting antibodies at 4° C and 22° C. Thus, patient’s blood group was confirmed to be B positive (Table 1). The polyspecific direct antiglobulin test (DAT) was done on thoroughly washed red cells and was strongly positive with 4+ reaction (Control Neg) (Figure 1). DAT profiling revealed presence of IgG and C3d on red cells and IgM showed a mixed field (MF) reaction. (Table 2, Figure 2).The Rh profile of the patient was also done and the results are shown in (Table 3, Figure 3). The antibody screening and identification [Biorad ID Diacell I, II, III and Diapanel (11 cell panel) respectively] showed a panagglutinating reaction in Coomb’s phase at 37 °C. Autocontrol was 4+ positive indicating the presence of autoantibodies (Figure 4). The titres for IgM antibody at 4 °C in saline phase were found to be clinically non significant. Simultaneously, multiple units put up for the cross match were all incompatible. As the patient was in impending congestive heart failure, she was given profile matched (“ c” neg ,“E” neg and “Kell” neg), least incompatible blood. Instructions were given to transfuse blood very slowly, under strict clinical supervision, after the consent of the clinician. Presently the Hb of the patient is 11.7 g/dL & serum ferritin levels are 1435 µg/L. Patient received intravenous immunoglobulin and is presently on T. Desirox along with prednisolone 10 mg, folic acid & calcium supplements.

Discussion & Conclusion

Blood transfusion is the mainstay of treatment in the thalassemic patients. Autoimmunization in thalassemic child makes transfusion management complicated. Most of the autoantibodies formed are pan-agglutinins in nature and react with all the cells of the panel, as was in our case. Also, in presence of autoantibodies there is increased transfusion rate and need for immunosuppressive

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Table 1: Extended Blood Grouping at 3 temperatures ( 4°C, 22°C, 37 °C ) TEMP.

ANTI-A

ANTI-B

ANTI-D1 ANTI-D2

A CELLS B CELLS O CELLS A/C

CORD BLOOD

BLOOD GROUP

4° C

Weak

Neg

Invalid

22° C

Weak

weak

Neg

Invalid

37° C

Neg

B+

Table 2: DAT profile of the patient IgG

IgA

IgM

C3d

C3c

CONTROL

Neg

Trace

Neg

Table 3: Rh profile of the patient C

Control

Dp (4+/neg)

Neg

Fig. 1 : Blood Group ( B+) and DCT of the patient (DCT- 4+, CONTROL- Negative)

Fig. 2 : DAT profile of the patient

Fig. 3: Rh profile of the patient

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Fig. 4: Antibody identification [Biorad Diapanel (11 cell panel)] - Panagglutinating reaction in Coomb’s phase at 37 °C with autocontrol 4+ positive

drugs, splenectomy/ alternate treatment regimes.[2] Alloimmunisation in thalassemia is a well recognised phenomenon and frequency ranges from 3.7% - 30% worldwide. [2-8] However, autoantibodies have been less frequently reported in literature. Harikrishan Dhawan etal.[3]in 2014, studied frequency of red blood cell alloimmunization and autoimmunization among 319 multiple transfused patients with β thalassemia major and reported very high rate of autoimmunization 28.2 %. A cross sectional study was also conducted at our Regional Blood Tranfusion Center in 2010 among 211 multitransfused thalassemic children, and the frequency of alloimmunzation and autoimmunization was found to be 3.79 % and 0.47% respectively.[4] In 2008, Singer etal.[2]reported very high rates of alloimmunization and erythrocyte autoimmunization in 64 transfusion dependent thalassemia patients of predominantly Asian descent i.e22% (14/64) and 25 % (16/64) respectively. Out of these 16 patients with autoantibodies, 3 had severe hemolytic anemia, 11 had IgG while 5 had IgM antibodies These high rates were mainly due to heterogeneity of red cell antigens between donors and recepients (white donors and Asian recepients). Similar study was done by Noor Haslina etal.[5] in 2006, and studied antibody formation in 58 Malaysian multiple transfused thalassemic patients and found overall frequency of alloantibodies and autoantibodies to be 8.6% and 1.7% respectively; with only a single panagglutinating autoantibody. In 2004, Khalid Hassan et al. [6]conducted a study on 75 cases of multiply transfused thalassemia major patients

and found alloimmunization in 22.7% of the patients with absence of autoantibodies. Ameen etal. [7] in2003, studied 190 transfusion dependent thalassemia patients in Kuwait for red cell alloimmunization and autoimmunization and reported clinically insignificant autoantibodies in 11% (21/109). Among them, 48% had both IgG and C3d while 52.38% patients had only IgG. All these patients with autoantibodies had underlying alloantibodies except in one case. The high incidence of autoantibodies was due to heterogeneity of population and transfusion of ABO and Rh (D) only matched blood. Michail-Merianou et al. [8]in 1987, compared the frequency of alloimmunization between the usual–match group [ABO and Rho(D) antigens] and the better-match group (ABO, CcDEe & K antigens) which was found to be 23.43% and 14.28% respectively. They did not comment on autoantibodies. Erythrocyte autoantibodies may cause clinically significant hemolysis by decreasing the red cell survival in vivo. They may lead to difficulty in cross matching blood and delay in transfusion. Das et al.[9] opined that decision to transfuse patients with autoimmune hemolytic anemia should be based on the clinical condition of the patient. No critical patient should be denied blood transfusion due to serological incompatibility. They have suggested that alloimmunization and splenectomy increases the incidence of formation of autoantibodies. Leucoreduction has definitive proven role in reduction of alloimmunzation and autoimmunization. Rh and Kell matching (better match) has been recommended by several authors to reduce the risk of alloimmunization.[8]

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Pahuja et al. We need to frame the guidelines for transfusion policy for thalassemia children. All transfusion services should follow the definite protocols and perform the minimum test required to issue safe and best match leucoreduced packed red blood cells.

Acknowledgements Nil

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Funding Nil

Competing Interests

Nil

References:

1. Weatherall D. The Thalassemias: The Role of Molecular Genetics in an Evolving Global Health Problem. American Journal of Human Genetics 2004;74(3):385-392. 2. Singer ST, Wu V, Mignacca R, Kuypers FA, Morel P, Vichinsky EP. Alloimmunization and erythrocyte autoimmunization in transfusion-dependent thalassemia patients of predominantly asian descent. Blood. 2000 Nov 15;96(10):3369-73. 3. Dhawan HK, Kumawat V, Marwaha N, Sharma RR, Sachdev S, Bansal D, Marwaha RK, Arora S. Alloimmunization and autoimmunization in transfusion dependent thalassemia major patients:

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Study on 319 patients. Asian J Transfus Sci. 2014 Jul;8(2):84-8. Pahuja S, Pujani M, Gupta SK, Chandra J, Jain M. Alloimmunization and red cell autoimmunization in multitransfused thalassemics of Indian origin. Hematology. 2010 Jun;15(3):174-7. Noor Haslina MN, Ariffin N, Illuni Hayati I, Rosline H. Red cell immunization in multiply transfused Malay thalassemic patients. Southeast Asian J Trop Med Public Health. 2006 Sep;37(5):1015-20. Hassan K, Younus M, Ikram N, Naseem L, Zaheer HA.Red Cell Alloimmunization in Repeatedly Transfused Thalassemia Major Patients. International Journal of Pathology. 2004; 2(1):16-19. Ameen R, Al-Shemmari S, Al-Humood S, Chowdhury RI, Al-Eyaadi O, Al-Bashir A. RBC alloimmunization and autoimmunization among transfusion-dependent Arab thalassemia patients. Transfusion. 2003 Nov;43(11):1604-10. Michail-Merianou V, Pamphili-Panousopoulou L, Piperi-Lowes L, Pelegrinis E, Karaklis A. Alloimmunization to red cell antigens in thalassemia: comparative study of usual versus better-match transfusion programmes. Vox Sang. 1987;52(1-2):95-8. Chaudhary RK, Das SS. Autoimmune hemolytic anemia: From lab to bedside. Asian J Transfus Sci. 2014 Jan;8(1):5-12.

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Case Report Membranous Nephropathy: A Rare Association with Renal Vein Thrombosis Ankur Mittal1, Ruchi Gupta2, Pavneet Selhi1, Harpreet Kaur1, Neena Sood1 and Jasvinder Sandhu3 Dept of Pathology, Dayanand Medical College & Hospital, Ludhiana (Pb) India 2 Dept of Pathology, GMC, Amritsar (Pb), India 3 Dept of Nephrology, Dayanand Medical College & Hospital, Ludhiana (Pb) India 1

Keywords: Membranous Nephropathy, Renal Vein Thrombosis, Nephrotic Syndrome

ABSTRACT Background: Thromboembolic events are a known complication of nephrotic syndrome and occur with a frequency varying from 10% to 45% depending upon the underlying disease. Case Presentation: A 25 year male presented with history of decreased urine output , groin pain and edema lower limbs. Laboratory tests showed Urea 30mg/dL; Creatinine 0.66mg/dL; albumin 2.3g/dL; proteinuria 3+ and negative viral serology. An abdominal ultrasound with Doppler showed features suggestive of partial revascularization. Renal biopsy showed membranous nephropathy. Conclusion: Our case shows that renal vein thrombosis (RVT) is a complication of membranous nephropathy.

*Corresponding author: Dr Ankur Mittal, Flat No 202, Plot No 330R, Rajrajeshwari apartment, Model Town, Ludhiana (Punjab) â&#x20AC;&#x201C; 141001, India Phone: +91 09815494331 E-mail: drankur03@yahoo.co.in

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Mittal et al.

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Introduction

Thromboembolic events are a known complication of nephrotic syndrome and occur with a frequency varying from 10% to 45% depending upon the underlying disease. The renal vein is a classic site for thrombosis in nephrotic patients, being unusual in healthy subjects. Deep vein thrombosis, especially renal vein thrombosis, is reported to occur more frequently amongst patients with membranous nephropathy (MN) than other nephrotic diseases.[1] Dahler et al [2] concluded that, in patients with nephrotic syndrome, the prevalence of DVT is high. RVT can be unilateral or bilateral; it can extend through inferior vena cava and can be associated with pulmonary embolism. The risk of thrombosis is proportional to the severity of nephrotic syndrome, mainly with serum levels of albumin lower than 2g/dL and proteinuria higher than 10g/day.[3]

Fig. 1: Right kidney normal in size and shows normal echogenicity. Corticomedullary differentiation is maintained. Renal pelvis is mildly prominent. On color Doppler, renal artery shows normal filling and renal vein shows patchy flow â&#x20AC;&#x201C; suggestive of partial obstruction

The majority of RVT cases are insidious and assymptomatic, usually discovered incidentally or when a workup for the source of pulmonary embolism reveals it. This paper describes a case of membranous nephropathy complicated with RVT.

Case Report

A 25 year old male presented with a history of decreased urine output for ten months associated with groin pain and easy fatigubility. After two month of initial symptoms, he had an episode of hemoptysis. Odema of feet and lower limbs along with dysopnea of NYHA II was also noted for the last 10days. At admission, physical examination was unremarkable other than odema lower limbs and feet. Laboratory tests showed Hemoglobin 15.8g/dL; White blood count 13,700/mm3; INR 1.04; Urea 30mg/dL; Creatinine 0.66mg/dL; K+ 4.02mEq/L; Na+ 140mEq/L; albumin 2.3g/dL and proteinuria 3+. Antinuclear antibodies(ANA) and viral serology were negative. A renovascular duplex ultrasound was performed which showed normal sized kidneys, with maintained echotexture. A patchy flow was seen in right renal vein along with an echogenic material in inferior vena cava suggestive of partial revascularization. (Figure 1). X-ray chest showed the features of right sided pleural effusion.

Fig. 2: Light microscopy shows a unremarkable glomerulus (H&E, 400X)

histologically

A renal biopsy was performed. Light microscopy revealed 26 glomeruli all of which were histologically unremarkable. Tubules showed degenerative changes (Figure 2). On immunoflourescence granular IgG deposits were noted along the glomerular capillary loops, compatible with the diagnosis of Early Membranous Nephropathy. (Figure 3).

Fig. 3: Immunoflourescence showes granular IgG deposits along the glomerular capillary loops (IF, 400X)

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Membranous Nephropathy: A Rare Association...

Discussion

The incidence of both venous and arterial thrombosis, is high in patients with nephrotic syndrome than in the general population.[2] The risk of thrombosis varies according to the type of glomerulopathy. It is higher in membranous nephropathy, followed by membranoproliferative glomerulonephritis and minimal change disease.[4] We showed a case of membranous nephropathy complicated by RVT. Risk factors for thromboembolism in adult patients with nephrotic syndrome are a high urine protein to serum albumin ratio (predictive factor for venous thrombosis) as well as age, hypertension, diabetes and smoking (predictive factors for arterial thrombosis). The reasons underlying the hypercoagulable state in nephrotic patients are not clearly understood. There is evidence of ongoing subclinical coagulation as the measures of hemostasis activation, such as the level of plasma fibrinopeptide A and D-dimer are increased, Multiple hemostatic abnormalities have been described, including decreased levels of antithrombin and plasminogen (due to urinary losses), increased platelet activation, hyperfibrinogenemia, inhibition of plasminogen activation, and the presence of high-molecular-weight circulating fibrinogen moieties [5]. RVT can be unilateral or bilateral and can extend to inferior vena cava.[6] The course of RVT is more frequently chronic, but acute forms can occur. Although it has been suggested that a chronic RVT may lead to worsening proteinuria or kidney function, this has never been clearly documented [7]. RVT can be asymptomatic[2] or present with symptoms of renal infarction: flank pain, scrotal edema, microhematuria, increased levels of LDH and increase in size of kidneys on ultrasound.[8] Pathologic changes that suggest RVT include vascular congestion (particularly interstitial capillaries), disproportionate interstitial edema and later fibrosis, and vascular thrombosis [9]. Even though, gold standard method for the diagnosis of RVT is renal venography, less invasive procedures like ultrasound with Doppler, magnetic resonance or angiotomography are more frequently used. [10] We preferred to use ultrasound as it is easily accessible and risks of using contrast media are absent. The role of prophylactic anticoagulation (especially in patients with membranous nephropathy with severe nephrosis) has been debated [11]. A better understanding of

the risk of VTE in patients with MN is critical to balance the risks and benefits of prophylactic anticoagulation. [1] Thromboembolic events are a preventable cause of morbidity and mortality in patients with the nephrotic syndrome, especially MN.[12] The treatment of renal vein thrombosis includes anticoagulants and thrombolytic therapy [13]. Patients with RVT should be treated with heparin initially followed by warfarin, as in the cases of pulmonary embolism.[14] Oral anticoagulation is recommended for a period of 6 to 12 weeks and can be prolongated if the patient continues to be symptomatic.[2]

Conclusion

This case highlights that renal vein thrombosis should always be considered in the differential diagnosis of flank pain and hematuria. The diagnosis can be rapidly made with renal venography. A renovascular duplex ultrasound or magnetic resonance angiography can also be performed in patients with renal insufficiency. More importantly, delay in the diagnosis and treatment of renal vein thrombosis can result in significant morbidity, including loss of renal function, extension into the IVC, and pulmonary embolus. Treatment is anticoagulation therapy.

References

1. Lionaki S, Derebail VK, Susan L. Hogan, Barbour S, Lee T, et al. Venous Thromboembolism in Patients with Membranous Nephropathy. Clin J Am Soc Nephrol 2012; 7:43–51. 2. Daher EF, Junior GBS, Galdino GS, Eduardo DS, Wanderley TP, Lopes TS. Renal Vein Thrombosis and Membranous Nephropathy – Report of 2 Cases. J Medicine 2011; 12:73-6. 3. Andrassy K, Ritz E, Bommer J. Hypercoagulability in the nephrotic syndrome. Klin Wochenschr 1980; 58: 1029-36. 4. Llach F, Papper S, Massry SG. The clinical spectrum of renal vein thrombosis: acute and chronic. Am J Med 1980; 69: 819-27. 5. Radhakrishnan J. Venous Thromboembolism and Membranous Nephropathy: So What’s New? Clin J Am Soc Nephrol 2012; 7: 3–4. 6. Cade R, Spooner G, Juncos L, Fuller T, Tarrant D, Raulerson D, et al. Chronic renal vein thrombosis. Am J Med 1977; 63: 387-97. 7. Wagoner RD, Stanson AW, Holley KE, Winter CS: Renal vein thrombosis in idiopathic membranous glomerulopathy and nephritic syndrome: incidence and significance. Kidney Int 1983; 23: 368–74.

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8. Llach, F. Hypercoagulability, renal vein thrombosis, and other thrombotic complications of nephrotic syndrome. Kidney Int 1985; 28: 429-39. 9. Glen S. Markowitz Membranous Glomerulopathy: Emphasis on Secondary Forms and Disease Variants. Adv Anat Pathol. 2001 May;8(3):119-25. 10. Singhal, R, Brimble, KS. Thromboembolic complications in the nephrotic syndrome: pathophysiology and clinical management. Thromb Res 2006; 118: 397-407. 11. Glassock RJ: Prophylactic anticoagulation in nephrotic syndrome: A clinical conundrum. J Am Soc Nephrol 2001; 18: 2221–5.

12. Kendall AG, Lohmann RC, Dossetor JB: Nephrotic syndrome. A hypercoagulable state. Arch Intern Med 1971;127: 1021–7.

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13. Markowitz GS, Brignol F, Burns EB, et al. Renal vein thrombosis treated with thrombolytic therapy: case report and brief review. Am J Kidn Dis 1995;25(5):801–6. 14. Wu CH, Ko SF, Lee CH, Cheng BC, Hsu KT, Chen JB, et al. Successful outpatient treatment of renal vein thrombosis by low-molecular weight heparins in 3 patients with nephrotic syndrome. Clin Nephrol 2006; 65: 433-40

Case Report Brunner’s Gland Hamartoma - A Rare Cause of Upper GI Bleed: Report of Two Cases Emphasising Pathogenesis

Rakesh Kumar Gupta1, Ravindra Kumar Saran1*, Shivakumar Varakanahalli2 and Amarender Singh Puri2 Department of Pathology, G B Pant Institute of Postgraduate Medical Education and Research, Jawaharlal Nehru Marg, New Delhi, India 1 Department of Gastroenterology, G B Pant Institute of Postgraduate Medical Education and Research, Jawaharlal Nehru Marg, New Delhi, India 1

Keywords: Brunner’s gland, Hamartoma, Endoscopy, Gastrointestinal

ABSTRACT Brunner’s gland hamartoma (BGH) is a rare benign tumor of duodenum and mostly presents as an incidental finding. However, large lesions may cause obstructive symptoms and bleeding. Herein, we report two cases of BGH, one with upper gastro-intestinal bleed and other with recurrent vomiting which were managed successfully by endoscopic resection.

*Corresponding author: Dr Ravindra Kumar Saran, MD, DNB, Department of Pathology, Acaedmic Block, G B Pant Institute of Post graduate Medical Education and Research, Jawahar Lal Nehru Marg, New Delhi-110002. INDIA Phone: +91 9718599074 E-mail: ravindraksaran@hotmail.com

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Gupta et al.

Introduction

Brunnerâ&#x20AC;&#x2122;s gland hamartoma (BGH) is also known as Brunneroma and Brunner gland adenoma. It is a rare benign tumor of the duodenum which commonly occurs in fifth to sixth decade of life. It usually presents as an incidental finding. However, large polyps can present with obstructive features or gastrointestinal bleed. Duodenal bulb is the most common site of occurrence.[1] We report two cases of successful endoscopic removal of a large BGH arising from the first part of duodenum.

Case report 1

A 47-year-old male, referred to our hospital with the history of 2 discrete episodes of black tarry stools 3 weeks ago. Blood loss in each episode was approximately 500 ml followed by giddiness, palpitations and sweating. For these symptoms the patient was transfused three units of packed cells. No history of prolonged NSAID use was documented. Systemic examination was unremarkable except for mild pallor. Upper gastrointestinal endoscopy done revealed a large polypoidal mass measuring 3x3 cm with overlying erosions on the anterior wall of the duodenal bulb (Figure 1a). Contrast enhanced computer tomography showed a polypoidal mass in the first part of the duodenum measuring 26x16 mm (Figure 2). Polypectomy was attempted elsewhere but failed and so the patient was referred to our hospital. Polypectomy was attempted at our hospital using a forward viewing endoscope (Fujinon, Japan). The polyp was snared using polypectomy snare (Wilson-cook, USA). Post-operatively the patient had two episodes of hematemesis with malena which was managed with transfusion of two units of blood and two units of fresh frozen plasma. A relook endoscopy

C-191 done the next day showed a clean based ulcer with no signs of active bleed (Figure 1c). The patient was asymptomatic at the next follow-up visit 4 weeks later.

Case report 2

A 52-year-old female, presented to our hospital with the history of intermittent, non-retention vomiting since 1.5 years. No history of upper gastro-intestinal bleeding, prolonged NSAID use was documented. Upper gastrointestinal endoscopy done revealed a large polypoidal mass measuring 3x2 cm with overlying erosions on the anterior wall of the duodenal bulb. Rest of the duodenum, esophagus and stomach were unremarkable. Polypectomy was done using a forward viewing endoscope and the polyp was snared. Post-operatively patient was stable, discharged next day and later lost to follow-up. Pathological Findings: Both the specimens showed a similar histomorphology. Grossly, both the polyps were large, smooth surfaced with focal ulceration and congestion (Figure 1b). No stalks were identified in both the specimens. On microscopy, a polypoidal structure comprising of innumerable proliferating benign Brunnerâ&#x20AC;&#x2122;s gland (BG) separated by thick fibrocollagenous and fibromuscular septae and focal ulceration of the overlying mucosa was seen (Figure 3a). At places the septae showed adipose tissue, smooth muscle bundles and infiltration by aggregates of mature lymphocytes (Figure 3c). Focal gastric foveolar metaplasia was seen (Figure 3b). Helicobacter pylori infection was not detected in Giemsa stain. The BGs were diffusely and uniformly positive for MUC1 (Thermo-scientific), while few draining ducts and focal overlying surface epithelium with gastric foveolar metaplasia were MUC5AC (Neomarkers) positive (Figure 3d).

Fig. 1: Endoscopic photomicrographs of case 1. a: showing a polyp with focal surface ulceration, b: Gross image of polyp measuring 3 cm, c: post-operative day 3 image showing healing ulcer with clean base.

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Brunner’s Gland Hamartoma

Fig. 2: CECT images showing a polypoidal mass in the first part of the duodenum measuring 26x16 mm (arrow).

Fig. 3: Histomorphological and immunohistochemistry photographs a: A polypoidal structure comprising of innumerable proliferating Brunner’s gland (HEx40), b: Focal gastric foveolar metaplasia (arrow) of the overlying epithelium and submucosal duct (HEx40), c: Thick fibrocollagenous and fibromuscular septae with focal adipose tissue elements and mature lymphocytes (Hex100), d: Diffuse and uniform MUC1 positivity in Brunner’s gland and MUC5AC in the gastric foveolar metaplastic epithelium (x200).

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Discussion

Since its first description by Cruveilhier about 200 cases of BGH have been reported in the literature. [2] The mean size of the tumor is 2-3 cm but larger tumors can also occur which have a higher tendency for obstruction and bleeding. BGs are compound tubular submucosal glands exclusively found between the pylorus and the ampulla of Vater. Their major function is secretion of mucus-rich alkaline fluid protecting the intestinal epithelium and maintaining the pH for the functioning of the intestinal enzymes. The exact pathogenesis of the lesion is not known. However, some reports hypothesised that any cause of gastric hyperacidity or accelerated gastric emptying can leads to BG hyperplasia to counteract the damage to intestinal mucosa. [2, 3] Though, this hypothesis cannot be completely justified because such a cause will leads to a more generalized phenomenon of BGs hyperplasia rather an isolated polypoidal growth. Also, rarity of this lesion is not explained by numerous cases of gastric hyperacidity or in cases of short gastric emptying time. So, some other factors, still unknown, are likely to interact with the proliferating BGs leading to isolated polypoidal presentation. Absence of response to proton pump inhibitors (PPI) in the form of reduction of hyperplasia or decrease in size of BGH, in clinical setting is against the above hypothesis. By definition, BGH should have accompanying mesenchymal elements and their presence cannot be explained. Stolte et al suggested that diffuse hyperplasia of the duodenal glands is likely to be an adaptive reaction to the exocrine insufficiency of the pancreas or gastric hyperacidity and accelerated emptying of the stomach caused by chronic pancreatitis. [4] The secretion of urogastrone an inhibitor of gastric acid secretion by the BGs supports this hypothesis. A role of Helicobacter pylori (H. pylori) in the pathogenesis of BGH has been also described. [5]

C-193 absolutely benign. Hamartoma will be a more appropriate term for all such lesions revealing disorganization of the BGs contributed by admixed mesenchymal elements. Endoscopic resection is the treatment of choice; however in large polyps open surgery may be required. Recurrence after endoscopic or surgical excision has not been reported. However, one case with malignant transformation has been reported. [10]

Conclusion

To conclude, we report two symptomatic case of BGH treated successfully by endoscopic resection.

References

Chong et al subclassified BG lesions based on size and presence of mesenchymal elements. [9] They used the term’ hyperplasia’ for lesions <1cm; ‘adenoma’ for lesions >1cm and term ‘hamartoma’ if it contains mesenchymal elements too. However, in gastro-intestinal tract ‘adenoma’ is a selfexplanatory term used for a lesion with dysplastic change, and it should not interchangeably used for BGHs which are

1. Levine JA, Burgart LJ, Batts KP, Wang KK. Brunner’s gland hamartomas: clinical presentation and pathological features of 27 cases. Am J Gastroenterol. 1995;90:290-4. 2. Sen R, Gupta V, Sharma N, Chawla N, Kumar S, Malik S. Brunner gland hamartoma masquerading as malignancy; a rare case report. Middle East J Dig Dis. 2014;6:237-40. 3. Peison B, Benisch B. Brunner’s gland adenoma of the duodenal bulb. Am J Gastroenterol. 1982;77:276-8. 4. Stolte M, Schwabe H, Prestele H. Relationship between diseases of the pancreas and hyperplasia of Brunner’s glands. Virchows Arch A Pathol Anat Histol. 1981;394:75-87. 5. Kovacevic I, Ljubicic N, Cupic H et al. Helicobacter pylori infection in patients with Brunner’s gland adenoma. Acta Med Croatica. 2001;55:157–160. 6. Kim K, Jang SJ, Song HJ, Yu E. Clinicopathologic characteristics and mucin expression in Brunner’s gland proliferating lesions. Dig Dis Sci. 2013;58:194–201. 7. Sakurai T, Sakashita H, Honjo G, Kasyu I, Manabe T. Gastric foveolar metaplasia with dysplastic changes in Brunner gland hyperplasia: possible precursor lesions for Brunner gland adenocarcinoma. Am J Surg Pathol. 2005; 29:1442–1448. 8. Akaki M, Taniguchi S, Hatakeyama K, Kushima R, Kataoka H. Duodenal mucosal damage is associated with proliferative activity of Brunner’s gland hamartoma: a case report. BMC Gastroenterol. 2014;14:14. 9. Chong KC, Cheah WK, Lenzi JE, Goh PM. Benign duodenal tumours. Hepatogastroenterology. 2000;47:1298-1300. 10. Zanetti G, Casadei G. Brunner’s gland hamartoma with incipient ductal malignancy: report of a case. Tumori. 1981;67:75-8.

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Gastric foveolar metaplasia is a frequent finding noted in BGHs, which may further progress to the development of dysplastic changes. [6, 7] Our case also delineated focal gastric foveolar metaplasia of the overlying surface epithelium and few ducts which were highlighted by MUC5AC positivity. Akaki et al found that gastric foveolar differentiation is possibly related to the proliferative activity of Brunner’s glands. [8]

Case Report An Extremely Rare Distant non Communicating Uterine Horn Mimicking Parasitic Leiomyoma: A Diagnostic Dilemma Abhijit Das1, Nidhi Verma1*, Sompal Singh1, Namrata Nargotra1 and Rakesh Kumar Deepak2 1

Department of Pathology, North Delhi Municipal Corporation Medical College & Hindu Rao Hospital, Delhi, India 2 Department of Pathology, All India Institute of Medical Sciences, New Delhi, India Keywords: Unicornuate Uterus, Mullerian Anomaly, Pedunculated Leiomyoma, Parasitic Leiomyoma.

ABSTRACT Unicornuate uterus is a rare congenital anomaly which occurs due to partial development of mullerian duct system. Patient with unicornuate uterus usually present with dysmenorrhoea, hematometra and endometriosis during their reproductive period. In the absence of these symptoms, proper identification and diagnosis of mullerian anomaly becomes difficult. Sometimes during laparoscopy it can mimic parasitic leiomyoma, another rare benign condition, originated from a subserosal pedunculated leiomyoma. Even on imaging study it becomes difficult to differentiate a rudimentary uterine horn from a parasitic leiomyoma. We present here a case of a patient with a rare distant non communicating uterine horn, mimicking parasitic leiomyoma, which needed proper clinical history, radiological evaluation and histopathological examination to arrive at a correct diagnosis.

*Corresponding author: Dr Nidhi Verma, Department of Pathology, North Delhi Municipal Corporation Medical College & Hindu Rao Hospital, Delhi, India Phone: +91 8527271104 E-mail: drnidhikamal@gmail.com

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Introduction

Utero-vaginal anomalies are uncommon findings, among which unicornuate uterus is an extremely rare congenital anomaly, comprising 2-10% of all types of utero-vaginal anomalies.[1] Partial development of mullerian duct system causes various degrees of uterine horn, either connected or separated from the opposite horn. Rudimentary cavitary non communicating horn represents the A1b variants by class II of unicornuate uterus and it comprises of 7-40% of unicornuate uterus with rudimentary horn.[2] Subseroasal leiomyomas are present beneath the endometrial surface. They may be sessile or pedunculated or exophytic. When these leiomyomas disseminate from the uterus and develop an auxiliary blood supply and lose their original attachment from the uterus, they are called parasitic leiomyoma. Rarely may they also be iatrogenic, [3] and they become torsive causing an acute abdomen.[4] Sometimes these wandering leiomyomas may be confused with distant non-communicating rudimentary horn of uterus. We present here a case of distant non-communicating rudimentary horn of uterus which mimicked parasitic leiomyoma.

Case Report

A 22 year female presented with chronic pelvic pain and dysmenorrhoea. Per abdomen examination showed diffuse tenderness in the right lower abdomen however no definite mass was palpable. Her routine investigations were within normal limits. MRI was performed and on the basis of findings a possibility of rudimentary non communicating uterine horn was kept, however a differential diagnosis of parasitic leiomyoma was also kept because the mass was quite away from the uterus (Fig 1). Patient was planned

for surgery and laparoscopy showed a well circumscribed globular mass completely separated and 3cm away from uterus (Fig 2). The mass was completely excised and sent for histopathology. Grossly the mass was 4.5x3x2.5 cm in size, well circumscribed, firm and globular with smooth external surface (Fig 3a). Cut sections showed myometrium like tissue with a central small cavity (? rudimentary endometrial cavity) measuring 0.8x0.5x0.5 cm in size, filled with blood clots. Myometrium also showed multiple brownish black areas (?adenomyotic foci) ranging in size from 0.1 to 0.3 cm (Fig 3b). Microscopy showed endometrial cavity lined by functional endometrium and normal myometrium with thick walled blood vessels (Fig 4 and 5). Sections taken from the brownish black foci showed areas of adenomyosis (Fig 6). Presence of endometrial cavity lined by functional endometrium and absence of well circumscribed areas with long interlacing fascicles and presence of thick walled blood vessels ruled out the possibility of a parasitic leiomyoma. Thus a final diagnosis of distant non communicating rudimentary horn of uterus with extensive adenomyosis was given. The post operative recovery was uneventful and the patient was relieved of her complains. At 4 months follow up patient was absolutely well.

Discussion

Abnormalities of embryogenesis of mullerian duct system resulting in various female congenital anomalies are relatively common, however exact incidence is difficult to ascertain, as they are not always clinically symptomatic. [5] Their prevalence could be higher among women with infertility or obstetric complications.[6]

Fig. 1: MRI of pelvis shows a well circumscribed mass completely separated from the uterus.

Fig. 2: Laparoscopy shows same distant well circubscribed globular mass.

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Fig. 3a: Grossly circumscribed globular tissue with smooth external surface.

Fig. 3b: Cut section of the specimen shows an atrophic cavity filled with blood clot. Myometrium shoes numerous adenomyotic foci of variable sizes.

Fig. 4: Microscopy from the cavitary area shows myometrium lined by functional endometrium (H&E,100X).

Fig. 5: Microsection from the myometrium shows many thick walled vessels (H&E,100X).

Fig. 6: Microsection from the myometrium shows many adenomyotic foci (H&E,100X).

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Unicornuate uterus is a type II mullerian anomaly according to the American Fertility Society classification system [5] that occurs due to complete or partial failure of development of one mullerian duct and incomplete fusion with the contralateral side. In complete failure, failed mullerian duct leads to formation of an isolated hemiuterus without a contralateral structure,[7] whereas in partial failure it causes various degrees of rudimentary uterine horn. Again rudimentary horn is sub classified into communicating or non communicating with or without uterine cavity.[5-7] This rare condition is often related to poor reproductive function with greater incidence of primary infertility, preterm labor and pregnancy loss.[8] Various gynaecological conditions which are associated with unicornuate uterus include dysmenorrhoea (in 70% cases), hematometra (in 50% cases) and adenomyosis (in 20-40% cases).[9] Presence of dysmenorrhoea and adenomyosis are not very helpful in the diagnosis of rudimentary horn as these are frequently encountered in women of reproductive ages. However presence of hematometra in association with dysmenorrhoea and adenomyosis is an important clue to the diagnosis of mullerian anamoly.

Conclusion

Proper preoperative identification and diagnosis of mullerian anomaly is important to decide the right surgical approach, because surgical procedure is extremely influenced by the specific subtype and at the same time by the anatomical architecture of the uterus.[10] In our case MRI of pelvis showed a completely separated hypoechoic mass that was quite consistent with leiomyoma, focally a hyperintense area is noted on T2 weight imaging suggesting endometrial tissue. For these reasons it became difficult to arrive at a correct diagnosis and possibility of both uterine horn and parasitic leiomyoma were kept on imaging. Since parasitic leiomyomas are separated from the uterus they can be easily mistaken for an adnexal tumor. An ultrasonographic diagnosis of pedunculated leiomyoma can be established if a pedicle is demonstrated between leiomyoma and uterus. A parasitic leiomyoma where pedicle gets detached from the uterus may have the clinical and radiological presentation just like any other adnexal tumors. In our case the correct diagnosis was given only after histopathological examination. Identification of normal myometrium with presence of thick walled vessels and absence of long fascicles of smooth muscles with cigar shaped nuclei exclude the possibility of leiomyoma. Presence of well formed endometrial cavity with functional endometrial lining along with numerous adenomyotic foci established the diagnosis of rudimentary horn of uterus with adenomyotic foci.

None Declared

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In conclusion besides rarity, there are several interesting aspects of this case, firstly it mimicked parasitic leiomyoma on imaging and during laparoscopy it appeared quite away from the uterus unlike usual rudimentary horn. Another rare thing was the presence of adenomyosis in this distant non communicating rudimentary horn of uterus. Histopathology was helpful in reaching the correct diagnosis. One should keep a differential diagnosis of rudimentary horn of uterus in patients of reproductive age group presenting with dysmenorrhoea along with hematometra. Proper clinical history, investigations and histopathology may be helpful to arrive at a correct diagnosis.

Acknowledgements

We would like to thank our consultants from the department of Pathology, NDMCMC & HRH for their guidance, otherwise it would have been difficult to work on this topic.

Funding None

Competing Interests Reference

1. Fenton AN, Singh BP. Pregnancy associated with congenital abnormalities of the female reproductive tract. Am J Obstet Gynecol. 1952 Apr;63(4):744-55. 2. Buttram VC Jr, Gibbons WE. MĂźllerian anomalies: a proposed classification. (An analysis of 144 cases). Fertil Steril. 1979 Jul;32(1):40-6. 3. G Ravichandra , B Harishchandra, Upadhyaya K Krishnaraj, Acharya Devdas, Madhurkar Rohit. Parasitic uterine leiomyoma; years after hysterectomy: a case report. International journal of medical and applied sciences. 2013; 2(3): 222-225. 4. Yeh HC, Kaplan M, Deligdisch L. Parasitic and pedunculated leiomyomas: ultrasonographic features. J Ultrasound Med. 1999;18(11):789-94. 5. R. Iverson, A. DeCherney, and M. Laufer, â&#x20AC;&#x153;Clinical manifestations and diagnosis of congenital anomalies of the uterus,â&#x20AC;? Uptodate, 2012. 6. Chopra S, Keepanasseril A, Rohilla M, Bagga R, Kalra J, Jain V. Obstetric morbidity and the diagnostic dilemma in pregnancy in rudimentary horn: retrospective analysis. Arch Gynecol Obstet. 2009; 280 (6): 907-10. eISSN: 2349-6983; pISSN: 2394-6466

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7. Reichman D, Laufer MR, Robinson BK. Pregnancy outcomes in unicornuate uteri: a review. Fertil Steril. 2009 May; 91(5): 1886-94. 8. Morelli M, Venturella R, Mocciaro R, Lico D, Zullo F. An unusual extremely distant noncommunicating uterine horn with myoma and adenomyosis treated with laparoscopic hemihysterectomy. Case Rep Obstet Gynecol. 2013; 2013:160291.

9. Heinonen PK. Unicornuate uterus and rudimentary horn. Fertil Steril. 1997; 68(2): 224-30. 10. Fedele L, Bianchi S, Zanconato G, Berlanda N, Bergamini V. Laparoscopic removal of the cavitated noncommunicating rudimentary uterine horn: surgical aspects in 10 cases. Fertil Steril. 2005 Feb; 83(2): 432-6.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Case Report Primary Sino-Nasal T Cell Lymphoma in a Young Female: A Rare Case Report

Rumpa Das1*, Gorakh Nath2, Sangita Bohara1, Ritu Sharma1, Aarti B. Bhattacharya1 and Vivek Gupta1 Department Of Pathology, Hind Institute of Medical Sciences, Barabanki-225003, India 2 Department Of Otorhinolaryngology, Nath ENT Centre. Faizabad, India

Keywords: Sino-nasal Lymphoma, NHL, T cell NHL, Locally Destructive Mass, Rare Tumor

ABSTRACT Sino-nasal lymphoma is a rare tumor of the elderly, rare in females and extremely rarely seen in young patients. It usually appears as a locally destructive mass with non specific symptoms and diagnosed by histopathology and immunohistochemistry. The tumor is treated with a combination of radiotherapy and chemotherapy and has a poor prognosis. We present an extremely rare case of T cell NHL in a girl of 16 years age. Present case highlights the importance of clinical suspicion in sino-nasal NHLs of young patients because early management can significantly improve the prognosis and survival of the patient.

*Corresponding author: Dr Rumpa Das. Department Of Pathology. Hind Institute of Medical Sciences, Barabanki - 225003. INDIA Phone: +91 8795132000, 8756860132 E-mail: dr.rumpadas@gmail.com

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

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Sino-nasal T Cell Lymphoma

Introduction

Malignant lymphomas can arise in any part of the body and they are classified into hodgkins and non-hodgkins type. Primary sino-nasal lymphoma is a rare entity and usually of non hodgkins type. 10% of all primary non hodgkins lymphomas (NHL) arise in the head and neck. [1] Among the subtypes seen, natural killer (NK)/T cell NHLs are more common in Asian population and B cell lymphomas are more common in western population. The tumor usually affects the adults and elderly with a male predominance but rarely can affect the childrens and young adults. Sinonasal lymphomas have a worst prognosis compared to other lymphomas occurring in other part of the body. Early diagnosis of the tumor is important because large size of the tumor is directly related to its poorer prognosis. We present a rare case of a young girl with primary sinonasal T cell NHL of the maxillary sinus and briefly discuss the management.

Case Report

A sixteen years old girl came to our otorhinolaryngology clinic with complaints of unilateral nasal obstruction, pain and puffiness in half of her face.(Fig 1a) Anterior rhinoscopy revealed a mass present in the right nasal cavity, which was whitish in color and firm in consistency. The patient didnâ&#x20AC;&#x2122;t have any lymphadenopathy or any other mass present in her body. CT scan showed a large heterogenous enhancing mass present in the right maxillary sinus, extending to infratemporal fossa, involving massater muscle with loss of interface of pterygoid muscle, extending into apex of orbit, retro-orbital fossa with loss of interface of rectus muscle, optic nerve and with erosion and destruction of posterolateral, anterior and medial wall of maxillary sinus. (Fig 1b & c) The mass was also involving maxillary antrum, right nasal cavity, nasopharynx and pterigopalatine fossa. FNAC was done from the mass palpable through cheek and the smears showed heterogenous population of lymphoid cells comprising of small lymphocytes and some atypical looking lymphoid cells, some histiocytes and some neutrophils. The atypical lymphoid cells had enlarged, hyperchromatic and convoluted nuclei with scanty cytoplasm.(Fig 1d) A possibility of atypical lymphoproliferative disorder was suggested on the basis of cytological findings and the patient was advised an early biopsy. Histopathological section of the biopsy specimen showed diffuse proliferation of small to medium lymphoid cells having convoluted nuclei and coarse nuclear chromatin.(Fig 2a) The tumor was positive for CD 45 and CD 3 but was negative for CD 56 and CD 20. ( Fig 2b, 2c & 2d) Depending upon the histopathological and

immunohistochemical findings the tumor was diagnosed as T cell NHL of maxillary sinus which was extending into the nasal cavity. No tumor extension was noted on peripheral blood examination, bone marrow examination and radiological examination of chest and abdomen. The patient received a combination of radiotherapy and chemotherapy. She responded well and her tumor was reduced in size without any evidence of distant metastasis after six months of follow up.

Discussion

Primary malignant lymphomas of nose and paranasal sinuses are rare and accounts for 8% of all paranasal malignancies and 2% of all extranodal NHLs.[1] Extranodal lymphomas predominantly arise in the liver, soft tissue, dura and bone marrow.[1] Nasal NK/T cell lymphoma is a locally destructive tumor and was previously called as lethal midline granuloma, polymorphic reticulosis, midline malignant reticulosis, idiopathic midline destructive disease and lymphomatoid granulomatosis. The common presenting symptoms of sino-nasal NHLs are nasal obstruction, nasal discharge, epistaxis, unilateral cheek swelling and headache. Though not in our case, non-specific symptoms are commonly observed in NK/T cell lymphomas. These include - fever, weight loss, weakness, night sweat, increased susceptibility to infection, peripheral lymphadenopathy and splenomegaly. These non-specific symptoms are due to cytokine secretion by tumor cells and more commonly observed in NK/T cell lymphomas.[2] Early diagnosis of the tumor is difficult because the tumor develops in an anatomical space. The tumor remains undetectable until it becomes progressive and starts involving adjacent structures[1] as also seen in in our case. The tumor is located in the submucosa. It is non ulcerative on inspection and easily differentiated from squamous cell carcinoma but smooth surface makes it difficult to differentiate from minor salivary gland tumors and soft tissue sarcomas.[2,3] Possible etiological associations are immune deficiency, Epstein-Barr virus infection and infection by Human T lymphotropic virus.[4,5,6] CT scan of the tumor shows mild to moderate enhancement in the involved soft tissue. C. H Ou et al. conducted a study on CT and MRI findings of nasal NK/T cell lymphomas and documented that MRI is better than CT scan in demonstration of tumor lesion.[7] Cytological smears of the tumor shows a monomorphic population of atypical lymphoid cells and histopathology of the tumor shows a diffuse proliferation of small sized atypical lymphoid cells. The tumor cells have rounded hyperchromatic nuclei and

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Fig. 1: (a) Clinical photograph of the case showing puffiness in half of her face. (b & c). CT scan showing a large heterogenous enhancing mass present in the right maxillary sinus, extending into apex of orbit, retro-orbital fossa, right nasal cavity and nasopharynx. (d.) FNAC smear of the mass showing atypical lymphoid cells having enlarged, hyperchromatic and convoluted nuclei with scanty cytoplasm (H & E 400X) (d. Inset) : H & E 1000X ).

Fig. 2: (a) Histopathological section showing diffuse proliferation of small to medium lymphoid cells having convoluted nuclei and coarse nuclear chromatin. (H & E 100X) (a. Inset : H & E 400X ). (b) CD 45 positivity by tumor cells. (c) CD 3 positivity by tumor cells. (d) CD 56 negativity by tumor cells.

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Sino-nasal T Cell Lymphoma

coarse nuclear chromatin. The tumor shows positivity for CD 45 and CD 3 along with negativity for CD 20, indicating T cell origin. Sinonasal NK/T cell lymphomas show positivity for CD56 along with other T cell markers. Our case also showed positivity for CD 45 and CD 3, confirming the origin as T cell NHL. The microscopic differential diagnosis of sino-nasal T cell lymphomas are myeloid sarcoma, plasmacytoma, undifferentiated carcinoma and amelanotic melanoma.[1] Microscopically, myeloid sarcoma is composed of medium to large hematopoietic cells with fine nuclear chromatin and prominent nucleoli; whereas plasmacytoma is composed of plasmacytoid cells. Undifferentiated carcinoma consists of uniform tumour cells with ovoid vesicular nuclei and prominent nucleoli along with an inflammatory component comprising of predominantly lymphocytes; whereas amelanotic melanoma is composed of epithelioid or spindle cells. On histological sections, plasmacytoid cells or any epithelioid or spindle cell morphology was not seen in our case and thus the possibility of plasmacytoma and amelanotic melanoma was ruled out; however the possibility of myeloid sarcoma and undifferentiated carcinoma could not be ruled out on histopathology. Immunohistochemically all of the tumours show negativity for CD45 and our case was easily differentiated from these tumours by immunohistochemistry as our case showed positivity for CD 45.

and its early diagnosis is important for better outcome and long term survival of the patients.

After diagnosis is made, a through physical examination, blood examination, radiological examination of chest and abdomen and bone marrow examination should be done to determine the tumor invasion.[2]

5. Dictor M, Cervin A, Kalm O, Rambech E. Sinonasal T- cell lymphoma in the differential diagnosis of lethal midline granuloma using in situ hybridization for Epstein-Barr virus RNA. Mod Pathol 1996; 9(1): 7-14.

The treatment of choice of large sino-nasal NHL is a combination of radiotherapy and chemotherapy. The most common chemotherapeutic regimen is cyclophosphamide, doxorubicin, vincristin and prednisone (CHOP) regime. [6,8] Long term survival rate of the tumor is poor but a combined treatment of radiotherapy and chemotherapy has a better prognosis.[3]

Conclusion

To conclude, primary sino-nasal T cell NHL is a rare tumor of elderly male and its occurrence in a young female is extremely unusual. The tumor shows a poorer prognosis

Funding None

Competing Interests None Declared

Reference

1. Pai S, Vaidya KA, Thada N, Raj SMD, Sukesh. Primary sinonasal diffuse large B cell lymphoma masquerading as a benign nasal polyp: A rare case report with review of literature. International journal of basic and applied medical sciences 2014; 4(1):180-84. 2. Dalirasani Z, Mohtasham N. T cell lymphoma of palate with nose and maxillary sinus involvement: A case report. IJMS 2010; 35(3): 254-58. 3. Chalastras T, Elefteriadou A, Giotakis J, Soulandikas K, Korres S, Ferekidis E. Non-Hodgkins lymphoma of nasal cavity and paranasal sinuses. A clinicopathological and immunohistochemical study. Acta otorhinolaryngologica italic 2007; 27: 6-9. 4. Castro VLDS, Ferreira JB, Gimaraes VDC, Nery GV, Aires TFC, Alves W. Nasosinusal lymphoma of T natural killer cells: case report. Intl Arch Otorhinolaryngol 2011; 15(1): 102-05.

6. Reddy S, Kumar A, Allugohu R, Uppin M, Ramgopal K. Sinonasal T-cell cell lymphoma invading the brain: A case study. Asian J Neurosurg 2014; 9(4):235. 7. Ou CH, Chen CCC, Ling JC, Chai JW, Wu CH, Chen WH, Hung HC, Lee T, Ho TL. Nasal NK/T cell lymphoma: Computed tomography and magnetic resonance imaging findings. J Chin Med Assoc 2007; 7(5): 207-12. 8. Adwani DG, Arora RS, Bhattacharya A, Bhagat B. Non-Hodgkins lymphoma of maxillary sinus: An unusal presentation. Ann Maxillofac surg 2013; 3(1): 95-97.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Case Report Cytological Diagnosis of Fibromatosis Coli: A Rare Presentation of Infantile Neck Swelling

Abhijit Das*, Namrata Nargotra, Ila Tyagi, Ritu Arora and Sompal Singh Department of Pathology, North Delhi Municipal Corporation Medical College & Hindu Rao Hospital, Delhi, India Keywords: Fibromatosis Coli, Mesenchymal, Birth Trauma, Self Limiting.

ABSTRACT Fibromatosis coli is a rare benign mesenchymal lesion, usually presenting with neck mass in infant. Although etiology is not known, about half of the patients have history of birth trauma. It is self limiting fibroblastic lesion. Fine needle aspiration cytology is used as first line of investigation. If the diagnosis is made early, it shows an excellent prognosis. Most cases are managed conservatively. Here we present two interesting cases of fibromatosis coli which were diagnosed cytologically & showed complete regression after conservative management.

*Corresponding author: Abhijit Das, Department of Pathology, North Delhi Municipal Corporation Medical College & Hindu Rao Hospital, Delhi, India Phone: +91 9266456814 E-mail: das.abhijit.email@gmail.com

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

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Cytological Diagnosis of Fibromatosis Coli

Introduction

Fibromatosis coli also known as sternocleidomastoid tumor of infancy, is a benign self limiting fibroblastic lesion. Its occurrence is rare and around 0.4% of live birth.[1] The tumor is seen characteristically between 2-8 weeks of life as a firm mass of around 2-3 cm in diameter in the mid or lower part of sternocleidomastoid muscle.[2] Male infants are affected more commonly than females.[2] In about 50% of the cases there is history of complicated delivery.[2] One fourth of them may present with mild torticollis. Various congenital anomalies may be associated with fibromatosis coli. These tumours show spontaneous regression & eventually disappear completely with conservative management. Cytological diagnosis is non invasive, non expensive & invaluable to differentiate it from other inflammatory or neoplastic conditions of infancy.

Case Report(S)

We present two cases of fibromatosis coli one in a 3 weeks old female and other in a 17 days old male infant where both the cases were diagnosed on FNAC. Case 1: A 3 weeks old female infant presented with neck swelling in the middle part of sternocleidomastoid muscle, noticed by her mother for one week. The swelling was firm; measuring 1x1 cm. There was history of preterm labour with breech presentation. On clinical examination, there was restricted neck movement on the affected side. There were no other congenital anomalies. FNAC was advised. Cytology showed bland appearing fibroblasts with oval to spindle nuclei and muscle giant cells (Fig 1, 2) in a clear background. Mother was reassured about the management. The baby was treated conservatively in the form of physiotherapy, passive stretching of muscles. The swelling started reducing after 2 weeks of physiotherapy & neck movements returned to normal. After 3 months of treatment the mass disappeared completely. Case 2: A 17 days old male infant presented with torticollis, noticed by his parents for 2 weeks. On clinical examination there was a firm, fusiform swelling, 2x1.5 cm in diameter in the lower part of sternocleidomastoid muscle. There was no history of prolonged labor or birth trauma. Contact history of tuberculosis was positive. Clinically diagnosis of both tuberculous lymphadenitis & sternocleidomastoid tumor of infancy were suggested. Ultrasonography of neck was advised, which was suggestive of cervical lymphadenopathy. For confirmation FNAC was done. Cytosmear showed benign looking oval to spindle fibroblasts, degenerated atrophic muscle & muscle giant cells (Fig 3, 4, 5). Diagnosis of fibromatosis

coli was given. Conservative management including gentle passive stretching was advised. Parents were educated about the techniques. After one week of physiotherapy, the swelling started to reduce with returning to near normal neck movements.

Discussion

Sternocleidomastoid tumour of infancy is an infrequent lesion presenting as neck mass. Fibromatosis coli is seen around 0.4% of all newborn. It usually occurs in the middle or lower part of the sternocleidomastoid muscle.[3] Etiology is not clearly understood, although 50% of cases are associated with a history of birth trauma.[4] Most infants present to OPD with congenital torticollis.[5] It usually appears at 2-4 weeks of age with a male predominance.[6] One of our patients is a female. Most of cases are diagnosed before 6 months of age. Both our patients were in the same age group. Causative factors are not known, however various theories have been proposed, which include fetal malposition, birth trauma, ischemic necrosis following vascular compression during birth, infections & presence of other endogenous factors.[7] Among these birth trauma is more commonly associated with fibromatosis coli, which was present in our first case. Another study by Kurtyez et al also showed the similar findings.[8] These tumors are sometimes associated with other congenital anomalies like club foot, congenital dislocation of hip etc,[1] however no such congenital anomalies were seen in our cases. Differential diagnosis of fibromatosis coli include congenital lesions like branchial cyst, thyroglossal cyst, inflammatory lesion like tuberculous lymphadenitis, benign neoplastic lesions like hemangioma, cystic hygroma or malignant neoplasms like neuroblastoma, rhabdomyosarcoma & lymphoma.[1] FNAC is the first line of investigation & helps to exclude

Fig. 1: Cytosmear shows bland appearing fibroblasts and muscle giant cells (Giemsa, 100X).

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Fig. 2: Cytosmear shows bland appearing fibroblasts with oval to spindle nuclei (Giemsa, 400X).

Fig. 3: Cytosmear showed benign looking oval to spindle fibroblasts, degenerated atrophic muscle & muscle giant cells (Giemsa, 40X).

Fig. 4: Cytosmear shows benign looking oval to spindle fibroblasts, degenerated atrophic muscle & muscle giant cells (Giemsa, 100X).

Fig. 5: Cytosmear showing degenerated atrophic muscle & muscle giant cells (Giemsa, 400X).

other differentials, as both our cases were diagnosed on FNAC. FNAC is cheap, non invasive, reliable diagnostic tool that helps to avoid nore invasive & costly procedures. Imagings like ultrasonography, computed tomography may be of help in diagnosis of fibromatosis coli, however high cost & less availability limit their roles.[9] There are few studies available in the literature that described cytological features of fibromatosis coli.[1,10,11] The characteristic cytological features described are bland appearing fibroblasts, degenerative atrophic skeletal muscles & muscle giant cells in a clear background. Similar findings are also seen in our cases. These rare infantile lesions are mostly managed by conservative treatment. Managements include passive stretching of the muscle & physiotherapy in early diagnosed cases, while for children greater than

one year or refractory cases, surgical interventions may be required.[12] Late diagnosed cases show worst prognosis.[1] Both of our patients were diagnosed confidently at an early stage & following conservative treatments both are doing well and are disease free.

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Conclusion

Fibromatosis coli is a self limiting fibroblastic lesion of infancy & it has a characteristic clinical presentation & history. These cases highlight vital roles of FNAC in diagnosis & reassurance of anxious parents. Cytology avoids unnecessary surgical intervention & guides for conservative managements. Hence fibromatosis coli should always be considered in the differentials of neck swelling in infants.

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Acknowledgements

We would like to thank our consultants of Department of Pathology, Hindu Rao Hospital for their guidance, otherwise it would not have been possible to work on it.

Funding None

Competing Interests None Declared

Reference

1. Khan S, Jetley S, Jairajpuri Z, Husain M. Fibromatosis colli - a rare cytological diagnosis in infantile neck swellings. J Clin Diagn Res. 2014;8(11):FD08-9. 2. McLeod DL, Geisinger KR, Hopkins MB, Silverman JF: Fine needle aspiration cytology of fibromatosis: A clinical and cytopathologic assessment (abstr). Acta Cytol 1987;31:68. 3. Baisakh MA, Mishra M, Narayanan R, Mohanty R. Cytodiagnosis of sternocleidomastoid tumour of infancy. J Cytol. 2012;29:149-51. 4. Goldblum JR, Weiss SW. Fibrous tumours of infancy and childhood. In: Goldblum JR, Weiss SW. (Eds):Enzeinger and Weiss’s Soft tissue tumours. 4th ed, St.Louis,Mosby, 2001; 273-75.

5. Ostrowski ML, Bradshaw J, Garrison D: Infantile myofibromatosis: Diagnosis suggested by fine-needle aspiration biopsy. Diagn Cytopathol 1990;6:284–288. 6. Porter SB, Blund BW. Pseudotumour of infancy and congenital muscular torticollis. Am Family Phys. 1995;52:1731-36. 7. Kumar B, Pradhan A. Diagnosis of sternomastoid tumour of infancy by fine needle aspiration cytology. Diagn Cytopathol. 2011;39:13-17. 8. Kurtycz DF, Logrono R, Hoerl HD, Heatley DG. Diagnosis of fibromatosis colli by fine needle aspiration. Diagn Cytopathol. 2000;23:338-42. 9. Chakrabarti I, Bandopadhyay A, Goswami BK. Fine Needle Aspiration Cytology of Fibromatosis Colli. Turkish Journal of Pathology. 2010;26:243-44. 10. Sharma S, Mishra K, Khanna G. Fibromatosis Colli in Infants. A cytologic study of eight cases. Acta Cytol. 2003;47:359-62. 11. Rajalakshmi V, Selvambigai G, Jaiganesh. Cytomorphology of fibromatosis colli. J Cytol. 2009;26:41-42. 12. Raab SS, Silverman JF, McLeod DL, Banning TL, Geisinger KR: Fine needle aspiration biopsy of fibromatosies. Acta Cytol. 1993;37(3):328-28.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Case Report Primary Splenic Lymphoma Presenting As Splenic Abscess: A Rare Entity

Anshu Jain1, Noora Saeed1*, Veena Maheshwari1 and Abdul Bari2 Department of Pathology, JNMC, AMU, Aligarh, U.P, INDIA, Department of Radiodiagnosis, JNMC, AMU, Aligarh, U.P, INDIA, 1

Keywords: Primary, Lymphoma, Spleen, Abscess.

ABSTRACT Primary splenic lymphoma are rare lymphomas which comprise less than 1% of the all malignant lymphoma. We present a case of 35 years old female with fever and pain in abdomen for a period of 20 days. The main purpose of this paper is to report a rare occurrence of primary splenic lymphoma and to demonstrate the possibility of it being misdiagnosed as splenic abscess clinically and on imaging.

*Corresponding author: Dr Noora Saeed, Department of Pathology, JNMC, AMU, Aligarh,U.P, INDIA, Phone: +91 9997240844 E-mail: dr.noorasaeed@gmail.com

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Primary Splenic Lymphoma presenting as Splenic Abscess

Introduction

nucleus, prominent nucleolus, focally prominent and atypical mitotic activity. Section also showed large areas of ischemic necrosis mixed with neutrophilic infiltrate (figure 2b&2c). Immunohistochemistry revealed positivity for CD 20 (figure 2d) while tumor cells were negative for CD5 and CD10. Following this a final diagnosis of DLBCL was made. The patient had received six cycles of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) postoperatively and was disease free and was on regular follow up for seven months.

Non-Hodgkin lymphoma (NHL) originates from lymphovascular system and can be classified as B cell lymphomas (about 90% of all NHL) and T cell lymphomas (10% of all NHL).[1] Since spleen is the major site for filtration of blood so primary tumors of spleen are generally haematological malignancies, in which most are lymphomas.[2] However primary splenic lymphoma (PSL) is an extremely rare neoplasm with a reported incidence of less than 1%.[3] To define primary splenic lymphoma, the lymphoma should be confined to the spleen and/or splenic hilar lymph nodes, although few authors also accept the presence of bone marrow involvement.[4] Histomorphologically great majority of cases are of B-cell type, with diffuse large B-cell lymphoma being the most common.[5] PSL of spleen is difficult to diagnose with the common differentials being hemangioma, lymphangioma, abscess, hamartoma, infarct and metastatic disease.[6] The patients usually present with abdominal pain or mass due to splenomegaly, with or without fever, systemic upset, or thrombocytopenia.[4]. Here, we present a case of PSL in a 35-year-old female patient, which was initially diagnosed as splenic abscess.

Case Report

A 35-year-old female patient presented with fever and pain abdomen for 20 days. General examination revealed the presence of mild pallor. On abdominal examination, spleen was palpable approximately 7 cm below the left costal margin. On palpation spleen was nodular and firm in consistency. No other significant systemic finding was noted. Complete blood count of patient revealed microcytic hypochromic anemia (hemoglobin level - 10 g/dL). The total and differential counts were within normal limits. ESR was found to be raised. Other routine investigations including liver function test, renal function test, and lactate dehydrogenase levels were within the normal limits. An ultrasonogram of the abdomen revealed single well circumscribed, lobulated hypoechoeic lesion which favoured the diagnosis of splenic abscess. The computed tomographic findings of abdomen revealed a circumscribed rounded, low attenuated lesion (figure 1a&1b). For splenomegaly the patient underwent splenectomy (figure 1c). Grossly spleen measured 12x10x9cms and weighed 1.3kg. Cut section revealed a single white nodule mixed with necrotic areas, involving almost whole of the spleen and measuring 11x 9x9cm (figure 1d). Imprint cytology of the specimen showed many degenerated atypical cells with prominent nucleoli mixed with inflammatory cells and necrosis (figure 2a). Histopathology showed diffuse sheets of large sized cell with central vesicular

Discussion

The definition of PSL is variable and remains a matter of controversy. The earliest definition was given by Dasgupta et al., who defined PSL as a condition confined to spleen or hilar lymph nodes with no recurrence of disease for at least 6 months after splenectomy.[7] In the present case, the patient fulfilled the criteria of this definition. On the other hand, Skarin et al [8] and Kehoe J et al [9] suggested that the diagnosis of PSL be made if splenomegaly is a predominant feature in any lymphoma involving the spleen. While Kraemer et al [10] recommended the diagnosis of PSL, as patients with splenomegaly, cytopaenia of at least two haematological cell lines and the absence of peripheral adenopathy. The etiology of splenic lymphoma largely remains unknown. However it has been suggested that chronic hepatitis due to hepatitis C virus infection along with some poorly defined genetic and environmental factors play a significant role in its development.[11] Symptoms of PSL may be variable, with few presenting as fever, weight loss, weakness and left upper quadrant discomfort, mimicking clinically as splenic abscess.[12] In the present case, the patient presented with fever along with pain abdomen and splenomegaly. Among the lab findings, significant features include cytopenia or increased ESR or b2 microglobulin level. [13] In our case also the patient had increased ESR along with microcytic hypochromic anemia. The radiological investigation of choice for the diagnosis is contrast enhanced computed tomography (CECT), in which the lesions appear hypoechoic, but sometimes anechoic areas may be seen, which suggest liquefactive necrosis thereby causing confusion with splenic abscess.[8] In our case both clinical presentation and imaging studies mimicked splenic abscess. The common differential diagnoses of solitary splenic lesion include hemangioma, lymphangioma and hamartoma.

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Fig. 1a: Computed tomography showing massive splenomegaly (axial section); 1b- Computed tomography showing massive splenomegaly (coronal section); 1c- Per opereative photo showing ligation of splenic hilum; 1d- Cut Section shows single large grayish white lesion with abundant central necrosis and a rim of normal splenic parenchyma at the periphery.

Fig. 2a: Imprint cytology showing occasional degenerated scattered atypical lymphoid cells in a hemorrhagic background (H&Ex 40x); 2b- Low power view showing sheets of monotonous tumor cells mixed with areas of necrosis (H&Ex 10x); 2cHigh power view showing tumor cells with prominant nucleoli (H&Ex 40x); 2d- Immunohistochemistry of primary splenic lymphoma showing CD20 positive tumor cells (x40x)

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However in an afebrile and symptomatic patient the likely diagnosis is a metastatic lesion. In cases of acute ischemic lesion, the condition is usually painful and clinical evidence of the predisposing cause for example trauma, is usually present. Febrile or immunocompromised patients, splenic abscess are a possible diagnosis. As the patients of PSL usually have no lymphadenopathy, and they have a normal general blood picture, diagnosis becomes difficult. [8]

with immunophenotyping. On microscopic examination of spleen, small, mature lymphocytes surrounding the germinal centers is seen in the white pulp and sometimes infiltrating the red pulp. Immunophenotyping shows expression of B-cell antigens and surface immunoglobulin, along with lack of expression of CD5, CD10, CD43 and CD103. [22]

Grossly primary splenic lymphoma has been classified into four categories: (1) Homogeneous enlargement without masses, (2) miliary masses, (3) 2-10 cm masses and (4) large solitary mass. Our case was fitting in the last category i.e. a large solitary tumor occupying almost 3/4th of the spleen.[14] When considering the type of primary splenic lymphoma, most are of B cell origin, with the common subtypes (seen in almost 50Â % of cases) being low-grade or intermediate-grade lymphomas.[15] Diffuse large B-cell lymphoma (DLBCL) of spleen is the most common type, accounting for almost 1/3 of the primary splenic lymphoma cases. [16]Next in frequency come the splenic marginal zone lymphomas (SMZL), [17] while less common one include mantle cell lymphoma, Tcell rich large B cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, follicular lymphoma and hepatosplenic T cell lymphomas. DLBCL of spleen usually presents as a single or multiple confluent nodules with majority being derived from the white pulp.[18] Other lymphomas with similar pattern of involvement are those in which spleen is secondarily involved. These include SMZL, follicular lymphoma and small lymphocytic lymphoma.[19] DLBCL usually occur in sixth and seventh decade of life, more commonly presenting as a single tumor mass, which is large and occupies more than 50Â % of the spleen; this pattern however, is not specific to primary DLBCL as secondary involvement by DLBCL will also have the same pattern. Bone marrow involvement is usually negative at the time of presentation.[16] Microscopically DLBCL have large cells with vesicular to clumped chromatin, containing one to many nucleoli and relatively abundant cytoplasm. They are positive for B cell markers CD19 and CD20 with variable expression of germinal centre markers CD10 and BCL6. [20] Next common differential of splenic lymphoma is SMZL. It is characterized by cytopenias, splenomegaly or detection of middle sized mature villous lymphocytes on peripheral blood smear [21]. Diagnosis is usually made by histological examination of the spleen/ bone marrow, along

Other lymphomas with similar pattern of splenic involvement are follicular lymphoma, which on histopathology reveals preserved or mild alteration of germinal center, having centrocytes (i.e., small, mature and cleaved lymphocytes) often with CD 10 positivity and overexpression of BCL2; mantle cell lymphoma, which presents with mature lymphocytes having irregular nucleus and positivity for CD5 while negative for CD23 along with universal overexpression of cyclin D1; and lastly small lymphocytic lymphoma, which presents as expanded and effaced white pulp areas due to small lymphocytes with condensed chromatin, high nucleus-to-cytoplasm ratio and positivity for both CD5 and CD23. [23] The present case was diagnosed as NHL - large cell type on histopathology. However final diagnosis of diffuse large B-cell type NHL was rendered only after immunohistochemistry showed CD20 positivity only. A staging system has also been proposed for PSL: Stage I - Tumor confined to spleen only; Stage II - Involvement of spleen and hilar lymph nodes; Stage III - Involvement of extra splenic nodes or liver[14]. In this case since the hilar lymph nodes were not involved, the patient had Stage I disease. There are controversies with regard to the treatment protocol for PSL. The proposed methods include splenectomy only, splenectomy followed by chemotherapy, splenectomy followed by radiotherapy or a combination of chemotherapy and radiotherapy.[24] The standard chemotherapeutic regimen used is CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Some authors suggest that the overall survival rates are better with early splenectomy, followed by multidrug chemotherapy compared with splenectomy alone or splenectomy, followed by single agent chemotherapy.[25]

Conclusion

Primary splenic lymphoma is an extremely rare entity. Both radiologically and clinically they can mimic splenic abscess that may delay the proper diagnosis and management. Hence, it is important that the clinicians keep this differential diagnosis in their minds while dealing with similar cases and undertake necessary steps like biopsy and immunohistochemical analysis to reach the correct diagnosis.

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Acknowledgements

We would like to acknowledge the technical staff of the histopathology lab and the departmental head for their general support.

Funding

None Declared

Competing Interests None Declared

Reference

1. Coiffier B. Monoclonal antibody as therapy for malignant lymphomas. C R Bio. 2006;329(4):241254. 2. Giovagnoni A., Giorgi C., Goteri, G. Tumours of the spleen. Cancer Imaging. 2005;5(1): 73-77. 3. Li M, Zhang L, Wu N, Huang W, Lv N. Imaging findings of primary splenic lymphoma: A review of 17 cases in which diagnosis was made at splenectomy. PLoS One 2013;8:e80264 4. Falk S, Stutte H J. Primary malignant lymphomas of the spleen. A morphologic and immunohistochemical analysis of 17cases. Cancer. 1990;66:2612-2619 5. Ambulkar I, Kulkarni B, Borges A et al. Primary non-Hodgkin’s lymphoma of the spleen presenting as space occupying lesion: a case report and review of literature. Leuk Lymphoma. 2006;47: 135-139 6. Konstantiadou I, Mastoraki A, Papanikolaou IS, Sakorafas G, Safioleas M. Surgical approach of primary splenic lymphoma: Report of a case and review of the literature. Indian J Hematol Blood Transfus. 2009;25:120-4. 7. Dasgupta T, Coombes B, Brasfield RD. Primary malignant neoplasms of the spleen. Surg Gynecol Obstet. 1965;120:947-60. 8. Skarin AT, Davey FR, Moloney WC. Lymphosarcoma of the spleen. Results of diagnostic splenectomy in 11 patients. Arch Intern Med. 1971; 127(2): 259–65. 9. Kehoe J, Straus DJ. Primary lymphoma of the spleen. Clinical features and outcome after splenectomy. Cancer. 1988; 62(7):1433–8. 10. Kraemer BB, Osborne BM, Butler JJ. Primary splenic presentation of malignant lymphoma and related disorders. A study of 49 cases. Cancer. 1984; 54(8):1606–19. 11. Cotta-Pereira R, Acar R, Mello CE, Iglesias AC, Basílio-de-Oliveira CA. Primary spleen lymphoma associated with hepatitis C virus infection. BMJ Case Rep. 2011;2011. www.pacificejournals.com/apalm

12. Kim JK, Hahn JS, Kim GE, Yang WI. Three cases of diffuse large B-cell lymphoma presenting as primary splenic lymphoma. Yonsei Med J. 2005;46:703-9. 13. Morel P, Dupriez B, Gosselin B, Fenaux P, Estienne MH, Facon T, et al. Role of early splenectomy in malignant lymphomas with prominent splenic involvement (Primary lymphomas of the spleen)- A study of 59 cases. Cancer. 1993;71(1):207–15. 14. Ahmann, D.L., Kiely, J.M., Harrison, E.G. Jr., Payne, W.S. Malignant lymphoma of the spleen: a review of 49 cases in which the diagnosis was made at splenectomy. Cancer. 1996;19(4):461-469. 15. Gobbi P, Grignani G, Pozzetti U, Bertoloni D, Pieresca C, Montagna G, et al. Primary splenic lymphoma: Does it exist? Haematologica. 1994;79:286–293. 16. Torlakovic E: Bone Marrow Workshop Prague 2012 17. Kraus MD, Fleming MD, Vonderheide RH. The spleen as a diagnostic specimen: a review of 10 years’ experience at two tertiary care institutions. Cancer. 2001 Jun 1;91(11):2001-9. 18. Kashimura M, Naro M, Akikusa B, et al. Primary splenic DLBCL manifesting in red pulp. Virchows Arch. 2008;453:501–509. 19. Mollejo M, Algara P, Mateo MS, Menárguez J, Pascual E, Fresno MF, et al. Large B-cell lymphoma presenting in the spleen: identification of different clinicopathologic conditions. Am J Surg Pathol. 2003;27(7):895-902. 20. Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood. 2008;111(8):3941-67. 21. Arcaini L, Lazzarino M, Colombo N, Burcheri S, Boveri E, Paulli M, et al. Splenic marginal zone lymphoma: a prognostic model for clinical use. Blood. 2006;107:4643-49. 22. Arcaini L, Paulli M. Splenic marginal zone lymphoma: hydra with many heads? Haematologica. 2010;95:534-537. 23. Sternberg, Stephen S., Stacey E Mills, and Darryl Carter. Sternberg’s Diagnostic Surgical Pathology. 5th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins, 2010. 24. Kim JK, Hahn JS, Kim GE, Yang WI. Three cases of diffuse large B-cell lymphoma presenting as primary splenic lymphoma. Yonsei Med J. 2005;46:703-9. 25. Healy NA, Conneely JB, Mahon S, O′Riardon C, McAnena OJ. Primary splenic lymphoma presenting with ascites. Rare Tumors. 2011;3:e25. eISSN: 2349-6983; pISSN: 2394-6466

Case Report Persistent Eosinophilia in Patients with Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma and TP53 Deletion is a Potential Predictor of Variant Richter’s Transformation Madhu M. Ouseph1, James N. Butera2, Rogers C. Griffith1, Dariusz Stachurski1 and Diana O. Treaba1* Department of Pathology and Laboratory Medicine, Rhode Island Hospital/The Warren Alpert School of Medicine at Brown University 2 Divison of Hematology/Oncology, Rhode Island Hospital/The Warren Alpert School of Medicine at Brown University

Keywords: Eosinophilia, Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, Classical Hodgkin Lymphoma

ABSTRACT Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) progression to diffuse large B-cell lymphoma (Richter’s syndrome) is noted in approximately 3 - 16% of the patients. However, transformation to classical Hodgkin lymphoma occurs in only 0.5% of the patients and has no clinically well-defined predictors. We report three patients with CLL/SLL and TP53 deletion in whom persistent eosinophilia preceded the Hodgkin lymphoma transformation.

*Corresponding author: Diana O. Treaba, MD, Department of Pathology and Laboratory Medicine, Rhode Island Hospital, APC 12, 593 Eddy Street  Providence, RI, 02903 Phone: 401-444-8897 E-mail: dtreaba@lifespan.org

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In July 1928, Dr. Maurice Richter published the first case of chronic lymphocytic leukemia transformation to a large cell tumor, later recognized as diffuse large B-cell lymphoma (DLBCL). Transformation to DLBCL (Richter’s syndrome) has an incidence of approx. 3-16% in patients with CLL/SLL and represents the most commonly seen secondary malignancy in these patients. Classical Hodgkin lymphoma (CHL) transformation in patients with CLL/SLL, second in occurrence after DLBCL, sometimes designated variant Richter’s transformation, has a 10-year cumulative incidence of 0.5%.[2,3] Unlike more commonly seen transformation to diffuse large B-cell lymphoma, clinical and laboratory features predictive of transformation are not well defined in the variant transformation. Though peripheral blood eosinophilia is seen frequently in patients with CHL, the utility of eosinophilia as a marker for variant transformation is unknown. Here we report three patients with CLL/SLL all with associated TP53 deletion, who had or developed sustained peripheral blood eosinophilia at the time of their transformation to CHL. This unusual association suggests that peripheral blood eosinophilia could potentially be used as a marker for early identification of transformation of CLL/SLL to CHL. [1]

Case Reports

The first patient is a 59-year old male with a 6-year history of CLL/SLL, with initially reported deletion 13q and trisomy 12, who subsequently developed TP53 deletion and was started on chlorambucil (switched from fludarabine, cytoxan and rituxan due to intolerance) but continued to show progression of his retroperitoneal lymphadenopathy and was diagnosed with progression to CHL on bone marrow and presacral biopsies. The Hodgkin/ Reed-Sternberg (HRS) cells were CD30, PAX-5 and CD15 positive and were negative for CD45 and CD20 expression. His blood counts were remarkable for lymphocytosis and eosinophilia (fig.1A) at the time of variant Richter’s transformation. His CHL was treated with ABVD and his eosinophilia reverted in proportion to the response of CHL to chemotherapy. He died of sepsis 16 months after his diagnosis of variant Richter’s transformation.

Epstein-Barr virus latent membrane protein 1 (LMP1) and negative for CD15, CD20 and CD45 while the associated CLL/SLL lymphoid cells had a classic CLL/SLL immunophenotype. Two months before her CHL diagnosis the patient developed eosinophilia that disappeared after ABVD therapy. Interestingly, the biopsy sections showed an increased population of eosinophils in the residual CLL/ SLL areas (fig.1C). The patient died within a year after her diagnosis of CHL.

Fig. 1: (left): Patient #1, A. Peripheral blood smear, lymphoid cells and an eosinophil (Wright-Giemsa stain, Ob. 50x, immersion oil); Patient #2, B. Hodgkin-Reed Sternberg cells (Hematoxylin and eosin stain, Ob. 50x, immersion oil); Patient #2, C. Area of CLL/SLL with increased population of eosinophils (Hematoxylin and eosin stain, Ob. 50x, immersion oil).

The second patient is a 79-year old female, with a clinical history of thymoma (58 years ago), and invasive breast carcinoma (17 years ago) recently diagnosed with CLL/ SLL with associated deletions of chromosome 13q and 17p (TP53). She underwent therapy with bendamustine and progressed to CHL 2 years after her CLL/SLL diagnosis. The / HRS cells (fig. 1B) were positive for CD30 and

Fig. 2: (right): Patient #3, A. Area of CHL transformation, Ob. 50x, immersion oil, Hematoxylin and eosin stain; Patient #3, B. CD30 positive HRS-cells (Hematoxylin and eosin stain, Ob. 50x, immersion oil).

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Eosinophilia and Variant Richter’s Transformation

The third patient is a 60-year old male with CLL/SLL diagnosed 7 years before, who was started on ibrutinib after being on conservative management for six years due to disease progression and identification of TP53 deletion. He was diagnosed with CHL transformation (fig.2A) while on ibrutinib. The HRS cells in this patient had a classic immunophenotype: CD30+ (fig.2B), CD15+, CD45- and CD79a- with a subset being positive with the anti-Epstein Barr virus latent membrane protein 1 (LMP1) antibody. The patient was also noted to have persistent eosinophilia for three months before his diagnosis of transformation, which reverted after initiation of treatment for Hodgkin lymphoma (R-ABVD and obinutuzumab). He is alive after he underwent bone marrow transplantation.

Discussion

CLL/SLL, though generally thought to have an indolent course, can progress to a higher-grade lymphoma in 1-11% patients at rate of 0.5 to 1% per year.[3,4]Most of these patients fall into category of well-characterized classic Richter’s transformation with development of diffuse large B-cell lymphoma (DLBCL).[4,5] The “variant” transformation, the transformation of CLL/SLL to CHL has a lower incidence, but has been recognized in various case reports and case series in the literature[2,6-19] with approximately 100 cases reported in the medical literature to date. CHL can occur in CLL/SLL patients with active disease as well as in patients in remission.[] with a median time of diagnosis from CLL/ SLL diagnosis of 4.3 to 6.2 years (range 0-26 years).[3,20-21] Similar to Richter’s transformation, variant transformation is associated with an overall significant reduction of median survival (0.8 to 1.7 years after diagnosis of CHL) in comparison to de novo classical Hodgkin lymphoma cases, survival being worse in patients who had prior treatment for CLL.[3,19-20] The variant transformation consists most often of the mixed cellularity subtype and may be associated by Epstein-Barr virus.[19] The cell of origin is not well established, but is hypothesized to be transformed B lymphoid cells arising in V(H) mutated CLL, though clonal similarity studies have shown conflicting results, unlike in cases of transformation to DLBCL, where up to 80% of the cases arise from the same clone.[9,20,22-24]” Patients with clonally unrelated DLBCL, have prognosis comparable to DLBCL arising de novo, in contrast to the cases with related clones”. [22] Whether a similar difference is present in cases of variant transformation is unclear, but it may be important in management decisions.[20] Furthermore, the cellular milieu of RS cells in CHL can consist of CLL/SLL cells or a background of inflammatory cells. It is still unclear whether clonal similarity between CHL and CLL/SLL

cells has any impact on the cellular milieu[19,23,25-27] It might appear from the current literature that at least few of the instances of CHL arising in CLL/SLL could represent a CLL/SLL independent neoplasia. In the single case published of systemic eosinophilia accompanying CHL transformation in CLL, the results of fragment length comparison analysis of IGHV PCR suggested no clonal relationship between the two components.[28] Irrespective of whether all the cases represent transformation of CLL/ SLL clone, early identification of CLL/SLL patients who are developing CHL is important. While different molecular risk factors like homology of immunoglobulin heavy chain, absence of del13q14, aberrant high CD38 and ZAP70 expression as well as presentation with high CLL/SLL tumor burden (defined by lymph nodes, Binet stage and serum LDH) are significant predictors of future transformation to DLBCL, no similar well-defined predictors are known for progression to CHL. [4,29] Similarly, TP53 loss/mutations and MYC amplification/ translocations are thought to be high risk factors for future DLBCL transformation, while the role of these factors in context of variant transformation is unclear.[22] The patients with variant transformation presented in this study had TP53 deletion, but this observation needs to be validated. Primary systemic eosinophilia is known to be associated with clonal lymphoid and myeloid disorders, with systemic eosinophilia being seen in up to 15% of patients with Hodgkin lymphoma. Eosinophilia has been reported in 2% of the patients with non-Hodgkin lymphomas and is considered reactive in nature.[30-34] Based on the current literature, eosinophilia is not a common accompaniment to treatment of naïve CLL/SLL. Andersen et al. evaluating the association of peripheral blood eosinophilia with future diagnosis of lymphoreticular malignancies demonstrated an odds ratio of 2.57 for mild and 5.0 for severe eosinophilia with CLL.[35] In the same study, the incidence of eosinophilia was low (0.11%) and was noted in a small group of patients who either had (89%) or developed CLL later. The clinical course of CLL/SLL in these patients, especially whether any of them underwent transformation to CHL, is not known. Eosinophilia is reported as a response to fludarabine treatment, and it is not clear if any of the patients in the above series were undergoing such treatment. [36-38] Patients with prior fludarabine treatment for CLL/SLL are known to have poor survival after transformation to CHL, fludarabine treatment by itself does not appear to increase the incidence of CHL transformation. [20,39-42] One of our patients received one course of fludarabine - based chemotherapy. However, in his case, the eosinophilia appeared prior to the treatment and persisted even after discontinuation of the drug.

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Conclusion

Association of systemic eosinophilia and risk of CHL transformation in CLL/SLL patients with eosinophilia has been previously reported to our knowledge in only one case of variant Richter transformation diagnosed postmortem. This association, though it needs to be validated in a larger series, appears to be of significance as it was found in all three patients with CLL/SLL with TP53 deletion and variant Richter’s transformation.

10.

11.

Acknowledgements None

12.

Funding None

Competing Interests

13.

None To Declare

Reference

1. Richter MN. Generalized Reticular Cell Sarcoma of Lymph Nodes Associated with Lymphatic Leukemia. Am J Pathol. 1928 Jul; 4(4): 285–292.7. 2. Rossi D, Cerri M, Capello D, et al. Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome. Br J Haematol. 2008 Jun;142(2):202-15. 3. Parikh SA, Habermann TM, Chaffee KG, et al. Hodgkin transformation of chronic lymphocytic leukemia: Incidence, outcomes, and comparison to de novo Hodgkin lymphoma. Am J Hematol. 2015;90 (4):334-338. 4. Parikh SA, Kay NE, Shanafelt TD. How we treat Richter syndrome. Blood. 2014;123(11):1647-1657. 5. Richter MN. Generalized Reticular Cell Sarcoma of Lymph Nodes Associated with Lymphatic Leukemia. Am J Pathol. 1928;4(4):285-292 287. 6. Krause JR, Drinkard LC, Keglovits LC. Hodgkin lymphoma transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma. Proc (Bayl Univ Med Cent). 2013;26(1):16-18. 7. Tseng D, Jones CD, Anderson M, Warnke R, Zehnder JL, Miklos DB. Clonally identical Hodgkin’s disease develops after allogeneic hematopoietic cell transplant for CLL. Bone Marrow Transplant. 2011;46(12):1576-1578. 8. Vasilj A, Kojic-Katovic S, Maricevic I, et al. Hodgkin’s lymphoma variant of Richter’s syndrome. Coll Antropol. 2010;34(1):295-299. 9. de Leval L, Vivario M, De Prijck B, et al. Distinct clonal origin in two cases of Hodgkin’s lymphoma www.pacificejournals.com/apalm

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variant of Richter’s syndrome associated With EBV infection. Am J Surg Pathol. 2004;28(5):679-686. Nemets A, Ben Dor D, Barry T, et al. Variant Richter’s syndrome: a rare case of classical Hodgkin’s lymphoma developing in a patient with chronic lymphocytic leukemia treated with fludarabine. Leukemia & lymphoma. 2003;44(12):2151-2154. Tzankov A, Fong D. Hodgkin’s disease variant of Richter’s syndrome clonally related to chronic lymphocytic leukemia arises in ZAP-70 negative mutated CLL. Med Hypotheses. 2006;66(3):577-579. Fabbri A, Cencini E, Bocchia M. Hodgkin’s lymphoma as Richter’s transformation in chronic lymphocytic leukemia. Med Oncol. 2014;31(4):904. Kazmierczak M, Kroll-Balcerzak R, Balcerzak A, et al. Hodgkin lymphoma transformation of chronic lymphocytic leukemia: cases report and discussion. Med Oncol. 2014;31(1):800. Fayad L, Robertson LE, O’Brien S, et al. Hodgkin’s disease variant of Richter’s syndrome: experience at a single institution. Leukemia & lymphoma. 1996;23(34):333-337. Dujardin F, Lefrancq T, Blechet C, et al. [Hodgkin’s disease variant of Richter’s syndrome. Two cases and literature review]. Ann Pathol. 2008;28(4):311-316.

16. Jamroziak K, Grzybowska-Izydorczyk O, JesionekKupnicka D, Gora-Tybor J, Robak T. Poor prognosis of Hodgkin variant of Richter transformation in chronic lymphocytic leukemia treated with cladribine. Br J Haematol. 2012;158(2):286-288. 17. Momose H, Jaffe ES, Shin SS, Chen YY, Weiss LM. Chronic lymphocytic leukemia/small lymphocytic lymphoma with Reed-Sternberg-like cells and possible transformation to Hodgkin’s disease. Mediation by Epstein-Barr virus. Am J Surg Pathol. 1992;16(9):859-867. 18. Tadmor T, Shvidel L, Goldschmidt N, et al. Hodgkin’s variant of Richter transformation in chronic lymphocytic leukemia; a retrospective study from the Israeli CLL study group. Anticancer Res. 2014;34(2):785-790. 19. Tsimberidou AM, O’Brien S, Kantarjian HM, et al. Hodgkin transformation of chronic lymphocytic leukemia: the M. D. Anderson Cancer Center experience. Cancer. 2006;107(6):1294-1302. 20. Bockorny B, Codreanu I, Dasanu CA. Hodgkin lymphoma as Richter transformation in chronic lymphocytic leukaemia: a retrospective analysis of world literature. Br J Haematol. 2012;156(1):50-66. eISSN: 2349-6983; pISSN: 2394-6466

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Eosinophilia and Variant Richter’s Transformation

21. Rabe KG, Ding W, Call TG, et al. Hodgkin Transformation Of Chronic Lymphocytic Leukemia (CLL): Mayo Clinic Experience. Blood. 2013;122(21):1642-1642.

31. Desenne JJ, Acquatella Stern R, Muller A, Sanchez M, Somoza R. Blood eosinophilia in Hodgkin’s disease: a follow up of 25 cases in Venezuela. Cancer 1992; 1;69(5):1248-53

22. Rossi D, Spina V, Deambrogi C, et al. The genetics of Richter syndrome reveals disease heterogeneity and predicts survival after transformation. Blood. 2011;117(12):3391-3401.

32. Roufosse F, Garaud S, de Leval L. Lymphoproliferative disorders associated with hypereosinophilia. Semin Hematol. 2012;49(2):138-148.

23. Ohno T, Smir BN, Weisenburger DD, Gascoyne RD, Hinrichs SD, Chan WC. Origin of the Hodgkin/ Reed-Sternberg cells in chronic lymphocytic leukemia with “Hodgkin’s transformation”. Blood. 1998;91(5):1757-1761. 24. van den Berg A, Maggio E, Rust R, Kooistra K, Diepstra A, Poppema S. Clonal relation in a case of CLL, ALCL, and Hodgkin composite lymphoma. Blood. 2002;100(4):1425-1429. 25. Shin SS, Ben-Ezra J, Burke JS, Sheibani K, Rappaport H. Reed-Sternberg-like cells in low-grade lymphomas are transformed neoplastic cells of B-cell lineage. Am J Clin Pathol. 1993;99(6):658-662. 26. Pescarmona E, Pignoloni P, Mauro FR, et al. Hodgkin/ Reed-Sternberg cells and Hodgkin’s disease in patients with B-cell chronic lymphocytic leukaemia: an immunohistological, molecular and clinical study of four cases suggesting a heterogeneous pathogenetic background. Virchows Arch. 2000;437(2):129-132. 27. Isikdogan A, Ayyildiz O, Buyukbayram H, Muftuoglu E.”Hodgkin’s disease variant of Richter’s transformation: a case report. Med Oncol. 2002;19(2):109-112. 28. Nagy V, Dommann-Scherrer, Tzankov A, Pless M. Systemic eosinophilia with skin and pulmonary infiltrates in a patient with chronic lymphocytic leukemia: A case report. Case Reports in CLinical Pathology. 2016;3(2):15-21. 29. Rossi D, Cerri M, Capello D, et al. Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome. Br J Haematol. 2008;142(2):202-215. 30. Vaughan Hudson B, Linch DC, Macintyre EA, et al. Selective peripheral blood eosinophilia associated with survival advantage in Hodgkin’s disease (BNLI Report No 31). British National Lymphoma Investigation. J Clin Pathol. 1987;40(3):247-250.

33. Koeffler HP, Levine AM, Sparkes M, Sparkes RS. Chronic myelocytic leukemia: eosinophils involved in the malignant clone. Blood. 1980;55(6):1063-1065. 34. Müller-Hermelink HK et al. Chronic lymphocytic leukemia/small lymphocytic lymphoma in Swerdlow SH et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, Fourth Edition. World Health Organization; 2008:180-182. 35. Andersen CL, Siersma VD, Hasselbalch HC, et al. Eosinophilia in routine blood samples and the subsequent risk of hematological malignancies and death. Am J Hematol. 2013;88(10):843-847. 36. Larfars G, Uden-Blohme AM, Samuelsson J. Fludarabine, as well as 2-chlorodeoxyadenosine, can induce eosinophilia during treatment of lymphoid malignancies. Br J Haematol. 1996;94(4):709-712. 37. Sezer O, Schmid P, Hallek M, et al. Eosinophilia during fludarabine treatment of chronic lymphocytic leukemia. Ann Hematol. 1999;78(10):475-477. 38. Voutsadakis IA. Fludarabine-induced eosinophilia: case report. Ann Hematol. 2002;81(5):292-293. 39. Bockorny B, Codreanu I and Dasanu CA: Hodgkin lymphoma as Richter transformation in chronic lymphocytic leukaemia: A retrospective analysis of world literature. Br J Haematol 2012;156: 50-66. 40. Dasanu CA: Intrinsic and treatment-related immune alterations in chronic lymphocytic leukaemia and their impact for clinical practice. Expert Opin Pharmacother 2008;9: 1481-1494. 41. Rubin D, Hudnall SD, Aisenberg A, Jacobson JO, Harris NL. Richter’s transformation of chronic lymphocytic leukemia with Hodgkin’s-like cells is associated with Epstein-Barr virus infection. Mod Pathol. 1994;7(1):91-98. 42. Dolcetti R, Carbone A. Epstein-Barr virus infection and chronic lymphocytic leukemia: a possible progression factor? Infect Agent Cancer. 2010;5:22.

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Case Report Balantidium Coli in Urine Microscopy: A Mere Coincidence or More Pratibha Mane, Jyoti Sangwan* and A.K. Malik Department of Microbiology, SHKM GMC, Nalhar, Mewat, Haryana, India Keywords: B.coli, Urine, UTI, Elderly Male

ABSTRACT Balantidium coli (B. coli) is a trophic ciliate of low virulence which can cause dysentery in humans. The organism is cosmopolitan in nature and can be found wherever pigs are present. Disease is mostly seen in developing countries, where water sources are frequently contaminated with porcine or human feces. B. coli can become an opportunistic parasite in individuals with immunosupression in urban environments, where pigs do not play a role in infection. We herein describe a case where trophozoites of B. coli were detected in urinary sediment examination of an elderly male presenting with urinary tract infection. The parasites were identified by their characteristic morphology and rapid spiraling motility. To best of our knowledge,this is only the fifth case described in Indian literature to detect B. coli in urine sediment.

*Corresponding author: Dr Jyoti Sangwan, Assistant Professor, Dept of Microbiology, SHKM GMC, Nalhar, Mewat, Haryana,122107. INDIA Phone: +91 8199972271 E-mail: jyolathwal@yahoo.co.in

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Introduction

Balantidium is the only ciliated protozoon known to infect humans and is the largest protozoon infecting humans and nonhuman primates. Balantidiosis is a zoonotic disease and is acquired by humans via the fecal-oral route from the normal host, the pig, where it is asymptomatic. Water is the vehicle for most cases of balantidiosis. Human-to-human transmission may also occur. Balantidium’s habitats in humans are the cecum and colon. Humans may remain asymptomatic, as does the pig, or may develop dysentery. In developing countries with undernourished and over parasitized populations, it can make the difference between a healthy life and chronic debilitation.[1]

Case Report

In July 2013 a 60 year old male presented with complains of fever, increased frequency and burning micturition associated with lower abdominal pain radiating from loin to groin on right side of seven days duration. Also he complained of decreased urine output for last three days. On examination, he was febrile with temperature 102° F, pulse was 100 per min and BP was 130/90mm Hg and had BMI of 16.8kg/cm2. Pallor was present. Abdomen was soft, tenderness was positive in renal angle. Other systems were within normal limits. A complete haemogram showed microcytic hypochromic anaemia with an Hb level of 9.0 gm%. Serology done for HIV and HBsAg were negative. His kidney functions were found to be deranged, with urea -50 mg/dl and creatinine -1.8 mg/dl. His liver function and blood sugar were found to be within normal limits. His fresh mid stream urine (MSU) sample was sent for routine and microscopic examinations and culture sensitivity. The urine physically appeared as mildly turbid. Urinalysis, by dipstick, showed pH 5.0, specific gravity 1.008 and negative albumin, hemoglobin, leukocyte esterase and nitrites. Urine proteins were absent. Microscopic examination of its sediment showed a few red cells with 2-4 pus cells per HPF. Also, ovoid to oblong ciliated parasites which were approximately 70 x 50 μm, were seen swimming rapidly across the slide [Fig-1] Its body was covered with short, delicate cilia which were all of uniform length. The cilia which lined the mouth part appeared to be longer than others. The morphology and swimming pattern were characteristic of Balantidium coli. Subsequent culture grew Escherichia coli, which was sensitive to ciprofloxacin, imipenem and amikacin. Patient was prescribed oral ciprofloxacin 500 mg twice/day for five days and advised to come for review with ultrasound report after a week.

Fig. 1: Microscopic examination showing ovoid to oblong ciliated parasite (Magnification10x 40).

A week later on review patient complained of persisting symptoms and his USG report showed presence of mild hydronephrosis on right side and no calculi. A repeat MSU sample was sent for routine, microscopy and culture. Urine was mildly turbid. Microscopic examination of its sediment showed 2-4 red cells with 2-4 pus cells per HPF. Again presence of ciliated trophozoites of Balantidium.coli was noticed with their characteristic motility. We did not observe any cyst. This time culture was sterile. After these findings, a search of B. coli in the feces was performed, which turned out to be negative in three consecutive samples. On questioning, the patient denied direct contact with pigs but admitted of bathing in contaminated pond water which they also use for their routine chores as he was from lower socio-economic background. He gave history of recurring diarrhea in the past for which he has taken treatment on and off. The patient was treated with tetracycline 500 mg four times daily and metronidazole 250 mg three times daily, for a total duration of seven days. His MSU sample was re-examined after a week, following treatment completion, in which no B. coli trophozoites were observed, probably due to successful therapy. Even following up after a month patient did not have recurrence in symptoms which again point towards successful therapy.

Discussion

Balantidium coli are low virulence pathogenic parasites with worldwide distribution. Usual mode of transmission is ingestion of infective cysts through water contaminated with porcine feces, though human to human transmission may also occur. Ingested cysts liberate trophozoites which reside and replicate by binary fission in the large bowel. [1] This patient had no contact with pig, but had history of bathing in the pond; hence he might have been infected

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from contaminated water of the pond. Many patients remain asymptomatic but some have persistent diarrhea and a few develop more fulminant dysentery.[ 2] Intestinal hemorrhage, ulceration, focal necrosis and perforation can occur and mediated by the production of balantidial proteolytic enzyme. Extra intestinal spread has also been described. The organism may have invaded from the colonic mucosa to the urinary bladder or directly from the anal area. [3-4] Sharma and Harding opine that infection of genitourinary sites occur due to direct spread from the anal area or secondary to rectovaginal fistula creation by the parasite. [3] The stool examination in this patient was non contributory possibly due to intermittent treatment with metronidazole. Immunocompromised individuals, malnutrition and alcoholism appear to act as important contributory factors for balantidiosis. [5-6] Our patient was malnourished (16.8kg/cm2) and anaemic (9.0 gm%).

Acknowledgements

Laboratory diagnosis of B. coli is relatively easy because of its large size and spiraling motility. In most of the cases morphology of the trophozoite has been described in dysenteric stool and the cyst phase commonly in formed stool. [1] In this patient excellent morphology of the parasite could be demonstrated in urine sample by light microscopy. Tetracycline and metronidazole are the drugs of choice for B. coli. [1] Ensuring clean uncontaminated water supply is probably the most efficient strategy to prevent human infection. Not much data is available on the prevalence of urinary Balantidiosis in India or worldwide. An internet search revealed only five cases which were reported, one each from Italy and Tehran and three from India[.4, 7-9] Though many cases of B. coli infection in stool have been reported from India, both in humans and animals, urinary balantidiosis is still a rare entity.[ 9, 10] To the best of our knowledge this is the fifth case report of urinary Balantidiosis from India.

4. Umesh S. Balantidium coli on urine microscopy. Natl Med J India 20:270.

Conclusion

None

Funding Nil

Competing Interests None Declared

Reference

1. Schuster FL, Ramirez-Avila L. Current world status of Balantidium coli. Clin Microbiol Rev 21:626–638. 2. Dodd LG. Balantidium coli infestation as a cause of acute appendicitis. J Infect Dis 163:1392. 3. Sharma S, Harding G. Necrotizing lung infection caused by theprotozoan Balantidium coli. Can J Infect Dis 14:163–166.

5. Anargyrou K, Petrikkos GL, Suller MTE, Skiada A, Siakantaris MR, Osuntoyinbo RT, Pangalis G, Vaiopoulos G. Pulmonary Balantidium coli infection in a leukemic patient. Am J Hematol 73:180–183. 6. Cermenõ JR, Hernańdez de Cuesta I, Uzca´tegui O, Paéz J, Rivera M, Baliachi N. Balantidium coli in a HIV-infected patient with chronic diarrhea. AIDS 17:941–942 7. Bandyopadhyay A, Majumder K, Goswami BK. Balantidium coli in urine sediment: report of a rare case presenting with hematuria. J Parasit Dis. 2013; 37 (2): 283-5. 8. Maino A, Garigali G, Grande R, Messa P, Fogazzi GB. Urinary balantidiasis: diagnosis at a glance by urine sediment examination. J Nephrol. 2010;23:732-7.

In conclusion, B. coli though a rare urinary pathogen, should come in the differential diagnosis in elderly debilitated patients presenting with urinary tract infection. Microscopic examination of fresh urine sediment can easily diagnose this large parasite by its characteristic morphology and rapid spiraling motility.

9. Maleky F. Case report of Balantidium coli in human from south of Tehran, Iran. Indian J Med Sci. 1998;52:201-2.

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10. Singh NK, Singh H, Jyoti, Haque M, Rath SS. Prevalence of parasitic infections in cattle of Ludhiana district, Punjab. J Parasit Dis. 2012;36(2):256-9.

Case Report Cholesterol Granuloma of Paratesticular Tissue: Report of a Rare Case Amoolya Bhat1*, Vijaya C1, D.H. Biradar2, Safarulla and Suraksha Rao B1 Department of Pathology, Sapthagiri Institute of Medical Sciences and Research Centre, Bangalore, India 2 Department of Surgery, Sapthagiri Institute of Medical Sciences and Research Centre, Bangalore, India

Keywords: Cholesterol Clefts, Cholesterol Granuloma, Fibrinous Necrosis, Paratesticular Tissue

ABSTRACT Cholesterol granuloma (CG), also known as cholesterol cyst was first reported by Manasse in 1894. It is as an expansible benign mass that contains brownish-yellow debris with cholesterol crystals. Clinically it has a slow growth. It can affect any organ or tissue, though the temporal bone, chiefly the petrous apex is the most common site. We report an unusual case of cholesterol granuloma that developed in the paratesticular tissue in a 61 year old man.

*Corresponding author: Dr. Amoolya Bhat, Assistant Professor, Department of Pathology, #15, Chikkasandra, Hesarghatta Main Road, Sapthagiri Institute of Medical Sciences and Research Centre, Bangalore â&#x20AC;&#x201C; 90. INDIA Phone: +91 9480315066 E-mail: amoolyabhat@rediffmail.com

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Introduction

Cholesterol granuloma is a fibrogranulomatous cystic lesion that develops from foreign body reaction to cholesterol crystals and exhibits the accumulation of foreign body giant cells along with mononuclear cells. Commonest sites of occurrence are air-filled spaces of the middle ear region of the temporal bone[1]. It is known to arise in the kidney, testis, epididymis, tunica vaginalis, tunica albuginea [2] breast, orbit, lung, peritoneum, subcutaneous tissues, parotid gland, liver, and spleen [3]. It is a benign inflammatory lesion, sometimes referred to as a pseudotumor and is not classified as a true tumor as it lacks a definitive lining epithelium. We present a very rare case of cholesterol granuloma of the paratesticular tissue.

Case Report

A 61 years old man came to the surgery OPD with the chief complaints of painless swelling in the left side of the scrotal sac since past 20 years. There was no history of trauma or sudden increase in size of the swelling. On examination the lesion was round to oval and measured about 8x6cm. It was non- tender and firm in consistency. Transillumination test was negative. The right inguinal region showed an indirect reducible hernia. The right testis was unremarkable. He did not have fever, caugh or any other swellings in the body. A fine needle aspiration cytology of the swelling on left side of scrotum yielded haemorrhage and scanty necrotic material. Hence a definitive cytological diagnosis was not rendered. A provisional clinical diagnosis of left paratesticular tumor was made and excision biopsy was carried out along with excision of the left testes as the mass was adherent to the testes [Figure 1a]. Right sided inguinal hernioplasty was also performed. Grossly the mass measured 7.5x6.5x4.5cm and the detached testis measured 6.5x4.5x4cm. Cut section of the mass showed a cyst with intraluminal brownish friable necrotic material. The wall thickness of the cyst was 0.5cm. Inner surface of the cyst wall showed ragged appearance. Cut surface of the testes showed thickened covering layers along with unremarkable testicular tissue and epididymis [Figure 1b]. Histopathological examination showed cyst wall composed of dense fibrocollgenous tissue and smooth muscle cell bundles along with small foci of calcifications. Dilated and congested blood vessels were seen surrounded by dense mononuclear cell infiltrates [Figures 1c&d]. The luminal aspect did not exhibit a definite lining epithelium [Figure 2a]. Lumen showed abundant fibrinous necrotic material and numerous cholesterol clefts [Figure 2b]. Foreign body type of giant cells in aggregates surrounding the cholesterol clefts [Figure 2c] were seen along with www.pacificejournals.com/apalm

haemosiderin laden macrophages [Figure 2d]. Areas of haemorrhage and cystic degenerations were seen. There was no atypia or increased mitosis. Acid fast stain was negative for acid fast bacilli. The patientsâ&#x20AC;&#x2122; post operative course was uneventful. His complete haemogram, urine examination findings, chest x ray and ultrasonography of the abdomen and pelvis was unremarkable. The features were suggestive of cholesterol granuloma. His serum lipid levels were evaluated and were within normal limits.

Discussion

Cholesterol granuloma is a benign fibrogranulomatous lesion that develops secondary to a foreign body reaction to cholesterol crystals. It usually affects the middle ear and paranasal sinuses, however it can affect any site. Its etiopathogenesis is unknown. It has been hypothesized that an inflammatory reaction induces ischemic necrosis, granulomatous reaction, and fibrosis. Ischemia induced rupture of blood vessels results in extravasation of blood containing cholesterol, fibrin, and hemosiderin. The cholesterol crystals elicit a foreign body reaction forming giant cells and granulomatous tissue [1-4]. Leakage from the ectatic ducts in breast lesions and trauma in case of middle ear and air sinuses has been postulated as etiologic factors[3]. It is sometimes referred to as a pseudotumor as it lacks lining epithelium. Also , it can present as a cystic or partially cystic lesion. Elevated serum lipid levels and hypercholesterolemia have been suggested as etiologic factors by some authors [5]. Our patient did not have features of dyslipidemia. The scrotal lesions are clinically asymptomatic. The commonest presenting complaint is painless lump in the scrotal sac [2,4]. Occasionally it can present as acute scrotum [7] or may be just incidental findings at general health check up or autopsy [3]. Histopathologically it shows fibrinous necrotic material and abundant cholesterol clefts surrounded by foreign body giant cells monocytes and lymphocytes. Haemosiderin laden macrophages and areas of haemorrhage are seen. The cyst wall doesnâ&#x20AC;&#x2122;t exhibit lining epithelium and shows only dense fibrocollagenous tissue along with bundles of smooth muscles[2-4]. Our case showed focal areas of calcifications in the cyst wall in addition to these findings. Occasional report has described osseous metaplasia within the cholesterol granuloma[6]. Their clinical differential diagnoses include spermatocoele, adenomatoid tumour and benign /malignant paratesticular neoplasms. Other granulomatous lesions that can arise in this location include tuberculosis, sarcoidosis, malakoplakia and spermatic granuloma [4,7]. eISSN: 2349-6983; pISSN: 2394-6466

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Cholesterol Granuloma

Fig. 1: a) Paratesticular mass adherent to testis. b)Specimen consisted of cystic lesion with intraluminal brownish material and ipsilateral testis. Photomicrographs showing c) cyst wall with bundles of smooth muscle cells d) dense fibrocollagenous tissue and foci of calcifications.

Fig. 2: Photomicrographs showing a) intaluminal aspect of the cyst lacking the lining epithelium b)fibrinous necrotic material and numerous cholesterol clefts c) cholesterol clefts (arrow) surrounded by foreign body giant cells and mononuclear cells d) haemosiderin laden macrophages (arrow).

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Tuberculosis shows well formed granulomas composed of epithelioid histiocytes and Langhans giant cells with central caseation necrosis surrounded by peripheral cuff of lymphocytes and fibrosis. Acid fast bacilli may be positive. Sarcoidosis shows non caseating granulomas with giant cells showing asteroid bodies. Malakoplakia shows sheets of foamy histiocytes, chronic inflammatory cells and Michaelis Gutman bodies within the giant cells. Cholesterol crystals are lacking in all these lesions. Spermatic granulomas can be painful and grow up to several centimeters. However they show central area of degenerated sperms surrounded by well formed granulomas and develop in men after vasectomy [8]. Spermatocoeles are the most common cysts of the scrotum. They are formed from retention of a tubule of rete testis and head of the epididymis. They are painless and grow upto several centimeters in size. They are transilluminaton test positive and show cyst wall line by ciliated columnar cells and intraluminal proteinaceous fluid and spermatozoa[8]. The adenomatoid tumor of epididymis arises from the mesothelial cells and has multiple microscopic appearances. The three basic patterns are tubules, cords, and small nests. The tumor cells are cuboidal with vacuolated cytoplasm. Lymphatic infiltration may be seen. Gaping spaces without lining epithelium, representing necrotic tubular components, and smaller spaces, representing remnants of the typical vacuolar spaces, are major clinching points to the diagnosis[9]. Histologically, papillary cystadenoma shows encapsulated tumor composed of multiple cystic spaces with intracystic papillary projections lined by clear cells, mimicking renal cell carcinoma. It is of mesonephric derivation[10]. Conservative management is the treatmnet of choice for cholesterol granuloma; however, if there is a clinical suspicion of tumor, surgical exploration with biopsy and histopathological examination may be considered[3,6].

Conclusion

Cholesterol granuloma arising in the paratesticular region is an extremely uncommon. It can grow into large dimension in this unusual site unlike in head and neck areas and lead to diagnostic dilemma. In our case the preoperative diagnosis was extremely challenging, considering the unusual site, and unusually large size. Cytodiagnosis can also be misleading due to the presence of necrotic material.

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In this area, it can be mistaken for tumors of the testes and epididymis. Hence it should be considered as a differential diagnosis in evaluation of patients with scrotal masses.

Funding None

Competing Interests None Declared

Reference

1. Nikolaidis V, Malliari H, Psifidis D, Metaxas S. Cholesterol granuloma presenting as a mass obstructing the external ear canal. BMC Ear, Nose and Throat Disorders 2010, 10:4. 2. Unal D, Kilic M, Oner S, Erkinuresin T, Demirbas M, Coban S, MAydos M,Cholesterol granuloma of the paratesticular tissue: A case report. Can Urol Assoc J 2015;9(5-6):E390-2. 3. Luckraz H, Coulston J, Azzu A. Cholesterol Granuloma of the Superior Mediastinum. Ann Thorac Surg 2006;81:1509 â&#x20AC;&#x201C;10. 4. Lam CY, Lin CM, Tsai SW, Hsieh TS. Cholesterol granuloma of the epididymis mimicking a paratesticular tumor. JTUA 2009;20:89-91. 5. MendonĂ§a R, Peron C S, Stefani MA, Gallo P. Cerebral cholesterol granuloma. Arq Neuropsiquiatr 2007; 65(2-B):540-541. 6. Ezzat TF, Alowami S. Cholesterol granuloma of the anterior mediastinum with osseous metaplasia. Rare Tumors 2012;4: e47. 7. Spajic B, Cupic H, Stimac G, Brigic I, Kruslin B, Kraus O. Cholesterol granuloma of the right epididymis mimicking an acute scrotum. Asian J Androl 2006;8(6):749- 50. 8. Rosai J, Ed. In: Rosai and Ackermanâ&#x20AC;&#x2122;s Surgical Pathology , Mosby an imprint of Elsevier, 9edition, 2009. 9. Kontos S, Fokitis I, Karakosta A, Koritsiadis G, Mitsios K, Koutsikos S, Koritsiadis S. Adenomatoid tumor of epididymidis: A case report. Cases Journal 2008, 1:206 doi:10.1186/1757-1626-1-206. 10. Odrzywolski K J and Mukhopadhyay S. Papillary Cystadenoma of the Epididymis. Archives of Pathology & Laboratory Medicine 2010; 134: 630-3.

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Case Report Unusual Cause of Splenomegaly: Splenic Cyst Anitha Chakravarthy*, Kishan Prasad H. L., Chandrika Rao and Jayaprakash Shetty K Department of Pathology, K. S. Hegde Medical Academy, INDIA Keywords: Splenic Cyst, Splenomegaly, Epithelial Cyst, Marsupialization

ABSTRACT Cysts in the spleen are rare, however the true incidence is not documented. There are very few cases reported in the literature. The diagnosis of splenic cyst is incidental, since majority of patients remain asymptomatic. If symptomatic, they present with a palpable mass in the left upper quadrant. The diagnosis of splenic cysts have increased due to the advancement of computed tomography(CT) and the various management options. We discuss two cases of splenic cysts as they are rare and the diagnosis and management has always been a challenge to a pathologist and the treating surgeon.

*Corresponding author: Dr Anitha Chakravarthy, Resident, Dept. of Pathology, K. S. Hegde Medical Academy, INDIA E-mail: anithachakravarthy@gmail.com

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Chakravarthy et al.

Introduction

Epithelial cysts of the spleen are the most commonly seen primary splenic cysts in clinical practice. They constitute 10-15% of benign non parasitic cysts[1]. A palpable mass in the left upper quadrant of the abdomen is the most common presentation[2]. We report two cases, a 15 year old female who presented with mass per abdomen. Computed Tomography(CT) scan revealed the possibility of a splenic cyst. Histopathology confirmed the diagnosis of primary epithelial splenic cyst. A 35 year old female presented with recurrent pain in the abdomen. Diagnosis of haemorrhagic splenic cyst was made on CT scan. Histopathology confirmed the diagnosis of a pseudo cyst of the spleen.

Case Series

CASE 1: A 15 year old female presented with a mass in the upper abdomen with pain and epigastric discomfort. On examination, a large globular mass was palpable in the epigastrium measuring 20cm x 18cm extending to the left hypochondrium. Haematological (Hb-11.8g%, TLC – 9500c/cmm, Platelets – 2,85,000c/cmm) and biochemical( Serum Creatinine–0.56mg/dl, Urea – 2.7mmol/L, Liver function test, and electrolytes) parameters were normal. Ultrasonography(USG) and CT scan of the abdomen revealed a large well encapsulated fluid filled splenic cyst measuring 20x15cmin the left upper quadrant. The cyst was thick walled and was not delineated from the spleen and pancreas.The diagnosis of splenic cyst was made. Laparoscopic Marsupialization was performed. The membranous splenic cyst mass was sent for histopathology. Microscopy showed a cyst wall withfibro-collagenous tissue lined by low cuboidal epithelium and centrally located benign nuclei [Figure 1A]. Numerous gamna-gandy bodies were present [Figure 1B]. The diagnosis of primary epithelial cyst of the spleen and chronic venous congestion was made. Immunohistochemistry profile was not done. The patient was discharged without any complications.

C-225 cystic in consistency. Cut surface exuded haemorrhagic serous fluid. Solid and cystic areas were noted. Some areas showed honey comb appearance [Figure 2B].Surrounding splenic tissue appeared normal and no calcification was seen. Microscopically, cyst wall was lined by fibrous tissue with areas of calcification. Some areas showed necrotic tissue with haemorrhage [Figure 2C]. The diagnosis of pseudo cyst of the spleen was made. The patient was discharged without any complications.

Discussion

Splenic cysts are not frequently encountered in everyday surgical practise[1]. The cysts can be due to benign disease like infections, malignancy and metastasis[2].Clinically,most of the cysts are asymptomatic and diagnosed incidentally[2,3]. They present as a palpable mass in the left upper quadrant of the abdomen[4]. When they are oversized(5cm),they can manifest with symptoms which cause atypical pain and heaviness in the left hypochondrium. The other nonspecific symptoms are nausea, vomiting, diarrhoea and flatulence. If the spleen is oversized they cause pressure

CASE 2: A 35 year old female, presented with recurrent pain in the left upper abdomen. On examination, a globular mass was palpable in the hypogastrium. Haematological (Hb–12.4g%, TLC-7500 c/cumm, Platelets – 4,10,000c/ cumm and biochemical(Serum creatinine-0.74mg/dl, Urea-2.96mmol/L, Liver function test and Electrolytes) parameters were normal. CT scan of the abdomen revealed a large fluid attenuated cystic lesion arising from the spleen measuring 16.3cm x 10.8cm x 17.5cm. It showed multiple thin septations and peripheral punctate calcification [Figure 2A]. Diagnosis of haemorrhagic splenic cyst was made and splenectomy was performed. The spleen was sent for histopathology. Grossly, the spleen was enlarged measuring 18cm x 6cm x 4cm.Outer surface appeared congested and

Fig. 1A:H & E stained section. High power view showing features of primary epithelial cyst, lined by low cuboidal epithelium.

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Splenic Cyst

Fig. 1B:H &E stained section. High power view showing presence of Gamna gandy body indicating chronic venous congestion of spleen.

Fig. 2A: CT Scan of the abdomen showing enlarged spleen with cystic changes, multiple septations and peripheral rim of calcification.

Fig. 2B: Cut surface of the enlarged spleen showing cystic areas with focal areas showing honey comb appearance.

Fig. 2C: H &E stained section.High power view showing features of pseudo cyst, lined by fibro-collagenous tissue and absence of lining epithelium

symptoms on the secondary organs causing chest pain and dyspnoea[2,3]. The cases reported, presented with a palpable mass in the upper quadrant of the abdomen with no secondary symptoms. The findingscorrelate with literature. It is essential to exclude other causes of splenomegaly which include infections like schistosomiasis, infectious mononucleosis, chronic liver disease, vascular disorders, metabolic disorders like Gaucherâ&#x20AC;&#x2122;s disease and NiemannPick disease, benign and malignant infiltrations like lymphoma and leukaemia[2].

cystic mass with an ill-defined wall. Magnetic resonance imaging shows a typical cystic lesion with hyper-intense signalling2. Microscopically they show the presence of a lining epithelium which can be columnar, cuboidal or stratified epithelium. The first case presented with the similar manifestations as described above. Primary cysts can be classified as parasitic and non-parasitic. NonParasitic cysts can further be classified as congenital, neoplastic, degenerative and traumatic[3,4].

Splenic cysts can be classified as primary and secondary cysts. Primary cysts are true cysts, most commonly seen in the first decade. They can be sporadic or familial[3]. They are asymptomatic. On USG, they are well demarcated and anechoic cystic mass[2,3]. CT scan shows a low density

Secondary splenic cysts are pseudocysts, lacking an epithelium and replaced by thick fibrous wall which is often calcified[2,3]. They are commonly seen following inflammation, infarction and thrombosis. On USG, peripheral echogenicity with distal shading and calcification of the wall is noted. CT scan shows a well demarcated

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Chakravarthy et al. homogenous non contrasting cystic mass[2]. The second case presented similar to the findings mentioned above. CA 19-9 and carcinoembryonic antigen levels are increased in primary epithelial cysts. Serum levels of CA 19-9 have been shown to reduce after cyst removal, offering a screening test to indicate recurrences in case of spleen preserving surgeries. These levels were not estimated in our case since histopathology confirmed the diagnosis of primary epithelial cyst with cuboidal lining epithelium5.

C-227 the patient is asymptomatic.Differentiation of splenic cysts should be made preoperatively. Preservation of the spleen is important to preserve the functions of the organ. The two cases discussed above highlight the features which help differentiate true and pseudo cysts and the role of histopathology in the diagnosis and the various management strategies available.

References

There are numerous causes of splenomegaly. It is important to consider splenic cyst as a differential diagnosis, since

1. Arif H, Hakim I, Fazl P, Rubina L. Non Parasitic Splenic cyst; A Case Report. Acta Medica Iranica 2012; 50(12): 849 â&#x20AC;&#x201C; 51. 2. Galyfos G, Touloumis Z, Palogos K, Stergios K, Chalasti M, Kavouras N et al. Oversized pseudocysts of the spleen: Report of two cases. IntSurg Case Reports 2014;5(2):104-7. 3. Fatih A, Enis D, Taner K, Tolga E, Osman Nuri D. Pseudocyst of Spleen: An uncommon clinical entity, although common theoretically. Eur J Gen Med 2012; 9 (1): 43-6. 4. Y SK, K BS, L KPH. Rare case of splenic epidermoid cyst. Adv Lab Med Int. 2011; 1(3): 52-54. 5. Vijayaraghavan R, Chandrashekar R, Aithal S, Rashmi MV, Belagavi CS. Mesothelial cyst of the spleen in an adult: a case report. BMJ Case Rep. 2010 Sep 9;2010 6. Van L M, Hessels R, Kremer G, Jaspers C. A splenic cyst and a high serum CA 19-9: a case report. . Eur J Gen Med 2000;11(2):104-7. 7. Wu HM, Kortbeek JB. Management of splenic pseudocysts following trauma: a retrospective case series. Am J Surg 2006;191:631-4. 8. Morgenstern L. Nonparasitic splenic cysts: pathogenesis, classification, and treatment. J Am Coll Surg 2002;194: 306-14.

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With the advancement of USG,CT scan and successful management options available, various techniques are adopted for management of splenic cyst[7]. They are aspiration of cysts by injecting tetracycline and alcohol into the cyst wall for destroying the cyst lining[4].Resection of the cyst and a portion of the parenchyma is another treatment option available but highly unsuccessful[6]. Marsupialization is creating an opening into the cyst wall for drainage. It can be done internally into the peritoneal cavity or externally through a purposefully created cystocutaneous fistula. The first case report was managed by the above method. But the chances of recurrence is very high. The other formof marsupialization is a near total splenic resection. It is simple, rapidly performed procedure. The chances of cyst recurrence is high because a portion of cyst lining is left intact[8]. Splenectomy is the most commonly performed surgical technique. The first attempt was performed by Jules Pean. Due to the awareness of the immunological functions of the spleen, spleen sparing surgeries are being adopted[3,8].

Conclusion

Case Report Type I (Non-neuronopathic) Gaucher’s Disease with Bone Marrow Involvement: A Rare Case Report Mallikarjun Adiveppa Pattanashetti* and Ramesh Yashwantrao Chavan Department of Pathology, KLE University’s Jawaharlal Nehru Medical College, Belagavi – 590001, INDIA Keywords: Gaucher, Glucocerebrosidase, Non-neuronopathic.

ABSTRACT Gaucher’s disease an uncommon autosomal recessive sphingolipid lysosomal storage disease characterized by the deposition of glucocerebroside in cells of the macrophage-monocyte system from the deficient activity of the lysosomal hydrolase β-glucosidase( glucocerebrosidase). In the absence of enzyme, glucosylceramide accumulates in lysosomes of macrophages of tissues with multisystem organ involvement viz. liver, spleen, bone marrow, lungs and central nervous system.Serum β glucosidase levels <15% of mean normal activity confirms the diagnosis, enzyme replacement being the only definitive treatment. We report this case of 1 year 6 month male child who presented with delayed milestones, purpuric rashes and massive hepatosplenomegaly. Hematological workup revealed pancytopenia. Bone marrow study showed the characteristic gaucher cells and liver biopsy also showed evidence of Gauchers disease. Hence diagnosis of Type I (non-neuronopathic) Gaucher disease was given. With the advent of enzyme replacement therapy and substrate reduction therapy the natural history of the disease has been changed with a marked decrease in morbidity. The incidence of Type I Gaucher’s disease is 1 in 60,000 births but this non-neuronopathic type presenting with pancytopenia due to bone marrow involvement is rare entity with very few cases in India.

*Corresponding author: Dr. Mallikarjun A. Pattanashetti, Plot No 295, RS No 183, 3rd Main, 2nd Stage, Hanuman nagar, Belagavi – 590001, INDIA Phone: +91 9739462156 E-mail: mallikarjun2030@gmail.com

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

Pattanashetti et al.

Introduction

Gaucher’s disease is a lysosomal storage disorder characterized by the accumulation of glucosylceramide in cells of the macrophage/monocyte system. It results from a hereditary deficiency of the lysosomal acid β-glucosidase enzyme. Gaucher’s disease is due to mutations involving the β-glucosidase gene, localized in the large arm of chromosome 1.[1] Gaucher’s disease was first described by Gaucher in 1882, and the storage of glucocerebroside was first recognized by Epstein in 1924.[2] The metabolic defect, which is the deficiency of the lysosomal hydrolase β-glucosidase, or β-glucocerebrosidase, was identified by Brady et al. There are three clinical subtypes, which are delineated by the absence or presence and progression of neurologic involvement. Type 1 or non-neuronopathic form; Type 2, the infantile-onset, acute neuronopathic form; and Type 3, the juvenile-onset neuronopathic form. All three subtypes are inherited as autosomal recessive traits. Type I is the most common form of Gaucher’s disease with an incidence of approximately 1/40–60,000 in the general population. The incidence varies between 1/40,000 in Central Europe and 1/2,000 in some non-European countries, such as Israel. It is most prevalent in the Ashkenazi Jewish population (1/450).[3] This Non-neuronopathic type presenting with bone marrow involvement is rare entity with very few cases reported in India.

Case Report

A one and half year old male child born of second degree consanguinity presented with chief complaints of poor appetite, poor feeding orally, poor weight gain and failure to thrive. Patient also had persistent intermittent low grade fever since birth as informed by the mother and she noticed distension of abdomen of the child since 2 months. Child had birth weight of 3 kg and immunization was complete for age. Developmental history revealed delayed milestones. On examination, heart rate was 106/ minute, respiratory rate was 24 breaths/ minute and child was afebrile. Child was moderately nourished. Pallor was present with no edema or cyanosis. Purpuric rash was present on left thigh. On CNS examination, child was conscious and playful. Per abdomen examination findings included hepatomegaly with liver span of 7 cm and moderate splenomegaly. Hematological investigations including bone marrow examination, biochemical tests, Ultrasonography abdomen and liver biopsy was performed with due consent. Investigations revealed hemoglobin – 10.2 gm% ; RBC count – 4.74 million/cumm, Reticulocyte count 0.4 %, Total leucocyte count - 6460 / cumm, Platelet count - 82000/cumm. Peripheral smear examination showed www.pacificejournals.com/apalm

C-229 normocytic hypochromic anemia with thrombocytopenia. Liver function tests showed raised enzymes with AST - 102 IU/L, ALT – 150 IU/L, ALP – 326 IU/L . Blood urea and serum creatinine were within normal limits. USG abdomen showed hepatomegaly with increased echotexture and gross splenomegaly with no focal lesions/collaterals at hilum. Bone marrow examination and liver biopsy was advised based on the above clinical findings and basic investigations. Bone marrow aspiration showed cellular smear which was satisfactory for evaluation. Myeloid series were normal in morphology and distribution. Moderate erythroid hyperplasia was noted and megakaryocytes were increased in number 5-7/ lpf in marrow particles. Many megakaryocytes were hypolobated. Scattered good number of foamy histiocytes were seen amidst marrow particles having abundant wrinkled ‘tissue paper like’ cytoplasm resembling gaucher cells which are highly characteristic of Gaucher’s disease were present [Figure 1A and 1B]. Hence an opinion of Megakaryocyte hyperplasia with erythroid hyperplasia and probable Gauchers disease was given. Subsequently liver biopsy was performed which showed sheets of gauchers cells disrupting the liver architecture [Figure 2A and 2B]. Periodic Acid Schiff (PAS) stain was intensely positive. [Figure 3]. The patient was diagnosed with Lysosomal storage disorder, Type I(Non neuronopathic) Gauchers disease based on clinical examination, bone marrow examination and liver biopsy. Parents were advised further confirmation by enzyme studies. But due to financial constraints of parents, further evaluation and confirmation of this case was not possible.

Discussion

Gaucher’s disease results from deficiency of a lysosomal enzyme glucocerebrosidase also known as acid betaglucosidase. The glucocerebrosidase gene is located on chromosome 1q21. The enzyme acts on the substrate glucocerebroside which is a component of the cell membrane. In the normal lysosome, protein Saposin C presents glucocerebroside to GBA which activates the enzyme. This enzyme is responsible for hydrolytic breakdown of glucosylceramide to glucose and ceramide. Deficiency of the enzyme leads to accumulation of glucosylceramide and other glycolipids in the lysosomes of macrophages, primarily in the spleen, liver, bone marrow, brain, osteoclasts and less often the lungs, skin, kidneys, conjunctiva and heart. The deacylated form of glucosylceramide, glucosylsphingosine, is elevated in neuronopathic disease and correlates more with phenotype severity compared to glucosylceramide.[4] Three types of Gaucher’s Disease (GD) have been described based on the clinical features, ethnicity and eISSN: 2349-6983; pISSN: 2394-6466

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Type I Gaucherâ&#x20AC;&#x2122;s Disease

Fig. 1A and 1 B: Bone marrow aspiration smear shows numerous Gauchers cells (Wright Stain ,100 X, 400 X )

Fig. 2 A and 2 B : Liver biopsy showing sheets of Gauchers cells (H&E stain :100 x and 400x)

Fig. 3 : Liver biopsy showing sheets of Gauchers cells with PAS positive material (PAS stain, 400x).

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Pattanashetti et al. the natural history of the disease. Type 1 GD patients do not have neurological involvement, Type 2 is the acute neuronopathic and Type 3 is the chronic neuronopathic type. Type 3 is further subdivided in to 3 subtypes a, b and c depending on the clinical features. Gaucher disease can also present as hydrops in the perinatal period which is often lethal. Type 1 GD or the non-neuronopathic type presents with symptoms which may first present in infancy to late adulthood. It is characterized by painless hepatosplenomegaly, which often leads to massive abdominal distension with anemia and thrombocytopenia. Fatigue, nose bleeds, and easy bruising are a manifestation of the cytopenias. Some patients are transfusion dependent. Cytopenias are secondary to hypersplenism, bone marrow infiltration by Gaucher cells and an intrinsic haemopoietic defect in cells. These patients with Type 1 GD are at increased risk of bleeding tendency which is related to thrombocytopenia, defective platelet function and coagulation abnormalities.[4] Differential Diagnosis include hematological malignancies and storage disorders like Niemann–Pick disease. Most of these disorders have characteristic clinical, radiographic, or laboratory features that distinguish them from GD. Atypical Gaucher disease or a variant of GD is known which is severe disorder due to deficiency of Saposin C.[5] Diagnosis of Gaucher disease is made on the basis of clinical history, physical examination, laboratory test and confirmed by a blood test showing deficient glucocerebrosidase enzyme which was not possible in this case and genetic mutation studies when the diagnosis is doubtful. History of consanguinity and family history of suspected or proven GD will support the diagnosis. Gaucher’s cells are large, round or oval, and have pale blue cytoplasm with a wrinkled appearance due to the presence of many fibrillar structures. The cytoplasm is Sudan Black B- and PAS-positive. Gaucher cells also stain positively for nonspecific esterase and tartrate-resistant acid phosphatase. Stains for iron give weak positive reactions. In histologic sections, Gaucher cells are often found in clumps or sheets and their abundant cytoplasm has a crumpled appearance. The affected marrow may show an increase in reticulin and collagen. The gold standard for diagnosing Gaucher disease is measurement of glucocerebrosidase enzyme activity in leucocytes or skin fibroblasts on a skin biopsy. [6] The specific acid β-glucosidase gene mutation may be determined for possible genotype/phenotype correlations. Electron microscopy reveals that the cytoplasm is packed with large elongated sacs containing characteristic tubes, 30–40 nm wide, each of which is made up of spirally www.pacificejournals.com/apalm

C-231 arranged fibrils. Imaging studies for organomegaly and skeletal survey for long bones is done. Other additional tests include Chest X-ray and 2D echo are also desirable to rule out lung parenchymal involvement and pulmonary hypertension. Prognostic biological marker Chitotriosidase correlates with the disease burden and is useful in monitoring therapy. It is a chitinase and reflects “alternative”type macrophage activation that is overexpressed by the Gaucher cell.[7] Prenatal diagnosis is performed by enzyme analysis of fetal cells obtained by chorionic villous sampling or amniocentesis at 16 weeks of pregnancy. [8] The treatment of Gaucher disease is by Enzyme Replacement Therapy (ERT) and has now become the

standard of care. Administration of the enzyme

reduces splenomegaly, hepatomegaly, improvement of anemia and thrombocytopenia. Severely ill patients with Type 1 Gaucher disease are generally started with a dose of 60 Units (U) of enzyme per kg of body weight every other week. As patients improve, the dose may be reduced to 30 U per kg of body weight, and in many cases to 20 U per kg of body weight every other week. The frequency of administration of enzyme has been extended to every 3 weeks in a number of patients who have been maintained in good condition with this regimen. Long term benefit of ERT in patients with type 1 GD has been clearly documented. Velaglucerase alfa , a newer enzyme therapy has been approved for treatment in Type 1 GD in both adults and children. Oral drugs as substrate reduction therapy with Miglustat and Eliglustat tartrate are used for milder disease where treatment with ERT is not possible.[9] These drugs are best used as maintenance therapy after the therapeutic goals have been achieved with ERT. The potential for cure of GD is Bone marrow transplantation. Future therapies include Enzyme enhancement therapy with chaperones, gene therapy, retroviral vector transfer of the GBA gene into cultured bone marrow cells of Gaucher patients results in expression of physiologic levels of enzyme.[4]

Conclusion

Gaucher’s disease may be under-diagnosed in India due to the paucity of the sources for performing the genetic evaluation and analysis. Hematologists need to have greater awareness of Gaucher disease because they have a unique opportunity to make an early diagnosis and provide optimal treatment for this disease. This increase in diagnosis is also related to the availability of ERT. ERT is profoundly expensive and virtually beyond the means of any patient. However, with increasing number of patients eISSN: 2349-6983; pISSN: 2394-6466

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Type I Gaucher’s Disease

being diagnosed, it is becoming difficult to support all patients with the disease. Hence, the government should intervene in assisting this group of patients in diagnosis and therapy.

Acknowledgements

We thank the patient and the parents for their co-operation and the Department of Pediatrics, JNMC , Belagavi for the support.

Funding None

Competing Interests None Declared

References

1. Shehi B, Boçari G, Vyshka G, Xhepa R , Alushani D. Gaucher’s Disease in Albanian Children: Casuistics and Treatment. Iranian Journal of Pediatrics 2011; 21( 1): 1-7. 2. Epstein E. Beitrag zurchemie der Gaucherschen krankeit. Biochem Z. 1924;145:398–402. 3. Jain VV, Yelwatkar S. Unusual presentation of adult Gaucher’s disease: A long and difficult road to diagnosis.Indian J Endocr Metab 2011;15:224-6.

4. Nagral A. Gaucher Disease. J Clin Exp Hepatol. 2014 Mar; 4(1): 37–50. 5. Schnabel D, Schroder M, Sandhoff K. Mutation in the sphingolipid activator protein 2 in a patient with a variant of Gaucher disease. FEBS Lett. 1991;284:57–9. 6. Ho M.W., Seck J., Schmidt D. Adult Gaucher’s disease: kindred studies and demonstration of a deficiency of acid beta-glucosidase in cultured fibroblasts. Am J Hum Genet. 1972;24:37–45. 7. Giraldo P., Cenarro A., Alfonso P. Chitotriosidase genotype and plasma activity in patients type 1 Gaucher’s disease and their relatives (carriers and non carriers) Hematologica. 2001;86:977–984. 8. Svennerholm L., Håkansson G., Lindsten J., Wahlström J., Dreborg S. Prenatal diagnosis of Gaucher disease. Assay of the beta-glucosidase activity in amniotic fluid cells cultivated in two laboratories with different cultivation conditions. Clin Genet. 1981;19:16–22. 9. Di Rocco M, Giona F, Carubbi F, Linari S, Minichilli F, Brady RO, et al. A new severity score index for phenotypic classification and evaluation of responses to treatment in type I Gaucher disease. Haematologica 2008;93:1211-8.

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Case Report Acute Oxalate Nephropathy in a Young Boy Due to Ingesting Averrhoa Bilimbi; Case Report and Literature Review of an Under-recognized Cause of tropical Renal Disease Niranthi Ruwini Perera1*, Hiranya Dulanjalie Tennekoon1 and Randula Ranawaka2 Department of Pathology, Faculty of Medicine, University of Colombo, Sri Lanka Department of Paediatrics, Faculty of Medicine, University of Colombo, Sri Lanka

1 2

Keywords: Acute Oxalate Nephropathy, Averrhoa Bilimbi, Dietary Hyperoxaluria

ABSTRACT The occurrence of acute oxalate nephropathy (AON) as a result of consuming foods rich in oxalates is well -recognized. Although renal injury has been extensively recognized and described following the ingestion of star fruit (Averrhoa carambola), reports implicating â&#x20AC;&#x2DC;bilimbiâ&#x20AC;&#x2122; (Averrhoa bilimbi), are far less common and were found in adults following ingestion of bilimbi juice. This report describes possibly the first case of AON occurring in an otherwise healthy eight year old boy following the consumption of several bilimbi fruit. Features which favoured dietary hyperoxaluria as the cause for his acute renal injury included the development of acute gastro-intestinal symptoms after ingesting the oxalate rich fruit, the presence of oxalate crystals in the urine full report and renal biopsy and the episodic reversible nature of his illness. Dehydration is likely to have contributed to his AON. The patient was managed medically, did not require dialysis and recovered completely within two weeks. It is important to note that dietary hyperoxaluria causing oxalate nephropathy may be an under-recognised cause of renal disease.

*Corresponding author: Dr Niranthi Ruwini Perera, Department of Pathology, Faculty of Medicine, University of Colombo, Sri Lanka E-mail: niranthiperera@hotmail.com

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Acute Oxalate Nephropathy Due to Averrhoa Bilimbi

Introduction

Acute oxalate nephropathy (AON) as a result of consuming foods rich in oxalates is well recognized [1]. However, the fact that commonly available fruit are rich in oxalates and therefore have the potential to cause renal damage, is less well known. Although renal injury has been described following the ingestion of star fruit (Averrhoa carambola), reports implicating ‘bilimbi’ or ‘biling fruit’ (Averrhoa bilimbi), are far less common. Moreover, in these reported cases, the renal injuries described had occurred in adults following the ingestion of bilimbi juice. We describe what is possibly the first case of AON occurring in an otherwise healthy young boy, following the consumption of several bilimbi fruit, a common tropical fruit.

Case Report

A previously healthy 8 year old boy presented with acute onset vomiting, lower abdominal pain and gross haematuria. He had eaten six bilimbi fruit the same day, while playing cricket for about four hours in the hot sun on a humid day, without water. He became anuric for more than 18 hours with the serum creatinine increasing to 508µmol/l. The urine full report revealed proteinuria, red blood cells and calcium oxalate crystals. A renal core biopsy stained with haematoxylin and eosin showed acute tubular destruction and inflammation with polymorphonuclear leucocytes on histologic sections (Figure 1). The renal tubules showed dilatation and destruction and were clogged by numerous fractured crystals which were polarisable (Figure 2). Interstitial fibrosis was notably absent. He developed acidosis, hyperkalaemia and hypertension which were managed medically. His urine output improved following a high

Fig. 1: Microphotograph showing acute tubular destruction and inflammation with polymorphs. Note the irregular oxalate crystals within and around tubules (H&Ex200).

dose of loop diuretics and he was started on steroids. His renal function improved gradually over the next two weeks and he recovered completely without the need for dialysis.

Discussion

Hyperoxaluria may be primary or secondary. Primary hyperoxaluria is metabolically inherited and characterized by the early appearance of renal failure and a family history of recurrent nephrolithiasis. Secondary hyperoxaluria may be due to increased oxalate absorption following the ingestion of oxalate-rich foods including herbs [ 1,2 ], due to Crohn’s disease and follow gastric by-pass surgery or due to increased production of oxalate in the body as a result of prolonged Vitamin C ingestion or accidental ethylene glycol ingestion [ 3 ]. In Sri Lanka, two commonly consumed tropical fruit rich in oxalates are bilimbi and the star fruit. Ingesting star fruit juice has been well-documented as a cause of AON [4,5,6]. However, reports implicating bilimbi as a cause of AON due to hyperoxaluria are far less common and therefore not as well known. Averrhoa bilimbi or the ‘biling tree’is widely cultivated in Sri Lanka, India and Malysia [7,8]. Its sour fruit only rarely eaten raw, is yellowish –green in colour and contains a high oxalate concentration ranging from 8.57 to 10.32 mg/g. Pickling and dilution processes are known to reduce the oxalate content markedly [8]. A literature search for AON secondary to bilimbi consumption revealed only a few biopsy-proven cases, a series of ten patients from five hospitals and two similar cases, both reported from Kerala, India in 2014 and a third case from Bangladesh in 2015. All cases were described in adults secondary to consuming large amounts of

Fig. 2: Numerous fractured deposits of oxalate crystals in the renal tissue under polarized light (x200)

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

Perera et al. fresh bilimbi juice as a presumptive, traditionally accepted herbal medication for the treatment of hypertension, diabetes and dyslipidaemia [9,10,11,12]. Many of these patients required dialysis during the acute stage of the disease. Our patient was different as he was an otherwise healthy child, who had consumed six bilimbi fruit, (not juice) while playing cricket in the hot sun for several hours. Features which favoured dietary hyperoxaluria as the cause for his acute renal injury included the development of acute gastro-intestinal symptoms within several hours of ingesting the oxalate rich fruit, the presence of diagnostic oxalate crystals in both the urine full report and the renal biopsy (with accompanying tubular damage) and the episodic reversible nature of his illness.

C-235

Funding None

Competing Interests None Declared

References

The authors wish to thank the technical staff of the Department of Pathology, Faculty of Medicine, Colombo, Sri Lanka for preparing the histopathology slides.

1. Jah V, Parameswaran S. Community acquired acute kidney injuries in tropical countries. Nature Reviews Nephrology. 2013;9:278-290 2. Syed F, Mena-Gutierrez A. A Case of Iced-Tea Nephropathy. N Engl J Med 2015;372:1377-1378 3. Glew RH, Sun Y, Horowitz BL, Konstantinov KN, Barry M, Fair JR. Nephropathy in dietary hyperoxaluria: A potentially preventable acute or chronic kidney disease. World J Nephrol 2014;3:122-42 4. Chen CL, Fang HC, Chou KJ, Wang JS, Chung HM. Acute oxalate nephropathy after ingestion of star fruit. Am J Kidney Dis. 2001;37:418-22 5. Abeysekera RA, Wijetunge S, Nanayakkara N, Wazil AWM, Ratnatunga NVI, Jayalath T et al. Star fruit toxicity: a cause of both acute kidney injury and chronic kidney disease: a report of two cases. BMC Res Notes, 2015;8: 796-801 6. Getting JE, Gregoire JR, Phul A, Kasten MJ. Oxalate nephropathy due to ‘juicing’: a case report and review. Am J Med. 2013; Sep;126(9):768-72. 7. Morton JF, Dowling CF. Bilimbi, Averrhoa bilimbi. In: Fruits of Warm Climates: Miami FL Winterville, N.C.Distributed by Creative Resources Systems, ©1987 8. De lima VLAG, Mélo EDA, Lima LDS. Physicochemical characteristics of Bilimbi (Averrhoa bilimbi L). Revista Brasileira de Fruticultura. 2001;23:421-23 9. Bakul G, Unni VN, Seethaleksmy NV, Mathew A, Rajesh R, Kurien G et al. Acute oxalate nephropathy due to ‘Averrhoa bilimbi”fruit juice ingestion. J Nephrol 2013;23:297-300 10. Nair S, George J, Kumar S, Gracious N. Acute Oxalate Nephropathy following ingestion of Averrhoa bilimbi Juice. Case Reports in Nephrology, 2014; Article ID 240936 5 pages 11. Billah MM, Rahuman MA, Rahim MA, Swarna AT, Mitra P, Chowdury TA et al. Acute Kidney Injury following Ingestion of Averrhoa bilimbi Juice. Bangladesh Crit Care J September 2015; 3 (2):71-73 12. Saini S. A review on phytochemistry and pharmacology of averrhoa bilimbi linn. International Education and Research Journal 2016; 2:71-76 13. Neto MM, ed. Star fruit as a cause of acute kidney injury. J Bras Nephrol 2014;36(2)118-120.

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While ‘unbound oxalate’ is readily absorbable in the gastrointestinal tract, the divalent cations calcium and magnesium present in other foods, form ‘oxalate salts’ which are poorly absorbable. [2,13]. A ‘safe’ dose of oxalate consumption has yet to be determined and is likely to vary among individuals [3]. Dehydration and fasting are recognized predisposing factors for hyperoxaluria, both of which were present in our patient [6,13]. Oxalate is eliminated almost exclusively by the kidneys and diuresis is known to promote urinary oxalate excretion in individuals with normal renal function. In hyperoxaluria, the crystals are thought to cause direct nephrotoxicity to renal tubular epithelial cells by inhibition of cell proliferation and by apoptosis . They are also thought to stimulate specific genes in renal tubular cells including the connective tissue growth factor gene, leading to interstitial fibrosis [3,9]. In renal biopsies, oxalate as a cause of acute or chronic nephropathy is diagnosed by identifying the characteristic intratubular polarizable crystals.

Conclusion

In Sri Lanka and other tropical countries where the bilimbi fruit is regularly consumed, identifying its potential as a cause of AON has many implications. As long term hyperoxaluria is known to cause nephrolithiasis, nephrocalcinosis, acute and chronic kidney disease and end stage renal disease, investigating the effects of chronic dietary hyperoxaluria on the kidney and assessing the extent, if any, of renal functional impairment becomes important. Dietary hyperoxaluria may well be an underrecognised cause of renal disease which requires careful epidemiological, biochemical and histologic consideration.

Acknowledgements

Letter to Editor Nucleic Amplification Testing: Individual Verses Minipool, What to choose? Rateesh Sareen Department of Pathology & Transfusion Medicine, Santokba Durlabhji Hospital & Reseach Center, Jaipur, India

Dear Sir,

The commonly used NAT testing technologies include Polymerase Chain reaction (PCR) and transcription mediated amplification[1]. TMA is a transcriptionamplification process, using two enzymes- Reverse transcriptase & RNA polymerase to produce millions of copy of targeted RNA sequences. It comprises of three steps- target capture, amplification & detection. TMA allows for simultaneous testing of multiple viruses in a single test tube.[2] Today there are two NAT tests availableCOBAS Ampliscreen HBV test/ COBAS Taq screen MPX- a multiplex assay for HBV, HCV & HIV- both of these are manufactured by Roche Molecular system Inc and Procleix Ultrio assay a multiplex assay for HBV, HCV & HIV manufactured by Gen Probe Inc. (Novartis) (Chiron in US) [3] The sero prevalence of anti HIV-1, anti HCV and HBsAg in Indian blood donors is 0.5%, 0.4% and 1.4% respectively [4] compared to 0.0097%, 0.3% & 0.07% in blood donors in USA.5 The NAT yield NAT+/Ab- in USA was 230 for HCV, 18 for HIV and 5 for HBV1. If we extrapolate Indian prevalence to expected NAT yield in our country as the technology is the same as followed in US, we get stunning figures for HIV- 5154, HCV-133 and HBV -2000, this emphasizes the importance of NAT in India with high prevalence of viremia. Japan was the first country to implement routine HBV NAT in addition to HCV & HIV-1 NAT 6. UK, USA, Australia, Japan, Austria, Belgium, Canada, Finland, France, Germany, Italy, Poland, Netherland, New Zealand, Singapore, Poland, Portugal , Norway, Slovenia & Hong kong are countries where there is 100% NAT testing of donor blood. The developing nations like India, China, Brazil, Thailand, Spain, Korea, Greece and others have a portion of their blood supply which is

NAT tested[7]. The decision to perform NAT in mini pool or Individual format is largely based on the balance between cost quality & resources available with blood centers. The implementation of NAT using minipools was a necessary compromise in light of the cost and complexity of NAT technologies and the massive scope of blood screening. There are studies from India by Makroo et al[8] & Chatterjee et al[9] suggesting that use of ID NAT in blood banks in India would ensure safer blood transfusion. On the other hand, another recent case report[10] from Australian Red cross Blood service where 11, 13, 288 donations were screened as pools of 24 and an additional 32,003 donations were screened in Individual NAT format and further 294474 donations exclusively on Individual format showed minor differences in Individual & minipool strategies with excellent specificities however when Individual NAT was performed at Minipool site, a potential for contamination limiting optimal processing of Individual NAT was observed. In a country with limited resources and where cost economics playing vital role we have to delicately strike a balance between cost, quality , manpower & infrastructural constraints as well as opening for new testing technologies prevalent in the world. In order to keep pace with the growth and safety for blood receipts we need to gear up for acceptance and incorporation latest testing facilities for our blood banks. The policy makers need to self retrospect. Recently, a study by Australian Red cross[10] emphasized on trained, skilled manpower requirement for such a technically demanding procedure. The most prudent approach in todayâ&#x20AC;&#x2122;s scenario is not debating the issue of Minipool NAT - versus Individual NAT rather it should be to fill the gap between those who

*Corresponding author: Dr Rateesh Sareen, Department of Pathology & Transfusion Medicine, Santokba Durlabhji Hospital & Reseach Center, Jaipur, India Phone: +91 0141-2603401 E-mail: drrateeshsareen@yahoo.co.in

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

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Minipool NAT Verses Individual NAT.

can afford everything and those who can afford nothing. At the drop of a hat, we need to think seriously on how to make available NAT testing facilities for our blood recipients that is not taxing on the patients, keeping high quality affordable at reasonable cost else we will be in a jaywalk situation.

Acknowledgements

Dr G N Gupta Head of the Department for his support.

Funding None

Competing Interests None Declared

Reference

1. Saiki RK, Gelfand DH, Stoffel S, et al. Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988; 239 (1): 488-491. 2. Giachetti C, Linnen JM, Kolk DP, et al. High sensitive multiplex assay for detection of HIV type-1 and hepatitis C virus RNA. J clin Microbiol. 2002;40(7): 2408-2419. 3. Euro survelliance Vol-10 Inc Jan â&#x20AC;&#x201C;Mar, 2005. 4. Bhatia R, Weber M, Seifried E: Feasibility & efficacy of routine PCR screening of blood donations for Hepatitis C virus, Hepatitis B virus & HIV-1 in blood bank setting. Lancet 1999; 353(9150): 359-363.

5. Dodd RY, Notan EP: Current prevalence if incidence of infectious disease markers & estimated window period risk in American Red cross blood donor population. Transfusion 2002: 42: 975-9. 6. Mine H et al: High through put screening of 16 million serologically negative blood donors of Hepatitis B, Hepatitis C & HIV virus by NAT with specific & sensitive multiplex reagent in Japan. J virol methods 2003; 112:145-151. 7. W.K Roth et al-International survey on NAT testing of blood donations: expanding implementation and yield from 1999 to 2009.Vox Sanguinis(2012)102,82-90. 8. Makroo N, Choudhury N, Jagannathan L, et al. Multicenter evaluation of individual donor NAT for simulataneous detection of HIV-1 , Heapatitis B & C virusesin Indian blood donors. Indian J Med Res. 2008;127,140-147. 9. Chatterjee K, Coshic P et al: Individual Donor NAT for blood safety against HIV-1 and Hepatitits B&C viruses in a tertiary care hospital. Nat Med J India; 25(4) : 207-9. 10. Mison L, Seed CR, Margaritis AR, Hyland C; Australian Red cross Blood services. NAT screening of Australian blood donors for Heaptitis C & HIV-1 RNA: Comparison of two high performance testing staregies. Vox Sang, 2003 Jan;84(1): 11-9.

Annals of Pathology and Laboratory Medicine, Vol. 03, No. 04, October - December 2016

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