APALM 4.2 (2017) by PaGe

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Annals of Pathology and Laboratory Medicine March-April 2017; Vol. 4, Issue 2

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DOI : 10.21276/apalm

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

Annals of Pathology and Laboratory Medicine Co-Editor in Chief

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

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Editorial Board Members

Advisory Editors

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

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Contents

Original Article Re-evaluation of conventional cervical smears with particular reference to revised A199-A125 Bethesda system criteria: a 5 years blind retrospective and prospective study Sumit Prakash Rathore, Manish Kumar, Rana K. Sherwani

A cytopathological study of malignant thyroid lesions and its implications in a A126-A130 referral center. Shifa Syed Ibrahim, Sharnila Thilagavathy Narayanan, Meenakumari Gopalakrishnan A Descriptive Study of Histopathological Patterns of Lymph Node Biopsies In A A131-A136 Tertiary Care Hospital Rajshri P Damle, Kishor H Suryawanshi, N V Dravid, D V Newadkar, Prashant N Deore Prevalence of Transfusion Transmitted Infections among Blood Donors at a Tertiary A137-A141 care Teaching Hospital in Southern Rajasthan Preeti Balkisanji Agrawal, Suraj Jain, Sajjan S Surana, Sashi Sujanani A Study of D2-40 Immunohistochemical Expression in Colorectal Carcinomas Sarvek Bajaj, Gururajaprasad C, Suchitha Satish

A142-A147

Impact of Iron Deficiency Anemia on Glycated Hemoglobin (HbA1c) Levels in A148-A152 Diabetics with Controlled Plasma Glucose Levels Lavanya Rajagopal, Sundaram Arunachalam, Shivashekar Ganapathy, Balaji Ramraj A study on Histomorphological Spectrum of Nasal Polyp K Pushpalatha, Sreedevi CH, Soujanya R

A153-A156

Diagnostic utility of fine needle aspiration cytology of sensory cutaneous nerve in A157-A162 leprosy Reena Naik, Dilip Kumar Sa, Kishori Moni Panda Histomorphological Study of Gestational Trophoblastic Lesions in a Tertiary Medical A163-A169 Centre: A Prospective Study Nidhi Rajendra, Fouzia Kauser, Prashanth Madapura V, Doddikoppad. M M Identification and antimicrobial susceptibility testing of microorganisms from A170-A177 positive blood cultures by a combined lysis-centrifugation method with MALDI-TOF MS and VITEK2 System Donatella Maria Rodio, Filomena Febbraro, Gianluca Puggioni, Camilla Paradisi, Flavia Stangherlin, Carla Prezioso, Guido Antonelli, Maria Trancassini, Valeria Pietropaolo Analysis of morphological changes in liver in obstructive jaundice with special A178-A186 emphasis on fibrosis Rachana Amit Chaturvedi, Jayashri Popat Chaudhari, Mayura Kekan, Apurv Deshpande, Ramkrishna Prabhu, Amita Suresh Joshi Comparative study of cell block versus centrifuged smear examination from aspirates A187-A194 of cystic lesions S R Niveditha, Thejasvi Krishnamurthy, Sumitha Prakash Impact of intervention on awareness of biomedical waste disposal among medical A195-A202 students Dhananjay Shrikant Kotasthane, Vaishali Dhananjay Kotasthane, Shanmugasamy K, Ancy A Study of incidence of microalbuminuria among first diagnosed diabetic patients and A203-A207 its correlation with body mass index and coexisting hypertension in a tertiary care hospital Esakki Muthuvel, Vimal Chander, Sowmiya Balu Blood transfusion profile of Beta-Thalassemia Major patients attending a tertiary A208-A211 care hospital Mallikarjun Adiveppa Pattanashetti, Ganga S Pilli, Laxmi Pattanashetti

III

A comparison of effect of Iron Deficiency Anemia on HbA1c levels in controlled A212-A218 diabetics and non-diabetics: A cross sectional analysis of 300 cases Lavanya Rajagopal, Sundaram Arunachalam, Shivashekar Ganapathy, Balaji Ramraj, Veena Raja Cytological and Histomorphological Correlation of Salivary Gland Lesions- An A219-A223 Experience at Rural Tertiary Healthcare Hospital Thangam R, Vaishali Dhananjay Kotasthane, Dhananjay Shrikant Kotasthane, Koteeswaran G, N S Kannan

Case Report

Synchronous Primary Carcinoma of Cervix and Endometrium Divya Madhala, Gouthaman Shanmugasundaram, Sai Shalini Chinnathambi Narayanan, Jaya Vijayaraghavan

C33-C36

Large hamartomatous polyp presenting with profuse rectal bleeding in colo-colic intussuception in a child: a case report Kamal Nain Rattan, Shruti Bansal, Roomi Yadav, Gurupriya J, Neha Singh

C37-C39

Cavernous hemangioma of the testis mimicking as torsion of testis: a case report. Shreekant Bharti, Narrendran AP

C40-C42

Non-Immune Hydrops Foetalis due to Congenital Toxoplasmosis: a rare case report with review of literature. Mallikarjun Adiveppa Pattanashetti, Vijayalaxmi V Suranagi, Hema B Bannur

C43-C46

Cholesterol granuloma of hydrocele sac mimicking testicular tumour Nehal Ahmad, Sabina Khan, Mohd Jaseem Hasan, Sujata Jetley, Abhinav Jain

C47-C49

Primary Neuroendocrine Carcinoma of Breast: an uncommon variant. Sunil V Jagtap, Vidya C Aher, Suresh J Bhosale, Atul Hulwan, Anup N Gosavi

C50-C52

Inflammatory Myofibroblastic Tumor Of Urinary Bladder Ronica Baruah, Abhijit Kalita, Phanindra Mohan Deka

C53-C55

A case series of congenital ovarian cyst in a Tertiary Care Hospital in Mumbai, India Bhavana Madhukar Bharambe, Saroj Ashok Bolde, Sushma Chandeshwar Bharti, Neha Satyanarayan Somani

C56-C58

Occult Primary Thyroid Carcinoma presenting as lateral cervical mass: Report of two cases Shalini Bahadur, Priyanka Anand, Kamlesh Prajapati, Namrata Nargotra

C59-C62

Cytodiagnosis of a testicular epidermoid cyst in a young male: A rare entity. Dilip Kumar, Anupama Arya, Shruti Mahawar, Poonam Das, Nitin Dayal

C63-C65

Letter to editor Trichilemmal Cyst at Wrist: A Rare Site of Occurence

Ruchi Sinha, Iffat Jamal, Shashikant Kumar, Purushottam Kumar

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L7-L9

Original Article DOI: 10.21276/APALM.987

Re-evaluation of Conventional Cervical Smears with Particular Reference to Revised Bethesda System Criteria: A 5 Years Blind Retrospective and Prospective Study

Sumit Prakash Rathore1*, Manish Kumar1 and Rana K. Sherwani2 Department of Pathology, Jhalawar Medical College , Jhalawar, India Department of Pathology, J.N. Medical College, A.M.U., Aligarh, UP, India 1

ABSTRACT Background: The revised Bethesda System of reporting Pap smears in addition to defining the diagnostic categories also develops criteria for determining the adequacy and quality of Pap smears. Methods: The present retrospective and prospective study was conducted in department of pathology after approval from institutional ethics committee which included retrospective cases from 2007 to 2009 and prospective cases from 2010 to 2011. Prospective cervical smears were stained by using Papanicolaou stain. After staining, slides were mounted, screened and reported by two consultant pathologists by both Conventional system and the 2001 Bethesda system. Smears which were already reported by the Conventional system were also reviewed retrospectively by the 2001 Bethesda system. Cytohistopathologic correlation was done wherever available. Result: Results on adequacy of the specimens showed 98.0% smears as satisfactory and 2.0% were found to be unsatisfactory for evaluation. Pap smear reporting by both the systems showed similar number of cases with inflammatory findings. 18.3% premalignant lesions were reported by the Conventional system but it was significantly higher (31.8%) when reviewed by the Bethesda system. Numbers of malignant lesions were found similar by both systems i.e. 3.6%. Conclusion: Reporting should be done by the Bethesda System as it improves producibility and helps in diagnose of various intraepithelial lesions and invasive lesions at an early stage and helps to manage them properly. Keywords: Pap Smear, Bethesda System, Premalignant Lesions, Malignant Lesions

Introduction

The highest incidence of cervical cancer occurs in Latin America, the Caribbean, Africa (Tropical Sub-Sahara), and South - East Asia. Around 80% of the cases occur in developing countries and just 20% in developed countries. [1] India, which accounts for one sixth of the world’s population, also bears one fifth of the world’s burden of cervical cancer.[2] There are approximately 132,082 new cases of cervical cancer in India per year and the disease is reported to be responsible for almost 20 percent of all female deaths.[3] India’s cervical cancer age-standardized incidence rate (30.7 per 100,000) and age-standardized mortality rate (17.4 per 100,000) are the highest in South Central Asia.[3]

of opportunist screening favors the continuance of this unfavourable situation and indicates the urgent need for the public health authorities to find a solution.[4] Cervical cytology was introduced by George N Papanicolaou into clinical practice in 1940.[5] In 1945, the Papanicolaou smear received the endorsement of the American Cancer Society as an effective method for cancer screening and prevention of cancer. Papanicolaou introduced a numeric classification 1-5 to communicate the degree of confidence that cancer cells were present in the specimen. However, this classification was unable to reliably communicate clinically relevant information. It did not reflect the current understanding of cervical neoplasia with no place for noncancerous entities.

Socioeconomic and cultural aspects are the factors in unequal distribution of cervical neoplasia around the world. However, a preponderant factor in the areas of low incidence is the level of information from the feminine population regarding the disease and the continual screening of this population. On the other hand, in developing countries, the low level of awareness of the problem, and the use

In 1988, the National Cancer Institute (NKI), National Institute of Health, Bethesda, Maryland, sponsored an open workshop including – cyopathologists, cytotechnologists, histopathologists, family practitioners, gynaecologists, public health physicians and epidemiologists to develop a uniform descriptive terminology for cervicovaginal cytology.[6] The recommendations of the 1988 workshop

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Re-evaluation of Conventional Cervical Smears

received widespread acceptance in practice. The Bethesda System has also replaced the three levels of dysplasia and carcinoma in situ with two levels, Low grade squamous intra epithelial lesion (LSIL) and High grade squamous intra epithelial lesion (HSIL). A second workshop was held in 1991 to modify the Bethesda system based on the actual laboratory and clinical experience after its implementation. The latest revision of this system was completed in April 30-May 2, 2001 and published in April 2002. Therefore, the present study was undertaken for the better understanding of the method of reporting of the Pap smears and to facilitate the effective and precise communication between pathologists and clinicians at our centre.

Material and Methods

The present study was conducted in the department of Pathology in a tertiary care teaching hospital. The study design was both retrospective and prospective including retrospective cases from 2007 to 2009 and prospective cases from 2010 to 2011. After approval from institutional ethics committee the study was conducted on the patients attending the indoor wards and the OPDs of the Departments of the Obstetrics and Gynecology. Cervical smears were collected by the residents of the OBG Department using Ayer’s spatula along with an endocervical cytobrush after taking informed consent from the patients. These were labeled, fixed in 95% ethanol and were sent to the Department of Pathology. These smears were then stained by using Papanicolaou stain. After staining, slides were mounted with DPX (distrene dibutyl phthalate xylene), screened and reported by two different consultant pathologists by using both the Conventional system and the 2001 Bethesda system. Retrospectively also, smears which were already reported by the Conventional system were reviewed by using the 2001 Bethesda system. A total of 1000 cases were studied and from each year 200 cases were taken randomly. Cytohistopathologic correlation was done wherever available. All the data were recorded in percentage and the sensitivity and specificity of Bethesda system was used to detect premalignant and malignant lesions.

Results

A total of one thousands cases were compared by conventional system and Bethesda system. As per the Bethesda system 2001, all the cervical smears were rescreened and commented on their adequacy. Results on adequacy of the specimens showed 980 smears (98.0%) as satisfactory for evaluation. Presence of T-zone component was seen in 449 smears (44.9%) and absence in 551 (55.1%) smears. All of the specimens were processed properly and none was rejected due to the reasons like broken slide or

incompletely filled form etc. Twenty smears (2.0%) were found to be unsatisfactory for evaluation. (Table 1) Inflammation was the most common finding seen in 896 smears (89.6%). Out of these inflammatory smears, 865 smears (86.5%) showed nonspecific inflammation, while 31 smears (3.1%) had organism specified inflammation of which Candida was found in 1.2% of cases, Bacterial vaginosis in 1.0%, Trichomonas vaginalis in 0.5% of cases and Herpes and Leptothrix in 0.2% cases respectively. HPV related changes were seen in 1.7% cases. Reactive cellular changes due to inflammation were seen in 231 cases (23.1%), while reactive changes due to radiation therapy and repair were noted in 13 cases (1.3%). Atypical squamous cells of undetermined significance (ASC-US) was reported in 2.3% of the smears, whereas atypical squamous cells- Can’t exclude HSIL (ASC-H) was seen in 4 smears (0.4%). Low grade squamous intraepithelial lesion (LSIL) was reported in 187 smears (18.7%). Out of these 187 smears, HPV related changes were seen in17 cases (1.7%). HSIL (high grade squamous intraepithelial lesion) was reported in 60 smears (6.0%). Squamous cell carcinoma was noted in 26 cases (2.6%). AGUS (atypical glandular cells of undetermined significance) was reported in 35 smears (3.5%) whereas AGC (atypical glandular cells–favouring neoplastia) was noted in 9 smears (0.9%). One smear (0.1%) was reported as adenocarcinoma-insitu (AIS), while adenocarcinoma was noted in 4 smears (4%), adenosquamous carcinoma in 1 smear (1%) and undifferentiated carcinoma was reported in 4 smears (4%). In one smear (0.1%) of a reproductive age female, endometrial cells were noted. (Table 2) For ‘specimen adequacy’ out of total 386 cases, presence of endocervical cells was noted in 269 smears (69.7%), presence of squamous metaplastic cells in 98 smears (25.4%), and 19 (1.9%) smears was found unsatisfactory for evaluation. The term satisfactory is also not used in the conventional reporting, unsatisfactory smears are mentioned in the reports. In our study 19 (1.9%) smears were unsatisfactory by conventional reporting and 20 (2.0%) on reviewing by the Bethesda system. Satisfactory smears were 980 (98.0%) by the Bethesda system which was similar to that by the Conventional reporting i.e. 981 (98.1%). (Table 3) Pap smear reporting by both the systems showed similar number of cases with inflammatory findings. Nonspecific inflammation was reported in 85.2% and 86.5% by the Conventional system and the Bethesda system respectively. Trichomonas, Herpes, and Leptothrix were found in 0.5%, 0.2%, and 0.2% cases respectively by both systems, while Candida was seen in 0.8% cases by the conventional

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system and in 1.2% of cases by the Bethesda system. Bacterial vaginosis was noted in 0.9% smears by the Conventional and in 1.0% cases by the Bethesda system. But the significant difference was noted in the number of premalignant lesions when studied by the two different systems i.e. 18.3% premalignant lesions were reported by the Conventional system but it was significantly higher i.e

31.8% when reviewed by the Bethesda system. Numbers of malignant lesions were found similar by both systems i.e. 3.6%. (Table 4) In our study, the Bethesda system was found to be 100% sensitive and 100% specific in detecting malignant lesions while it was 36.5% more sensitive and 45.5% more specific in detecting premalignant lesions as compared to conventional system of reporting.

Table 1: Specimen adequacy criteria based on the revised Bethesda system, 2001. Cytological findings

Number of cases (%) N=1000

Satisfactory for evaluation

980 (98%)

T-zone component

Present

449 (44.9%)

Absent

551 (55.1%)

Specimen rejected/not processed

Specimens processed and examined but unsatisfactory for evaluation due to

Scant cellularity

05 (0.5%)

Air drying artifact

04 (0.4%)

Completely obscured by inflammation

08 (0.8%)

Completely obscured by blood

03 (0.3%)

Table-2: Findings according to the revised Bethesda system, 2001 Number of cases (%) N=1000

Cytological findings Smears within normal limits

52 (5.2%)

Inflammatory (nonspecific)

865(86.5%)

Inflammatory (Organism specified)

Trichomonas

05 (0.5%)

Bacterial vaginosis

10 (1.0%)

Candida

12 (1.2%)

Herpes

02 (0.2%)

Leptothrix Reactive cellular changes

Due to inflammation Due to radiation and repair

02 (0.2%) 231 (23.1%) 13 (1.3%)

Atypical squamous cells of undetermined significance (ASC-US)

23 (2.3%)

Atypical squamous cells- canâ&#x20AC;&#x2122;t exclude HSIL (ASC-H)

04 (0.4%)

Low grade squamous intraepithelial lesion (LSIL)

Human papilloma virus(HPV)

17 (1.7%)

Mild dysplasia

170 (17%)

High grade squamous intraepithelial lesion (HSIL)

60 (6%)

Squamous cell carcinoma

26 (2.6%)

Atypical glandular cells of undetermined significance (AGUS)

35 (3.5%)

Atypical glandular cells (favours neoplastic)

09 (0.9%)

Adenocarcinoma-in-situ

01 (0.1%)

Adenocarcinoma

04 (0.4%)

Adenosquamous carcinoma

01 (0.1%)

Undifferentiated carcinoma

04 (0.4%)

Endometrial cells

01 (0.1%)

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Table 3: Distribution of satisfactory and unsatisfactory smears according to the Conventional Pap and the Bethesda system, 2001. Conventional system Bethesda system Findings Number of smears (%) N=1000 Number of smears (%) N=1000 Satisfactory 981 (98.1%) 980 (98%) Unsatisfactory 19 (1.9%) 20 (2%) Table 4: Comparative study on Pap smear findings by Conventional Pap smear reporting and Bethesda system (n=1000). Conventional System Bethesda system Pap smear findings No. of smears (%) No. of smears (%) Nonspecific inflammation 852 (85.2%) 865(86.5%) Trichomonas Vaginalis 05 (0.5%) 5(0.5%) Candida 08 (0.8%) 12(1.2%) Inflammatory Bacterial vaginosis 09 (0.9%) 10(1.0%) (Organism specified) Herpes 02 (0.2%) 2(0.2%) Leptothrix 02 (0.2%) 2(0.2%) Premalignant lesions 183 (18.3%) 318(31.8%) Malignant lesions 36 (3.6%) 36 (3.6%)

Fig. 1: Smear showing Trichomonas Vaginalis infection â&#x20AC;&#x2DC;TV bodiesâ&#x20AC;&#x2122;(pap,40x).

Fig. 2:-smear showing filaments of Leptothrix infection (Pap,40X).

Fig. 3:-smear showing branching species (pap40X).

Fig. 4:-smear showing ASCUS-Favour Neoplasia (PAP,40X).

hyphae of candida

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Fig. 5:-smear showing LSIL (PAP,40X)

Fig. 6:-smear showing HSIL (PAP,40X)

Discussion

total of 18.7%. HSIL (high grade squamous intraepithelial lesion) was reported in 6.0%. Squamous cell carcinoma was noted in 2.6%. AGUS (atypical squamous cells of undetermined significance) were reported in 3.5% whereas AGC-favour neoplastic were noted in 0.9%. One smear (0.1%) was reported as Adenocarcinoma-in-situ (AIS). Adenocarcinoma was noted in 0.4%, adenosquamous carcinoma in 0.1% and undifferentiated carcinoma also was reported in 0.4%.

Reporting of Pap smear is done by the Conventional system and by the Revised Bethesda system 2001. The Bethesda System in addition to defining the diagnostic categories also develops criteria for determining the adequacy and quality of Pap smears. Assessment of the adequacy of a specimen is an integral part of the overall evaluation of a Pap smear. The purpose of designating smears as unsatisfactory is to alert clinicians that the particular smear might not be reliable for detecting preneoplastic or neoplastic conditions. A longitudinal study found that unsatisfactory Pap smears were more often from high-risk patients.[7] All the smears were commented regarding their adequacy on reviewing with the revised Bethesda system. 98.0% were found satisfactory for evaluation, T-zone component was present in 44.9% and absent in 55.1%. All of the specimens were processed properly and none was rejected due to any reason. 2.0% smears were found to be unsatisfactory due to the reasons like scant cellularity, air drying artefact, completely obscured by inflammation or completely obscured by blood. Like the Conventional reporting, inflammation (89.6%) was the most common finding with the Bethesda system too. Out of these 89.6% inflammatory findings, 86.5% had nonspecific inflammation while 3.1% had organism specified inflammatory smears. 5.2% were found to be within normal limits. Atypical squamous cells of undetermined significance (ASC-US) was reported in 2.3%, whereas Atypical squamous cells- canâ&#x20AC;&#x2122;t exclude HSIL (ASC-H) was reported in 0.4%. Low grade squamous intraepithelial lesion (LSIL) was reported in a www.pacificejournals.com/apalm

Our findings are discordant with that of Dhruva et al study, who reported 4.5% smears as unsatisfactory for evaluation and 37% smears were in normal limits. 1.3% smears had atrophic changes, inflammatory (47.5%), ASCUS (0.8%), LSIL (5%), HSIL (2.8%) and 0.8% were squamous cell carcinoma in their study.8 Our findings are also discordant with the study of Ranabhat SK et al, who reported 26% with nonspecific inflammation and 2.5% with reactive cellular changes. Organism specific infections were detected in 9.4%. In their study 3.1% smears were unsatisfactory for evaluation. ASC-US, AGC and Squamous cell carcinoma were noted in the 0.2% each, whereas LSIL was noted in 0.3% and HSIL was seen in 0.7% smears in their study.[9] Khan MS et al in their study on 546 patients found 9.5% to be inadequate. 22.7% were normal, 55.3% showed inflammatory changes and atrophic changes were seen in the 7.3%. 1.8% had LSIL, and 1.3% had HSIL while Carcinoma in situ, was seen in 2.0%.[10] Our findings are concordant with the study by Sherwani RK et al, who reported LSIL in 20.0%, HSIL in 4.4% smears and invasive carcinoma in the 3.75% respectively. [11] another study done by Abdullah LS, have reported 2.8% smears as unsatisfactory and 97.2% as satisfactory eISSN: 2349-6983; pISSN: 2394-6466

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Re-evaluation of Conventional Cervical Smears

smears; out of which 5% were abnormal and these were further classified as ASC-US in 40%, ASC-H in 2%, LSIL in 22%, HSIL in12%, AGC in 11% and invasive squamous cell carcinoma in 9%. They also reported 77% smears as negative for squamous epithelial abnormalities while 18% as inflammatory/reactive changes. No endocervical or endometrial cells, which favour neoplastic or adenocarcinoma in situ were reported in their study.[12] Although in the Conventional reporting there is no term like ‘specimen adequacy’ but the following findings may be used to explain it, as we did in our study; presence of endocervical cells was noted in 26.9%, presence of squamous metaplastic cells was seen in 9.8% and unsatisfactory for evaluation which was noted in 1.9% smears. These findings could not be compared with other studies as no such study was found in literature. In our study 1.9% smears were unsatisfactory on conventional reporting and 2% smears on reviewing them by the Bethesda system. Satisfactory smears were 98.0% by the Bethesda system which were found to be similar to that by Conventional reporting i.e. 98.1%. These findings are in concordance with the study of Shorey et al, who reported 96.8% smears as satisfactory and 3.3% as unsatisfactory.[13] Similar results of around 3.8% unsatisfactory smears were also reported by Sankaranarayanan R et al.[14] On comparing the cytological findings of the Conventional system and the Bethesda system, inflammation was found to be the most common finding in both the systems. Nonspecific inflammation was seen in similar number of cases by both the systems of reporting i.e 85.2% by the Conventional method and 86.5% by the Bethesda system. Trichomonas, Herpes, and Leptothrix and bacterial vaginosis were reported approximately similar in both the systems. Candida was found slightly higher in Bethesda system. But the significant difference was noted in the number of premalignant lesions by the Conventional system (18.3%) and significantly higher by the Bethesda system (31.8%). Malignant lesions seen by both the systems of reporting were similar i.e. 3.6%. This could not be compared with any study as none was found mentioning the comparison of two systems.

Conclusion

It was concluded that reporting should be done by the Bethesda System as it improves producibility and helps in identification of ASCUS and AGUS lesions and plays a key role to diagnose various intraepithelial lesions and

invasive lesions at an early stage and helps to manage them properly. Variation in reporting system for cervical smear results can lead to difficulty in communication between pathologists and clinicians, difficulty in comparing results from different centers and in some cases difficulty in selecting the proper treatment for given patients.

References 1.

Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55(2):74-108.

Sankaranaryanan R, Buduk AM, Rajkumar R: Effective Screening Programmes for Cervical Cancer in low- and middle-income developing countries. Bull World Health Organ 2001;79(10): 954-62.

Ferlay J, Bray F, Pisani P, Parkin DM. GLOBOCAN 2002: Cancer Incidence. Mortality and Prevalence Worldwide. IARC Cancer Base No. 5, version 2.0., Lyon: IARC Press 2004.

Canfell K, Sitas F, Beral V. Cervical cancer in Australia and the United Kingdom: comparison of screening policy and uptake, and cancer incidence and mortality. Med J Aust 2006;185:482-6.

Papanicolaou GN. Introduction of Pap smear in early detection of cervical malignancies. Am J Clin Path 1940;19:301-8.

National Cancer Institute Workshop. The 1988 Bethesda System for reporting cervical/vaginal cytological diagnoses. JAMA. 1989;262:931-34.

Ransdell JS, Davey DD, Zaleski S. Clinicopathologic correlation of the unsatisfactory Papanicolaou smear. Cancer Cytopathol 1997;81:139-43.

Dhruva GA, Agravat AH, Bhojani KR. Analyses of cervical cancer in Rajkot population : Electronic Journal of Pharmacology and Therapy 2011;4:15-20.

Ranabhat SK, Shrestha R, Tiwari M. Analysis of abnormal epithelial lesions in cervical Pap smears in Mid-Western Nepal: Journal of Pathology of Nepal 2011;1: 30-3.

10. Khan MS, Raja FY, Ishfaq G, Tahir F, Subhan F, Birjees MK et al. PAP Smear Screening for Pre-cancerous Conditions of the Cervical Cancer. Pak J Med Res 2005; 44:111-3. 11. Sherwani RK, Khan T, Akhtar K, Zeba A, Siddiqui FA, Rahman K et al. Conventional Pap Smear and Liquid Based Cytology for Cervical Cancer Screening – A Comparative Study: Journal of Cytology 2007;24(4):167-72.. 12. Abdullah LS. Pattern of abnormal Pap smears in developing countries: A report from a large referral hospital in Saudi Arabia using the revised 2001 Bethesda System. Ann Saudi Med 2007; 27(4 ):268-72.

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Rathore et al. 13. Shorey G, Shorey P, Kurien A, Joshi VR, Mallick AK.Â Can the use of ayerâ&#x20AC;&#x2122;s spatula and cytobrush in combination improve the adequacy of cervical smears: International Journal of Applied Biology and Pharmaceutical Technology 2011; 2(2):111-16.

A-125 14. Sankaranarayanan R, Thara S, A Sharma, Roy C, Shastri S, Mahe C, et al. Accuracy of conventional cytology: results from a multicentre screening study in India. J Med Screen 2004;11:77-84.

*Corresponding author: Dr Sumit Prakash Rathore, Assistant professor, Department of Pathology , Jhalawar Medical College , Jhalawar, Rajasthan, India Phone: +91 7737295268 Email: drsumitrathore@gmail.com Date of Submission : 25.07.2016 Date of Acceptance : 27.01.2017 Financial or other Competing Interests: None. Date of Publication : 03.04.2017

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eISSN: 2349-6983; pISSN: 2394-6466

Original Article DOI: 10.21276/APALM.1063

A Cytopathological Study of Malignant Thyroid Lesions and Its Implications in A Referral Center Shifa Syed Ibrahim*, Sharmila Thilagavathy Narayanan and Meenakumari Gopalakrishnan Department of Pathology, Madurai Medical College, Madurai. India

ABSTRACT Background: Fine needle aspiration [FNA] is the well-established diagnostic modality. It has a positive predictive value of 99% in diagnosing malignant thyroid lesions. The incidence of various thyroid malignancies in our department was evaluated using FNA and they were compared to those available in the literature. Methods: During our study period, cases those were referred to our lab for FNAC thyroid were retrieved from the records. From that, malignant cases reported during our study period were enumerated and reexamined. The prevalence of malignant cases, its age and sex incidence was calculated. Increase in the incidence of undifferentiated thyroid carcinoma cases in our setup was recognized and our experience was compared with those available in the literatures. Result: During our study period, 339 thyroid cases were reported. The incidence of papillary carcinoma was 26.3% and anaplastic carcinoma was 58% in our study. Peak age incidence was around 50-60 years. Female predominance was noted in all age groups except in the 50-60 year category where male predominated. When the tumor was staged based on TNM classification, anaplastic carcinoma presented with a higher stage. Conclusion: Thyroid FNAC has a high predictive value in the diagnosis of malignancies. The incidence of undifferentiated carcinoma was higher in our setup. The reason for the increase in the incidence needs further evaluation. Keywords: Papillary, Insular, Anaplastic, Medullary.

Introduction

Benign thyroid nodules are commonly encountered in a fine needle aspiration [FNA] clinic. The incidence of malignant thyroid nodule is 5% [1]. The positive predictive value of FNA in diagnosing malignant thyroid lesion is 99% [2]. Among the malignant tumors of the thyroid papillary carcinoma of thyroid [PTC] is the most common constituting around 80% of all the thyroid malignancy [3]. Poorly differentiated thyroid carcinomas [PDTC] accounts for 4% to 7% of thyroid carcinomas, undifferentiated (or anaplastic) carcinoma of the thyroid [UTC] represents less than 5% of malignant thyroid tumors, medullary thyroid carcinoma [MTC] accounts for 5% to 10% of all thyroid carcinomas, squamous cell carcinoma [SQC] of the thyroid accounts for 1% or less of thyroid cancers and metastatic tumors to the thyroid are constitute 0.1% to 0.3% of thyroid aspirates [4, 5]. Our study was done to calculate the incidence of various malignant tumor in our setup and to compare it with the literature reports.

Materials and Methods

During our study period for seven months FNA cases that were referred to us for thyroid lesions were retrieved from

the records. As both FNA procedure and the interpretation were carried out by the pathologist the accuracy of our diagnosis was more [6]. Age, sex, clinical details including thyroid profile, duration of the lesion, family history of thyroid cancer, previous head and neck irradiation, rapid growth, hardness or adherence of the lump to surrounding structures and the presence of associated lymphadenopathy and clinical examination of the thyroid including size were retrieved from the records. Age and sex incidence were calculated. The size [based on ultrasound findings] based split of malignant lesions was done to evaluate the stage at which each thyroid carcinoma presented in our study. The slides were reexamined by three cytopathologists as recommended by Bethesda reporting of thyroid cytology [TBSRTC] and lesions were categorized according to Bethesda recommendations. Malignant lesions were categorized into type based on the morphology. Because malignancy in follicular neoplasm cannot be made on cytology, these lesions were not included in our study. The incidence of various thyroid malignancies was calculated. These findings were compared with the available literature reports. As our study used fine needle aspiration as a single diagnostic modality as it do have a higher sensitivity and specificity, neither histopathological correlation nor follow up was done.

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Ibrahim et al.

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Result

carcinomas constitute about 26.3% and anaplastic carcinoma constitutes about 58%. PTC was seen below 20 years and rest of the tumors peaked around 50-60 years. The incidence of malignant neoplasm in 50-60 years was 36.8% [Table 2].

During our study period for seven months from January 2016 to July 2016, 1999 cases were referred to our FNA clinic for cytopathological diagnosis. 339 cases were referred for thyroid FNA [Table 1]. Those cases were reported based on Bethesda recommendations.

When sex incidence was calculated, in the age group 50-60 years male predominance was noted [M: F: 2.5:1]. In all other age groups female predominance was noted [Table 2]. When stage of the tumor was compared with the type of tumor, higher stage was noticed in anaplastic carcinoma [Table 1].

Eighty five percentages of the thyroid cases were benign. 1.8% of the cases were diagnosed as follicular neoplasms and malignant lesion constitute around 5.6% in our study. Among the malignant thyroid lesions, papillary

Table 1: Stratification of various thyroid lesions we had encountered in our study based on Bethesda recommendation. Benign Malignant Non Suspicious Follicular diagnostic or Benign Lymphocytic for Undifferentiated Total Neoplasm Papillary thyroid Medullary thyroid Unsatisfactory follicular (Hashimoto) Malignancy (anaplastic) carcinoma carcinoma nodule thyroiditis carcinoma 10

268

T3 T4 T1 1

T3 T4 T1 T2

T3 T4

339

Table 2: Age and sex wise stratification of the malignant thyroid lesions. <20 Years

21-30 years

31-40 years

41-50 years

PTC

UTC

SOM

PTC

MTC

UTC

61-70 years

>71 years

SOM

UTC

51-60 years

Male

Female

PTC- Papillary carcinoma thyroid, MTC- Medullary carcinoma thyroid, UTC- Undifferentiated carcinoma thyroid, SOM- Suspicious of malignancy.

Fig. 1: A: Show oval cells arranged in papillary pattern with anatomical bordering [Black arrow in the inset picture, H&E, 4x] - Papillary carcinoma thyroid.

Fig. 2: Show pleomorphic tumor cells arranged in sheets and neutrophils are seen in the background [H&E, 10x] Undifferentiated carcinoma thyroid.

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Cytopathological Study of Malignant Thyroid Lesions

Fig. 3: Show rhabdoid tumor cells arranged in sheets and scattered individually [H&E, 10x] - Undifferentiated carcinoma thyroid.

Fig. 4: Show scattered and monolayered sheets of oval to spindle tumor cells in a hemorrhagic background [H&E, 10x]. Inset shows tumor cells exhibiting dispersed chromatin [H&E, 40x] – Medullary carcinoma Thyroid.

Discussion

Pediatric PTCs and PTCs that occur in young adults have some unique features. They are usually larger in size, metastasize early and often present with cervical metastasis [14]. These tumors show high expression of sodium-iodide transporter [NIS]. This NIS expression results in increased response to radio iodine therapy resulting in better prognosis [15]. The incidence of papillary carcinoma in Sinna E.A and Ezzat N’s study was 72.4%, whereas in our study the incidence of PTC was 26.3% [16].

Thyroid malignancy is commonest among endocrine tumors. Annual incidence ranges from 1.0 to 2.9 cases per 100,000 men and 3.4 to 9.1 cases per 100,000 women [7] . Thyroid FNA is a rapid, safe, cost-effective modality to diagnose thyroid lesions [8]. Cytology has a sensitivity and specificity of up to 94% and 98% respectively for the diagnosis of malignant lesions [9]. TBSRTC recommends six diagnostic categories which includes – Non diagnostic, benign, atypia of undetermined significance or follicular lesion of undetermined significance, follicular neoplasm or suspicious for a follicular neoplasm, suspicious for malignancy and malignant. Malignancy is more common in solitary nodule and the prevalence of malignancy in solitary cold nodules ranges from 10% to 44.7% [10]. Malignant thyroid tumors are broadly divided into well differentiated- PTC and follicular carcinoma, poorly differentiated- insular carcinoma and undifferentiatedanaplastic carcinoma. Papillary carcinoma is the most common malignancy among the thyroid carcinoma. It arises secondary to head and neck radiation and it is associated with BRAF, RAS mutation and RET/PTC rearrangements [11].There is a female preponderance with female to male ratio 4:1 and seen in all ages [12]. In our study female: male was 3:1 in concordance with Schlumberger M‘s study [12]. The incidence of PTC peaks around 43+_ 12 years [13]. In contrast, the peak age group in our study was 16 years.

Grossly, the tumor is solid grey white and cystic changes are seen in 10% of the cases. Multicentricity and occult tumors less than one centimeter in size are also common in PTC. Histologically, PTC is identified by its nuclear features including, enlarged oval irregular nucleus, overlapping and crowding of the nucleus, grooves, intracytoplasmic inclusions and Orphan Annie nucleus. Nuclear cytoskeletal abnormality leads on deformable nucleus hence the irregular contour, grooves and pseudo inclusions seen in the nucleus [17]. Cytologically, PTC shows all the nuclear features except nuclear clearing. Instead the nucleus show dispersed chromatin pattern. Anatomical bordering, nuclear grooves, nuclear overlapping and crowding, intranuclear inclusion and dispersed chromatin are diagnostic of PTC in the cytological smears. All the features should be there to diagnose a PTC [Fig 1]. IHC markers that are specific for PTC includes HBME-1 membranous positivity, cytoplasmic CK19 positivity, CD44, p63 and galectin 3.

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Ibrahim et al. Insular carcinoma is seen in elderly age group with a female predominance. Grossly, tumors are necrotic and have an invasive margin. Histologically, the tumor cells are small arranged in an insular pattern with foci of necrosis. Cytologically, the cells are smaller, with dispersed chromatin, convoluted nuclei, increased mitotic figures [more than 3/10 hpf] in a necrotic background [18]. Immunohistochemical markers that are positive include TTF-1, thyroglobulin and synaptophysin. Insular carcinoma was not encountered by us during our study period. Undifferentiated carcinoma [anaplastic carcinoma] [UTC] constitutes around 17.2% of all the thyroid malignancies [14] . In all other studies the incidence were <5% of all the thyroid malignancies [19, 20]. In contrast, in our study group the incidence was 58% which was higher than all the study groups seen in the literatures. It is a tumor of elderly females secondary to nodular goiter, FA [Follicular adenoma], FTC [Follicular carcinoma thyroid] and PTC [21]. In our study male: female was 1.2:1 in contrast to LiVolsi V A, et al’s study [19]. Two cases in our study presented with features of UTC secondary to PTC. It was invasive at presentation and it was associated with both local and distal metastasis. Extra thyroidal extension in UTC tumors was seen in 91% of our cases [Table 3]. Histologically, this tumor shows both malignant epithelial and spindle cell morphology. Increased mitotic figures and areas of necrosis were also noted. Cytologically, the tumor exhibits malignant spindle component with neutrophilic infiltrates, epithelioid [Fig 2] or rhabdoid [Fig 3] morphologies. This tumor is positive for cytokeratin and PAX8. P53 mutation, RET and RAS mutations are observed in UTC. Medullary carcinoma thyroid constitutes around 5-10% of all the thyroid neoplasm [16, 22]. Likewise in our study too the incidence of MTC was 5.3%. It is associated with RET and RAS mutations. The cell of origin is the parafollicular C cells. It is sporadic or familial. Familial MTC may be isolated familial disease or associated with multiple neuroendocrine syndromes [MEN 2A &2B]. Sporadic cases show female predominance and familial cases show equal sex distribution. In our study a single case was reported in a male. Histologically, the tumor cells are polygonal or spindle arranged in sheets separated by a fibrovascular septa. The tumor cells show dehiscence and nucleus show stippled chromatin and amyloid is seen in 80-85% of the cases. The sensitivity of FNAC in detecting MTC is higher than PTC [23]. Cytologically, polygonal or spindle shaped cells are arranged individually or form loose monolayer sheets [Fig 4]. Amyloid is also seen. Tumor cells are positive for CK, chromogranin, CEA, calcitonin and TTF-1. www.pacificejournals.com/apalm

A-129 Thyroid malignancies contribute about 1.5–3% of all carcinomas in children and adolescent group [24]. In our study group, PTC was seen predominantly in younger age groups less than twenty years of age constituting 10.5% of all the thyroid malignancies. Family history of thyroid malignancies and irradiation are considered to be the risk factors associated with thyroid malignancies in young adults. The increased incidence of PTC in young adults in our study group needs further evaluation. The incidence of UTC was much higher than all the study groups. The reason for increased incidence of UTC in our study group may be because of the following reasons: UTC might have developed from undiagnosed PTC which was seen predominantly in younger age groups in our study. May be more studies related to the genetic instability associated with PTC might give us a clue. UTC might have arisen in the background of some unknown gene mutations prevalent in our area which needs further evaluation.

Conclusion

FNAC is the cheapest available diagnostic tool in classifying a thyroid nodule. There was a female predominance in our study group. The incidence of UTC is 58% in our study group which needs further analysis.

Reference 1.

Mortensen JD, Woolner LB, Bennett WA. Gross and microscopic findings in clinically normal thyroid glands. J Clin Endocrinol Metab. 1955;15:1270-1280.

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(3):450–6.

Rosai J, Carcangiu ML, DeLellis RA. Tumors of the thyroid gland. Atlas of Tumor PathologyWashington, DC: Armed Forces Institute of Pathology; 1992 Fascicle 5, 3rd series.

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; 2004:49-134.

Schmid KW, Hittmair A, Ofner C, Totsch M, Ladurner D. Metastatic tumors in fine needle aspiration biopsy of the thyroid. Acta Cytol. 1991;35:722-724.

Sangalli G, Serio G, Zampatti C, Bellotti M, Lomuscio G. Fine needle aspiration cytology of the thyroid: a comparison of 5469 cytological and final histological diagnoses. Cytopathology 2006;17: 245–50.

Chan JK. Tumors of the thyroid and parathyroid glands. In: Fletcher CDM. Diagnostic histopathology of tumors. 4th eds, Saunders, Philadelphia, Churchill Livingstone; 2013. 1177-1289.

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A-130 8.

Cytopathological Study of Malignant Thyroid Lesions

Olson MT, Clark DP, Erozan YS, Ali SZ. Spectrum of Risk of Malignancy in Subcategories of ‘Atypia of Undetermined Significance’. Acta Cytol 2011;55(6):518–25.

16. Sinna EA, Ezzat N. Diagnostic accuracy of fine needle aspiration cytology in thyroid lesions. Journal of the Egyptian National Cancer Institute 2012; 24: 63–70.

9. Yang J, Schnadiq V, Logrono R, Wasseman PG. Fineneedle aspiration of thyroid nodules: a study of 4703 patients with histological and clinical correlations. Cancer 2007;111:306–15

17. Papotti M, Manazza AD, Chiarle R, Bussolati G. Confocal microscope analysis and tridimensional reconstruction of papillary thyroid carcinoma nuclei. Virchows Arch. 2004;444(4):350-355.

10. Ashcroft MW, van Herle AJ. Management of thyroid nodules. I. Head, Neck Surg 1981;3:216–30. 11. Henderson YC, Shellenberger TD, Williams MD, El-Naggar AK, Fredrick MJ, Cieply KM, Clayman GL. High rate of BRAF and RET/PTC dual mutations associated with recurrent papillary thyroid carcinoma. Clin Cancer Res 2009;15:485–91. 12. Schlumberger M J Papillary and follicular thyroid carcinoma. N Engl J Med 1998; 338: 297-306 13. Huang FJ, Fang WY, Ye L, Zhang XF, Shen LY, Han R.L et al. BRAF mutation correlates with recurrent papillary thyroid carcinoma in Chinese patients. Curr Oncol, 2014; 21(6): e740-747. 14. Pasieka JL, Thompson NW, McLeod MK, Burney RE, Macha M, Reeve TS. The incidence of bilateral well differentiated thyroid cancer found at completion thyroidectomy, World Journal of Surgery1992:16(4): 711–716. 15. Patel A, Jhiang S, Dogra S , Terrell R, Powers PA, Fenton C, et al. Differentiated thyroid carcinoma that express sodium-iodide symporter have a lower risk of recurrence for children and adolescents. Pediatric Research, 2002: 52( 5); 737–744.

18. Volante M, Collini P, Nikiforov YE, Sakamoto A, Kakudo K, Katoh R et al. Poorly differentiated thyroid carcinoma: the Turin proposal for the use of uniform diagnostic criteria and an algorithmic diagnostic approach.Am J Surg Pathol 2007;31:1256–64. 19. Nel C J, van Heerden J A, Goellner J R, Gharib H, McConahey WM, Taylor WF, et al. Anaplastic carcinoma of the thyroid: a clinicopathologic study of 82 cases. Mayo Clin Proc 1985:60: 51-58 20. Tan R K, Finley R K III, Driscoll D, Bakamjian V, Hicks WL, Jr., Shedd DP. Anaplastic carcinoma of the thyroid: a 24-year experience. Head Neck 1995:17:41-47 21. LiVolsi VA, Brooks JJ, Arendash-Durand B. Anaplastic thyroid tumors. Immunohistology. Am J Clin Pathol 1987:87:434-442 22. Moo-Young TA, Traugott AL, Moley JF. Sporadic and familial medullary thyroid carcinoma: state of the art. Surg Clin North Am 2009;89:1193–204. 23. Papaparaskeva K, Nagel H, Droese M. Cytologic diagnosis of medullary carcinoma of the thyroid gland. Diagn Cytopathol 2000; 22: 351-358. 24. Greenlee R T, Hill-Harmon M B, Murray T, Thun M. Cancer statistics, 2001 Cancer Journal for Clinicians 2001: 51(1):15–36.

*Corresponding author: Dr. S. Shifa, Department of Pathology, Madurai Medical College, Madurai. Phone: +91 9486669274 Email: shifafrin@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 03.09.2016 Date of Acceptance : 30.12.2016 Date of Publication : 02.04.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Original Article DOI: 10.21276/APALM.1089

A Descriptive Study of Histopathological Patterns of Lymph Node Biopsies In A Tertiary Care Hospital Rajshri P. Damle*, Kishor H. Suryawanshi, N.V.Dravid, D.V.Newadkar and Prashant N. Deore Department Of Pathology, A.C.P.M. Medical College, Dhule, Maharashtra. India.

ABSTRACT Background: Lymph node is affected by various types of lesions, both non-neoplastic (inflammatory) or neoplastic. Due to non-specific symptoms, the clinical diagnosis are usually delayed thus lymph node biopsy is mandatory to arrive at specific diagnosis for appropriate management. Objective: 1) To study the spectrum of lymph node pathology with clinical correlation. 2) To study the different histological patterns of lymph node lesions and classify them into major categories. Methods: The present study was a prospective observational type of study and included 331 patients who presented with lymph node enlargement in a tertiary care hospital over a period of 3 years from Jan 2013 to Dec 2015. Detailed clinical history was noted. The specimens were routinely processed and stained by Haematoxylin and eosin. Special stains along with immunohistochemistry were done whenever required. Result: A total of 331 lymph node biopsies were studied. Age distribution varied from 4 to 81 years with male to female ratio of 1:1.4. Non –neoplastic lesions comprised of maximum cases (80.06%) while neoplastic lesion were present in (19.93%) cases. Reactive lymphadenitis was the predominant non-neoplastic finding followed by granulomatous lymphadenitis. Neoplastic lesions were included 3.61% cases of lymphoma and 16.31% cases of metastatic lesions. Conclusion: Lymphadenopathy is not uncommon in our region so lymph node biopsy is an important tool for early diagnostic and prognostic purpose. Reactive lymphadenitis was the most common cause of lymphadenopathy followed by granulomatous lymphadenitis. Keywords: Lymphadenopathy, Biopsy, Histopathological Examination, Diagnostic

Introduction

Materials and Methods

Lymphadenopathy is a common clinical condition which may be neoplastic or non-neoplastic, affects any age group and can be diagnosed on clinical history, physical examination and fine needle aspiration cytology (FNAC). FNAC is commonly used to establish the etiological diagnosis of lymph node lesions but the limitation of this technique is unable to diagnose suspected or grey zone of various lesions and lymphohematogenous malignancy. So excision biopsy and histopathological examination of the lymph node remains the ‘Gold standard’ for diagnosis. [2,3]

Eligibility criteria adopted in our study:

Lymph nodes are the most widely distributed and easily accessible lymphoid tissue and are frequently examined for diagnostic purpose. The degree and pattern of the morphologic changes are dependent on the inciting stimulus and the intensity of the response. Trivial injuries and infections induce subtle changes while more significant infections inevitably produce nodal enlargement. [1]

Hence this study was undertaken to study the different histological patterns in detail of lymph node biopsies.

The present study was carried out in Department of pathology ACPM Medical College Dhule from Jan 2013 to Dec 2015 and included 331 cases of lymph node biopsies. Detailed clinical history like age, sex, site, presenting complaints and clinical diagnosis was noted. Relevant findings and investigations were recorded. The study was approved by institutional ethical committee. 1) Inclusion criteria: all type of lymph node specimens and all age groups. 2) Exclusion criteria: inadequate samples and poorly preserved tissue were excluded. All cases of lymph node biopsies were received in 10% formalin. The gross morphological features like size of node, shape, color, consistency, presence of necrosis and matting were noted and taken sections from each. The specimens were processed in automated tissue processor and 4-5 micron thick paraffin embedded sections were taken and stained by Haematoxylin and

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Histopathological Patterns of Lymph Node Biopsies

eosin. Special stain including Ziehl Neelsen, periodic acid Schiff (PAS) and reticulin were used where indicated. Immunohistochemistry was performed whenever required. Each slide was carefully examined by two pathologist and the histopathological patterns of lymph node biopsies were reported and categorized.

Results

In the present study, a total of 331 lymph node biopsies were received to the department of pathology and reviewed cases were categorized into four main groups; 175 cases (52.87%) of reactive lymphadenitis, 82 cases (24.77%) of granulomatous lymphadenitis, 66 cases (19.93%) of neoplastic disease and 8 cases (2.41%) of miscellaneous group . [Table- 1] Out of these, 135 (40.78%) were males and 196 (59.21%) were females giving a male to female ratio was 1:1.4. Most of the cases (78%) presented with clinical features of pain and tenderness. Age group of patients ranged from 4 years to 81 years. Maximum no. of patients were in the age group of 21-30 years (22.05%) followed by 31-40 years (18.42%) and least cases were seen in the age group over 70 years (2.11%)[Table -2]. In the present study, non-neoplastic lesions were more common comprising 265 cases (80.06%) and neoplastic lesions were 66 cases (19.93%). The most common sites were observed from the cervical lymph node followed by axillary lymph node representing (65.86%) and (15.70%) respectively. [Fig.1] Among non-neoplastic lesions, reactive lymphadenitis were the most common accounting for 175 cases (52.87%) and maximum number of cases was observed in 21-30 yrs age group. Out of 175 cases, chronic non-specific lymphadenitis (36.85%) was more common followed by follicular hyperplasia in (9.96%)[Fig.2d],sinus histiocytosis

in (3.62%)[Fig.3a] and paracortical hyperplasia in (2.41%). Majority of cases of chronic non-specific lymphadenitis were in the age group of 21-30 yrs followed by 31-40 yrs of age group. In our study, granulomatous lymphadenitis was the second most common cause of lymphadenopathy. Tuberculosis was the most frequent cause accounting for 67 cases (20.24%) of granulomatous lesions [Fig-2a]. The most common age group was 31-40 years of age with slight female predominance. AFB was positive in 46 cases (68.5%) [Fig-2b]. Other 15 cases of granulomatous lesions included only 2 cases of cat scratch disease and 13 cases were not otherwise specified or categorized. Among neoplastic lesions, primary neoplasm of lymph nodes were 12 cases (3.61%). Out of which 4 cases of Hodgkinâ&#x20AC;&#x2122;s lymphoma [Fig-3b] were diagnosed and in which 3 cases were observed in 41-60 years of age group and only single case was observed in 20 years of female patient. There were 8 cases of Non-Hodgkinâ&#x20AC;&#x2122;s lymphoma and diagnosed after the age group of 30 years and peak between 41-50 years with slight male predominance. Out of 8 cases, 5 cases were diffuse small lymphocytic lymphoma [Fig-3d] and 3 cases were follicular lymphoma [Fig-3c]. All cases of primary neoplasm of lymph node were confirmed by immunohistochemistry. Secondary neoplasm of lymph node were 54 cases (16.31%) in which 15 were male and 39 were female patients and most common age group was 51-60 years followed by 3140 years. Out of 54 cases 37 cases were infiltrating ductal carcinoma of breast [Fig- 4a], 9 cases were squamous cell carcinoma [Fig- 4b] and 8 cases were adenocarcinoma. Miscellaneous lesions comprised of Kikuchi disease [Fig2c], Castleman disease and Kimura disease were diagnosed as 1.20%, 0.90%, 0.30% respectively of all cases.

Table 1: Histopathology of lymph node biopsies. Histological diagnosis 1) Reactive lymphadenitis

No. of case

175

52.87%

a) Chronic non-specific lymphadenitis

122

b) Follicular hyperplasia

c) Sinus histiocytosis

d) Paracortical hyperplasia

2) Granulomatous Lymphadenitis

a) Tuberculosis

b) Cat scratch disease

c) Others

24.77%

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Histological diagnosis

No. of case

3) Neoplastic disease

19.93%

a) Lymphoma

-Hodgkin’s

-Non-Hodgkin’s

b) Metastatic disease

4) miscellaneous group

a) Kikuchi disease

b) Castleman disease

c) Kimura disease

Total

2.41%

331

100%

Table 2: Age wise distribution of different types of lymph node biopsies. Age

Reactive lymphadenitis

Granulomatous Lymphadenitis

Hodgkin’s

Non Hodgkin’s

metastatic disease

Miscella neous

Total

< 10

11-20

21-30

31-40

41-50

51-60

61-70

>71

Total

175

331

Fig.1:-Site of lymph node biopsies.

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Histopathological Patterns of Lymph Node Biopsies

Fig. 2: Lymph node lesions (a) HPE -Tubercular Lymphadenitis showing necrotic background with epithelioid cell granuloma (H&E; x100) (b) special stain (ZN) AFB positive bacilli (c) HPE- Kikuchiâ&#x20AC;&#x2122;s lymphadenitis (H&E; x400) (d) HPE- Follicular hyperplasia (H&E; x400).

Fig. 3: Lymph node lesions (a) HPE - sinus histiocytosis (H&E; x400) b) HPE - Hodgkinâ&#x20AC;&#x2122;s lymphoma (H&E; x400) (c) follicular lymphoma (H&E; x400) and (d)HPE- diffuse small lymphocytic lymphoma (H&E; x400).

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Discussion

Lymph node is one of the major anatomic components of the immune system. Because normal immune response leads to proliferation and expansion of one or more of the cellular components of lymph nodes, it leads to significant lymph node enlargement. [4] Lymphadenopathy is a common clinical problem and lymph node biopsies are usually carried out to determine the cause of nodal enlargement, which may be non-neoplastic or neoplastic. Fine needle aspiration is commonly used for diagnosis of lymphadenopathy though excision biopsy is gold standard especially in suspected cases of lymphoproliferative disorders or in those cases where diagnosis cannot be reliably reached on clinical grounds or fine needle aspiration cytology. The present study was carried out over a period of 3 years to evaluate the different histopathological patterns of lymph node biopsies and its clinicopathological correlations. In our study majority of patients were female with male to female ratio of 1:1.4 and study included patients of all age group. Similar results of female predominance were also reported by Kamat GC [5], Tiwari M [6], Kim LH [7] and Rahman Md A [8] . In our study the most common clinical features were pain and tenderness which were confirmed as of infectious origin on histopathological examination. Cervical lymph nodes are involved most often in all types of lymphadenopathy in adults as well as children. This is because these lymph nodes draining the most commonly affected and inflamed region of the body as well as due to easy accessibility of the cervical lymph node for biopsy. In our study predominant site of lymph node biopsy was cervical followed by axillary lymph nodes. This observation was consistent with most of the recent studies like OluEddo AN [9], Saraswat A et al [10] and Vachhani A [11]. In present study non-neoplastic lesions (80.0%) were much more common than neoplastic lesions (19.93%) and these results were consistent with Kamat GC et al (88.92%) [5] , Rahman Md A (70.2%) [8] Saraswat A et al (90.9%) [10] , Vacchani A et al (75%) [11], and Rao MN et al (56%) [12] . Reactive lymphadenitis (52.87%) comprised of the predominant pattern in non-neoplastic lesions. This may be due to reactive hyperplasia in lymph nodes draining the site of malignant lesion, which do not shows metastatic deposit due to early diagnosis and surgical excision. This was consistent with study of Moore SW et al found 47.8% 0f reactive lesions [3]. Other non-neoplastic lesions comprised of (36.85%) were chronic non-specific lymphadenitis, (9.96%) follicular hyperplasia, (3.64%) sinus histiocytosis and (2.41%) of paracortical hyperplasia. Higher percentage of chronic www.pacificejournals.com/apalm

A-135 non-specific lymphadenitis might be due to persistent bacterial, Viral and other infections in the tissue drained by the affected lymph nodes. In a study by Mohan A et al [13] non-specific lymphadenitis was 35.6%, which is consistent with the present study. Saraswat A et al [10] reported high percentage of chronic non-specific lymphadenitis. In a study by Reddy MP et al [14] included 63 cases of reactive lymph nodes, 54 cases (85.71%) were non-specific reactive hyperplasia and 9 cases (14.29%) were follicular hyperplasia. In a study by Kamat GC et al [5] included 75 cases of reactive lymphadenitis, 52(69.3%) were follicular hyperplasia, 16 (21.40%) were sinus histiocytosis and 7 (9.3%) were paracortical hyperplasia. In our study, tuberculosis was the second most common pattern constituting 20.24% of cases but several authors have reported tuberculosis as the predominant cause of lymph node enlargement. [5, 6, 8, 10] Among neoplastic lesions (19.93%), there were 12 cases (3.61%) of lymphoma. Out of which 4 cases (1.20%) were Hodgkinâ&#x20AC;&#x2122;s lymphoma and 8 cases (2.41%) were NonHodgkinâ&#x20AC;&#x2122;s lymphoma. Remaining 16.31% accounted as metastatic lesions. In a study conducted by Sibanda EN et al [2], Kamat GC et al[5] and Tiwari M et al [6] reported 7%, 3.6% and 2% of cases were lymphoma and which were comparable with our study. Malignancies have been the predominant cause of lymphadenitis in developed countries than developing countries like India because of racial and genetic factors. But in contrast in a study by Roy A et al [15], Sinclaire S et al [16] and Mohan A et al [13] constituted 44.5%, 63.29% and 25.9% cases of lymphoma which were very higher incidence than present study because of these studies included large number of cases and conducted in research centre or onco institute. Metastatic malignancy were found in 54 cases (16.31%) of which 34 cases were metastatic infiltrating breast carcinoma, 12 cases were metastatic squamous cell carcinoma and 8 cases were metastatic adenocarcinoma. Majority of cases were seen in 51-60 years followed by 31-40years of age group. In comparision to various studies in the literature our findings were similar to findings of Vachhani A et al (23%) and Tiwari M et al (11%) [11,6]. Metastasis of breast carcinoma in lymph node is due to adopting western lifestyle and cultural factors. Similarly squamous cell metastasis is due to consumption of tobacco in various forms in our area leading to malignancy in aerodigestive tract.

Conclusion

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lymphadenopathy. So more attention should be given to lymph node swelling for the appropriate clinical management of patients. Reactive lymphadenitis was the most common cause of lymphadenopathy followed by granulomatous lymphadenitis in our study.

Md Atiqur R, Md Mamun AB. Histopathological evaluation of lymph node biopsies: A hospital based study. J Enam Med Col 2012 ; 2(1) : 8-14.

Olu-Eddo AN, Ohanaka CE. Peripheral lymphadenopathy in Nigerian adults.J Pak Med Assoc 2006 ;56 : 405-8.

References

10. Saraswat A, Rajender A, Purohit K. Lymph node biopsy: Spectrum and clinical significance as diagnostic tool at tertiary care centre.J of Evolution Med and Dent Sci. 2015; 4(6): 1008-14.

Robbins S, Cotran R. Diseases of the immune system. In: Kumar V, Abbas AK, Sausto N, Aster JC (eds). Robbins and Cotran pathologic basis of disease.8th edn. Philadelphia: Elsevier Saunders, 2010: 235-249. Sibanda EN, Stanczuk G. Lymph node pathology in Zimbabwe: A review of 2194 specimens. Q J Med 1993;86:811-7.

Moore SW, Schneider JW, Schaaf HS. Diagnostic aspects of cervical lymphadenopathy in children in the developing world: A study of 1877 surgical specimens. Pediatr Surg Int 2003;19:240-4.

Longo, Fuci et al, Harrisonâ&#x20AC;&#x2122;s Internal Medicine 17th edition. Ch-60, p-370-372.

Kamat GC. A ten-year histopathological study of generalized lymphadenopathy in India. S Afr Fam Pract 2011; 53(3): 267-270.

Tiwari M, Aryal G, Shrestha R. Histopathologic diagnosis of lymph node biopsies. Nepal Med Coll J2007;9(4) : 259-61.

Kim LH, Peh SC, Chan KS. Pattern of lymph node pathology in a private pathology laboratory. Malays J Pathol 1999 ;21(2) :87-93.

11. Vachhani A, Bhuva K, Jasani J, et al. Histopathological study of lymph node biopsy. International Journal of Biomedical and Advance Research 2013; 4 (11) : 790-5. 12. Rao MN, Raju YS, Prasad AK, et al. Evaluation of lymphadenopathy at a referral centre. JAPI ; 50 : 1488-1489. 13. Mohan A, Reddy MK, Phaneendra BV, Chandra A. Aetiology of peripheral lymphadenopathy in adults: analysis of 1724 cases seen at a tertiary care teaching hospital in Southern India. Natl Med J India 2007; 20(2): 78-80. 14. Reddy MP, Moorchung N, Choudhary A. Clinicopathological profile of pediatric lymphadenopathy. Ind J Paediatr 2002; 69 : 1047-1051. 15. Roy A, Kar R, Basu D, Badhe BA. Spectrum of histopathologic diagnosis of lymph node biopsies: a descriptive study from a tertiary care center in South India over 5Â˝ years. Indian J Pathol Microbiol 2013; 56(2) : 103-8. 16. Sinclair S, Beckman E, Ellman L. Biopsy of enlarged superficial lymph nodes. JAMA 1974; 228(5): 602-603.

*Corresponding author: Dr. Rajshri P. Damle, Department Of Pathology, A.C.P.M. Medical College, Dhule, Maharashtra. India. 424005 Phone: +91 9767637624 Email: rajshriborase@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 04.10.2016 Date of Acceptance : 21.01.2017 Date of Publication : 02.04.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Original Article DOI: 10.21276/APALM.1095

Prevalence of Transfusion Transmitted Infections among Blood Donors at a Tertiary care Teaching Hospital in Southern Rajasthan Preeti Agrawal*, Suraj Jain, Sajjan S.Surana and Sashi Sujanani Department of Pathology, Pacific Medical College and Hospital, Udaipur, Rajasthan, India

ABSTRACT Background: Safe blood transfusion is of utmost importance as an unsafe blood transfusion bears lot of burden on human life and economy. Amongst the blood transfusion complications, transmission of certain infections like HIV, Hepatitis B, Hepatitis C and Syphilis are most significant for the long term detrimental side effects. The present study was carried out with an aim to assess the trend and seroprevalence of transfusion transmitted infections (TTIs) among blood donors at our blood bank. Methods: The present retrospective study was carried out at blood bank attached to Pacific Medical College and Hospital, Udaipur, Rajasthan after ethical committee approval. Data regarding sex of the donor, type of donors and screening test results were collected from the records of blood bank over a period of 3 years (May 2014 - June 2016). Result: Total 2015 donors were registered of which 1944 (96.5%) were males and 71 (3.5%) were females. Replacement donors (68.7%) were more compared to voluntary donors (31.3%). Overall prevalence of TTIs in our blood bank is 2.38% of which prevalence of Syphilis (1.2%) was highest followed by HBV (0.89%), HIV (0.14%), and Malaria (0.1%). Conclusion: TTIs were more common in replacement donors than in voluntary donors, hence more voluntary donations need to be encouraged. Extensive donor screening by more sensitive methods to detect infections early can reduce the risk of TTIs. Keywords: TTIs, Voluntary Donors, Replacement Donors, Blood Bank, HIV, HBV, HCV, Syphilis, Malaria.

Introduction

Banking of blood is important as it is one of the most precious commodities. Safe blood transfusion is of utmost importance as an unsafe blood transfusion bears lot of burden on human life and economy. Providing safe and adequate blood should be an integral part of every countryâ&#x20AC;&#x2122;s national health care policy and infrastructure.[1] Amongst the blood transfusion complications, transmission of certain infections (TTIs) like HIV, Hepatitis B and C and Syphilis are most significant for the long term detrimental side effects. Meticulous pre-transfusion screening and testing particularly for transfusion transmissible infections (TTIs) is the need of the hour.[2] India has a population of more than 1.2 billion with 5.7 million Human Immunodeficiency Virus (HIV) positive, 43 million HBV positive and 15 million HCV positive persons. The risk of transfusion transmission of these viruses may be alarming due to high seroprevalence of HIV, HCV, and HBV (0.5%, 0.4%, and 1.4% respectively) among the blood donors.[2] Over period of time, availability of newer and more sensitive screening tests with strict implementation of

testing rules has significantly reduced the incidence of TTIs in most developed countries while on the other hand, scenario in developing countries has not changed much. Poor health infrastructure, lack of health awareness in people, and failure to implement strict norms of screening result is increasing prevalence and incidence of the infections in the population.[3] Thus the present study was carried out with an aim to assess the trend and seroprevalence of TTIs among blood donors at the blood bank attached to Pacific Medical College and Hospital, Udaipur, Rajasthan.

Materials and Methods

The present retrospective study was carried out at the blood bank attached to Pacific Medical College and Hospital, Udaipur, Rajasthan after approval from the ethical committee of our institute. Data over a period of 3 years (May 2014 - June 2016) was collected from the records of blood bank. All donors who donated blood at blood bank as well as at various blood donation camps organized by our blood bank were included in this study. Exclusion of donors for the blood donation was done as per NACO guidelines. The screening methods for the detection of TTIs

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at our blood bank is HIV – Tridot (immunoassay); HBV – HEPACARD (for HbsAg detection : Immunoassay based on sandwich principle); HCV – Tridot (immunoassay); VDRL– ASPEN Syphilis Rapid test (qualitative membrane based immunoassay); and Malaria – Satya 2.0 Pf/Pv Malaria Antigen (Card test) The confirmatory testing methods for the detection of TTIs at our blood bank is HIV (Erba Lisa HIV Gen 4); HBV (Erba Lisa SEN HbsAg); HCV (Erba Lisa HCV Gen 3) Data regarding sex of the donor, type of donors, and screening tests results were collected and tabulated in MS excel sheet. Statistical analysis of the collected data was carried out.

Result

In the present study a total of 2015 donors donated blood in three years. Most of the donations 1384 (68.7%) were replacement compared to 631 (31.3%) voluntary donation (Table 1). When Sex wise distribution of the donors was studied, it was found that majority of the donors are males 1944 (96.5%) and rest 71(3.5%) were females (Table 2). Overall seroprevalence of TTIs in our blood bank is 2.38%. Table 3 shows the year wise proportion of different TTIs among blood donors. With respect to individual TTI, it was observed that Syphilis (26 cases, 1.29%) was the most common infection among blood donors followed by HBV (17 cases, 0.84%), HIV (3 cases, 0.15%), and Malaria (2 cases, 0.09%). None of the donor in our study was positive for HCV. When year wise prevalence trend of TTIs was plotted, it was found that peak prevalence for Syphilis was during the

year 2014 (2.36%) followed by decline in the year 2015 (0.69%), and with slight upsurge during the year 2016 (1.06%). With respect to HBV infection peak prevalence was noted during the year 2014 (1.09%), then declining trend in the years 2015 (0.83%) and 2016 (1.06%). In case of HIV infection, the same was 0.18% during the year 2014, nil in the year 2015 (0%), followed by an upsurge in the year 2016 (0.26%). However prevalence of malaria (0.28%) was noted only in the year 2015 (Table 3, Figure 1 and 2). The pattern of TTIs with respect to the type of donor is depicted in Table 4. Higher prevalence of TTIs was noted in replacement donors compared to voluntary donors. Even with respect to individual TTI, HBV (0.72%) and Syphilis prevalence (1.4%) was high among replacement donors (Table 4).

Discussion

TTIs threaten the safety of recipients and community as a whole and are a subject of real concern worldwide.[3] Despite the pre-donation counseling and medical fitness test, the presence of TTIs is inevitable in blood donation since a person can transmit infection during its asymptomatic phase (window period), transfusion can contribute to an ever widening pool of infection in the population.[1] In the present study, out of total donors, replacement donors constituted 68.7% and voluntary donors constituted 31.3%. Similar predominance of replacement donors was noted in other studies[3-8] while on the contrary, Sunderan S et al[1], Bhattacharya P et al[9], Shah N et al[10] noted predominance of voluntary donors in their studies. Out of 2015 donations,

Fig. 1: Trends of HBV and Syphilis prevalence in Blood Donors.

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Fig. 2: Trend of HIV Prevalence in Blood Donors. Table 1: Sex wise yearly distribution of blood donor types. REPLACEMENT DONORS

VOLUNTARY DONORS

Year

Male

Female

Male

Female

Total

2014

311

224

549

2015

537

159

715

2016

525

188

751

Total

1373

571

2015

Table 2: Sex wise yearly distribution of all blood donors. Year

Male

Female

Total

2014

535

549

2015

696

715

2016

713

751

Total

1944

2015

Table 3: Year wise Trends in Seroprevalence of TTIs. Year

HBV

HIV

VDRL

Total

2014

06 (1.09%)

01 (0.18%)

13 (2.36%)

2015

06 (0.83%)

05 (0.69%)

02 (0.28%)

2016

05 (0.66%)

02 (0.26%)

08 (1.06%)

Total

17 (0.84%)

03 (0.15%)

26 (1.29%)

02 (0.09%)

Table 4: Prevalence of TTIs among Donors types Type of donation

HBV

HIV

Syphilis

Replacement (2.52%)

10 (0.72%)

03 (0.21%)

20 (1.4%)

02 (0.14%)

Voluntary (2.06%)

07 (1.10%)

06 (0.95%)

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males contributed to a larger percentage (96.5%) compared to females (3.5%), which is in concordance with the data given by a similar other study.[3] Syphilis (1.2%) was the most common TTI in the present study, which is in contrast to the data of other studies[1-5, 10-13] where HBV was the most common TTI, and where as in the study by Kaur H et al[14] HCV was most the common TTI. None of the donor in our study was positive for HCV, but all other studies noted HCV prevalence of which Kaur H et al[14] had reported the highest HCV prevalence (1.79%). (Table 5) In the present study, higher prevalence of TTIs was noted among replacement donors as compared to the voluntary donors, which is in concordance with the other studies.[1, 4,

Where as only Kakkar N et al[7] reported marginally higher prevalence among voluntary donors. 6, 10-13, 15]

In order to reduce the risk of these infections and to improve the blood safety, stringent measures need to be taken for the blood donor screening by using more sensitive methods to detect the infections early. Better screening methods are required to decrease the window period of TTIs for example testing of hepatitis B core antigen, along with the already existing Enzyme Linked Immunosorbent Assay (ELISA) tests for detection of hepatitis B surface antigen will aid in better detection of HBV. However, now with the advent of Nucleic Acid Amplification Techniques (NAT), western countries have decreased the risk of TTIs to a major extent. NAT has added benefits but its high cost is of concern especially in developing countries like India.

Table 5: Seroprevalence of individual TTIs among different studies. Studies Sunderam S et al

HBV

HIV

HCV

Syphilis

Malaria

0.14%

0.03%

0.33%

1.01%

0.08%

[2]

2.21%

0.51%

1.11%

0.17%

Yadav BS et al[3]

1.77%

0.14%

0.09%

0.04%

1.7%

0.3%

1.0%

0.9%

Shaikh M et al Arora D et al

[1]

[4]

Pahuja S et al

2.23%

0.56%

0.66%

Shah N et al[10]

0.97%

0.16%

0.10%

0.23%

Mandal R et al[11]

1.24%

0.42%

0.62%

0.65%

0.004%

Philip CJ et al[12]

1.7%

0.7%

0.3%

Pallavi P et al

1.27%

0.44%

0.23%

0.28%

Kaur H et al

[5]

[13]

0.75%

0.16%

1.79%

0.67%

Fernandes H et al[15]

0.34%

0.06%

0.11%

0.01%

Present Study

0.84%

0.15%

1.29%

0.09%

[14]

Conclusion

To conclude, overall seroprevalence of TTIs in our study is 2.38% with Syphilis being the most common TTI. TTIs were more common in replacement donors as compared to the voluntary donors implying that voluntary donations are more safer. Hence more voluntary donations need to be encouraged. Extensive donor screening by more sensitive methods to detect infections early can reduce the risk of TTIs. The major limitation of our study is that there was no previous data available from Rajasthan for comparison and analysis of trends. Hence, we recommend for future studies with larger sample size to look into the trends of TTIs from this geographical area.

Reference

1. Sunderam S, Karir S, Haider S, Singh SB, Kiran A. Seroprevalence of Transfusion Transmitted infections among blood donors at Blood Bank of Rajendra Institute of Medical Sciences, Ranchi. Healthline Journal 2015; 6(1): 36-40. 2.

Shaikh M, Bhople KS. Seroprevalence of Transfusion Transmitted infections in blood donors at a rural based tertiary care teaching hospital in India. IOSR Journal of Dental and Medical Sciences 2015; 14(10): 29-32.

Yadav BS, Varma AV, Singh P, Kumar R, Bandi PK. Seroprevalence of Transfusion-transmitted infections (TTIs) in blood donors: A study from Central India. Int J Med Sci Public Health 2016; 5(6): 1-5.

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.

Pahuja S, Sharma M, Baitha B, Jain M. Prevalence and trends of markers of hepatitis C virus, hepatitis B virus and

Acknowledgements

The authors wish to acknowledge the help received from our blood bank staff Mr Tejpal Chauhan and Mr Ganesh

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HIV in Delhi blood donors: A hospital based study. JP J Inf Dis 2007; 60: 389-91.

care centre in Darjeeling, India. Journal of Traditional and Complementary Medicine 2015; 1-6.

Singh B, Verma M, Kotru M, Verma K, Batra M. Prevalence of HIV and VDRL seropositivity in blood donors of Delhi. Indian J Med Res 2005; 122: 234-36.

Kakkar N, Kaur R, Dhanoa J. Voluntary donors need for a second look. Indian J Pathol Microbiol 2004; 47: 381-83.

12. Philip J, Sarkar RS, Kumar S, Pathak A. Changing trends of Transfusion transmitted viral infections among blood donors in the last decade - A 10 year study in a large tertiary care blood bank (2000-2009). MJAFI 2015; 68(1): 28-32.

Khageshan AP, Kulkarni KR, Baragundi MC. Seroprevalence of Co-infections among blood donors in a blood bank of a tertiary care health centre. APALM 2016; 3(1): A29-32.

Bhattacharya P, Chakraborty S, Basu SK. Significant increase in HBV, HCV, HIV and syphilis infections among blood donors in West Bengal Eastern India 2004-2005. Exploratory screening reveals high frequency of occult HBV infection. World J Gastroenterol 2007; 13: 3730-3733.

10. Shah N, Shah JM, Jhaveri P, Patel K, Shah CK, Shah NR. Seroprevalence of HBV, HCV, HIV and syphilis among blood donors at a tertiary care teaching hospital in Western India. Gujarat Medical Journal 2013; 68(2): 35-39. 11. Mandal R, Mondal K. Transfusion Transmissible infections among blood donors from a sub-Himalayan rural tertiary

13. Pallavi P, Ganesh CK, Jayshree K, Manjunath GV. Seroprevalence and trends in Transfusion Transmitted Infections among blood donors in a university hospital blood bank – A 5 year study. Indian J Hematol Blood Transfus 2011 Mar; 27(1): 1-6. 14. Kaur H, Mannan R, Manjari M. Seroprevalence of the blood borne infections in blood donors: Our 11 year (2001-2011) experience in a tertiary care teaching hospital at Amritsar (Punjab). International Journal of Advanced Research 2014; 2(6): 967-71. 15. Fernandes H, D’Souza PF, D’Souza PM. Prevalence of Transfusion Transmitted Infections in voluntary and replacement Donors. Indian J Hematol Blood Transfus 2010; 26(3): 89-91.

*Corresponding author: Dr. Preeti Agrawal, 174A/P Road, Bhupalpura, Udaipur, Rajasthan, 313001 India. Phone: +91 8854801375 Email: preetibagrawal79@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 07.10.2016 Date of Acceptance : 12.01.2017 Date of Publication : 06.04.2017

eISSN: 2349-6983; pISSN: 2394-6466

Original Article DOI: 10.21276/APALM.1122

A Study of D2-40 Immunohistochemical Expression in Colorectal Carcinomas Sarvek Bajaj*, Gururajaprasad. C and Suchitha S Department of Pathology, JSS Medical college, JSS university, Mysore, India

ABSTRACT Background: Colorectal cancer is one of the most common cancers. Factors affecting survival include nodal positivity, lymphovascular invasion (LVI) and lymphangiogenesis (role in metastasis). Most endothelial markers stain lymphatics and blood vessels without discrimination. Recent marker D2-40 is specific for lymphatic endothelium. This study aims to interpret the utility of D2-40 in detecting LVI, which would be missed on H&E; and in analysing lymphatic vessel density and LVI as predictive markers for the risk of nodal metastases in colorectal carcinomas. Methods: Study was conducted on 40 specimens of colorectal carcinomas. Immunohistochemistry was performed with D2-40. Stastical analysis was by Spearmanâ&#x20AC;&#x2122;s correlation and Chi square test Result: Mean age was 55.50 years. Of 40 cases, majority were in ascending colon followed by rectum, sigmoid, transverse and descending colon. 80.0% were adenocarcinomas, 15.0% mucinous adenocarcinomas and 5.0% signet-ring cell carcinomas. Tumors were moderately differentiated in 80.0% and poorly in 20.0%. Most patients were in stage T3 followed by T2 and T4. Most common nodal stage was N0 followed by N1, N2 and N3. LVI was detected in eleven more cases on D2-40 than H&E and correlated with lymphatic density and nodal status. Lymphatic density correlated with nodal status and pT. Peritumoral lymphatics were found in 95% cases and intratumoral lymphatics in 90%. Intratumoral lymphatics correlated with nodal status. Conclusion: D2-40 increased the detection rate of LVI as compared to H&E and showed the value of lymphangiogenesis in disease progression and metastasis. Keywords: Colorectal Adenocarcinoma, D2-40 Immunohistochemistry, Lymphovascular Invasion, Lymphatic Density

Introduction

Globally, nearly 800,000 new colorectal cancer cases occur each year (10% of all cancers).[1] Colorectal cancer (CRC) is the third commonest in men and second in women.[2] Multiple prognostic factors affect the survival of patients. The most important are metastases, local tumor extent, nodal positivity and status, lymphovascular invasion and the status of resected margins. Lymph node metastases is an important prognostic indicator for disease progression and crucial for therapeutic strategies. Not much is known regarding the mechanisms through which tumor cells enter the lymphatic system. Some investigators have suggested that lymphangiogenesis plays an active role in metastasis. The study of lymphangiogenesis, growth and production of new lymphatic vessels under several physiological and pathological conditions has gained more attention in the recent years. Presently, there is no consensus whether the major pathway of lymphatic spread is through the

development of lymphatic vessels intra or peritumorally, with studies published supporting each of the possibilities Lymphangiogenesis has been difficult to investigate because there was a lack of specific antibodies recognizing the lymphatic endothelium. Most available endothelial markers used to assess intratumoral micro vessel density, a reflection of tumor angiogenesis, stained both lymphatics and blood vessels without discrimination. [3] Recently, lymphatic endothelial specific markers have become available, which facilitate the analysis of lymphangiogenesis in cancer.[4] D2-40 (also known as antipodoplanin), an IgG2a monoclonal antibody, was generated against an oncofetal membrane antigen M2A and identified in ovarian carcinoma cell lines and germ cell neoplasia.[5] It has shown to stain endothelium of lymphatic vessels and lymphangiomas but negative in hemangiomas.[6] D2-40 highlights the lymphatics and outlines the tumor emboli otherwise indiscernible by hematoxylin and eosin stain and increases the detection rate of lymphatic invasion.

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In the absence of effective immunohistochemical markers of lymphatic endothelium in paraffin sections, lymphatic invasion is identified on the basis of conventional hematoxylin and eosin (H&E) staining. Pitfalls in the technique arise mainly from the difficulty in visualizing the lymphatic vessel wall following H&E staining.[6] [7]

Aims and Objectives

To assess the lymphatic microvessel density and lymphovascular invasion using D2-40 immunostaining in colorectal carcinomas To evaluate the association of lymph vessel density (LVD) (peritumoral and intratumoral) and lymphovascular invasion with tumor size, stage (pT), grade and lymph node status. To compare the detection of lymphovascular invasion (LVI) by hematoxylin and eosin staining with D2-40 immunostaining

Materials and Methods

Study Population: 40 patients with colorectal carcinoma were studied between September 2013 and Aug 2016 in the Department of Pathology, JSS Medical College, Mysore. The following data were reviewed: age, gender, tumor location, size, histological type, grade, TNM staging and LVI. Type of Study: Descriptive study; One year of retrospective (September 2013 - August 2014) and 2 years of prospective study (September 2014 - August 2016) Inclusion Criteria: Adenocarcinomas. Exclusion Criteria: Other malignancies. Histology and TNM Classification: Specimens were fixed in 10% of formalin for 24-48 hours and embedded in paraffin. Deparaffinzed sections were stained with H&E and classified according to WHO. American Joint Committee Of Cancer (AJCC), seventh edition, was followed for TNM staging. Immunohistochemical Staining: The paraffin blocks of the tumor with maximum depth of invasion were selected. 4 µm sections were taken on Poly-L-Lysine slides. After deparaffinizing and removing endogenous peroxidase, ready-to-use primary antibody ((DAKO Monoclonal Mouse Anti-D2-40, Code IS072) was applied and antigen retrieval was done using a pressure cooker followed by secondary antibody (DAKO). Subsequently DAB-chromogen was added. Slides were stained with hematoxylin and mounted with DPX. Tonsil was used as positive control. For negative controls, primary antibody was omitted. www.pacificejournals.com/apalm

Method of Reporting by Immunohistochemistry (IHC): Staining pattern of D2-40 is cytoplasmic and membranous and it stains only lymphatic endothelial cells. After scanning at low magnification (x40), three areas with greatest number of distinct intratumoral lymphatic foci (hot spots) were selected by two observers and independently evaluated for microvessel (MV) counts using x400 magnification without knowing the patient status. In the absence of apparent hotspots, three or more randomly selected areas were counted. The highest number of lymphatic vessels counted was recorded and used for statistics. Intratumoral lymphatic vessels were defined as vessels within the main tumor mass, surrounded by tumor cells, with no RBCs in lumina. Vessels more than one high power field (HPF) away from the tumor front were considered as peritumoral lymphatic vessels. Single immunoreactive endothelial cells, or endothelial cell clusters separate from other MVs, were counted as a vessel. The highest number of vessels counted were recorded. LVD was classified into low (<10 vessels / HPF), moderate (10-15 vessels / HPF) and high (>15 vessels / HPF). Lymphovascular invasion was considered evident if at least one tumor cell cluster was clearly visible inside the vascular space.[4], [8] Stastical Analysis: The association of clinicopathological data with LVD and LVI on D2-40 was evaluated using Chi-square test and Spearman’s coefficient of correlation as appropriate. The difference between LVI on D2-40 and H&E was tested with McNemar test. Statistical analyses was performed with SPSS software 23. P < 0.05 was considered statistically significant.

Result

40 patients were from 25-82 years with a mean of 55.50 ± 14.122, maximum number (12) between 41-50 and least number (2) between 81-90 years. 23 (57.5%) cases were males and 17 (42.5%) were females with a male to female ratio of 1.35:1. 12 tumors (30%) were located in the ascending colon, 05 (12.5%) in transverse colon, 03 (7.5%) in descending colon, 9 (22.5%) in sigmoid colon, and 11 (27.5%) in the rectum. Tumor size ranged from 2.5 to 11 cm with a mean of 5.67 ± 2.37 cm. Based on size, 17 cases were <5cm in the greatest dimension, 23 were ≥5cm in size. 32 cases (80.0%) were adenocarcinomas, 06 (15.0%) mucinous adenocarcinomas and 02 cases (5.0%) signet-ring cell carcinomas. 32 cases (80%) were moderately differentiated and 8 (20.0%) were poorly differentiated. 13 cases (32.5%) were in stage pT2, 21 (52.5%) in pT3 and 6 (15.0%) in pT4. 17 cases (42.5%) were N0, 13 cases (32.5%) were N1, 8 cases (20.0%) were N2 and 2 cases (5.0%) were N3. Metastasis could not be ascertained. eISSN: 2349-6983; pISSN: 2394-6466

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Out of 40 cases studied, 8 (20%) showed perineural invasion. Amongst them, 6 cases were moderately differentiated and 2 were poorly differentiated. 16 (40%) out of 40 cases were positive for LVI by H&E. 27 (67.5%) showed LVI with D2-40. 10 (25%) showed both lymph node involvement and lymphatic infiltration on H&E compared to 22 (55%) on D2-40. D2-40 detected 11 (27.5%) more cases with lymphatic invasion than H&E. The difference between both the methods of assessing vascular invasion was significant by McNemar test (P = 0.0127) D2-40 lymphatic microvascular counts ranged from 9 to 18 with a mean of 13.53 Âą 2.552. 27 (67.5%) cases had moderate lymphatic microvascular density (LMVD), 10 (25%) had high and 3 (7.5%) had low LMVD. LVI detected by D2-40 correlated significantly with LMVD (r = 0.473, P = 0.002) and nodal status (r = 0.663, P= 0.000003).

Lymphatic microvessel counts correlated significantly with nodal status (r = 0.462, P = 0.003) and stage (pT) (r = 0.327, P = 0.040). LVI showed no significant correlation with stage (pT) (P = 0.211), grade (P = 0.623), size (P = 0.326) or perineural invasion (P = 0.744). LMVD showed no significant correlation with grade (P = 0.80), size (P = 0.381) or perineural invasion (P = 0.804). Peritumoral lymphatics were found to be present in 38 (95%) and intratumoral lymphatics were present in 36 (90%) cases. Intratumoral lymphatics showed significant correlation with nodal status (r = 0.357, P = 0.024) but peritumoral lymphatics showed no such correlation (P = 0.127). Intratumoral and peritumoral lymphatics showed no significant correlation with tumor size (P = 0.757, P = 0.831), stage (pT) (P = 0.325, P = 0.500), grade (P = 0.304, P = 0.481) or perineural invasion (P = 0.799, P = 0.48 ).

Fig. 1 (A): Adenocarcinoma colon. Grade moderately differentiated (H&E, x100). (B): Signet ring cell carcinoma colon. Grade poorly differentiated (H&E, x100). (C): Mucinous adenocarcinoma colon. Grade poorly differentiated (H&E, x100). (D): A case of colorectal adenocarcinoma showing perineural invasion (H&E, x100).

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Fig. 2 (A): Section from tonsil taken as positive control showing immunostained lymphatics (D2-40, x40). (B): Section from colorectal carcinoma showing no apparent lymphovascular invasion (H&E, x40). (C): Corresponding section showing lymphatic invasion (arrow) on immunostaining with D2-40 (x40). (D): Higher magnification of the same showing lymphovascular invasion with clearly outlined lymphatic endothelium on D2-40 (x200). (E): Blood vessel on H&E (x100). (F): Blood vessel is not stained with D2-40 whereas surrounding area shows lymphatics stained with D2-40 (x100).

Fig. 3 (A): Intratumoral lymphatics (D2-40 immunostaining, x100). (B): Peritumoral lymphatics (arrows) away from a focus of tumor (arrowhead) (D2-40, x100). (C): A case with an area showing <10 lymphatic vessels. Such areas were taken as low lymphatic microvascular density. (D2-40 IHC, x400). (D): A case with areas having 10-15 lymphatic vessels which were taken as having moderate lymphatic microvascular density. (D2-40 IHC, x400). (E): An area from a different case having >15 lymphatic microvessels that was counted as an area with high lymphatic microvascular density. (D2-40 IHC, x400).

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Discussion

Colorectal cancer is one of the commonest causes of cancerrelated deaths.[9] The lymphatics make up an important pathway of metastasis and extent of nodal involvement is an important prognostic factor.[8] Lymphatic invasion is an important finding in assessing primary tumors. Some studies have found it as a predictor of survival.[10] Studies have shown the prognostic value of lymphatic vascular density.[4] D2-40 is a specific marker for lymphatics to detect LVI and evaluate LMVD. [11],[12] In the present study, 40 cases were analyzed for LVI and LMVD by D2-40 and their correlation with various parameters. Lymphatic invasion: On H&E, LVI is detected as tumor emboli in spaces lined by a single layer of endothelial cells.[11] However, many difficulties arise in identifying lymphatic vessel walls by H&E. One is in detecting tumor emboli when the lumen of a lymphatic vessel is obliterated, the other is the impossibility of distinguishing retraction artifacts during fixation from true tumor emboli floating in lymphovascular spaces.[6], [13] Another problem with H&E is inability to know whether the involved space is lymphatic or vascular. These problems result in missing lymphatic invasion, false positivity for lymphatic invasion or misdiagnosing an invasion in blood vessel as lymphatic invasion. To decrease this inaccuracy, D2-40 immunostaining was used. In this study, LVI was detected in 11 (27.5%) more cases by D2-40 immunostaining as compared to H&E, which was significant (P = 0.0127), similar to studies by Kawaura et al[13] & Walgenbach et al[14]. This study showed a significant correlation between LVI on D2-40 and lymph node status and lymphatic microvascular density. Lymphatic Microvascular Density: Studies have evaluated the correlation of lymphangiogenesis in colon carcinoma with prognostic parameters[15], [16] but a few of them used D2-40 and observed a trend between lymphatic density and lymph node metastases, although not statistically significant.[15] Saad et al5 demonstrated that using D2-40 and CD31, microvessel counts correlated significantly with the presence of LVI and nodal metastases. Lymphatic microvascular counts ranged from 9 to 18 with a mean of 13.53 ± 2.552 in this study compared to Naik et al[17] (17±9.7) and Saad et al[4] (18±9). This study showed significant correlation of LMVD with LVI, nodal metastases and pT stage similar to Saad[4]. Intratumoral lymphatics were found in 90% of cases which

was similar to a study by Kuroyama.[15] In this study, intratumoral lymphatics showed significant correlation with lymph node status (P = 0.024). Peritumoral lymphatics were seen in 95% of the cases. Peritumoral lymphatics were enlarged and dilated and intratumoral lymphatics were small and flattened similar to other studies. [4], [18], [19], [20] D2-40 proved very useful in this study in overcoming the inaccuracy in identifying true LVI and evaluating LMVD, peritumoral and intratumoral lymphatics (which is not possible on H&E) and their correlation with histopathological parameters. This study also underlines the potential role of lymphangiogenesis and lymphatic invasion in tumor progression and lymph node metastasis.

Conclusion

Colorectal adenocarcinoma is one of the most common malignancies causing death worldwide. Lymph node status and lymphovascular invasion play an important role in the disease progression and outcome. 40 cases were studied for tumour morphology including the tumour size, location, histologic type, grade, pT stage, nodal status and immunohistochemistry was done with D2-40. Immunostaining with D2-40 significantly increased the detection rate of LVI as compared to H&E underlying the importance of D2-40 IHC as an investigative modality. In this study, LVI correlated significantly with nodal status suggesting its role in disease metastasis. LVI also showed significant correlation with LMVD. LMVD was more in cases with high pT stage and lymph node status as compared to cases with lower pT stage and nodal status indicating the value of lymphangiogenesis in disease progression and metastasis. D2-40 has the potential as a marker in better understanding the pathogenesis related to lymphatics in the colorectal cancer cases as well as in the prognosis of the disease. More studies are necessary to further evaluate and ascertain the prognostic value of lymphatic invasion and the induction of tumor lymphangiogenesis and its role in human cancer progression.

References 1.

Parkin DM, Pisani P, Ferlay J. Global cancer statistics. CA Cancer J Clin. 1999;49(1):33-64.

Ferlay J, Shin HR, Bray F et al. Estimates of worldwide burden of cancer in 2008. Int J Cancer. 2010;127(12):2893–917.

Parums DV, Cordell JL, Micklem K et al. JC70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections. J Clin Pathol. 1990;43:752-7.

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Saad RS, Kordunsky L, Liu YL et al. Lymphatic microvessel density as prognostic marker in colorectal cancer. Mod Pathol. 2006;19:1317-23.

Marks A, Sutherland DR, Bailey D et al. Characterization and distribution of an oncofetal antigen (M2A antigen) expressed in testicular germ cell tumors. Br J Cancer. 1999;80:569-78.

Kahn HJ, Bailey D, Marks A. Monoclonal antibody, D240: A new marker of lymphatic endothelium, reacts with Kaposi’s sarcoma and a subset of angiosarcomas. Mod Pathol. 2002;15:434-40. Niakosari F, Kahn HJ, Marks A et al. Detection of lymphatic invasion in primary melanoma with monoclonal antibody D2-40: a new selective immunohistochemical marker of lymphatic endothelium. Arch Dermatol. 2005; 141:440-444 Maghraby HK, Elsarha AI, Saad RS. Peritumoral lymphatic vessel density as a prognostic parameter in endometrial carcinoma: An immunohistochemical Study. Indian J Pathol Microbiol. 2010;53(3):465-9 Stewart B, Wild C. Colorectal cancer. In: Bosman F, Hamilton S, Lambert R, editors. World cancer report. Lyon: IARC; 2014. P. 392-99

10. Compton CC. Pathology report in colon cancer : what is prognostically important? Dig Dis. 1999;17(2):67-79 11. Kahn HJ, Marks A. A new monoclonal antibody, D2-40, for detection of lymphatic invasion in primary tumors. Lab Invest. 2002;82(9):1255-7. 12. Fogt F, Zimmerman RL, Ross HM et al. Identification of lymphatic vessels in malignant, adenomatous and normal colonic mucosa using the novel immunostain D2-40. Oncol Rep 2004;11(1):47–50

13. Kawaura K, Fujii S, Murata Y et al. The Lymphatic Infiltration Identified by D2-40 Monoclonal Antibody Predicts Lymph Node Metastasis in Submucosal Invasive Colorectal Cancer. Pathobiology. 2007;74(6):328-35 14. Walgenbach-Bruenagel G, Tolba RH, Varnai AD et al. Detection of lymphatic invasion in early stage primary colorectal cancer with the monoclonal antibody D2-40. Eur Surg Res. 2006;38(5):438-44 15. Kuroyama S, Kobayashi N, Ohbu M et al. Enzyme histochemical analysis of lymphatic vessels in colon carcinoma: Occurrence of lymphangiogenesis within the tumor. Hepatogastroenterology. 2005;52(64):1057-61 16. Parr C, Jiang WG. Quanitative analysis of lymphangiogenic markers in human colorectal cancer. Int J Oncol. 2003;23(2):533–9 17. Naik VR, Jaafar H, Seng CE. Lymphatic channel density in colorectal adenocarcinoma. Indian J Pathol Microbiol. 2010;53(1):12-4. 18. Gombos Z, Xu X, Chu CS et al. Peritumoral lymphatic vessel density and vascular endothelial growth factor C expression in early stage squamous cell carcinoma of the uterine cervix. Clin Cancer Res. 2005;11(23):8367-71. 19. Sipos B, Klapper W, Kruse ML et al. Expression of lymphangiogenic factors and evidence of intratumoral lymphangiogenesis in pancreatic endocrine tumors. Am J Pathol. 2004;165(4):1187-97 20. Padera TP, Kadambi A, di Thomaso E et al. Lymphatic metastasis in the absence of functional intratumor lymphatics. Science. 2002;296(5574):1883-6.

*Corresponding author: Dr. Sarvek Bajaj, 30-P RCC Area Model Town, Fatehabad, Haryana; INDIA; PIN: 125050 Phone: +91 9741860166 Email: drsarvek@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 24.10.2016 Date of Acceptance : 15.01.2017 Date of Publication : 07.04.2017

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Original Article DOI: 10.21276/APALM.1126

Impact of Iron Deficiency Anemia on Glycated Hemoglobin (HbA1c) Levels in Diabetics with Controlled Plasma Glucose Levels Lavanya Rajagopal1*, A. Sundaram1, Shivashekar1 and Balaji Ramraj2 1 Department of Pathology, SRM Medical College Hospital and Research Centre, Kattankulathur, Chennai India Department of community medicine, SRM Medical College Hospital and Research Centre, Kattankulathur, Chennai India

ABSTRACT Background: Glycated hemoglobin (HbA1C) is used as a gold standard for monitoring glycemic control. American Diabetes Association (ADA) has certified HbA1C ≥ 6.5% as a diagnostic criterion for diabetes mellitus (DM). Recent studies suggest that conditions affecting erythrocyte turnover like Iron deficiency anemia (IDA) alters HbA1C levels but their results are conflicting. However the effect of IDA on HbA1C is rarely reported. To determine the impact of IDA on HbA1C levels among controlled diabetics [Fasting plasma glucose (FPG) <126mg/dl since last 6 months] independent of blood glucose concentration and its variation according to the degree of anemia. Methods: This cross-sectional study carried out in SRM Medical College Hospital and Research Centre, Chennai includes totally 300 controlled diabetic patients –Type 2 DM (150 with IDA and 150 without IDA). Medical history recorded. HbA1C, complete hemogram and FPG were tested. Result: The mean HbA1C among controlled diabetics with IDA (7.86 ± 0.11%) was significantly higher than those without IDA (5.45 ± 0.038%) (P<0.05). HbA1C results were higher with the reduction of total hemoglobin (p< 0.05) Conclusion: IDA spuriously elevates HbA1C levels independent of blood glucose concentration in controlled-diabetics.HbA1C increases significantly as severity of anemia worsens. Thereby this study insists on the utter importance to exclude IDA and to correct it before any diagnostic or therapeutic decision is made based solely on HbA1C level. Keywords: HbA1C, Iron deficiency Anemia, Diabetes

Introduction

Glycated hemoglobin (HbA1C) is a form of hemoglobin that is measured to estimate the three-month average blood glucose concentration. It is widely used as a gold standard for monitoring glycemic control over the previous three months as this is the life span of red blood cell. HbA1C also serves as a predictor of complications of diabetes. Recently American Diabetes Association (ADA) has certified HbA1C ≥ 6.5% as a diagnostic criterion for diabetes mellitus.[1] However HbA1C levels can be influenced by a variety of other factors affecting erythrocyte turnover and glucose homeostasis.[2-5] One such condition affecting erythrocyte turnover is anemia. Anemia may be associated with rapid erythrocyte turnover conditions like acute or chronic blood loss, hemolytic anemia, sickle cell anemia, vitamin B12 deficiency, pregnancy that lower HbA1C levels (or) with slower erythrocytes turnover conditions like Iron deficiency anemia (IDA), alcoholism that increases HbA1C levels. However some but not all studies suggest that iron depletion is associated with increased glycation of hemoglobin leading to falsely high values of HbA1C independent of glycaemia.[6,7] To shed additional light on

this, in the present study we aimed to analyze the effect of IDA on HbA1C levels in controlled diabetics (fasting plasma glucose levels ≤ 126 mg/dl since last 6 months).

Materials and Methods

This is a cross-sectional study carried out in SRM Medical College Hospital and Research Centre, Chennai (February 2016 to September 2016) after obtaining approval from our institutional ethical committee. All diabetic patients (Type 2 DM) from both outpatient and inpatient departments of our hospital were included in the study evaluation. Totally 300 controlled diabetics whose FPG level is <126 mg/dl since last 6 months (150 with IDA and 150 without IDA) were included in this study. These two groups were matched for age, sex and plasma glucose levels. Their blood samples were tested for HbA1C, hemoglobin (Hb), hematocrit (Hct), Mean corpuscular volume(MCV), Mean corpuscular hemoglobin(MCH), Mean corpuscular hemoglobin concentration(MCHC), Peripheral smear, Serum iron, Ferritin and fasting plasma glucose (FPG) levels. Medical history was recorded. The anemic patients were selected based on their hemoglobin levels (Hb <13 gm% in males and <12

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gm% in females) based on definition of World Health Organization (WHO).[8] And those with predominantly microcytic red cell indices (MCV<76 fl), hypochromic red cell indices (MCH<27 pg/cell and MCHC<32 g/dl) and on their peripheral smear (microcytic hypochromic) were considered to have IDA which was confirmed by low serum iron (<59 µg/dl in males and <37 µg/dl in females) & low serum ferritin (<15 ng/ml in males and <9 ng/ml in females). Subjects having FPG >126 mg/dl (or) RPG >200 mg/ dl (or) 2 hour post prandial plasma glucose >200 mg/dl and patients with hypothyroidism, vitamin B12 deficiency, pregnancy and those having abnormal renal function tests (serum urea, creatinine), hemolytic anemia were excluded from the study. HbA1C was measured by HPLC method using Bio-Rad analyzer. Hb, MCV, MCH, MCHC were estimated by sysmex XT-1800 analyzer. FPG estimated by glucose oxidase/peroxidase method. Serum ferritin (Bio-Rad Quanimune Ferrin IRMA, Bio-Rad lab) & Serum iron (TPTZ method). Statistical analysis: The data are presented as mean ± S.D for continuous variables. A student’s t- test was applied for comparison of group means. Pearson’s co-efficient of correlation was calculated to determine correlation between two variables. P value <0.05 was considered statistically significant.

Result

Our study results show that mean HbA1C levels in controlled diabetics with IDA patients was 7.86±0.11% while that in controlled diabetic patients without IDA was 5.45±0.03%. The HbA1C levels were significantly higher in IDA patients than those without IDA (p<0.05). We also

observed a statistically significant difference (p<0.05) in mean Hb levels in controlled diabetics with and without IDA (10.33±0.15gm/dl and 14.06±0.10gm/dl respectively) These data are presented in [Table-1]. The mean Hct, MCV, MCH, MCHC, serum iron and ferritin levels in controlled diabetics with IDA and without IDA were 34.20 ± 0.44,72.67 ± 1.39, 25.52 ± 0.32, 30.20 ± 0.209,32.68±0.71,10.06±0.79 and 41.90 ± 0.32, 86.79 ± 0.39, 29.39 ± 0.12, 33.11 ± 0.08,75.26±0.79,45.19±1.11 respectively. These data show that Hct, MCV,MCH and MCHC levels were lower in controlled diabetic patients with IDA than in those without IDA and the observed difference was statistically significant as shown in [Table-2]. Additionally when patients were classified according to the degree of anemia, 55 presented with mild anemia, 70 presented with moderate anemia and 25 presented with severe anemia. HbA1C results were higher with the reduction of total hemoglobin (p<0.05) [Fig-1]. Additionally we classified study subjects into well controlled (FPG <100mg/dl) and controlled diabetics (FPG 100 – 126mg/dl) and compared HbA1C levels between anemic and not anemic groups. However HbA1C level was found to be negatively correlated with IDA in study subjects and positively correlated in control subjects (p<0.05) [Table-3]. The baseline characteristics of the study subjects were analyzed. Among 150 controlled diabetics with IDA , 89 were females (59%) and 61 were males (41%) and among those without IDA 92 were females (61%) and 58 were males (39%) as shown in [Fig-2]. The mean age of the IDA patients was 50.24±1.55 and those without IDA was 54.79±1.38.

Table 1: comparison of HbA1c% between anemic and not anemic controlled diabetics. Parameters

IDA

No anemia

T test

P value

Hb (g/dl)

10.33±0.15

14.06±0.10

-20.276

0.0001

HbA1c %

7.86 ± 0.11

5.45 ± 0.03

20.842

0.0001

Table 2: comparison of red cell indices. Parameters

IDA

Not anemic

T test

P value

Hct (%)

34.20 ± 0.44

41.90 ± 0.32

-13.922

0.0001

MCV (fl)

72.67 ± 1.39

86.79 ± 0.39

-9.718

0.0001

MCH (pg/cell)

25.52 ± 0.32

29.39 ± 0.12

-10.930

0.0001

MCHC (gm/dl)

30.20 ± 0.20

33.11 ± 0.08

-13.358

0.0001

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Impact of IDA on HbA1C

Table 3: MEAN HbA1C% GENDERWISE IN WELL CONTROLLED AND CONTROLLED DIABETICS. Females

Males

Total

IDA (n=89)

NA (n=58)

IDA (n=61)

NA (n=92)

IDA (n=150)

NA (n=150)

HbA1C in FPG < 100

7.88 ± 0.17

5.45 ± 0.06

7.62 ± 0.25

5.33 ± 0.06

7.77 ± 0.15

5.38 ± 0.04

HbA1C in FPG 100 - 126

7.93 ± 0.22

5.78 ± 0.04

7.95 ± 0.22

5.47 ± 0.06

7.94 ± 0.16

5.57 ± 0.05

T test

-0.183

-3.560

-0.983

-1.568

-0.775

-2.749

P value

0.855

0.001

0.330

0.120

0.439

0.007

Fig. 1: HbA1c VARIATION WITH DEGREE OF ANEMI.

ANOVA – 202.613, P – 0.0001 No anemia to mild anemia, T test - -7.591, P – 0.0001 Mild anemia to Moderate anemia, T test - -3.306, P – 0.001 Moderate anemia to Severe anemia, T test - -5.805, P – 0.0001

Fig. 2: AGE AND SEX DISTRIBUTION OF ANEMIA IN DIABETICS.

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Discussion

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia and its incidence is sharply increasing worldwide with many long term macrovascular and microvascular complications.[9] The HbA1C test is commonly used to diagnose diabetes and also as a marker of glycemic status over previous 3 months. Many studies suggest that anemia is twice common in diabetics when compared with non-diabetics.[10-12] Prevalence of anemia is estimated about 10-30% in patients with diabetes.[13] The etiology of anemia in diabetics is multi- factorial and includes inflammation, nutritional deficiency, concomitant autoimmune diseases, drugs, hormonal changes in addition to kidney disease. Approximately one-third of patients with anemia exhibit iron deficiency.[14] Though there are several studies on the role of anemia on HbA1C levels, only few studies have reported on the effect of iron deficiency anemia on HbA1C levels. Our study result suggested that IDA elevates HbA1C levels independent of plasma glucose concentration. This is in accordance with the study results of Brooks et al, Gram-Hansen et al and Coban et al who showed that iron therapy significantly reduces HbA1C levels in non-diabetic population.[6,7] Also our results positively correlates with results of Koga et al, Shanti et al who showed that HbA1C levels in patients with iron deficiency anemia (IDA) were higher than those of subjects with normal iron levels. [15,16] Our finding confirms the study results of Tarim et al, who reported that iron deficiency elevate HbA1C levels in diabetics when compared with iron-sufficient controls when matched for FPG levels.[1] This elevation of HbA1C in IDA may be explained by iron deficiency related changes in the quaternary structure of hemoglobin molecule increasing the glycation of globin chain.[6] Some studies proposed that the glycation of hemoglobin is a permanent process and hence the HbA1C levels in red blood cell will increase as the cellâ&#x20AC;&#x2122;s age increases. They also found that after treatment of IDA in patients with normal blood glucose levels, HbA1C concentration was reduced because of very young red cells. However if iron deficiency persists for a long time, production of red cells would fall, leading to a higher average age of circulating erythrocytes and therefore increased HbA1C levels.[16] However our study results differs with study results of E.S.Ford et al, Van Heyningen et al and Saudek et al who reported that there is no significant difference in mean HbA1C concentration according to IDA status.[6,17,18] www.pacificejournals.com/apalm

A-151 Few studies by Horton BF & Huisman TH, Rau et al and Sinha et al reported that HbA1C level decrease in IDA patients and this contradicts with our study result.[6,19] There are several strengths of this study. First we designed this study to reduce as much as possible confounding factors that could affect our HbA1C results like renal insufficiency, pregnancy, alcohol etc. Second we also analyzed HbA1C results in different degrees of anemia (mild, moderate and severe) and observed that HbA1C level increases as severity of anemia worsens. Limitations to this study were we couldnâ&#x20AC;&#x2122;t follow up patients after iron supplementation which might have given a new dimension to our study and since this was a cross-sectional study and the mechanism by which anemia affects HbA1c was not evaluated.

Conclusion

IDA spuriously elevates HbA1C levels independent of plasma glucose concentration. HbA1C level increases significantly with severity of anemia. Thereby this study insists on the utter importance to exclude IDA in diabetes and to correct it before any diagnostic or therapeutic decision is made based solely on HbA1C level.

Acknowledgements

I would like to thank our Dean Dr. A.Sundaram, SRM Medical College Hospital and Research Center for his support throughout the study. Am thankful to all the participating people for their cooperation.

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Herman WH, Ma Y, Uwaifo G, Haffner S, Kahn SE, Horton ES, et al. Differences in A1C by race and ethnicity among patients with impaired glucose tolerance in the Diabetes Prevention Program. Diab care. 2007;30:2453-7.

Nathan DM, International Expert Committee. International Expert Committee report on the role of the A1C assay in the diagnosis of diabetes. Diab Care. 2009;32:e160.

Soulimane S, Simon D, Herman WH, Lange C, Lee CM, Colagiuri S,et al. HbA1c, fasting and 2 h plasma glucose in current, ex-and never-smokers: a meta-analysis. Diabetologia. 2014;57:30-9.

Kalasker V, Kodliwadmath MV, Bhat H. Effect of iron deficiency anemia on glycosylated hemoglobin levels in nondiabetic Indian adults. Int J Med Hlth Sci. 2014;3(1):40-3.

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Coban E, Ozdogan M, Timuragaoglu A. Effect of iron deficiency anemia on the levels of hemoglobin A1C in nondiabetic patients. Acta Haematol. 2004;112(3):126-8.

World Health Organization, Worldwide prevalence of anaemia 1993–2005. WHO global database on anaemia, WHO, 2008

Kilpatrick ES, Bloomgarden ZT, Zimmet PZ. Is haemoglobin A1c a step forward for diagnosing diabetes. BMJ. 2009;339:b4432.

10. Thomas MC, MacIsaac RJ, Tsalamandris C, Power D, Jerums G. Unrecognized Anemia in Patients With Diabetes A cross-sectional survey. Diab care. 2003;26(4):1164-9. 11. Cawood TJ, Buckley U, Murray A, Corbett M, Dillon D, Goodwin B, et al. Prevalence of anaemia in patients with diabetes mellitus. Irish J Med Sci. 2006;175(2):25-7. 12. Stevens PE, O’Donoghue DJ, Lameire NR. Anaemia in patients with diabetes: unrecognised, undetected and untreated? Current medical research and opinion. 2003;19(5):395-401. 13. Hosseini MS, Rostami Z, Saadat A, Saadatmand SM, Naeimi E. Anemia and microvascular complications in patients with type 2 diabetes mellitus. Nephro-urol. 2014;6(4).

14. Wu AC, Lesperance L, Bernstein H. Screening for iron deficiency. Pediatrics in Review. 2002;23(5):171-8. 15. Koga M, Saito H, Mukai M, Matsumoto S, Kasayama S. Influence of iron metabolism indices on glycated haemoglobin but not glycated albumin levels in premenopausal women. Acta diabetologica. 2010;47(1):65-9. 16. Shanthi B, Revathy C, Manjula Devi AJ, et al. Effect of iron deficiency on glycation of haemoglobin in nondiabetics. J Clin Diagn Res. 2013;7(1):15–17. 17. Ford ES, Cowie CC, Li C, Handelsman Y, Bloomgarden ZT. Iron-deficiency anemia, non-iron deficiency anemia and HbA1c among adults in the US. J Diabetes. 2011;3(1):67-73. 18. Saudek CD, Herman WH, Sacks DB, Bergenstal RM, Edelman D, Davidson MB. A new look at screening and diagnosing diabetes mellitus. J Clin Endocrinol Metab. 2008;93(7):2447-53. 19. Sinha N, Mishra TK, Singh T, Gupta N. Effect of iron deficiency anemia on hemoglobin A1c levels. Ann Lab Med. 2012;32(1):17-22.

*Corresponding author: Dr. R. Lavanya Rajagopal M.D., 902, B-block, SRM Medical Staff Quarters, SRM Medical College Hospital and Research Centre, Kattankulathur, Chennai , Tamilnadu, India .603203 Email: drrlavan@yahoo.co.in Date of Submission : 26.10.2016 Date of Acceptance : 10.01.2017 Financial or other Competing Interests: None. Date of Publication : 07.04.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Original Article DOI: 10.21276/APALM.1173

A study on Histomorphological Spectrum of Nasal Polyp K Pushpalatha*, Sreedevi CH, Soujanya R Department of Pathology, Maheshwara Medical College, Sangareddy, India

ABSTRACT Background: Nasal Polyp is commonly encountered in clinical practice and important from clinical and pathological perspectives as they have varieties of histological patterns. The Polypoidal masses in nasal cavity form a complex group of lesions with wide spectrum of histopathological features, mainly grouped under allergic and inflammatory. Histopathological examination shows a spectrum of lesions ranging from the non-neoplastic ones to neoplastic tumors. Methods: Present study included 153 polypoidal lesions of the nasal cavity during a period of one year. All the tissues were fixed in 10% buffered formalin, processed, stained with H & E and studied for various histopathological patterns. Periodic acid Schiff’s and reticulin stains were used wherever necessary. Results: classifying the sinonasal lesions according to histo-pathological features into various types helps us to know the clinical presentation, treatment, clinical outcome and prognosis of the disease. Although most of nasal polyps sent for histopathology are inflammatory, secondary to infection or allergy, various benign and malignant lesions of nose may present as polypoidal masses, Conclusion: The study recommends, all polyps need histo-pathological examination. Keywords: Nasal Polyps, Sinonasal Lesions, Histo-Pathology.

Introduction

Sinonasal lesions are the common lesion encountered in clinical practice and important from clinical and pathological perspectives as they give rise to varieties of histological patterns and grades of malignancies. [1] The presence of mass in the nose is a seemingly simple problem; however, it raises numerous questions about differential diagnosis. Although neoplasms of the nose and paranasal sinuses are not common, they are of interest because of their various types. It has been found, the nose and paranasal sinuses account for less than 1% of all malignant tumors in general, not more than 3% of the head and neck region malignancies. Clinically sometimes, it becomes quite impossible to distinguish between inflammatory conditions presenting as simple polyps, polypoidal lesions due to specific disease and polypoid neoplasm (benign and malignant). Therefore it becomes important that all polyps and polypoidal lesions should be submitted for histopathological examination. Nasal polyps are defined as prolapsed lining of the nasal sinuses. They are essentially rounded projections of edematous membrane. They are often bilateral and multiple which lead to visible broadening of nose. The commonest site of origin is in the ethmoidal labyrinths, particularly from the mucosa of middle turbinate. In nasal cavity, tumors of various type have a tendency to become polypoid. Thus an epithelial papilloma of the nasal

cavity often resembles a nasal polyp.[5] Some lesions are specific to certain location, for e.g., epithelial papilloma of turbinate, juvenile angiofibroma of nasopharynx. Polypoidal masses of the nasal cavity form complex group lesions with a wide spectrum of histopathological features.[6] The true nasal polyps are the tumor like nonneoplastic polypoid masses arising from nasal cavity and sinuses, classified interms of Allergic nasal polyp, which constitutes 85-90%, Fibroinflammatory polyp characterized by chronic inflammation and metaplastic changesof the overlying epithelium. Other variants present with pronounced hyperplasia of seromucinous glands and Polyp with atypical stroma, rarely seen. The current study initiated to determine the Hisotopathological variations to categorize nasal polyps which are difficult to identify by clinical findings.

Material and Methods

A total number of 153 Specimens received in the histopathology section of the Dept. of Pathology of our institution with the clinical diagnoses of ‘Nasalpolyp’. The age and sex of the patients were recorded. The tissues were routinely processed for histopathological sections are made 4-6 mm on a rotatory microtome and were stained by H&E stain. Special stains by Reticulin and PAS methods were undertaken wherever applicable. The cases were classified into Nonneoplastic , Benign Neoplastic lesions

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and Malignant Neoplastic lesions. The Neoplastic lesions were further classified according to WHO classification on histopathological examination. Data Analysis: Data was analyzed by SPSS 20.0. Data was expressed as mean ± SD.

benign neoplastic cases (n=44) distribution, Adenomatous polyps are high with 40.91% (table 2). Among distribution in malignant neoplastic lesions, Squamous cell carcinoma is highest with 54.55% (table 3). Figure 2 presents allergic nasal polyp and Figure 1-3 shows the various Allergic nasal polyp inflammatory nasal polyp and Haemangioma.

Results

Discussion

Among Non-neoplastic lesions (n=98), allergic Nasal polyp cases were high with 56.12% (table 1). Among

Fig. 1: Illustrating Allergic nasal polyp.

Though nasal polyps are affecting about four percent of the population, their etiology is still not clear.[16] Polypoidal

Fig. 2: Illustrating Inflammatory nasal polyp

Fig. 3: Haemangioma. Table 1: Non-neoplastic lesions (n=98) Diagnosis Nasal polyp-Allergic Nasal polyp-inflammatory Rhnosporidiosis Rhino scleroma Mucocele Nasoalvelor cyst Tuberculous granuloma Fungal granuloma TOTAL

No of cases 55 30 9 0 1 0 1 2 98

Male 43 23 6 0 1 0 1 1 75

Female 12 7 3 0 0 0 0 1 23

Data was expressed as frequency and percentages.

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% 56.12% 30.61% 9.18% 0.00% 1.02% 0.00% 1.02% 2.04%

Soujanya R et al.

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Table 2: Benign neoplastic lesions (n=44). Diagnosis Epithelial

Mesenchymal

TOTAL

No of cases

Male

Female

Inverted papilloma

9.09%

Adenomatous polyps

40.91%

Squamous papilloma

4.55%

Haemangioma

31.82%

Angiofibroma

9.09%

Haemangiopericytoma

2.27%

Fibroma

0.00%

Schwannoma

0.00%

Osteiod osteoma

0.00%

Leiomyoma

2.27%

Data was expressed as frequency and percentages.

Table 3: Malignant neoplastic lesions (n=11). Diagnosis

No of cases

Male

Female

Epithelial

Squamous cell carcinoma

54.55%

Adenocarcinoma

27.27%

Adeniodcysticarcinoma

0.00%

Malignant Melanoma

9.09%

Mucoepidermoid carcinoma

0.00%

Neuroblastoma

0.00%

Mesenchymal

NonHodgkins lymphoma

9.09%

Angiosarcoma

0.00%

TOTAL

Data was expressed as frequency and percentages.

masses in the nasal cavity form a complex group of lesions with a wide spectrum of histopathological features.[6] While there are many nonneoplastic lesions including mainly the allergic and inflammatory one, there are also good number of neoplastic tumefaction in the nose and nasal sinuses. These lesions are often quite impossible to distinguish clinically and are labelled as nasal polyp.[7] Histopathological examination of such polypoidal masses show a spectrum of lesions ranging from nonneoplastic ones to neoplastic tumors including benign and malignant neoplasms.[8] The true nasal polyps are the tumor like nonneoplastic polypoidal masses arising from nasal cavity and sinuses. Two types are encountered- one is associated with nasal allergy and another with numerous inflammatory or granulomatous polyp.[9]

lesions, followed by Nasal polyp inflammatory lesions with 30.61%. These polyps are found to be common in male. In benign neoplastic cases, Adenomatous polyps with 40.91% found to highest, followed by Haemangioma with 31.82%. These type of polyps also mostly found in male.In malignant neoplastic cases, Squamous cell carcinoma with 54.55% found to be highest, followed by Adenocarcinoma with 27.27%. The incidence of nasal polyps was slightly higher in this study (82.06%) compared to the observations by Tondon et al (64%) and Anjali et al (62.85%). The age range of the patients was from 10 to 80 years.[10] Most commonly patients are in 2nd to 3rd decade which is comparable with Ghosh and Bhattacharya and Zafar et al.[11]

In our study, we have observed 98 cases were Nonneoplastic lesions, 44 cases were benign neoplastic lesions and 11 cases were malignant neoplastic lesions. Nasal polyp with 56.12% found to be highest in non-neoplastic

There is male preponderance with male to female ratio of 1.53:1 which was same as that observed by Zafar et al and Dasgupta et al. Although adolescence or early childhood is stated to be the commonest age of occurrence, there are reports of this disease occurring in all age groups.[1214] Nasal polyps were bilateral in 60% cases in our study, while according to Batsakis bilateralism was the rule. In

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our study, 2 cases (4.36%) of fungal infection were found in the age group of 20-60 years with male predominance which is comparable with Ghosh and Bhattacharya.[15] Hemangiopericytoma is a rare angiogenic tumor accounting for only 1% of total cases in our study. As the initiating cause of the nasal polyps are still not clear, it is essential to understand the Histomorphological Spectrum of Nasal Polyp. Our study attempted to add further knowledge about etiology of nasal polyps.

Conclusions

To conclude, classifying the sinonasal lesions according to histopathological features into various types helps us to know the clinical presentation, treatment, clinical outcome and prognosis of the disease. Although most of nasal polyps sent for histopathology are inflammatory, secondary to infection or allergy, various benign and malignant lesions of nose may present as polypoidal masses, so all polyps need histopathological examination.

Acknowledgements

We would like to acknowledge the staff of departments of both Pathology for extending their support and cooperation to carry out the study.

References 1.

Cotran RS, Kumar V, Stanly L. Robins Pathologic basis of disease, 6th edn. W.B. Saunders Company, 2000.

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

Drake Lee AB. Nasal polyps. Chapter 10 in Scott Browns Otolaryngology Rhinology 6th edn, 1984.

Newton JR, Kim Wong Ah-See. A review of nasal polyposis. Ther Clin Risk Manag. 2008 Apr; 4(2): 507–512.

Bateman ND, Fahy C, Woolford TJ. Nasal polyps: still more questions than answers. J Laryngol Otol. 2003;117 :1–9.

Dalziel K, Stein K, Round A, et al. Systematic review of endoscopic sinus surgery for nasal polyps. Health Technol Assess. 2003;7:1–159.

7. Baudoin T, Kalogjera L, Hat JI. Capsaicin significantly reduces sinonasal polyps. Acta Otolaryngol. 2000;120 :307–11. 8.

Luis CilloZenaida BA. “ Cancer of Nasal Cavity” Cancer. 1976; 37; 1458-1463.

Dalziel K, Stein K, Round A, et al. Systematic review of endoscopic sinus surgery for nasal polyps. Health Technol Assess. 2003;7:1–159.

10. Dasgupta A, Ghosh RN, Mukherjee C. Nasal polyps histopathologic spectrum. Indian J Otolaryngol Head Neck Surg. 1997;49(1):32-37. 11. Jyothi AR et al. Morphological spectrum of lesions in the sinonasal region. Journal of evolution of medical and dental sciences. 2013;37(2);7175-86. 12. Khan N, Zafar U, Afroz N, Ahmad SS, Hasan A. Masses of nasal cavity, paranasal sinuses and nasopharynx: A clinicopathological study Indian J Otolaryngol Head Neck Surg. 2006 Jul;58(3):259-263. 13. Modh S K, Delwadia K N, Gonsai R N. Histopathological spectrum of sinonasal masses- A study of 162 cases. Int J Cur Res Rev. 2013;5(3):83-9.1 14. Zafar U, Khan N, Afroz N et al.Clinicopathological study of nonneoplastic lesions of nasal cavity and paranasal sinuses. Indian J Pathol Microbiol. 2008;51(1):26–29. 15. Kulkarni AM, Mudholkar VG, Acharya AS, Ramteke RV. Histopathological study of lesions of nose and paranasal sinuses.Indian J Otolaryngol Head Neck Surg. 2012; 64(3):275-289. 16. Fokkens W, Lund V, Mullol J. European Position Paper on Rhinosinusitis and Nasal Polyps Group. EP3OS 2007: European position paper on rhinosinusitis and nasal polyps 2007. A summary for otorhinolaryngologists. Rhinology. 2007; 45:97–101.

*Corresponding author: Dr K Pushpalatha, Associate Professor, Department of Pathology, Maheshwara Medical College, Sangareddy, India

Financial or other Competing Interests: None.

Date of Submission : 21.11.2016 Date of Acceptance : 21.01.2017 Date of Publication : 07.04.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Original Article DOI: 10.21276/APALM.1178

Diagnostic Utility of Fine Needle Aspiration Cytology of Sensory Cutaneous Nerve in Leprosy Naik Reena1, Sa Dilip Kumar2, Panda Kishori M1* Dept. of Pathology, Govt Medical College (LSLAMMC), Raigarh, Chhattisgarh. India Dept of Skin &VD , Govt Medical College(LSLAMMC), Raigarh, Chhattisgarh, India

1 2

ABSTRACT Background: Peripheral neuropathy is a central feature of leprosy. Intraneural inflammation caused by M. leprae is the morphological hallmark of this disease, FNAC of sensory cutaneous nerve has proved to be a valuable diagnostic tool. Methods: The data of patients with sensory cutaneous nerve involvement were retrieved from our record for the period from Nov 2014 to Sept 2015. The hematoxylin and eosin (H and E)- and May-Grünwald-Giemsa (MGG) stained slides were screened for Schwann cells, granuloma, and necrosis. Modified Ziehl-Neelsen (ZN) stained smears were searched for single lepra bacilli and globi. Results: Twenty-five sensory cutaneous nerves were aspirated. Out of which 19 yielded diagnostic aspirate. Five cytologic pictures were seen - epithelioid cell granulomas (6), epithelioid cell granulomas with necrosis (3);necrosis + lepra bacilli (4); only lepra bacilli (2); and lymphocyte & macrophage infiltrate (4).Following the Ridley-Jopling classification, in our study there were 9 cases of TT, 4 of BT-TT, 1 of BB, 2 of BL, and 3 of LL. Conclusion: FNAC of sensory cutaneous nerve is useful in diagnosis and classification of leprosy on the R-J scale. Keywords: Peripheral Neuropathy, Fine-Needle Aspiration Cytology, Hansen’s Disease, Lepra Bacilli, Sensory Cutaneous Nerve.

Introduction

Leprosy is a chronic granulomatous infection caused by Mycobacterium leprae, affecting the skin and peripheral nerves and resulting in disabling deformities.[1] Pure neuritic leprosy (PNL) is a type of leprosy, which is clinically limited to peripheral nerves and constitutes 4-8% of all leprosy. [2].The clinical features of leprosy include anesthetic skin lesions, nerve enlargement, tenderness, pain and sensory motor impairment. These are not specific and may not always be present. So diagnosis remains difficult in early stages of leprosy including PNL cases. It is recommended that sensory cutaneous nerve fine needle aspiration cytology (FNAC) is a feasible, viable, effective, and relatively “nerve sparing” procedure, which can be done routinely as an outdoor procedure in the evaluation of leprosy patients.[3] Only a few studies have evaluated the role of fine needle aspiration cytology (FNAC) in the diagnosis of leprosy, especially in PNL cases.[2] This study was undertaken to evaluate the diagnostic role of nerve FNAC in leprosy and to evaluate the possible utility of cytology in classifying lesions of leprosy on the R-J scale.

Materials and methods

This retrospective study included leprosy patients attending the outpatient department of our medical college hospital during one-year period from Nov 2014 to Sept 2015.

FNAC was done from the enlarged thickened nerve in 25 cases, where diagnosis of leprosy was suspected clinically including relapse cases. All peripheral and cutaneous nerves were palpated for their number, size, nodularity and tenderness. These findings were entered in a chart. The cases were examined for most prominent site of thickened nerve. The area was cleaned with an alcohol swab. The prominent part of nerve was fixed by index finger and thumb of left hand and FNAC was done using 22 G needle fitted in 10 mL disposable plastic syringe. The suction was applied and aspiration was performed using a singlepuncture. The direction of the needle was always kept parallel to the length of the nerve so as to cause minimal damage to the nerve. The material aspirated was smeared on glass slides. Minimum three smears were made for each case. The wet smear was fixed in 95% ethanol and stained by Papanicolaou stain after 30 minutes of fixation. One of the dried smear was stained by Giemsa stain and the other dried smear was stained by modified acid fast bacilli (AFB for Lepra) stain demonstrate AFB. Both Papanicolaou and Giemsa stained smears were examined for cytological details. Smear stained by modified AFB (Lepra) stain was examined for the presence or absence of AFB. If the AFB was seen, it was quantified according to the presence of their number per high-power field. It was denoted as present (+), if occasional bacilli was seen after searching many high-

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power fields, and many (++) if many bacilli per high-power field were seen. Negative finding was denoted as absent (â&#x2C6;&#x2019;). Cytological criteria for sub-classification of leprosy were applied as defined by SinghÂ et al ( table 1).[4] Patients were classified according to RJ criteria into tuberculoid (TT), borderline tuberculoid (BT), mid-borderline (BB), borderline lepromatous (BL), and lepromatous (LL) types.

Results

Out of total 25 cases of FNAC done from sensory cutaneous nerves 19 cases (76%) yielded diagnostic aspirates. The sensory cutaneous nerves aspirated are detailed in [Table-2]. Most common nerve affected in the present study was right ulnar nerve. But the most common nerve aspirated was right posterior tibial nerve. Mononeuropathy

was seen in 6 cases and polyneuropathy was seen in 13 cases. Affected age group was 20 to 61 years [Table 3]. In cytology smears, in order to ascertain if the aspiration was done from a nerve, a search was made for schwann cells. Schwann cells may be present either singly or in fascicles along with other features like granuloma, necrosis, lymphocytic infiltrate and lepra bacilli [Figure1& 2].Five cytomorphologic patterns were observed in smears of nerve aspirates[Table 4].These are inflammation composed of epithelioid cell granulomas (6), epithelioid cell granuloma with necrosis (3); necrosis + lepra bacilli (4); only lepra bacilli (2); and lymphocyte & macrophage infiltrate (4). Following the Ridley-Jopling classification, in our study there were 9 cases of TT, 4 of BT-TT, 1 of BB, 2 of BL, and 3 of LL.

Fig. 1: (A) Showing Schwann cell in fascicle. (B) Epitheloid granuloma. (C) Dense lymphocytic infiltrate. (D) Epitheloid cell with giant cell.

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Fig. 2: Microphotographs showing (A) Necrosis. (B) Lepra bacilli in globi. (C) Foamy macrophages. and (D) Singly present lepra bacilli. Table 1: Cytological criteria (R-J) for sub-classification of leprosy. Diagnosis

Cellularity

Macrophages

Epitheloid cell granuloma

Lymphocytes

Tuberculoid- Borderline tuberculoid

good

absent

well formed

numerous

Mid borderline

moderate

few

diffuse

few

1+,2+

Borderline lepromatous

fair

fair number

absent

numerous

3+,4+

Lepromatous

heavy

foamy

absent

few

5+,6+

Lepra reaction

Neutrophil +

Neutrophils +

Fragmented

Table 2:Showing details of sensory cutaneous nerve aspirated(n=19) Sensory Cutaneous nerve aspirated

Right posterior tibial nerve

Left posterior tibial nerve

Right radial nerve

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Sensory Cutaneous nerve aspirated

Left radial nerve

Left ulnar nerve

Right ulnar nerve

Left common peroneal nerve

TOTAL

Table 3: showing clinical details and cytological diagnosis (n=19) S.No.

Age/Sex

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

40/F 34/F 33/F 22/M 38/M 61/M 20/F 31/M 27/M 38/M 42/M 42/F 42/F 48/M 25/M 30/M 40/M 29/M 22m

Involment N + + + + + + + + + + + + + + + + + + +

S+N + + + + + + + + + + +

Site of FNAC

Mono

Poly

Cytological diagnosis

Rt Post tibial Rt Post tibial Lt Post tibial Rt Radial Rt Post Radial Rt Post tibial Rt Post tibial Lt ulnar N Rt ulnar Lt ulnar Rt Post tibial Lt commopero Rt Post tibial Lt radial Rt Post tibial Lt radial Rt post tibial Rt Post tibial Left radial

+ + + + + + -

+ + + + + + + + + + + + +

BT-TT LL BT-TT BB TT LL TT TT LL BL TT TT TT TT-BT TT-BT TT TT TT BL

Table 4: Showing five cytologic patterns. Cytomorphological picture Epithelioid cell granuloma only Granuloma +Necrosis Necrosis+ lepra bacilli Only lepra bacilli Lymphocyte, macrophage infiltration

n=19 6 3 4 2 4

Table 5: Cytomorphological classification of leprosy according to Ridley-Jopling spectrum. Class

Singh et al. [4] (skin smear)

Prasad PV et al. [19] (skin smear)

Jaswal et al. [20] (skin smear)

Vijaikumar et al. [15] (nerve aspirate)

Cellular smears, cohesive epithelioid cell granulomas, numerous lymphocytes not infiltrating the granuloma, no stainable AFB

Cellular material with predominantly lymphocyte population and histiocytes without epithelioid transformation, no stainable AFB

Cellular smears, cohesive epithelioid cell granulomas, numerous lymphocytes not infiltrating the granuloma. BI 0–3+

Good cellular aspirate· Cohesive epithelioid cell granuloma or lymphocytic cell collection· Predominantly epithelioid cells with predominant to moderate number of lymphocytes. Occasional giant cells and neutrophils· BI 0-1+.

Same as TT

Cellular material with lymphocytes, histiocytes and epithelioid cells, foamy macrophages are not a feature, no stainable AFB.

Same as TT

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Class

Singh et al. [4] (skin smear)

A-161 Prasad PV et al. [19] (skin smear)

Moderate cellularity, singly dispersed macrophages with no epithelioid cells. Numerous lymphocytes diffusely scattered along with macrophages. BI 3-4+

Jaswal et al. [20] (skin smear)

Vijaikumar et al. [15] (nerve aspirate)

Fair cellular yields, poorly cohesive granuloma composed of an admixture of epithelioid cells and macrophages, few lymphocytes infiltrating the granulomas. BI 1-2+

Fair cellular aspirate· Mixed cellularity of predominantly nonfoamy macrophages, moderate number of epithelioid cells and lymphocytes. Macrophage granuloma· BI 2-3+.

Moderate cellularity, singly dispersed macrophages with negative images, no epithelioid cells, numerous lymphocytes diffusely admixed with macrophages. BI 3-4+

Fair cellular aspirate· Predominantly lymphocytes and moderate number of foamy macrophages. BI. 4-5+.

Heavy cellularity, numerous foamy Heavy cellularity, numerous Heavy cellularity, numerous macrophages in Fair to poor cellular foamy macrophages in foamy macrophages in fatty background aspirate·Predominantly foamy LL fatty background with a few fatty background with a few with intracellular and macrophages and few lymphocytes· BI lymphocytes. BI 5-6+ lymphocytes. BI 5-6+ extracellular negative 6+ images, few lymphocytes. BI 4–6+ TT, tuberculoid; BT, borderline tuberculoid; BB, borderline borderline; BL, borderline lepromatous; LL, lepromatous leprosy; BI, Bacillary index.

Discussion

The Ridley-Jopling (RJ) classification is used currently for classifying leprosy, which is based on clinical, bacteriological, immunological, and histological parameters [5-8] It divides the leprosy spectrum into ‘five’ clinical and histological groups. Use of the RJ scale in the classification of leprosy helps in understanding the immunology of the patient to know the prognosis and possible complications. Ridley used ZN stain in 1989, to study the cytological material in leprosy cases [9]. In 1994, Singh et al used FNAC to diagnose 30 leprosy cases including a case of nodular lepromatous leprosy[10] using same technique.[4] It is proved in many studies that FNAC of sensory cutaneous nerve helps in detection of leprotic inflammation especially granulomas and lepra bacilli.[11-17] Schwann cells arranged in fascicles could be seen along with granulomas. [13] These cytological features of nerve aspirates also helps in the categorization of leprous neuritis along the Ridley-Jopling scale.[11,14,15] Vijaikumar et al. studied cases with nerve involvement in leprosy and classified leprous neuritis into paucibacillary (PB), borderline borderline (BB), borderline lepromatous (BL), and polar lepromatous leprosy (LL) types.[15] (Table-5) describes the RidleyJopling classification as given by different authors.

However, a negative aspirate does not entirely rule out leprosy.[15] A strong concordance in tuberculoid (90%) and in lepromatous (93.7%) cases has been documented. Mid-borderline cases of leprosy show a problem in proper diagnosis.[17] Correlation of clinical diagnoses with FNAC examination has revealed varying results in different studies. In the present study, we were able to classify all nerve aspirates in 19 cases according to R-J criteria. We could observe organized granulomas as reported by Singh et al .[4] We did however, notice a very high correlation between clinical diagnoses and FNAC in all types of leprosy. We could not differentiate between TT and BT leprosy in four cases as reported by Singh et al .[4]. Correlation was also high in BL and LL types where there were scanty cellular infiltrates and more foamy macrophages. Thus, it was possible to distinguish tuberculoid types by the presence of epithelioid cells and lepromatous types by the presence of lymphocytes and foamy macrophages. Singh et al. opined that cytological features of LL showed negative images of M. leprae on MGG-stained smears, which were later confirmed by AFB staining,[4] and we could find the same in one case in the present study. Predominant lymphocytes are seen in cytology smears in the borderline types of the disease. In this study also, we found the largest number of lymphocytes in BL cases. [15]

The accuracy of cytological classification along the RidleyJopling spectrum in nerve aspirate was found in 92% cases.

Singh et al. [4] in 1995 attempted the cytological diagnosis and classification of leprosy and found 100% cytohistological concordance. Rao et al. [18] also evaluated the utility of

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FNAC in the classification of leprosy and found 90% concordance in cases of tuberculoid leprosy and 93.75% concordance was observed in lepromatous leprosy. They, however, observed difficulty in differentiating tuberculoid leprosy (TT) from borderline tuberculoid leprosy (BT) and borderline lepromatous leprosy (BL) from lepromatous leprosy (LL) on cytology. In this study, similar problem was seen in differentiating TT and BT cases.

Conclusion

Sensory cutaneous nerve FNAC is a quick & safe procedure, which can be done routinely as an outdoor procedure in the evaluation of leprosy patients. Not only it is useful in diagnosis of PNL, but also in patients having concomitant skin & nerve involvement. It is a sensitive tool for classifying the nerve lesions as per the Ridley-Jopling classification. It is also useful in patients with relapse.

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Alexander J., Mc Adam, Milner DA, Sharpe AH. Infectitious diseases. In: Kumar, Abbas, Aster editors. Robbins and Cotran Pathologic basis of disease. 9 th edition Saunders/ Elsevier, Philadelphia 2014:377

E. Wilder-Smith, “Diagnosis of pure neuritic leprosy,” Neurological Journal of South East Asia 2002;7: 61–63

Prasoon D, Mandal SK, Agrawal P. Sensory cutaneous nerve fine-needle aspiration in Hansen’s disease: A retrospective analysis of our experience. J Cytol 2015;32:170-75.

Singh N, Bhatia A, Gupta K, Ramam M. Cytomorphology of Leprosy across the Ridley Jopling spectrum. Acta Cytol 1996;40:719-23.

5. Jopling WH, McDougall AC. Definition, epidemiology and world distribution. In: Jopling WH, McDougall AC, editors. Handbook of Leprosy. 5 th ed.CBS Publishers, New Delhi 1996:1. 6.

Noordeen SK. The epidemiology of leprosy. In: Hastings RC, Opromolla DVA editors.Leprosy 2 nd ed. Churchill Livingstone, London 1994:29-45.

WHO Expert committee on leprosy 1988 Sixth report. Technical Report Series 768, Geneva: World Health Organization.

8. Ridley DS, Jopling WH. Classification of leprosy according to immunity: A five group system. Int J Lepr 1966;34:255-73. 9.

Ridley MJ. The cellular exudate: Mycobacterium leprae relationship and the critical reading of slit smears. Lepr Rev 1989;60:229-40.

10. Singh N, Arora VK, Ramam M. Nodular lepromatous leprosy: Report of a case diagnosed by FNA. Diagn Cytopathol 1994;11:373-5. 11. Siddaraju N, Yaranal PJ, “Use of fine needle aspiration cytology in leprotic lesions: a report of 4 cases,” Acta Cytologica 2007;51: 235–238 12. Theuvenet WZ, Miyazaki N, Roche PW, and Shrestha I, “Cytological needle aspiration of the nerve for the diagnosis of pure neural leprosy,” International Journal of Leprosy 1993; 61:597–599 13. Jayaseelan E, Shariff S, Rout P, “Cytodiagnosis of primary neuritic leprosy,” International Journal of Leprosy and Other Mycobacterial Diseases 1999;67: 429–434 14.

Siddaraju N, Sistla SC, Singh N et al., “Pure neuritic leprosy with nerve abscess presenting as a cystic, soft tissue mass: report of a case diagnosed by fine needle aspiration cytology,” Diagnostic Cytopathology 2009;37:355-8

15. Vijaikumar M, D’Souza M, Kumar S, Badhe B, “Fine needle aspiration cytology (FNAC) of nerves in leprosy,” Leprosy Review 2001;72:171–178 16. Singh N, Malik A, Arora VK, Bhatia A, “Fine needle aspiration cytology of leprous neuritis,” Acta Cytologica 2003;47: 368–372 17. Rao IS, Singh MK, Datta SK, Pandhi RK, Kapila K. “Utility of fine-needle aspiration cytology in the classification of leprosy,” Diagnostic Cytopathology 2001;24:317–321 18. Prasad PV, George RV, Kaviarasan PK, Viswanathan P, Tippoo R, Anandhi C, “Fine needle aspiration cytology in leprosy,” Indian Journal of Dermatology, Venereology and Leprology 2008;74:352–356 19. Jaswal TS, Jain VK, Jain V, Singh M, Kishore K, Singh S. “Evaluation of leprosy lesions by skin smear cytology in comparison to histopathology,” Indian Journal of Pathology and Microbiology 2001;44:277–281

*Corresponding author: Dr Kishori M. Panda, Prof. of Pathology, Dept of Pathology, Govt Medical College, Raigarh, Chhattisgarh, India Phone: +91 8763874020 Email: drkishoripanda@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 22.11.2016 Date of Acceptance : 26.12.2016 Date of Publication : 07.04.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Original Article DOI: 10.21276/APALM.1193

Histomorphological Study of Gestational Trophoblastic Lesions in a Tertiary Medical Centre: A Prospective Study Nidhi Rajendra1*, Fouzia Kauser2, Prashanth Madapura V 3, Doddikoppad.M M4 Department of Pathology, Dr. B R Ambedkar Medical College and Research Centre, KG Halli, Bangalore, Karnataka, India Department of Pathology, Clumax Diagnostics, The Banyan Apartments, Anjanapura, JP Nagar 9th Phase, Bangalore. India 3 Department of Paediatrics, M S Ramaiah Medical College and Hospital, MSRIT Post, Bangalore, India 4 Department of Pathology, Basaveshwara Medical College and Hospital, Chitradurga, India.

1 2

ABSTRACT Background: Gestational trophoblastic diseases(GTDs) consists of pregnancy related disorders ranging from benign Hydatidiform mole, Invasive mole to neoplastic conditions including Choriocarcinoma, Placental site trophoblastic tumor and Epitheloid trophoblastic tumor with varying potential for local invasion and metastasis. GTDs mimic growth pattern encountered in early normal placental development, nonmolar abortions and variety of nontrophoblastic lesions. Therefore an appreciation of different types of GTDs with its histomorphological manifestations are important for the confirmation of diagnosis. Thus the study was undertaken. Materials & Methods: The material for the study comprised of products of conception specimens received in the Department of Pathology, J.J.M. Medical college, Davangere, India during the period of 2 years. Detailed gross examination was done before fixation and subsequent microscopic analysis was done. Results: 65 cases of GTDs were diagnosed in 495 cases of products of conception. 37 cases of complete mole, 27 cases of partial mole and one case of Invasive mole were diagnosed. Diffuse hydropic swelling of the villi, round smooth villous outline, circumferential trophoblastic proliferation and focal hydropic swelling of the villi, irregular scalloping outline of the enlarged villi, focal trophoblastic cell proliferation were characteristic features of complete & partial mole respectively. Histomorphological features were analysed in all the cases and compared with studies done by others. Conclusion: The classification and histomorphological analysis of GTDs are important elements for better understanding of the disease. Studies are needed to look for ancillary markers in distinguishing different types of GTDs and for predicting the prognosis. Keywords: Gestational Trophoblastic Diseases, Complete Mole, Partial Mole, Invasive Mole.

Introduction

Gestational trophoblastic lesions (GTD) includes a heterogenous family encompassing various neoplastic and non neoplastic lesions arising from different type of villous and non villous trophoblast.[1] Trophoblast is an integral component of the human placenta for mediating the implantation of the embryo, protecting the fetus from the maternal immune system, delivering nutrient and removing waste products as well as producing vital pregnancy hormones.[1] Hertig referred to gestational trophoblastic neoplasms as “God’s first cancer and man’s first cure”.[2] Gestational trophoblastic disease constitutes a diverse group of lesions that includes Hydatidiform mole, benign non neoplastic lesions, and gestational trophoblastic neoplasms.[3] Gestational trophoblastic lesions arises from trophoblast, a tissue normally exhibiting features otherwise associated with malignant tumors like intense proliferation, anaplasia, infiltration of contiguous structures, invasion of blood

vessels and hematogenous dissemination. Thus it may be difficult to discriminate benign, even normal proliferating trophoblastic tissue from its malignant counterpart on morphologic grounds alone.[4] Multiple hCG related molecules are present in serum and urine samples of pregnancy and trophoblastic disease. These include degraded molecules, hyper glycosylated molecules, large subunits and their fragments, which are estimated in serum and urine using different combination of antibodies. While all are appropriate for normal pregnancy application, only certain types of assay may be correct for monitoring trophoblastic diseases.[5,6] With a half life of 24-36 hours, hCG is the most sensitive and specific marker for trophoblastic tissue. Current methods for hCG measurement do not reliably discriminate among pregnancy, GTD and non gestational trophoblastic tumors. However serial measurements of hCG have revolutionized the management of GTD for several reasons. The amount of hCG produced correlates with tumor volume so that a serum hCG of 5 IU/lt corresponds to approximately 104

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– 105 viable tumor cells. Consequently, these assays are several orders of magnitude more sensitive than the best imaging modalities available today. The hCG levels can be used to determine prognosis. Serial measurements allow progress of the disease or response to therapy to be monitored.[7,8,9]

single laboratory. Out of 65 GTD cases, serum β hCG levels reports were available in 55 cases. Serum β hCG levels reports in 7 cases of complete hydatidiform mole and 3 cases of partial hydatidiform mole was not done. We sorted out the serum β hCG levels based on their gestational age ( in weeks) at which it was done and also the type of GTD.

Gestational trophoblastic diseases mimic growth pattern encountered in early normal placental development, non molar abortions and a variety of nontrophoblastic lesions, therefore an appreciation of histomorphological manifestations of gestational trophoblastic lesions are important to avoid confusing with their mimickers. Thus the study is undertaken for histomorphological evaluation.

The mean values of the serum β hCG levels in that particular gestational age was found (table-1 ). The serum β hCG levels when plotted on the bar chart (y axis) against gestational age (x axis) at which the values were done (graph- 1). In graph it is evident that mean serum β hCG levels in complete mole was always on the higher side than in partial mole at that respective gestational age.

Material and Methods

Gross Features of GTD: Gross specimens were subjected for detailed examination to identify fetal parts. No fetal parts were observed in all suspected cases. All 65 cases showed multiple grape like vesicles and edematous change. The size of the specimens varied from 2cm to 15 cms.Vesicles were clear fluid filled, smooth surface, cystic in consistency and the color varied from grey white to grey brown. In order to appreciate the vesicles better, they were separated from blood clots and kept in normal water and photography was taken. Then the tissue was fixed in formalin. Specimens were subjected for detailed gross examination to identify the fetal parts.

This is a prospective study done during the period from July 2009 – June 2011 (2 years) in the department of Pathology, J.J.M Medical college Davangere, India. Brief clinical history and examination findings were obtained from treating Obstetrician. During the study period, total of 12188 specimens were received in the histopathology section, in the department of Pathology. Among these, 495 were products of conception. Gestational trophoblastic lesions were identified in 65 cases and included in this study. Serum β hCG level estimation was done during pre evacuation wherever it was possible. Permission and clearance was obtained from Institution’s Ethical and scientific committee. Written consent was obtained from each individual who were included in the study group. Inclusion Criteria: All specimens received at the Department of Pathology, J.J.M Medical college, Davangere showing trophoblastic tissue excluding term placenta during the span of 2 years(from July 2009- June 2011) Exclusion Criteria: Non gestational trophoblastic lesions.

Results

Sixty five GTD cases were histopathologically diagnosed in the present study and were classified according to WHO classification criteria. Serum β hCG level estimation ( single laboratory) was possible in 55 cases(Table 1: Mean serum β hCG levels done pre-evacuation) and ultrasound abdomen was done in all 65 cases. There were 37 cases of complete mole, 27 cases of partial mole and one case of Invasive mole. Of the total 65 cases of GTDs, 8 cases had previous history of abortions. Table 1: Mean serum β hCG levels (done pre-evacuation) Serum β hCG levels were done by in all suspected GTD cases before evacuation of products of conception in a

Complete Mole: Out of 37 cases of complete mole, 34 cases demonstrated diffuse hydropic swelling of the villi and circumferential trophoblastic proliferation. All cases showed round smooth villous outline. Three cases of early complete mole demonstrated focal trophoblastic proliferation without hydropic changes in the villi. In complete mole villi were of various sizes and even the smaller villi also showed edematous changes. The proliferating cytotrophoblast and syncytiotrophoblast showed circumferential growth from the villous surface. Mild to moderate degree of trophobastic proliferation was noted. 17 cases showed central cistern formation characterized by a prominent central space that is entirely acellular fluid filled spaces within the centre of the villi. 27 cases had myxoid intravillous stroma, 4 cases had hydropic intravillous stroma and 6 cases showed both myxoid and hydropic stroma.10 cases showed trophoblastic inclusions. Areas of haemorrhage and necrosis was seen in 15 cases. Decidual cells were seen in 22 cases. None of these cases showed blood vessels within the intravillous stroma or any embryonic tissue. 3 cases showed features of early complete mole characterized by redundant bulbous terminal villi, hypercellular villous stroma with primitive stellate cells and karyorrhexia, a labyrinthine network of villous stromal canaliculi and focal hyperplasia of cytotrophoblast and syncytiotrophoblast.

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Rajendra et al. Partial Mole: 27 cases were diagnosed as partial hydatidiform mole & all cases exhibited focal hydropic swelling and small normal sized villi along with irregular and scalloping outline of the enlarged villi. Focal proliferation of cytotrophoblast and syncytiotrophoblast cells was seen in all 27 cases of partial hydatidiform mole. Trophoblastic hyperplasia is less marked, focal, consisting of small, haphazard tufts of trophoblast, often syncytiotrophoblast, emanating from the surface of some of the abnormal villi. All 27 cases of partial mole had scalloped outline of the enlarged villi, yielding a pattern of trophoblastic invaginations into the villous stroma. 23 cases showed trophoblastic inclusions. Central cisterns are less conspicuous and present in 4 cases of partial mole. Fibrotic intravillous stroma was seen in 17 cases, myxoid intravillous stroma was seen in 8 cases and hydropic intravillous stroma was seen in 2 cases. Blood vessels was seen in 12 cases and were devoid of nucleated red blood cells. Intermediate trophoblasts in the intervillous spaces was seen in 8 cases. Decidual cells were seen in 14 cases and focal areas of haemorrhage was present in 10 cases. Partial mole should be distinguished from hydropic abortus. The hydropic abortus usually are smaller specimens. Villi in hydropic abortus are enlarged only slightly and do not assume the large dimensions found in complete or partial

A-165 mole. In hydropic abortus the villous swelling and central cistern formation are not present. The villous trophoblast is attenuated and trophoblastic atypia is absent or minimal. The trophoblast proliferating from the villous surface of an abortus shows polar distribution characterized by proliferation of trophoblast at the distal end of the villous that implants into the basal plate. Invasive Mole: This was seen in a multiparous woman. Her pre-operative serum Î˛ hCG levels was 126000 mIU/ ml. Total hysterectomy specimen measuring 9x7x5cms. Small grape like vesicles altogether measuring 5x4 cms were present within the endometrial cavity invading upto the serosa but not rupturing the uterus. The vesicles were small measuring <1 cm in size and were adherent to endometrial cavity. This case of invasive mole was diagnosed in a woman aged 39 yrs, which was thought to be preceded by Hydatidiform Mole. Microscopically it showed villi of varying sizes, lined by proliferating cytotrophoblasts and syncytiotrophoblasts. Transmural invasion of the myometrium by the villi was seen. Villi were enlarged, irregular in size and shape, with hydropic degeneration in the stroma. No mitosis was seen. Focal areas of haemorrhage and necrosis was seen. There was presence of molar villi and trophoblast cells deep in the myometrium.

Graph 1: Mean serum Î˛ hCG levels complete mole and partial mole.

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Table 1: Mean serum β hCG levels (done pre-evacuation). Gestational age (weeks)

Mean serum β hCG levels in complete mole (mIU/ml)

Mean serum βhCG levels in partial mole (mIU/ml)

79460

40100

85650

56060

87642

62598

89610

57826

82697

67006

98213

58322

101800

75450

97120

68460

Fig.1: Gross photograph showing edematous villi of varying sizes with membranous tissue.

Fig. 2: Edematous villi showing circumferential trophoblastic proliferation with myxoid intravillous stroma.(H&E 4x).

Fig. 3: Invasive mole - Uterine cavity completely fiilled with multiple grape like vesicles.

Fig.4: Chorionic Villi present within the myometrium.

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Discussion

Gestational trophoblastic lesions represents a spectrum of lesions characterized by an abnormal proliferation of trophoblasts which includes complete mole, partial mole, invasive mole, choriocarcinoma and placental site trophoblastic tumors. Some of these lesions are true neoplasms where as others represents abnormally formed placentas with a predisposition for neoplastic transformation of trophoblasts. In the study period of two years, among 12188 pathological specimens, 4.06% (495cases) were products of conception. We analysed 495 specimens of products of conception, received in our department for histopathological analysis. Of 495 specimens, 65 cases were diagnosed as GTDâ&#x20AC;&#x2122;s based on morphology and quantitative serum Î˛ hCG level estimation . All 34 cases of complete mole were characterized by diffuse hydropic swelling of the villi with round smooth villous outline and circumferential trophoblastic proliferation. A study conducted in a tertiary care hospital, Lahore on histopathological analysis of compete mole and partial mole in 2011 June stated that all cases of complete mole demonstrated diffuse swelling of the villi and exhibited circumferential proliferation of the trophoblast.[10] Another study done in Tokyo also defined 100% existence of villous edema in complete mole and also in partial mole.[11] Similar reports were seen in other studies.[12] Study done by Richard M C et al revealed that 65% of complete mole revealed uniform dilation of the villi and 35% had focal dilation.[13] The histomorphological features of 27 cases of partial mole were focal hydropic swelling of the villi, scalloping villous outline and focal trophoblastic proliferation and trophpblastic hyperplasia was less marked than that of complete mole. In a study done by Fukunaga et al on partial mole described that focal villous edema, focal syncytiotrophoblastic hyperplasia, villous scalloping outline , vacuolated syncytiotrophoblasts and trophoblastic inclusions were seen in all(100%) the cases of partial mole.[11] In a study done by Richard MC et al demonstrated focal dilation of the villi in 86%, scalloped villi in 83%and uniform dilation of the villi in only 10% of partial mole. [13] Trophoblastic inclusions are nothing but break in the continuity of outline of the villi and was seen in 27%( 10 cases) of complete mole. In a study done by Rozina et al and Richard MC et al, the incidence of trophoblastic inclusions in complete mole was 92.5% and 65%respectively.[10,13] Thus presence of trophoblastic inclusion is not a uniform feature. But in case of partial mole, trophoblastic inclusions www.pacificejournals.com/apalm

A-167 were found in 85.5%(23 cases) which can be attributed to commonly encountered scalloped margins which forms pseudo inclusions within the stroma. In a study done by Fukunaga et al, trophoblastic inclusions was seen in 100% of partial mole.[11] Richard MC et al described trophoblastic inclusions in 79% of the partial mole.[13] Thus presence of trophoblastic inclusions is a more consistent finding in partial mole than complete mole. Marked central cistern formation are predominant feature of complete mole which is attributed to widely separated broken strands of fibrillar material. Central cistern was seen in 50% in complete mole and 14.8% in partialmole in our study. Richard MC and others described 100% presence of central cisterns in both complete mole and partial mole.[13] However Fukunaga and others described in a different way, according to them 93% of partial mole had central cistern formation and central cistern was occasionally observed in complete mole.[11] Thus there exists different views about the central cistern formation . We found myxoid intravillous stroma in 72.9%(27 cases) of complete mole and 29.6%(8 cases) of partial mole and fibrotic intravillous stroma was described in 62.9% which was seen only in partial mole. This finding was consistent with the study done by Fukunga and others. In their study,intravillous fibrous stroma was seen in 57% of partial mole.[11] In our study, hydropic intravillous stroma was seen in less than 10%(4 cases) of complete mole and partial mole. Myxoid and hydropic intravillous stroma was seen in 16.2%(6 cases) of complete mole only. Intermediate trophoblasts in the intervillous space were seen in 40.5%(15cases) of complete mole and 29.6%( 8 cases) of partial mole. Blood vessels in the stroma were described in 44.4% (12 cases) of partial mole which were devoid of nucleated red blood cells. Fukunga and others described presence of blood vessels in 89% cases in partial mole. [11] Rozina Jaffer and others described a similar finding about the presence of blood vessels in 65% cases of partial mole.[10] Similar findings was observed in a study done by Young Ho De and others12. Blood vessels were absent in complete mole in our study. Similar finding was seen in other studies.[10,12] One case of invasive mole in 39 year old woman with gravid-5 presented with 2 months amenorrhoea and bleeding per vagina. The incidence of invasive mole was 1.53%. A study from Pakistan done in 2009, revealed a very high incidence of invasive mole (23.3%), [14] where the frequency of GTD was 28 per 1000 live birth which was alarmingly high. In another pathological study done in North India in 2007, revealed that the incidence of eISSN: 2349-6983; pISSN: 2394-6466

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invasive mole was 8.7%.[15] In our study, microscopically invasive mole showed villi of varying sizes lined by cytotrophoblasts and syncytiotrophoblast and were invading the myometrium. Transmural invasion of the myometrium by the viili was a similar finding to other studies.[16,17] The diagnosis of invasive mole rests on the demonstration of complete hydatidiform mole invading the myometrium or the presence of villi in the metastatic lesion.[16,17] 3 cases of early complete mole were diagnosed in our study which were characterized by redundant bulbous terminal villi, hypercellular villous stroma with primitive stellate cells and karyorrhexia, a labyrinthine network of villous stromal canaliculi and focal hyperplasia of cytotrophoblast and syncytiotrophoblast. Few studies suggested that complete vasculogenic differentiation is significantly retarded in very early complete mole, due to increased apoptosis in the precursor components of blood vessels. It may result in a lack of vascular drainage and cause progressive accumulation of vesicular fluid in the later gestational period.[18] Another study from the developed country revealed that bulbous villous pattern ,cellular stroma and focal trophoblastic proliferation are characteristic features of early complete mole.[19] Many histological features in early complete mole are shared with early developmental stage of normal villous stroma, but timely differentiation of stromal components is defective or arrested and immature features are for prolonged periods. These findings confirm that hydatidiform mole is not only a disease of trophoblastic proliferation, but is associated with the abnormal or incomplete maturation of villous stromal components. These findings were also reflected in a study done by Kyo.R.K et al.[20] Estimation of β hCG levels may be of value in diagnosing molar pregnancies. The clinical signiﬁcance of the accurate detection of GTD is in relation to development of persistent gestational trophoblastic neoplasm(pGTN), which is deﬁned as either clinically apparent invasive disease or persistently raised serum human chorionic gonadotropin (hCG) concentration due to gestational trophoblastic proliferation.[21] In women with a complete mole, the quantitative serum β-hCG level is higher than expected, often exceeding 100,000 IU/L. In case of a partial mole, the level of β-hCG is often within the wide range associated with normal pregnancy and the symptoms are usually less pronounced. For these reasons the diagnosis of a partial mole is often missed clinically and made from subsequent histologic assessment of the abortive material.[22] In our study the pre evacuation mean serum β hCG levels were higher in GTDs when compared to the β hCG

normogram levels.[23] It is also evident that mean serum β hCG levels in complete mole was always on the higher side than in partial mole at that respective gestational age.

Conclusion

Frequency of complete hydatidiform mole was higher as compared to partial hydatidiform mole. Histomorphological features of complete hydatidiform mole differs from partial hydatidiform mole on the basis of trophoblastic proliferation, villous contours, scalloping borders and central cisterns. The mean serum β hCG levels in complete hydatidiform mole was more than the partial hydatidiform mole at that gestational age. Further studies are needed to prove this. Studies are needed to look for ancillary markers in distinguishing complete hydatidiform mole and partial hydatidiform mole. Ethical Approval : The study was approved by institution ethical and scientific committee.

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Annie NY Chung. Gestational trophoblastic disease. In : Stanley JR, George LM, eds. Robboy’s pathology of female reproductive tract. Churchill Livginstone: Elsevier (Pub); 2009: 881-907.

Hertig AT. Human trophoblast coated by Charles B Hammond. Gestational trophoblastic neoplasia, history of current understanding. Obst & Gynec Clin of N-Am 1988;153:435-439.

Ie M S Michael TM, Robert JK. Gestational trophoblastic disease. In : Stacey EM, Jode KG, Harold AO, Victor R, Mart HS eds. Sternberg’s Diagnostic surgical pathology. Lippincot Williams and Wilkins (pub) 2010. pp2049-69

Liane D, Shirley GD, Donald PG. Gestational trophoblastic neoplasm. Morphology correlates of therapeutic response. Am J Obstet Gynec 1978;130(7):801-806.

Laurnence A. Cole. hCG, its free subunits and its metabolites role in pregnancy and trophoblastic disease. J Reprod Med 1998;43:3-10.

Michael JS, Neil SS, Ross SB. Gestational trophoblastic disease. Lancet 2010;376:717-729.

Michael JS, and Edward SN. Management of gestational trophoblastic disease. In: David MG, Gillian T, William PM, Martin G, Michael AQ eds. Gynecologic cancer . Churchill Livginstone: Elsevier (Pub); 2004:555-573.

George AH, Carl MH. Ectopic pregnancy. In: Larry JC eds. Text book of gynecology. W.B. Saunder’s Compnay; 1993 :242-260.

Ross SB, Donald PG. Gestational trophoblastic disease. In: Jonathan SB eds. Berek and Novak’s gynecology. Wolters Kluwer Health and Lippincott Williams and Wilkins 2006 pp.1581-1603

10. Rozina J, Rahat K, and Asmaa Q. Histopathological review of partial and complete hydatidiform moles in a tertiary care hospital, Lahore Pakistan, Biomedica 2011 June;27:76-80.

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Rajendra et al. 11. Fukunaga M, Shinichiro U and Yasuhiko E. Incidence of hydatidiform mole in a Tokyo Hospital : A 5 year (1989 to 1993) prospective, morphological and flow cytometric study. Human Pathol 1995;26(7):758-764. 12. Young HY, Hyun MK, Tchan KP, Chang KK and Yoo BL.Comparative cytogenetic and clinicopathologic studies on gestational trophoblastic neoplasia, especially hydatidiform mole. Yonsei Med J 1986;27(4):250-260. 13. Richard MC, Charles LH, Edwina JP, Henry JN, William F. Mc. Diagnostic considerations in molar gestations. Hum Pathol 1993;24(1);41-48. 14. Khairunnisa N, Guljareen H, Nizamuddin M, Ambreen H. Gestational trophoblastic disease: Experience at Nawabshah Hospital. J Ayub MedColl Abbottabad 2009;21(1):94-97. 15. Shakuntala C, Ambreen Q. Gestational trophoblastic neoplasms with special reference to invasive mole. Obstet Gynecol India 2007;57(2):124-127. 16. Amandeep KA, Shashi G, Vikram M, Rajat G, Rajni G. Invasive mole presenting as acute abdomen. J K Science 2011;13(1):35-36. 17. Debarmita M, Napur M, Ram PD, Ranu RB, Amiya KB, Subhash B.Partial invasive molar pregnancy – 2 case reports. Al Ameen J Med Sci 2010;3(1):91-93.

A-169 18. MiJK, KyuRK, JaeYR, JaniceML, HyangIL. Diagnostic and pathogenetic significance of increased stromal apoptosis and incomplete vasculogenesis in complete hydatidiform moles in very early pregnancy periods. Am J Surg Pathol 2006;30(3):362-69. 19. David K. Michael VZ, Terry H and Raymond WR. Very early complete hydatidiform mole. Hum Pathol 1996;27:708-713. 20. Kyu RK, Bong HP, Young OKH, Hyuck CK and Robboy SJ. The villous stromal constituents of complete hydatidiform mole differ histologically in very early pregnancy from the normally developing placenta. Am J Surg Pathol 2009;33:176-185. 21. Sebire.NJ.The diagnosis of gestational trophoblastic disease in early pregnancy: implications for screening, counseling and management Ultrasound Obstet Gynecol 2005; 25: 421–424. 22. Soper JT, Mutch DG, Schink JC. American College of Obstetricians and Gynecologists. Diagnosis and treatment of gestational trophoblastic disease: ACOG Practice Bulletin No. 53. Gynecol Oncol. 2004 Jun;93(3):575–85. 23. Overview of chemistry studies. In: Frances F, Marshall BD. A manual of laboratory and diagnostic tests. Lippincott Williams and Wilkins, and Wolters Kluwer Health (Pub) 2005 pp.338-482

*Corresponding author: Dr Nidhi Rajendra. MD (Path), Assistant Professor, Department of Pathology, Dr.B R Ambedkar Medical College and Research Centre, KG Halli, Bangalore-65, Karnataka, India Phone: +91 9535799579 Email: rnidhi25@gmail.com Date of Submission : 04.12.2016 Date of Acceptance : 02.01.2017 Financial or other Competing Interests: None. Date of Publication : 07.04.2017

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Original Article DOI: 10.21276/APALM.1212

Identification and Antimicrobial Susceptibility Testing of Microorganisms From Positive blood Cultures by a Combined Lysis-centrifugation Method with MALDI-TOF MS and VITEK2 System Donatella Maria Rodio1, Filomena Febbraro2, Gianluca Puggioni3, Camilla Paradisi1, Flavia Stangherlin1, Carla Prezioso1, Guido Antonelli4, Maria Trancassini1, Valeria Pietropaolo1* Department of Public Health and Infectious Diseases, “Sapienza” University Rome, Italy 2 Department of Pediatrics, “Sapienza” University Rome, Italy 3 Department of Clinical Medicine, “Sapienza” University Rome, Italy; 4 Department of Molecular Medicine and Pasteur Institute-Cenci Bolognetti Foundation, “Sapienza” University Rome, Italy 1

ABSTRACT Background: Rapid identification and the application of antimicrobial susceptibility testing (AST) to microorganisms causing bloodstream infections is pivotal to guide antimicrobial therapy. This study aims to: 1) utilize the Lysis-Centrifugation Method (LCM) not only for identification of microorganisms from positive blood culture bottles, but also for direct AST full panel by Vitek®2 system (bioMérieux, Inc. France) and by disc diffusion plate (Kirby Bauer Method) and 2) analyze the accuracy of these combined methods. Methods: 124 mono-microbial positive blood culture bottles were included in this study. An aliquot was subjected to LCM and used for the identification by the MALDI-TOF System. Moreover the microbial pellet was used for direct AST testing full panel by VITEK®2 system and by Kirby Bauer Method. Results: 123 isolates were correctly identified to the species level and 1 isolate was identified to the genus level. Comparing the two utilized AST methods, it was observed that Gram-positive isolates showed an agreement rate of 96.6% (58/60). Enterococcus faecalis was the only microorganism with a major error rate of 0.6% (2/324) related to erythromycin. Among the Gram-negative, the overall agreement rate was 93.3 (56/60). Klebsiella pneumoniae, Escherichia coli and Enterobacter spp. were the major cause of minor error rates (0.6%, 4/709) and major error rates (1.1%, 8/709). Among the yeasts, results showed an agreement rate of 100% (4/4). Conclusions: Our simple and cost-effective sample preparation method is very useful for rapid identification as well as AST of microorganisms directly from positive blood culture bottles in a clinical setting. Keywords: Bacteremia, LCM, MALDI-TOF MS, VITEK®2, AST.

Introduction

The presence of microorganisms in the blood or bloodstream infections (BSI), confirmed by a positive culture [1, 2], is a major cause of morbidity and mortality throughout the world [3-7]. During the period from 2000 to 2010, mortality from septicemia grew by 17% [8] and recent reports still show mortality to range from 34 to 52% [9]. Microorganisms enter the bloodstream through various portals, including dissemination from a previous or concomitant infection and access via surgical sites, intravenous catheters, and other vascular access devices [10, 11] . Bloodstream infections can be caused by a wide variety of microorganisms, commonly Escherichia coli, Klebsiella spp., Staphylococcus aureus, Enterococcus spp. and yeast. These infections can lead to increased mortality, longterm disability, excess length of stay in hospitals, large

additional costs for health systems, and high costs as well as loss of quality of life for patients and their families [7]. Rapid identification and the application of antimicrobial susceptibility testing (AST) to microorganisms causing bloodstream infections is pivotal to guide antimicrobial therapy [12], helping to reduce the detection time of the antimicrobial treatment and improving patient response. The standard protocol to diagnose a bloodstream infection involves liquid medium blood cultures that remains the gold standard to establish its etiology [13]. Currently, steps in routine phenotypic identification involves microscopic observation, sub-culturing and analysis of various biochemical reactions in order to establish an empiric treatment [14]. This process could take several days, especially if fastidious and slow-

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Rodio et al. growing microorganisms are present [15]. An alternative to the phenotypic identification, it is possible to perform a genotypic sequencing technology, such as 16S rRNA, but it is still expensive for a routinely diagnosis [16]. Matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) was recently introduced in the clinical microbiology for microorganisms’ identification. Although it identifies bacteria and yeasts within a few minutes, it requires isolated colonies, which takes 18-48 hr of incubation [17]. To reduce the time needed for identification and AST, some investigators tried to inoculate microorganisms present in positive blood culture bottles directly into MALDI-TOF MS system [18-20]. However, direct identification requires sample preparation steps since blood culture bottles contain proteins/debris which could interfere with the spectra of the microorganisms [21]. Therefore, lysis solutions [22], the lysis-filtration method [12] or commercial kits [19] were utilized for sample preparation. However, some of these procedures are laborious and/or expensive [23]. We previously published a study describing a LysisCentrifugation Method (LCM) that can be used for microbial identification by ID Vitek®2 System (bioMérieux, Inc. France) directly from positive blood cultures detected by Oxoid Signal Blood system and by BD BACTECTMFX [24] by MALDI-TOF MS system. Results of this research demonstrated that ID obtained by LCM method correctly identified a 91.4% of microorganisms responsible of the mono-microbial bacteremia with an agreement to the species and the genus level, if compared with the standard method Vitek®2 [24]. The present study aims to: 1) utilize LCM not only for identification of microorganisms from positive blood culture bottles, but also for direct antimicrobial susceptibility testing (AST) full panel by Vitek®2 system (bioMérieux, Inc. France) and by disc diffusion plate (Kirby Bauer Method) and 2) analyze the accuracy of these combined methods.

A-171 Oxoid Signal Blood Culture System Medium (OXOID S.p.A.). Only mono-microbial cultures were selected. Blood culture bottles signaling positive were removed from BD BACTECTMFX or visualized as CO2 production in the Oxoid Signal Blood Culture System Medium and an aliquot was taken for Gram staining, subjected to LCM and used for the identification by the MALDI-TOF System (Bruker Detection Corp, Bruker Nano GmbH Germany). Moreover the microbial pellet obtained from LCM was used for direct AST full panel by Vitek®2 system (bioMérieux, Inc. France) and by disc diffusion plate (Kirby Bauer Method). In parallel, an aliquot taken from positive blood culture bottles was sub-cultured on solid media. Isolates grown from such culture media were used for identification (MALDI-TOF System) and AST Vitek®2 System (bioMérieux, Inc. France). LCM for Positive Blood Cultures Detected by Oxoid Signal Blood System: 5 ml of blood culture was centrifuged to 3000 rpm for 10 minutes in order to obtain a bacterial suspension pellet that was finally resuspended in 4 ml of distilled water in order to lyse red blood cells [24]. LCM for Positive Blood Cultures Detected by BD BACTECTMFX : 5 ml of blood culture sample was centrifuged to 3000 rpm for 15 minutes. In order to obtain a bacterial suspension pellet without traces of blood, subsequent washings with 5 ml of 0.45% ammonium chloride solution (NH4Cl, Sigma-Aldrich Co. LLC) were carried out. Finally the pellet was resuspended in 5 ml of distilled water to remove traces ammonium chloride and protein residues. MALDI-TOF System Identification: The pellet was smeared on a MALDI-TOF target plate and processed for a rapid microbial identification. According to the microscopic observation the pellet was processed in the following way: •

Materials and Methods

Samples: This research was performed at the Laboratory of Microbiology (DLC01) of “Umberto I” Hospital in Rome. A total of 124 consecutive positive blood cultures, obtained from patients of the Department of “Anesthesiology and Intensive Care” and “Internal Medicine” of the same hospital in the period from April 2015 to March 2016, were included in this study. Out of 124 blood samples, 70 were collected and inoculated in BD BACTECTM Plus Aerobic/F culture vials and incubated in the automated system BD BACTECTMFX, whereas 54 samples were collected and inoculated in www.pacificejournals.com/apalm

•

For Gram-positive Bacteria: a small amount of the pellet was spread on two spots of a target plate both processed with 1 µl of 70% formic acid (HCOOH, Sigma-Aldrich Co.LLC) and subsequently with 1 µl of the acid α-cyano-hydroxycinnamic matrix solution (HCCA, Bruker Daltonik, GmbH). For Gram-negative Bacteria: a small amount of the pellet was spread on two spots of a target plate and processed with 1 µl of HCCA matrix solution. For Yeast: a small amount of the pellet was dissolved on two spots of a target plate and processed with 1 µl of 70% formic acid (HCOOH, Sigma-Aldrich Co. LLC). Subsequently 1 µl of HCCA matrix solution was added, as above mentioned. eISSN: 2349-6983; pISSN: 2394-6466

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The plate was placed in the mass spectrometer MALDITOF System and analyzed for protein mass patterns identification within a few minutes. Antimicrobial Susceptibility Testing: The microbial pellet obtained from LCM was utilized in order to obtain a suspension preparation adjusted to a McFarland standard of 0.5 for Gram-positive and Gram-negative bacteria and used for standard microdilution method AST Vitek®2 System. The same bacterial suspension was used to perform susceptibility disc diffusion test (Kirby Bauer Method). Yeast suspension was used to carry out antifungal susceptibility tests by Vitek®2 card and Sensititre YeastOne (Thermo-Fisher). AST-P632, AST-P592, AST-N202 (bioMérieux, Inc. France) were used for AST of staphylococci, enterococci and Enterobacteriaceae, respectively according to the identification results. The resulting minimum inhibitory concentration (MIC) values were classified into clinical categories of susceptible, intermediate or resistant following the European Committee on Antimicrobial Susceptibility Testing – EUCAST recommendations, 2014. For susceptibility, disc diffusion test (Kirby Bauer Method) antimicrobial discs were chosen among those recommended by EUCAST guidelines and common with the standard method AST Vitek®2. The comparison between AST Vitek®2 system and Kirby Bauer Method was expressed in terms of agreement, very major error (false susceptibility), major error (false resistance), or minor error (susceptible/resistance versus intermediate susceptibility). Statistical Analysis: We compared LCM to current standard practice in an effort to gain insight into how the process would improve clinical work flow. Fig. 1 shows a flow chart for identification and AST of microorganisms obtained directly from positive blood culture bottles.

identified with MALDI-TOF system by LCM: 123 isolates were identified to the species level and 1 isolate was identified to the genus level. The list of isolates are reported in Table 1 and Table 2. The identified microorganisms were then analyzed for direct antimicrobial susceptibility testing by disc diffusion plate (Kirby Bauer Method) and by microdilution method AST by Vitek®2 (bioMérieux, Inc. France). The comparison of the two antimicrobial susceptibility testing utilized methods was showed in Table 1. It was observed that Gram-positive isolates showed an agreement rate of 96.6% (58/60) (Table 1). Enterococcus faecalis (N = 2) was the only microorganism responsible of the disagreement with a major error rate of 0.6% (2/324) (Table 1). Among the Gram-negative, the overall agreement rate was 93.3 (56/60) (Table 1). Klebsiella pneumoniae (N = 2), Escherichia coli (N = 1) and Enterobacter spp. (N =1) were the major cause of this disagreement with a minor error rate of 0.6% (4/709) and a major error rate of 1.1% (8/709) (Table 1). In Table 3 the discordant antibiotics obtained from comparison by AST Vitek®2 and Kirby Bauer Systems are showed. Regarding Gram-positive, erythromycin (N = 2) was the only antibiotic with a major error rate related to Enterococcus faecalis (N = 2) (Table 3). Concerning Gram-negative [Klebsiella pneumoniae (N = 2), Escherichia coli (N = 1) and Enterobacter spp. (N =1)], Gentamicin (N = 2), Amikacin (N = 1) and Ceftazidime (N = 1) were the discordant antibiotics obtained from comparison by AST Vitek®2 System and Kirby-Bauer System. This discrepancy was classified as a minor error (Table 3). Moreover, amoxicillin/clavulanic acid (N = 3), Cefotaxime (N = 1), Ciprofloxacin (N = 2), Piperacillin/ tazobactam (N = 1) and Cefepime (N = 1) were an attribute of major error rates (Table 3).

Results

Among the yeasts, the comparison of antimicrobial susceptibility testing results between Sensititre YeastOne® System (Thermo Fisher Scientific comparison) and ASTyeast Vitek®2 System (bioMérieux, Inc. France) showed an agreement rate of 100% (4/4) with the absence of very major errors, major errors and minor errors (Table 2).

Within these selected positive blood cultures, 60 Grampositive, 60 Gram-negative and 4 yeasts were correctly

Finally, the Table 4 reported the AST by Vitek®2 and Kirby Bauer Method percentage comparison results according to the type of blood culture bottles included in the study. Out of 124 positive blood cultures no significative discordance was observed within the Oxoid Signal Blood and BD Bactec bottles: in fact there was an agreement of 98.1% and of 93% (p= 0.830) in the Oxoid Signal Blood and BD Bactec, respectively (Table 4).

Chi-square test or Fisher’s exact test was used for statistical comparisons. A P value < 0.05 was considered statistically significant. Out of 124 positive blood samples included in this study, 70 were collected and inoculated in BD BACTECTM Plus Aerobic/F culture vials and incubated in the automated system BD BACTECTM FX, whereas 54 samples were collected and inoculated in Oxoid Signal Blood Culture System Medium (OXOID S.p.A.).

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Table 1: Comparison of antimicrobial susceptibility testing results between the AST-Vitek®2 System and Kirby Bauer System from blood cultures Oxoid Signal Blood and BD BACTEC. Agreement N° of Antibiotics No Agreement Susceptibility Antimicrobial In (VITEK2-Kb) Test(VITEK2-Kb) Tested Agreement 58 2 324 322 (99.4%) 18 90 90 19 95 95 1 5 5 1 5 5 4 20 20 5 25 25 7 2 63 61 3 21 21 56 4 709 697(98.3%) 23 2 300 298 1 12 12 18 1 228 224 1 12 6 2 24 24 1 12 12 6 72 72 1 12 12 2 24 24 1 12 12 1 1 1 114 6 1033 1019

N° of Isolates

Microorganism Gram-positive Staphylococcus aureus Staphylococcus epidermidis Staphylococcus haemolyticus Staphylococcus lentus Staphylococcus hominis Staphylococcus capitis Enterococcus faecalis Enterococcus faecium Gram-negative Klebsiella pneumoniae Klebsiella oxytoca Escherichia coli Enterobacter spp. Acinetobacter baumanii Enterobacter cloacae complex Pseudomanas aeruginosa Pseudomanas oryzihabitans Proteus mirabilis Serratia marcescens Stenotrophomonas maltophilia TOTAL

60 18 19 1 1 4 5 9 3 60 25 1 19 1 2 1 6 1 2 1 1 120

Minor Error

Major Error

0 -

2 (0.6%) 2 8 (1.1%) 1 3 4 10

4 (0.6%) 1 1 2 4

Very Major Error 0 0 0

Table 2: Comparison of antimicrobial susceptibility testing results between Sensititre YeastOne® System (Thermo Fisher Scientific comparison) and AST-yeast Vitek®2 System (bioMérieux, Inc. France) from positive blood culture (OXOID SIGNAL BLOOD/ BACTEC ) after by using LCM. Microorganism

Agreement N° of N° of Antifungals Minor (Sensititre YeastOne – No Agreement Isolate Tested Error Ast-Yeast)

Major Error

Very Major Error

Yeasts

Candida albicans

Candida tropicalis

Table 3: Discordant antibiotics obtained from comparison by AST Vitek®2 and Kirby Bauer Systems. Antibiotics showing discrepancy MICROORGANISM

Very major error (0)

Major error (10)

Minor error (4)

Enterococcus faecalis (2)

Erythromycin (2)

Klebsiella pneumoniae (2)

Amoxicillin/clavulanic acid (1)

Gentamicin (1)

Cefotaxime (1) Amoxicillin/clavulanic acid (1) Ciprofloxacin (1)

Gentamicin (1)

Enterobacter spp. (1)

Piperacillin/tazobactam (1) Cefepime (1) Ciprofloxacin (1) Amoxicillin/clavulanic acid (1)

Amikacin (1) Ceftazidime (1)

TOTAL

Escherichia coli (1)

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Table 4: Percentage comparison results according to the type of blood culture bottles (Oxoid Signal Blood and BD Bactec) between the direct antimicrobial suscetibility testing by Vitek ÂŽ 2 and Kirby Bauer Method. Comparison results AST Vitek2 / Kirby-Bauer Oxoid Signal Blood

BD Bactec

P-value

AGREEMENT

53/54 (98.1%)

65/70 (93%)

0.830

NO AGREEMENT

1/54 (2%)

5/70 (7%)

0.193

Fig. 1: Flow chart for identification and AST of microorganisms obtained directly from Positive Blood Culture bottles.

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Discussion

The rapid identification and the application of AST to microorganisms causing bloodstream infections is pivotal to guide antimicrobial therapy, helping to reduce the detection time of the antimicrobial therapy and improving the patient response. In this study we evaluated the Lysis-Centrifugation Method for identification as well as antimicrobial susceptibility testing of microorganisms directly from positive blood cultures (Oxoid Signal Blood and BD Bactec bottles) by disc diffusion plate (Kirby Bauer Method) and by microdilution method (AST Vitek®2 System bioMérieux, Inc. France) analyzing the accuracy of these combined methods. During the study period, within the 124 selected positive blood cultures (60 Gram-positive, 60 Gram-negative and 4 yeasts), 123 microorganisms were correctly identified with MALDI-TOF system by LCM to the species level. Only 1 isolate was identified to the genus level. In this case, MALDI-TOF MS has not been able to precisely identify the species responsible for bacteremia (Enterobacter spp. instead of Enterobacter kobei). Our result is in agreement with what reported by other previous studies, in which MALDI-TOF MST accurately detects Enterobacter cloacae complex although it may not discriminate some species within this complex [25-27]. Excluding the only identification to the genus level, the concordance rate of identification with MALDI-TOF system by LCM was very reliable (99,2%, 123/124). Moreover, the application of 70% formic acid to Grampositive spots of a target plate improved the identiﬁcation of gram-positive bacteria with a perfect overlapping with classical ID using VITEK®2 system (bioMerieux, Inc. France). The evaluation of LCM for AST of microorganisms directly from the 124 positive blood cultures by disc diffusion plate (Kirby Bauer Method) and by microdilution method (AST Vitek®2 System bioMérieux, Inc. France) showed different results within the isolates. Comparing the two antimicrobial susceptibility testing utilized methods, it was observed that Enterococcus faecalis, within Gram-positive groups, was the only microorganism with a major error rate related to erythromycin.

A-175 Enterobacter spp, the discordant agents, as minor error rates, obtained from comparison by AST Vitek®2 System and Kirby-Bauer System were amikacin and ceftazidime. On the other hand, amoxicillin/clavulanic acid, cefotaxime, ciprofloxacin, piperacillin/tazobactam and cefepime were an attribute of major error rates in Klebsiella pneumoniae, Escherichia coli and Enterobacter spp. The percentage of minor and major errors was low among the 1033 microorganism-antimicrobial combinations tested. Although in literature, the coagulase negative staphylococci (CNS) that are common blood culture contaminants, exhibited the most errors among the Gram positive isolates [12, 28], our CNS (N= 30) isolates showed a perfect agreement susceptibility between the Kirby Bauer Method and AST Vitek®2 System. Among the Gram-negative isolates, previous studies reported the most errors for Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis isolates [29]. Our data are in agreement with these reports only for Escherichia coli. For Pseudomonas aeruginosa and Proteus mirabilis strains, the comparison of the two antimicrobial susceptibility testing utilized methods revealed no discrepancies. Among our Gram-negative isolates we observed the most errors in Klebsiella pneumoniae (N=25) and Enterobacter spp. (N=2) as just indicated by Wimmer et al. [29]. In the present study, we can conclude that the accuracy of AST was outstanding with both Gram-positive and Gram-negative isolates, since the antibiotics in agreement were 1019/1033 with only 4 minor errors and 10 major errors. This result was comparable or higher to that of other studies [12, 28]. During the study period, the number of positive blood cultures (Oxoid Signal Blood and BD Bactec bottles) for yeast isolates was very low, although the comparison of antimicrobial susceptibility testing results between Sensititre YeastOne® System and AST-yeast Vitek®2 System from positive blood culture after by using LCM was perfectly overlapping. Therefore, the limited number of yeasts strains was not utilized for statistical analysis, but it was included in the total results.

Among the Gram-negative, Klebsiella pneumoniae, Escherichia coli and Enterobacter spp. were the major cause of minor error rates and major error rates.

The final goal for using MALDI-TOF MS is to get early ID and AST for better use of antibiotics for treatment of infection and for patient management and hospital costs [30]. In fact, the average time to identification and antimicrobial susceptibility testing by using our LCM protocol for both Vitek®2 Method and Kirby Bauer method was 18-24 hours, directly from positive blood culture.

Klebsiella pneumoniae and Escherichia coli showed a minor error rates against Gentamicin. Regarding

The results obtained in this study support that our strategy reduced the times for species identification and

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susceptibility testing. In fact, particularly in Gram-negative sepsis, it is crucial that an appropriate antibiotic therapy must be administered as soon as possible and this could contribute to have a maximum benefit to patient care.

Further, this new technology may provide better definition of the epidemiology, pathogenesis, and antimicrobial susceptibility of unrecognized or misidentified microorganisms.

Hall M, Levant S, DeFrances C. Trends in inpatient hospital deaths: National Hospital Discharge Survey, 2000– 2010. NCHS Data Brief 118. National Center for Health Statistics, Centers for Disease Control and Prevention, 2013; Atlanta, GA.

Liu V, Escobar GJ, Greene JD, Soule J, Whippy A, Angus DC, Iwashyna TJ. Hospital deaths in patients with sepsis from 2 independent cohorts. JAMA 2014; 312: 90–2.

An important limitation is that polymicrobial infections were not included during the study period, since as just reported in literature, there is a difficulty in identifying polymicrobic infections.

Conclusion

Larger future studies should include more isolates, particularly more yeast to confirm our results. as well as polymicrobial infections, to check whether, within polymicrobial infections, a predominant microorganism could be evidenced by LCM without misidentifications. In summary, our simple and cost-effective sample preparation method is very useful for rapid identification as well as AST of microorganisms directly from positive blood culture bottles in a clinical setting.

References 1.

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10. Beekmann SE, Diekema DJ, Doern GV. Determining the clinical significance of coagulase- negative staphylococci isolated from blood cultures. Infect Control Hosp Epidemiol 2005; 26(6): 559–66. 11. Micklos L. Do Needle-Free Connectors Prevent CatheterRelated Bloodstream Infections in Patients Receiving Hemodialysis Treatments Using Central Venous Catheters. Nephrol Nurs J 2015; 42(4): 383–6. 12. Machen A, Drake T, Wang YF. Same day identification and full panel antimicrobial susceptibility testing of bacteria from positive blood culture bottles made possible by a combined lysis-filtration method with MALDITOF VITEK mass spectrometry and the VITEK2 system. PLoS One 2014; 9(2): e87870. 13. Suarez S, Nassif X, Ferroni A. Applications of MALDITOF technology in clinical microbiology. Pathol Biol 2015; 63: 43–52. 14. Bizzini A, Greub G. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolution in clinical microbial identification. Clin Microbiol Infect 2010; 16: 1614–9. 15. Bizzini A, Durussel C, Bille J, Greub G, Prod’hom G. Performance of Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for identification of bacterial strains routinely isolated in a clinical microbiology laboratory. J Clin Microbiol 2010; 48: 1549–54. 16. Bizzini A, Jaton K, Romo D, Bille J, Prod’hom G, Greub G. Matrix-assisted laser desorption ionization time-offlight mass spectrometry as an alternative to 16S rRNA gene sequencing for identification of difficult-to-identify bacterial stains. J Clin Microbiol 2011; 49: 693–6. 17. Jo SJ, Park KG, Han K, Park DJ, Park YJ. Direct Identification and Antimicrobial Susceptibility Testing of Bacteria From Positive Blood Culture Bottles by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and the Vitek 2 System. Ann Lab Med 2016; 36: 117–23. 18. Stevenson LG, Drake SK, Murray PR. Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 2009; 48: 444–7. 19. Kok J, Thomas LC, Olma T, Chen SC, Iredell JR. Identification of bacteria in blood culture broths using matrix-assisted laser desorption-ionization Sepsityper™ and time of flight mass spectrometry. PLoS One 2011; 6: e23285.

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Rodio et al. 20. Schneiderhan W, Grundt A, Wörner S, Findeisen P, Neumaier M. Work flow analysis of around-the-clock processing of blood culture samples and integrated MALDI-TOF mass spectrometry analysis for the diagnosis of bloodstream infections. Clin Chem 2013; 59: 1649–56. 21. Lavergne RA, Chauvin P, Valentin A, Fillaux J, RoquesMalecaze C, Arnaud S, et al. An extraction method of positive blood cultures for direct identification of Candida species by Vitek MS matrix-assisted laser desorption ionization time of flight mass spectrometry. Med Mycol 2013; 51: 652–6.

A-177 25. Harris P, Winney I, Ashhurst-Smith C, O’Brien M, Graves S. Comparison of Vitek MS (MALDI-TOF) to standard routine identification methods: an advance but no panacea. Pathology 2012; 4(6): 583–5. 26. Pavlovic M, Konrad R, Iwobi AN, Sing A, Busch U, Huber I. A dual approach employing MALDI-TOF MS and real-time PCR for fast species identification within the Enterobacter cloacae complex. FEMS Microbiol Lett 2012; 328(1): 46–53. 27. Patel R. Matrix-assisted laser desorption ionization-time of flight mass spectrometry in clinical microbiology. Clin Infect Dis. 2013; 57(4): 564–72.

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*Corresponding author: Valeria Pietropaolo, Assistant Professor, Department of Public Health and Infectious Diseases, Sapienza University, P.le Aldo Moro, 5, 00185 Rome, Italy. Email: valeria.pietropaolo@uniroma1.it

Financial: This work was supported by MIUR grant. Competing Interests: The Authors declare that they have no competing interests.

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Date of Submission : 01.12.2016 Date of Acceptance : 25.01.2017 Date of Publication : 13.04.2017

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Original Article DOI: 10.21276/APALM.1223

Analysis of Morphological Changes in Liver in Obstructive Jaundice with Special Emphasis on Fibrosis Rachana A Chaturvedi1*, Jayashri P. Chaudhari1, Mayura Kekan1, Apurv Deshpande2, Ramkrishna Prabhu2 and Amita Joshi1 1 Department of Pathology, Seth G. S. Medical College & KEM Hospital, Parel, Mumbai, India Department of Gastrointestinal Surgery, Seth G. S. Medical College & KEM Hospital, Mumbai, India

ABSTRACT Background: Biliary obstruction can present with distressing symptoms and increased morbidity which leads to liver fibrosis, cholestasis, portal inflammation and ductular proliferation. Experimental studies showed reversal of histological findings in liver after biliary decompression surgery; however only a limited data is available regarding the same. Methods: Prospective observational study of 28 liver biopsies from 14 patients of obstructive jaundice, who underwent decompression surgery and showed clinical deterioration at 6 weeks with normal HIDA scan. Patients were clinically evaluated. Both intra (1st bx) and postoperative (2nd bx) liver biopsies were studied for fibrosis, cholestasis, ductular proliferation and portal inflammation. Result: Patientâ&#x20AC;&#x2122;s age ranged from 24 to 75 years (8 Males and females 6), commonest symptom being jaundice. In 1st bx, most of the patients showed histological evidence of obstruction, which improved at least partially after surgery. There was no definite correlation of fibrosis with etiology. Fibrosis was less commonly seen with shorter duration of symptoms and younger males had higher prevalence. Increase/ static grades of fibrosis were seen in 35.71% patients each, while 28.57% showed regression. No correlation of age and etiology with status of fibrosis was observed. Regression was more common in males and with absence of cholangitis while progression was more common in females and with presence of cholangitis. Conclusion: We wonder whether younger males are more prone for fibrosis but males in general have better prognosis regarding the reversal. Also, cholangitis could be an important factor for deciding the further course of fibrosis. However we require larger data with multivariate analysis for the confirmation of the same. Keywords: Obstructive Jaundice, Reversal/ Regression, Liver Fibrosis

Introduction

Patients with biliary obstruction form an important subgroup presenting to gastroenterology. Its management usually depends on various factors like the cause of obstruction (benign/ malignant) and duration of symptoms, and requires a coordinated multidisciplinary approach involving gastroenterologists, radiologists, and pathologists.[1] The benign causes of biliary obstruction include gall stone disease, chronic pancreatitis, parasitic infestation of biliary tree and strictures following trauma including iatrogenic. Strictures may be asymptomatic, but if ignored, can cause life-threatening complications, such as ascending cholangitis, liver abscess, and secondary biliary cirrhosis.[2,3] Among malignant conditions, pancreatic cancer is the commonest cause[4,5] however most of these patients die due to complications of tumor invasion and metastasis rather than due to strictures per se. Nonetheless, both benign and malignant bile duct strictures are associated with distressing symptoms and increased morbidity. [6]

The liver in biliary obstruction is basically an innocent victim, suffering from the consequences of events like increased biliary pressure and bile stasis proceeding somewhere distally in the bile ducts. Over a period of time they initiate complex hepatic histopathological changes, which can result in progressive hepatic fibrosis.[7] These changes are modified by the duration, degree, cause of the blockage and complicating factors such as biliary infection which affect the immediate and long-term outcome of definitive surgical repair, therefore have prognostic and medico legal implications.[8] Patientâ&#x20AC;&#x2122;s recovery depends on the improvement in liver function, normal bile flow and reversal of pathological changes. A histological return of normal liver parenchyma is seen after relief of obstruction in both animal and human models, which correlates with the return to nearnormal liver function. However, only limited data is available regarding histological findings in liver following decompression surgery. [9,10,11,12] Hence we undertook this

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Chaturvedi et al. study to analyze morphological changes in liver secondary to biliary obstruction and following surgical correction with special emphasis on regression of fibrosis along with its clinical correlation.

Materials and Methods

Study Design: This is a prospective observational study carried out over the period of 1 year and 10 months (September 2014 to June 2016), in the Department of Pathology at a tertiary care centre. Institutional ethics committee permission was taken. Inclusion Criteria: Patients who underwent surgical intervention in gastrointestinal surgery department for obstructive jaundice and showed no clinical/ biochemical improvement or had deterioration after 6 weeks of decompression surgery. Exclusion Criteria: Patients with liver parenchymal disease, other than obstructive biliary pathology or with previous history of biliary decompression surgery. Study Procedure: Patients were evaluated for history with respect to age, gender, symptoms with duration and cause of obstruction along with detailed clinical examination. Preoperative Liver function tests (LFTs) including Serum bilirubin, Serum alanine transaminase (ALT), Serum aspartate transaminase (AST) and Serum alkaline phosphatase (ALP) were assessed along with radiological investigations. Patients underwent the required surgery, depending upon the cause and location of obstruction and an Intra-operative liver biopsy (1stBx) was taken to evaluate the morphological changes in the liver. Post-operative evaluation of LFTs was also done at various intervals up to 6weeks as by this time maximum recovery after surgery is expected. Then a HIDA (HepatobiliaryIminoDiacetic Acid) scan was done to check the functionality of hepato-enteric anastomosis. The patients showing no evidence of biliary obstruction but having clinical/ biochemical deterioration or no improvement underwent a second percutaneous liver biopsy (2ndBx). Three to four Âľm sections from the biopsy samples, fixed in 10% neutral buffered formalin solution, embedded in paraffin were obtained and stained with hematoxylineosin, Masson trichrome and reticulin. The histological features like fibrosis, cholestasis, ductular proliferation, portal inflammation or any other incidental pathological changes were assessed by two pathologists. (Table 1) Low grade was defined as 0-1 and higher grades as 2 and above. Findings were correlated clinically with respect to age, gender, cause and duration of symptoms. Longer duration was defined as > 6 months. www.pacificejournals.com/apalm

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Result

Of the 24 patients who underwent decompression surgery, 15 fulfilled the inclusion criteria, of which 8 were males (M) and 7 females (F). One female expired in 2 weeks following surgery, hence was excluded from the study. Of the total 14 patients, 2 each (14.28 %) were in the age group of 3rd and 6th decade, 7(50%) in 4th decade and 3(21.42%) in 7thdecade or older age group. The youngest patient was 24 years, eldest being 75 years old and 9 (64.28%) were below 40 yrs. Their presenting symptoms in decreasing order of frequency were Jaundice (85.71%), pain (64.28%), fever (57.14%) and itching (35.71%). One patient each had weight loss (7.14%) and portal hypertension (7.14%) and many had multiple symptoms. Duration between onset of symptoms and 1st biopsy ranged from 0.5 -36 months (mths) of which most patients (9/14, 64.28%) had duration of symptoms between 0-3 months, 4 (28.56%) between 3-6 months and only 1 (7.14%) had 36 months. At least one of the LFTs ( Both preoperative and 6 weeks postoperative) were deranged in all except one patient, who showed only postoperative derangement.Their range with mean values are given in Table 2. Radiological investigations like CT, MRI or MRCP were done in all. Various causes (benign/ malignant) of obstruction found are mentioned in Table 3. An analysis of the etiology was also done with respect to age and gender. Of the total 8 patients with benign cause, 87.50% (3M, 4F) were <40yrs and 12.50% (1F) was>40yrs old. Among 6 patients with malignant cause, 33.33% (2M) were< 40yrs and 66.67% (3M, 1F) were >40yrs old. Various surgical procedures done for relieving obstruction were hepaticojejunostomy in 12 and choledochoduodenostomy, cholecystojejunostomy and shunt surgery in 1 patient each. Total 28 liver biopsies, including one intra-operative and post-operative each from 14 patients were studied. The histopathological analysis of the biopsies was done to look for the grade of fibrosis, ductular proliferation, inflammation and cholestasis (Fig 1, 2) which is given in table 4. Fibrosis in 1stbx was low grade in 11 (78.5 %), none showed grade 2, and grade 3 was seen in 3 patients (21.42%), in 2ndbx, all patients (100%) had low grade fibrosis. Cholestasis in 1stbx was low grade in 7(50 %), rest showed grade 2, while in 2nd bx; cholestasis was low grade in all. Inflammation in 1stbx was low grade in 7(50%) while rest showed grade 2 or 3, in 2nd bx, 13 (92.85%) had low grade, one (7.14%) had grade 2 while none showed grade 3 inflammation and cholangitis was observed in 8 patients. eISSN: 2349-6983; pISSN: 2394-6466

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Ductular proliferation in1stbiopsy was low grade in 12 (85.71%) and rest had grade 2, in 2nd biopsy, all patients had low grade ductular proliferation. The grades of fibrosis were also analyzed with respect to the age and gender of the patients, cause of obstruction and duration of symptoms (Table 5-7). In 1st biopsy, in <40yrs age group, most males (80%, 4/5) showed presence of fibrosis (grade 1), while most females (75%, 3/4) showed no fibrosis. Grade 3 fibrosis was observed only in one patient (Male). In >40yrs age group also, most males (66.66 %, 2/3) while only 1 female (50%) showed fibrosis. In 2nd biopsy, most patients (92.85%, 13/14) showed evidence of fibrosis (grade 1) except one male of > 40 yrs age group. Of the total 8 patients with benign causes, 5 (62.50%) had fibrosis in 1st while all had fibrosis in 2nd bx. Of the total 6 with malignant cause, 4 (64.28%) had fibrosis in 1st while 5 (83.33%) had fibrosis in 2nd bx. Of the total 9 patients with < 3 mnth duration of symptoms, 5 (55.55%) had fibrosis, of the 4 with 3 to 6 mnth duration 3 (75%) had fibrosis while a single patient with > 6 mnth had fibrosis which was grade 3. Table 1: criteria used for grading. Portal/ Grade Fibrosis Periportal inflammation 0

Absent

Portal and periportal

Mild (in < one third of portal tracts)

Presence of numerous septa

Moderate (in one third to two thirds of portal tracts)

Cirrhosis

Severe (Dense packing of cells > two thirds of portal tracts)

Table 2: Biochemical Investigations: Investigation Preoperative range (mean value) AST 28-212 IU (69.14 IU) ALT 08-478 IU (75.92 IU) ALP 152-1400 IU (665.50 IU) Bilirubin 1-17 mg/dl (4.84 mg/dl) Table 3: Distribution according to the cause of obstruction: Causes Benign Benign Biliary Stricture Portal Biliopathy Chronic Pancreatitis Choledocholithiasis Malignant Carcinoma head of pancreas Ampullary carcinoma Distal CBD carcinoma

Correlation of change in status of fibrosis (Regression / static/ progression) following decompression surgery was also done with respect to age, gender, cause of obstruction and cholangitis (Table 8). Progression and static fibrosis was seen in 5 patients each (35.7%), of which 3 each showed association with benign and 2 each with malignant cause. Of those with progression of fibrosis (5), 4 were females, all showing association with cholangitis while single male had no cholangitis. Of those with static fibrosis (5), cholangitis was seen in 2 of 4 males and single female patients. Regression was seen in 4 patients ((28.6%) of which 2 each showed association with benign and malignant cause. There were 3 males of which 1 showed cholangitis and one female with no cholangitis. Three patients showed reversals from grade 3 to grade 1 and one showed from grade 1 to grade 0. Other incidental findings noted were, mild steatosis (Fig 3a) in 3 and single foreign body granuloma in portal tract (fig 3b) in 1 patient. Ductular proliferation Absent or mild

Cholestasis Absence Bile accumulation in centrolobular hepatocytes; bile accumulation in centrolobular and periportal hepatocytes or in portal tracts; the presence of bile infarcts (bile accumulation with hepatocyte necrosis surrounded by foamy histiocytes

Moderate Severe

Postoperative range (mean value) 28-55 IU (38.07 IU) 19-42 IU (29.07 IU) 90-1100 IU (376.00 IU) 0.8-2.3 mg/dl (1.30 mg/dl) Number of patients (n=14) 08 (57.14 %) 05 (35.71 %) 01 (7.14%) 01 (7.14%) 01 (7.14%) 06 (42.85) 2 (14.28%) 2 (14.28%) 1 (7.14%)

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Table 4: Histopathological analysis of liver biopsies Fibrosis

Grade

Cholestasis

inflammation

1stbx (n=14) 2ndbx (n=14) 1stbx (n=14) 2ndbx (n=14)

1stbx (n=14)

Ductular proliferation

2ndbx (n=14) 1stbx (n=14)

2ndbx (n=14)

5(35.71%)

1(7.10%)

6(42.85%)

11(78.51%)

1(7.14%)

4(28.56%)

5(35.71%)

6(42.85%)

13(92.85%)

1(7.14%)

3(21.42%)

6(42.85%)

9(64.28%)

8(57.14%)

9(64.28%)

7(50.00%)

5(35.71%)

1(7.14%)

2(14.28%)

3(21.42%)

2(14.28%)

Table 5: Grades of fibrosis with respect to age and gender of patients Age < 40yrs (n=9)

Grades

1stbx

Age >40yrs (n=5) 2ndbx

1stbx

2ndbx

Males (n=5) Females (n=4) Males (n=5) Female (n=4) Males (n=3) Females (n=2) Male (n=3) Female (n=2) 0

03 (21.42%)

04 (28.57%)

01 (7.14%)

01(7.14%)

01 (7.14%)

01(7.14%)

01 (7.14%)

02(14.28%)

02 (14.28%)

01 (7.14%)

05(35.71%) 04 (28.57%)

Table 6: Grades of fibrosis in relation to cause of obstruction (1st biopsy): Benign (n=8)

Grade

Malignant (n=6)

1stbx

2nd bx

1stbx

2ndbx

03 (37.50%)

02 (33.33%)

01 (16.67%)

03 (37.50%)

08 (100%)

03(50.00%)

05 (83.33%)

02(25.00%)

01(16.67%)

Table 7: Grades of fibrosis (1stbiopsy) in relation to duration of symptoms: Grade

<3mths (n=9)

3-6mths (n=4)

>6mths (n=1)

04 (28.57%)

01 (7.14%)

04 (28.57%)

02 (14.28)

01 (7.14%)

0 01 (7.14%)

01 (7.14%)

Table 8: Status of fibrosis after decompression surgery With respect to age and gender (n=14)

With respect to cause(n=14)

Grades

Overall status (n=14)

Regression

04 (28.57%)

2 (25.00%) 2 (33.33%)

Static

05 (35.71%)

Progression

05 (35.71%)

Benign (n=8)

Malignant (n=6)

<40 yrs

cholangitis

>40 yrs

Male (n=5)

Female (n=4)

Male (n=3)

Female (n=2)

01 (7.14%)

02 (14.28%)

01 (7.14%)

3 (37.50%) 2 (33.33%) 04 (28.57%)

01 (7.14%)

3 (37.50%) 2 (33.33%)

03 (21.42%)

01 (7.14%)

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Fig. 1: Grade 1 fibrosis, H &E and Massonâ&#x20AC;&#x2122;s Trichrome (a,b X 400) , grade 3 fibrosis, H& E and Massonâ&#x20AC;&#x2122;s trichrome (c,d X 100)

Fig. 2: (a) cholestasis (H & E X 400); (b)bile ductular proliferation (H & E X 100); (c) portal inflammation (H & E X 400) and (d )cholangitis (H&E X 400).

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Fig. 3: (a) Incidental steatosis (H & E X 400); (b) portal foreign body granuloma (H & E X 400).

Discussion

Obstructive Jaundice is a common surgical problem that occurs due to an obstruction to the passage of conjugated bilirubin from liver into intestine and can be due to intra or extrahepatic obstruction. Early diagnosis to determine the precise etiology is of great importance because pathological changes in liver can occur if obstruction is unrelieved. Symptoms of biliary obstruction include jaundice, clay colored stools, darker urine, abdominal pain and intense itching. Other symptoms vary depending on the underlying cause of the obstruction. Patient can present early or late, depending on the severity of the disease. [12] In our study, 14 patients of obstructive jaundice due to various etiologies with deranged post-operative LFTs were analyzed. The morphological changes in liver, occurring secondary to biliary obstruction, as well as following surgical correction were noted, along with its clinical correlation. There was slight male preponderance (M: F 1.33:1) among the patients with wide age variation with majority being below 40 years. Most common presenting symptoms were jaundice followed by pain and fever, itching was seen in few while none had history of clay colored stool. One of our patients had associated portal hypertension. In the studies of patients of obstructive jaundice by Hammel et al, [13] all were male between 34 www.pacificejournals.com/apalm

to 54 yrs and in a study by Sikora et al [9], there was a significant female preponderance (M: F ratio 1:9) with age range of 26 to 52yrs.Chalya et al [14] also studied patients of obstructive jaundice due to various etiologies and there was a slight female preponderance (M: F, 1:1.32)Â with age range from 12 to 78 years. However they analyzed only preoperative biopsies. The most common symptoms in their study were jaundice (48.4%) followed by clay colored stools (36.2%), other symptoms being itching, weight loss and abdominal pain similar to our study and abdominal mass in occasional case which was not seen in our patients. Their duration of symptoms ranged from 5 to 32 days as oppose to our study where few patients had symptoms even for several months (36mths). Elevated serum bilirubin level with a preponderance of the conjugated fraction is usually seen in obstructive jaundice. The transaminases might be raised many fold above normal and decrease rapidly once the obstruction is relieved. ALP and Îł-glutamyl transferase (GGT) are markers for cholestasis. [15] In our study too, various liver enzymes and bilirubin showed preoperative elevation in most patients and their levels significantly reduced after surgery. ALP levels reduced in most but remained significantly elevated in few patients even after surgery. However, GGT levels were not done in our study. eISSN: 2349-6983; pISSN: 2394-6466

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The causes of obstructive jaundice can be benign or malignant, stone disease being the commonest. Delay in presentation and treatment can lead to significant morbidity and mortality due to secondary infections causing cholangitis, liver abscess and systemic effect like malnutrition, weight loss, decreased immunity and renal insufficiency. [16] In our study too, benign causes were commoner, but post cholecystectomy biliary stricture was commonest as opposed to stone disease, seen in only one. In a study of 116 patients by Chalya et al, [14] higher number of patients (58.6%) had association with malignant etiology but choledocholithiasis was the commonest (62.5%) cause among benign (62.5%) and carcinoma of the head of pancreas (64.7%) among the malignant causes similar to our study. However, in the studies by Sikora et al, [9] Hammel et al [13] and Negi et al, [17] patients with only benign etiology were included. We also analyzed the cause of obstruction with respect to age and gender, benign causes were more prevalent in females and in younger (<40yrs) age group while malignant causes were more prevalent in males and in older age group. This analysis was not done in other studies. Surgical intervention is done with the intent to relieve obstruction and restore the normal bile flow. Various surgical procedures are done for the relief of obstruction [14] which can result into full recovery, normalization of liver function tests, to persistent abnormality and progressive hepatic failure. In our study, most common surgical procedure done was hepatico-jejunostomy which resulted into complete correction of physical obstruction as evident on HIDA scan; however patients continued to have biochemical derangement. Ongoing biliary obstruction is associated with progressive fibrosis leading to cirrhosis of liver. [6] Proliferation of ductular epithelial cells and inflammation secondary to obstruction and cholestasis are responsible for activation of hepatic stellate cells which leads to deposition of collagen and matrix proteins.[18] Various factors responsible are, cause of obstruction (benign/ malignant), associated infection and general condition of the patient. High grade fibrosis and cirrhosis at the time of surgery is considered poor prognostic indicator. [19] In our study, in 1st biopsy, on microscopy, most of the patients showed evidence of fibrosis, cholestasis, inflammation and ductular proliferation. In addition few patients also had mild steatosis and occasional portal granuloma however, none of them showed any significant change in their clinical course on follow up thus supporting there incidental nature. Most common grade of fibrosis was 1 followed by 3, similar to Sikora et al who studied 71 patients. [9] However,

in a study of 11 patients by Hammel et al, [13] most had grade 2 fibrosis followed by grade 1. The difference in prevalence of grades in above studies could be possibly due to variation in sample size, cause/ degree/ location of obstruction, associated infection or other complications. We further analyzed fibrosis according to the age and gender of the patients which has not been reported by other researchers. In our study, in1st bx, (in <40yrs age group), all males showed fibrosis (5/5) raising a possibility whether younger males are more prone for fibrosis following an obstruction. Nevertheless, larger studies are required for any definite conclusion. Grade of fibrosis also depends on cause of obstruction. Scobie et al [20] observed that cirrhosis was more commonly associated with benign etiology. In our study, in 1stbx, patients with both benign and malignant etiology showed fibrosis which was slightly more common in association with malignancy (66.66% vs. 62.50%). However in 2nd bx, all (100%) patients with benign causes showed presence of fibrosis including grade 3, which was also seen in more patients (25.00%) as compared to malignant cause (16.67%). The duration of obstruction is as an important factor deciding the grade of fibrosis in liver. In a study by Negi et al, [17] of 64 patients of post cholecystectomy strictures, duration of symptoms ranged from 1-120 months and a longer duration of biliary obstruction was found to be the most important predictor of advanced hepatic fibrosis. In our study, the range of duration of symptoms was shorter (0.5-36 moths) and fibrosis was less commonly seen with shorter duration of symptoms similar to above study. Though there was only one patient in our study with longer duration but he showed advanced fibrosis. Regression in fibrosis, ductular proliferation, inflammation and cholestasis is seen in patients of both benign and malignant etiology after decompression surgery. [1, 13, 20, 21] In our study too, overall improvement of all histological parameters from was observed (Table 4), which is also observed by Sikora et al [9] and Hammel et al. [13] This supports the fact that decompression surgery is beneficial in obstructive jaundice. In a study of by Hammel et al, [13] post-surgery biopsy (2ndbx), was done in all (11) patients and majority showed regression (54.55%) followed by static (36.36%) and increased fibrosis (9%). In the study by Sikora et al, [9] 2ndbx was done in only 5 and most patients showed either regression or static fibrosis (40% each), and only 20% showed progression. However, in our study, 2ndbx was available in more patients (14), however regression was

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Chaturvedi et al. seen in only 4, (28.57%), increased fibrosis was seen in more patients (35.71%) while prevalence of static fibrosis was quite similar to other studies. The difference could be due to many factors as discussed earlier, including sample size. We also did analysis of status of fibrosis in relation to age, gender, cause, duration and cholangitis which was not done in other studies. No correlation of age and etiology with status of fibrosis was observed, however males appeared to show more association with regression or stasis of fibrosis while progression was more common with females. But larger studies are necessary for the confirmation. Moreover, most patients with regression (3/4) did not have cholangitis while most with progression (4/5) had associated cholangitis. Thus cholangitis appears to an important factor for deciding the course of fibrosis. Also, most patients who showed regression from grade 3 to 1 (3/4, 75%) had duration of symptom < 6 months, suggesting shorter duration is associated with higher prevalence and degree of regression.

Conclusion

It is difficult to justify multiple liver biopsies in patients, especially if there is clinical improvement. Hence a large data is not available in literature regarding histological changes in liver after surgery. However our data is one of the largest regarding availability of post operative liver biopsy in obstructive jaundice. Our study supports the previous observation of possibility of reversal of histological changes after effective timely decompression surgery, including fibrosis which has lower prevalence when associated with shorter duration of obstructive symptoms. We observed no role of etiology for the occurrence as well as regression of fibrosis. However we wonder whether males have better prognosis regarding reversal of fibrosis and patients with cholangitis in general, as well as younger males are more prone for fibrosis. But we require larger data with multivariate analysis for the confirmation of the same.

Reference 1.

Madhusudhan KS, Gamanagatti S, Srivastava DN, Gupta AK. Radiological interventions in malignant biliary obstruction. World Journal of Radiology2016; 8(5):518-529.

Hanau LH, Steigbigel NH. Acute (ascending) cholangitis. Infect Dis Clin North Am. 2000; 14(3):521-46.

Hastier P, Buckley JM, Peten EP, Dumas R, Delmont J. Long term treatment of biliary stricture due to chronic pancreatitis with a metallic stent. Am J Gastroenterol. 1999; 94(7):1947-8.

Deviere J, Cremer M, Baize M, Love J, Sugai B, Vandermeeren A. Management of common bile duct

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Kamisawa T, Tu Y, Egawa N, Nakajima H, Tsuruta K, Okamoto A. Involvement of pancreatic and bile ducts in autoimmune pancreatitis. World J Gastroenterol. Jan 28 2006; 12(4):612-4.

Magistrelli P, Masetti R, Coppola R, Coco C, Antinori A, Nuzzo G et al. Changing attitudes in the palliation of proximal malignant biliary obstruction. J SurgOncol. Suppl1993; 3:151-3.

Portmann BC, NakanumaY. Diseases of the bile ducts. In: MacSweenRNM, Burt AD, Portmann BC, Ishak KG, Scheuer PJ, Anthony P. Peds. Pathology of the Liver, 4th ed. London, England, Churchill Livingstone, 2002;435-506.

PellegriniCA, Thomas MJ, Way LW. Recurrent biliary stricture: patterns of recurrence and outcome of surgical therapy. Am J Surg. 1984;147175-180.

Sikora SS, Shrikanth G, Agrawal V, Gupta RK, Kumar A, Saxena R et al. Liver histology in benign biliary stricture: fibrosis to cirrhosis and reversal? J GastroenterolHepatol. 2008;23: 1879-1884.

10. Kirkland JG, Godfrey CB, Garrett R, Kakar S, Yeh BM, Corvera CU. Reversible surgical model of biliary inflammation and obstructive jaundice in mice. J Surg Res. 2009; 164(2):221-7. 11. Franco D, Gigou M, Szekely AM, Bismuth H. Portal hypertension after bile duct obstruction: effect of bile diversion on portal pressure in the rat. Arch Surg. 1979;114:1064-7. 12. Kawasaki S, Imamura H, Kobayashi A, Noike T, Miwa S, Miyagawa S. Results of Surgical Resection for Patients With HilarBile Duct Cancer:Application of Extended Hepatectomy After Biliary Drainage and Hemihepatic Portal Vein Embolization. Annals of Surgery2003;238(1):84-92. 13. Hammel P, Couvelard A, O’Tootle D, Ratouis A, Sauvanet A, Flejou JF et al. Regression of liver fibrosis after biliary drainage in patients with chronic pancreatitis and stenosis of common bile duct. New Engl. J. Med. 2001;344: 418–23. 14. Chalya Philipo L, Kanumba ES, Mchembe M. Etiological spectrum and treatment outcome of Obstructive jaundice at a University teaching Hospital in northwestern Tanzania: A diagnostic and therapeutic challenges BMC Research Notes 2011;4:147. 15. Verma S, Sahai S, Gupta P, Munshi A, Verma S, Goyal P. Obstructive Jaundice- Aetiological Spectrum, Clinical, Biochemical And Radiological Evaluation At A Tertiary Care Teaching Hospital.The Internet Journal of Tropical Medicine 2010;7(2).

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16. Pitiakoudis M, Mimidis K, Tsaroucha AK, Papadopoulos V, Karaviannakis A, Simopoulos C. Predictive value of risk factors in patients with obstructive jaundice. J Int Med Res.2004;32:633-8. 17. Negi SS, Sakhuja P, Malhotra V, Chaudhary A. Factors Predicting Advanced Hepatic Fibrosis in Patients With Postcholecystectomy Bile Duct Strictures. Arch Surg. 2004; 139(3):299-303. 18. Aronson DC, De Haan J, James J et al.Quantitative aspects of the parenchyma-stroma relationship in experimentally induced cholestasis. Liver 1988; 8: 116-126.

19. Rothlin MA, Loppe M, Schlumpf R, Largiader F. Long_term results of hepaticojejunostomy for benign lesion of bile ducts. Am J Surg. 1998; 175:22-26 20. Scobie BA, Summerskill WHJ. Hepatic cirrhosis secondary to obstruction of the biliary system. American Journal of Digestive Diseases. 1965; 10:135-146. 21. Zimmerman H, Reichen J, Zimmerman A, Sägesser H, Thenisch B, Höflin F. Reversibility of secondary biliary fibrosis by biliodigestive anastomosis in the rat. Gastroenterology 1992; 103:579-89.

*Corresponding author: Dr Rachana Chaturvedi, Department of Pathology, 1st floor, college building KEM Hospital Parel, Mumbai 12, India Phone: +91 9967017267 Email: rachanachaturvedi@yahoo.co.in

Financial or other Competing Interests: None.

Date of Submission : 22.12.2016 Date of Acceptance : 11.01.2017 Date of Publication : 14.04.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Original Article DOI: 10.21276/APALM.1249

Comparative Study of cell Block Versus Centrifuged Smear Examination from Aspirates of Cystic Lesions S.R. Niveditha1*, Thejasvi Krishnamurthy1 and Sumitha Prakash2 Department of Pathology, Kempegowda institute of Medical sciences, Bangalore, India Department of Pathology, Rajarajeswari Medical College and Hospital, Bangalore, India

1 2

ABSTRACT Background: Cystic fluids encountered during routine FNA poses a diagnostic challenge to cytopathologists due to its low cell yield with high dispersal of cells on conventional centrifuged smears (CS). Cell Block (CB) technique enables retrieval of small tissue fragments from fluids, thereby providing scope for better morphology and material for ancillary techniques which help in improving the diagnosis. Aim: To compare the efficacy of CB versus CS, in cyto-diagnosis of cystic lesions. Methods: This observational study was conducted on a total of 50 fluid samples aspirated from cystic lesions during routine FNA and fluids aspirated peri-operatively from cystic ovarian lesions. Divided into two equal parts, one part was processed for CS and CB by Fixed Sediment Method and relevant immunohistochemistry was performed. CSs were categorized as positive for malignancy, benign diagnosis, Inadequate for opinion and suspicious for malignancy. CBs were categorized as; no material, Non-contributory (CS+, CB-), confirms the smear diagnosis and establishes a specific diagnosis. The comparison between CS and CB was analysed by Chi- square test & kappa test. Results: Out of the 50 cases, 35(70%) were given a benign diagnosis, 10 (20%) were positive for malignancy, 2(4%) were suspicious and 3(6%) were inadequate for opinion on CS. In CB out of 50 cases, 29 of them confirmed/established a diagnosis and 21 cases were non diagnostic / non-contributory. CB gave an improved diagnosis in 2 out of 10 (20% ) malignant cases and 2 out of 35 (5.7%) benign cases. ( p value = 0.00054, Kappa value =0.34) Conclusions: CBs complemented CS, more so in malignant lesions by preserved architecture. Aspirates from multiple sites of the cystic lesions (with/without radiological assistance) pooled as one specimen yielded better material for CBs and ancillary techniques like histochemistry and IHC. Keywords: Cystic Lesions, Cell Block, Centrifuged Smear, Fixed Sediment Method

Introduction

to compare the efficacy of cell block by formalin fixed sediment method versus the conventional centrifuged smears, in studying all cystic aspirates encountered during routine FNA procedure. Additionally, intact cysts received for frozen sections were also aspirated (Peri-operative) and included in the study.

Karnauchow et al were the first to emphasize that cell block technique could be used in thick tissue particles aspirated by FNA, which provided sufficient material for a good section, special stains and IHC.[3] Some authors also tried needle washes of aspirates from solid lesions with considerable success.[2,5-10]

Materials and Methods

One of the constraints of conventional FNA smear is the limited material available for adjuvant diagnostic investigations.[1,2]Cellblock technique has been applied to aspirated material [3] and since then has been used increasingly to improve the diagnostic accuracy of FNA. [4,5]

Following which many authors studied role of cell block in cystic fluid aspirates of jaw lesions, thyroid lesions, cystic lesions of head and neck and cystic pancreatic mucinous tumors. [11-16] Most of these studies were limited to single organ or anatomical region. Therefore, we formulated a study

This observational study was carried out in the cytopathology division of the department of pathology at a tertiary care hospital, over a period of two months. A total of 50 fluid samples aspirated from cystic lesions during routine FNA and fluids aspirated peri-operatively from cystic ovarian lesions were included in the study. Data was collected in a pretested proforma. All fluids (irrespective of volume), aspirated in the laboratory were processed at the earliest. Due to technical reasons, if the fluids could not be processed immediately, they were stored in a refrigerator at 4oC and processed later. The fluids were examined grossly for volume, color and appearance and findings were noted.

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

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The fluids were divided into two equal parts. One part was kept for conventional cytology (centrifuged smear â&#x20AC;&#x201C; CS) and the other part for cellblock (CB).[17] For conventional smear, the fluid was centrifuged at 2500 rpm for 10 minutes (REMI CENTRIFUGE) in plastic test tubes and supernatant decanted. Minimum of two thin smears were prepared from the sediment and PAP and H&E staining were done. The other portion of fluid specimen was processed by Fixed Sediment Method of Cellblock according to Nathan et al[18]The fluid specimen kept aside for CB, was fixed in ethanol formalin fixative (9 parts absolute alcohol & 1 part 10% formalin) in the ratio of 1:1 for one hour. After fixation the specimen was centrifuged at 2500 rpm for 10-15mins. Supernatant was poured off and sediment drained by inverting the tube on Whatman filter paper (No: 52, WR BALSTON LTD, 11cm disc). The sediment was then wrapped in the same filter paper and processed in histokinette as routine histopathological specimen. Multiple thin sections of 4-5 micron thickness from paraffin blocks were obtained, stained with Haematoxylin and Eosin stain and examined microscopically. Based on the cytological findings, relevant immunohistochemistry was performed wherever necessary. After studying all the available clinical data, based on morphology, the CS and CB were categorized as:[8] Centrifuged smear

Cell block

Positive for malignancy Benign diagnosis Inadequate for opinion Suspicious for malignancy

Non diagnostic / no material Non-contributory (CS+, CB-) Confirms the smear diagnosis Establishes a specific diagnosis

Binomial distribution was performed to assess the comparison between conventional smear and cellblock. SPSS 20.0 for Windows software package (SPSS Inc., Chicago, IL, USA) was used for analysis by Chi- square test, kappa test. P< 0.05 was considered to be statistically significant. Since this is a comparative study, for statistical purposes the CS and CB categories were grouped as: CS = 0 (Positive for malignancy & Suspicious for malignancy) CS = 1 (Benign diagnosis & Inadequate for opinion) CB = 0 (Non diagnostic/ Non-contributory) CB = 1 (Confirms/ Establishes diagnosis)

Results

Fifty (50) fluids from cystic lesions were included in the study,of which 37 belonged to fluids from routine FNAs while the remaining 13 belonged perioperative fluids. Among FNA fluids majority (27%) were from breast lesions

followed by lymphnodes (18.9%). Eight (8) fluids grouped as miscellaneous included 4 cases of hepatic abscess, 2 from nape of neck swellings, 1-glabellar swelling and 1-palm swelling. Among the peri-operative all were from ovarian cysts (table 1). Females (68%) were majority most of them in the age froup of 40-60 yrs (table 2). Volume of the fluid aspirated was >10 ml in 80% of the fluids by FNA while perioperative ovarian cyst fluids , were upto 100ml in majority. On centrifugation of the fluids , good pellet formation was seen in 66% of cases. On conventional smear (CS) examinaton, 70%(35/50) were given a benign diagnosis, 20% (10/50) were positive for malignancy, 4%(2/50) were suspicious and 6% (3/50) were inadequate for opinion (table 3). Of the benign lesions majority were ovarian cysts (10/35) followed by cystic lesions in the breast (7/35). On CB preparation from these 35 fluids (benign on CS) smear diagnosis was confirmed in 57% (20/35), while a specific/ improved diagnosis could be established in 2 cases (table 3).One case was of a breast aspirate diagnosed as benign breast disease on CS (fig 1,inset), however the CB of the same showed varying sized cysts surrounded by fibrosis and epitheliosis there by improving the diagnosis to fibrocystic disease (fig 1). The second case was of an ovarian neoplasm with cystic degeneration, was diagnosed as broad ligament leiomyoma with cystic change based on the scattered plump spindle cells on CS. CB of the same showed monotonous spindle cells with absence of whorling and hence was subjected to IHC for SMA which was negative(fig 2) improving the diagnosis to ovarian fibroma. (fig 2, inset). In the remaining 13 cases CB did not yeild material on CB. Hence CB yeilded material in 22/50 ie., 62.8% of benign cases. Among the 10 cystic fluids which were positive for malignancy by CS, in 5 cases CB confirmed the diagnosis. In 2 cases, CB established a specific diagnosis (table 2). They were: 1. Fluid from a cervical lymph node, on CS suggested metastatic poorly differentiated carcinoma which on CB showed squamous pearl, CK and EMA positivity on IHC thereby establishing diagnosis of metastatic squamous cell carcinoma (fig 3). 2. Fluid from a cystic ovarian mass was reported as positive for malignancy on CS, which on CB showed papillary clusters with pleomorphic cell morphology establishing a diagnosis of papillary cystadenocarcinoma - ovary. In the remaining three cases CB material was non-contributory. Hence in positive for malignancy, CB yielded material in 70% (7/10) of cases. Among the two cases which were diagnosed as suspicious for malignancy on CS, CB material did not contribute to either confirming or establishing the CS diagnosis.

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Of the 50 cases, three of them were inadequate for opinion even on CS. Of the remaining 47 having enough material on CSs, 29(61.7%)of them confirmed/established a diagnosis on CB. CB gave an improved diagnosis in 2 out of 10

(20% ) malignant cases and 2 out of 35 (5.7%) benign cases (table4,5). Chi square test yeilded 11.947 with a significant p value of 0.00054(p<0.05).Kappa test yeilded a value of 0.34 suggesting a fair degree of aggreement between CS and CB.

Table 1.:Distribution of cases among cystic fluids (n==50) Type of cystic fluids

Site Thyroid Breast Cervical lymph node Salivary gland Liver Lung Omental mass Rib swelling Inguinal swelling Arm swelling Palm swelling Nape of the neck swelling Glabellar region swelling Ovary

FNA

Miscellaneous

Peri-operative Total

Frequency (n) 2 10 7 5 4 1 1 1 1 1 1 2 1 13 50

Table 2: Age and sex distribution. Age in yrs 0-20 20-40 40-60 61-80 Total

Male 0 7 3 3 13

Female 1 9 20 7 37

Percentage 2% 32% 46% 20% 100%

Malignant/suspicious 0 3 2 0 5 2 12

Total 2 10 7 3 13 12 47

Table 3: Distribution of benign and malignant cases among cystic fluids Site Thyroid Breast Lymph Node Salivary Gland Miscellaneous Peri-op ovary Total

Benign 2 7 5 3 8 10 35

*3 cases were inadequate.

Table 4: Cross tabulation of CS vs CB in fluids from cystic lesions CB category Non-diagnostic/no material Non-contributory Confirms diagnosis

CS category

Total

Positive for malignancy

Benign

Inadequate

suspicious

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CB category Establishes diagnosis Total

CS category

Total

Positive for malignancy

Benign

Inadequate

suspicious

Table 5: Summary of cystic fluids (malignant cases) Sl No

TYPE OF FLUID

VOLUME

PELLET FORMATION

CS*

CB**

MORPHOLOGY

IHC

FNA CERVICAL LN

<10ml

Keratin pearl

FNA CERVICAL LN

<10ml

Clusters of cells

CK+ EMA+

3 4 5 6 7 8 9

FNA INGUINAL LN FNA BREAST FNA rib swelling FNA lung mass Cystic Ovarian mass Cystic Ovarian mass FNA BREAST

<10ml <10ml <10ml <10ml >100ml 10-100ml <10ml

+ + + + + + +

1 1 1 1 1 1 1

3 3 3 3 4 2 2

Clusters of melanoma cells Clusters of cells Cytoplasmic bridges Cell groups Papillary cluster Non-contributory Non-contributory

FNA OMENTAL MASS

<10ml

Non-contributory

11 12

FNA BREAST FNA arm swelling

10-100ml 10-100ml

+ +

4 4

1 1

Non-diagnostic Non-diagnostic

CS* categories: 1=positive for malignancy, 2=Benign, 3=Inadequate for opinion, 4=suspicious for malignancy CB**categories:1= non-diagnostic/nomaterial,2=noncontributory, 3=confirms smear diagnosis, 4=Establishes specific diagnosis

Fig. 1: CS on cystic fluid from breast showing only stromal fragments- PAP stain 40X(Inset) CB on cystic fluid from breast showing cysts surrounded by fibrosis- H&E 10X.Â

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Fig. 2: CB on fluid from cystic ovarian mass showing cluster of spindle cells – (H&E,400x) Inset- IHC on CB from cystic ovarian mass – SMA negative (IHC, DAKO, 400x).

Fig. 3: CK and EMA(inset) positivity on CB from cystic aspirate of cervical lymph node with metastatic carcinoma (IHC,DAKO, 400x).

Discussion

techniques like CB becomes important. [11] Oenning and Rivero et al, studied series of 17 and 33 cystic jaw lesions respectively, by aspiration and processing the material by the CB technique. [11, 12] Both were of the opinion that cell block technique is a complementary method which is simple, fast and cost–effective and can eliminate the need for incisional biopsy.

On many occasions, clinical and radiological examination can give a provisional diagnosis, which however needs to be confirmed /complemented by tissue diagnosis. The commonest procedure for obtaining material for tissue diagnosis is either an excisional or incisional biopsy. Biopsy may be a complex procedure at some sites such as maxilla-mandibular areas, oral cavity etc. and also difficult in patients with some systemic morbid conditions. [11, 12] In such situations FNA may be the only alternative. However, in aspirates of cystic lesions where the cell numbers are generally less, the technique of cell block is a great advantage. On FNA of cystic lesions, when aspirated fluid is centrifuged, concentration of lesion-typical cells with decreased cell dispersion occurs and improves diagnosis. [19, 20] CB procedure of such concentrated material, could replace biopsy thereby simplifying the diagnostic process. [10-12] Moreover, material obtained by FNA of some lesions accessed through newer procedures like endoscopy etc., maybe so little that CB on the little material gives added advantage of architectural details and additional material for special stains. Some Cystic lesions of the jaw have similar clinical and radiological findings. For example, Keratocystic Odontogenic tumors (KOTs) are developmental neoplastic lesions which need to be differentiated from aggressive lesions like ameloblastoma, as their treatment varies. Hence a good pre-operative diagnosis will help the clinician in therapeutic planning for such lesions and therefore ancillary www.pacificejournals.com/apalm

Hegazy et al studied 85 thyroid lesions by both CS and CB methods.[13] Their series included one thyroglossal cyst and two cases of branchial cleft cysts along with Cystic nodules in 5 cases (colloid goiters). Cystic degeneration may be observed in both benign and malignant thyroid nodules and the approximate malignancy rate within cystic thyroid nodules is 10%. They were of the opinion that conventional FNA of cystic nodules has a high rate of nondiagnostic and false negative results and CB may resolve this problem. In their series, inadequacy of smears was 15% but cell block reduced it to (5.8. %). They also found that CB gave good inter-relation of the cells together, forming follicles or papillae with the nuclear features becoming more clear and obvious. Their study showed CBs with an overall sensitivity of 91.6% and specificity of 97.2% among thyroid aspirates. [13] In our study we had 2 cases of cystic thyroid nodules diagnosed as colloid goiters on CS later confirmed by CB with better morphology. Xiao et al studied 17 cases of cystic pancreatric mucinous tumors on FNA and found that the yield of diagnostic cells was typically low. [16] Specific diagnosis was supported by cellblock and/or increased CEA. Narayan et al reported a eISSN: 2349-6983; pISSN: 2394-6466

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case of papillary cystic variant of acinic cell carcinoma in which CB aided in an accurate pre-operative diagnosis. [21] When cell count is low in CS smears it is difficult to discern whether it is a cyst or cystic degeneration. Our study had three aspirates of salivary gland diagnosed as benign lesions on CS, but CB helped in segregating them into two cases of pleomorphic adenoma with cystic degeneration and one case of true cyst later confirmed by histopathology. A specific diagnosis helped the surgeon in planning the surgery. Similarly in cystic breast aspirates, especially from malignant lesions, CB would help differentiating degenerative changes seen in cells(due to presence of exfoliated cells in fluids for long time) leading to false positive diagnosis, from anaplastic changes with the help from the surrounding architecture. Among the 10 breast aspirates 7 were benign on CS confirmed by CB. Remaining three were suspicious for malignancy on CS, of which only in one case CB, confirmed the diagnosis. In cystic lesions with infective etiology also, CB seems to complement the FNA /CS diagnosis. Kim et al reported CB in a case of hepatic hydatid cyst showing protoscolices, hooklets and fragments of laminated material along with inflammatory cells and amorphous necrotic debris complementing the CS diagnosis. [22] In our study 4 cases of hepatic abscess were aspirated and CB confirmed the CS diagnosis. Multiple serial sections could be obtained from the respective CBs for special stains to rule out amoebic and fungal etiology. Two lymph node aspirates diagnosed as tubercular cold abscess on CS were confirmed by ZN stain on CB too, with an added advantage of quicker screening of AFB due to less cell dispersion on CB.

still controversial, with some authors suggesting that aspiration biopsy of the ovary (except for the purpose of oocyte retrieval) is potentially dangerous and should not be regarded as a routinely acceptable clinical practice. [24] The arguments against the use of FNA include the possible spillage of malignant cells into the abdominal cavity, leading to the potential dissemination of tumor, as well as misdiagnosis related to sampling errors. [25] Having noted this opinion, it is important to point out that some of the literature also indicates that the use of needle aspiration of the ovary is valuable, safe, and even the standard of care in certain clinical settings, notably in young women who wish to preserve their ovarian function. We aspirated 12 intact ovarian cysts sent to us for frozen section, before cutting them open to take bits. The aspirated fluid was processed for both CS and CB. CB confirmed CS findings in eight benign cysts, bettered diagnosis in one (from ovarian leiomyoma with cystic degeneration to ovarian fibroma), where the CB gave additional material for smooth muscle actin (SMA) staining. Two cases were positive for malignancy in CS out of which CB gave better diagnosis by better architecture in one, but in the other case CB was non-contributory. In another ovarian cyst, suspicious

for malignancy on CS, the CB was non- contributory. Extrapolating this finding, may we suggest pre-operative USG guided aspirate of ovarian cyst with CBs to confirm their nature and plan for future surgical procedure? This appears promising especially in young women who wish to preserve their ovaries.

Khurana et al studied 20 cases of squamous lesions in the neck by cell block. Their study group included 7 cases of atypical squamous cells which were suspicious of squamous cell carcinoma (SCC) on conventional smears, which were later confirmed as SCC by p53 staining on CB. [15] Shaloo et al also shared similar experience in diagnosing

Panlanowitz et al in their study of utility of cell block preparation in cytologic specimens diagnostic of lymphoma, found cell block preparation of cytologic material extremely beneficial, with the potential for further reducing the need for surgical excision.[10] Similar to our study, Mayall et al reviewed 50 consecutive cytology cell block preparation in a large general hospital. They concluded that the use of CBs are reliable and technically unsophisticated which aid in the cytological examination.[26]

complementing smear diagnosis. We also observed better architecture in the form of well-developed keratin pearl on CB from a lymph node aspirate diagnosed as poorly differentiated carcinoma on CS. CB provided material for CK and EMA in another case of lymph node aspirate diagnosed as undifferentiated carcinoma/lymphoma on CS thereby improving the diagnosis to carcinoma. This highlights the role of CB in pre-operative diagnosis of metastatic cystic lesions.

Though CB complements CS diagnosis with added advantage of better architecture, availability of material for special stains and IHC in many cases, CB also has certain disadvantages. Delay in the diagnosis when compared to conventional smears due to additional time for CB preparation,[17] risk of losing material during processing and increased cost are some of the drawbacks.[26] Some authors like Wojick et al found that the additional studies of cell blocks is of little benefit after a study

recurrent SCC of oral cavity. [23] They suggested that CB is a forgotten tool these days and reemphasized it’s utility in

Despite the expanding literature and increasing use of the technique, the role of aspiration of the ovaries is

comparing CS and CB in recurrent gynecologic malignancies.[5]

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Karnauchow PN, Bounin RE. Cell block technique for fine needle aspiration biopsy. J Clin Pathol 1982;35:688.

needle aspirations from 844 superficial and deep seated

Kung IT, Yuen RW, Chan JK. Optimal formalin fixation and processing schedule of cell blocks from the fine needle aspirates. Pathology 1989;21: 143-5.

Wojcik EM, Selvaggi SM. Comparison of smears and cell blocks in the fine needle aspiration diagnosis of recurrent gynecologic malignancies. Acta Cytol 1991;35:773-6.

Yang GC, Wan LS, Papellas J, Waisman J. Compact cell blocks. Use for body fluids, Fine needle aspirations and Endometrial brush biopsies. Acta Cytol 1998;42:703-6.

Zito FA, Gadaleta CD, Salvatore C, Filatico R, Labriola A, Marzullo A et al. A modified cell-block technique for fine needle aspiration cytology. Acta Cytol 1995;39: 93-9.

Liu K, Dodge R, Glasgow BJ. Fine needle aspiration: comparison of smear, cytospin and cellblock preparations in diagnostic and cost effectiveness. Diagn Cytopathol 1998;19:70-4.

Saleh HA, Hammoud J, Zakaria R, Khan AZ. Comparison of Thin-Prep and Cell block preparation for the evaluation of Thyroid epithelial lesions on fine needle aspiration biopsy. CytoJournal 2008;5-3.

[8]

to those of cytospin and CB preparations ( from fine

lesions), to determine the cost effectiveness of each found

that smears were superior to either cytospin or CBs

in providing a diagnosis. However, they suggested that when the immediate smear evaluation is non-diagnostic, it is cost effective to obtain cell blocks.[8] Saleh et al, in their study of comparison of thin-prep and cell block preparation for the evaluation of 126 thyroid epithelial lesions on fine needle aspiration biopsy concluded that thin-prep slide preparation is superior to cell block preparation and is more likely to have greater cellularity for diagnosis and detect atypical/ neoplastic thyroid lesions, particularly those of follicular cell origin. They also suggested thin-prep slides to be used as complementary to direct smears.[9] Similarly in our study 18/50 cases (36%) CB was inadequate and in 3/50 (6%) cases CB was noncontributory. Among these were 3 cases diagnosed as positive for malignancy n and 2 suspicious for malignancy on CS but CB was false negative. To address these short comings authors have suggested a dedicated needle aspiration for cell block improves yield. [17] Mayall et al were of the opinion, that a highly experienced aspirator should perform FNA to obtain sufficiently cellular material for CB. [26] With our experience of CB with cystic lesions, aspirates from multiple sites of the cyst pooled as one specimen showed good pellet formation and thereby adequate material on CB. CB was diagnostic in aspirates done with radiological assistance (USG/CT). In summary, CB method is an excellent complementary tool for improving cyto-diagnosis in aspirates of cystic lesions. Though CBs were complementary to CS in the overall categorization of benign and malignant groups, they appeared to be more useful in diagnosis of malignancy by better preserved architectural patterns, as seen in corresponding histopathology sections. Aspirates from multiple sites of cystic lesions, pooled as one specimen yielded good pellet formation and thereby adequate material on CB. CB was diagnostic in aspirates done with radiological assistance (USG/CT). Thus CBs appeared to bridge cytology and histopathology along with providing excellent resource material for ancillary techniques like histochemistry and IHC.

References 1.

Ansari NA, Derias NW. Fine needle aspiration cytology. J Clin Pathol 1997;50 : 41-3.

Khan S, Omar T, Michelow P. Effectiveness cell block technique in diagnostic cytopathology. J of Cytol 2012;29:177-82.

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10. Panlanowitz L, Freeman J, Goulart RA. Utility of cellblock preparation in cytologic specimens diagnostic of lymphoma. Acta Cytol 2010;54:236-7. 11. Oenning ACC, Rivero ERC, Calvo MCM, Meurer MI, Grando L J. Evaluation of the cell block technique as an auxiliary method of diagnosing jawbone lesions. Braz Oral Res 2012;26:355-9. 12. Rivero ERC, Grando LJ, Manegat F, Claus JDP, Xavier F. Cell block technique as a complementaty method in the clinical diagnosis of cyst-like lesions of the jaw. J Appl Oral Scin 2011;19:269-73. 13. Hegazy RA, Hegazy AA. FNAC and cell block study of thyroid lesions. Universal Journal of Medical Science 2013; 1:1-8. 14. Nguyen CK, Lee MW, Ginsberg J, Wragg T, Bilodeau D. Fine needle aspiration of the thyroid: an overview. Cyto Journal 2005;2:12. 15. Khurana KK, Ramzy I, Truong LD. p 53 immunolocalization in cell block preparation of squamous lesions of neck: an adjunct to fine needle aspiration diagnosis of malignancy. Arch Pathol Lab Med 1999;123:421- 5. 16. Xiao GQ. Fine needle aspiration of cystic pancreatic mucinous tumor: oncotic cells as an aiding diagnostic feature in paucicellular specimens. Diagn Cytopathol 2009;37:111-6. 17. Koss LG. Effusions in the absence of cancer. In: Koss LG, Melamed MR, editors. Diagnostic Cytology and its Histopathologic Basis, 5th edition, Philadelphia: Lippincott Williams & Wilkins; 2006. p. 919-48.

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18. Nathan NA, Narayan E, Smith MA, Horn MJ. Cell block cytology: Improved preparation and its efficacy in diagnostic cytology. Am J Clin Pathol 2000;114:599-06. 19. Jain D, Mathur SR, Iyer V K. Cell blocks in cytopathology: a review of preparative methods, utility in diagnosis and role in ancillary studies. Cytopathology 2014;25:356-71. 20. Radhika S, Nijhawan R, Das A, Dey P. Ameloblastoma of the mandible: diagnosis by fine needle aspiration cytology. Diagn Cytopathol 1993;9:310-3. 21. Narayan SM, Padmini J, Parthiban R, Madhusmita J, Natarajan G, Revadi PS. Diagnosis of a case of papillarycystic variant of acinic – cell carcinoma on fine needle aspiration cytology: Myriad of cyto-morphological features. International Journal of Case Reports and Images 2014;5:18-22.

22. Kim AR, Park SJ, Gu MJ, Kim HJ. Fine needle aspiration cytology of hepatic hyadatid cyst: a case study. The Korean J Pathol 2013;47:395-8. 23. Dahima S, Hegde P, Shetty P. Cell Block a forgotten tool. J of Mol Path Epidemol 2015:1:1 24. Trimbos JB, Hacker NF. The case against aspirating ovarian cysts. Cancer 1993;72:828-31. 25. Sherman ME: Cytopathology. In: Kurman RJ (ed). Blaustein’s Pathology of the Female Genital Tract, 4th ed. New York: Springer-Verlag;1994. p. 1120–22. 26. Mayall F, Chang B, Darlington A. A review of 50 consecutive cytology cell block preparations in a large general hospital. J Clin Pathol 1997;50:985-90.

*Corresponding author: Dr S R Niveditha, Department of Pathology, Kempegowda institute of Medical sciences, Banashankari 2nd stage, Bangalore – 560070, India Phone: +91 9845485544 Email: srniveditha@gmail.com Date of Submission : 03.01.2017 Date of Acceptance : 02.02.2017 Financial or other Competing Interests: None. Date of Publication : 14.04.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Original Article DOI: 10.21276/APALM.1251

Impact of Intervention on Awareness of Biomedical Waste Disposal Among Medical Students Dhananjay Shrikant kotasthane*, Vaishali Dhananjay kotasthane, Shanmugasamy K and Ancy A Department of Pathology, Mahatma Gandhi Medical College and Research Institute, Pillaiyarkuppam, Pondicherry, India

ABSTRACT Background: The proper management of biomedical waste has become a worldwide humanitarian topic today. Hazards of poor management of biomedical waste have aroused the concern world over, especially in the light of its far-reaching effects on human, health and the environment. With this milieu, this interventional study was carried out on medical students aimed at assessing and creating the awareness of knowledge of Biomedical Waste Management (BMWM) among Second year MBBS Students. Methods: Multiple-choice-Question(MCQ) based Questionnaire was administered to the students. This was followed by a lecture on Biomedical Waste Management.The same MCQ based Questionnaire was administered to the students immediately after the lecture and after one month.The questions in the test were based on Four domains-“Existence of Biomedical Waste rules”, “Categories of Waste”, “Different colour codes used” and “Waste disposal methods” Mean scores were calculated for the entire tests as well as for these domains. Results: Mean scores in pre-lecture test indicated that awareness of Biomedical Waste Management amongst medical students was quite low. Mean scores were higher in post lecture test and post one month test, indicating that the knowledge of the various aspects of biomedical waste management significantly improved after the lecture conducted by the investigator of the study However, the knowledge did not sustain after one month, though it was still higher than the original baseline level. Friedman test and Wilcoxon signed rank tests were used to prove the statistical significance. Conclusion: This study shows that early sensitisation about BMWM in Second MBBS students who are future doctors will improve awareness of BMWM. Repeated sensitisation is needed for the knowledge levels to sustain. Keywords: Biomedical waste, Medical students, Awareness

Introduction

Hospital waste management is a global problem of immediate concern, due to rapid increase in the hospital acquired infection both by the general public as well as the health personnel.[1] Healthcare facilities create waste that may hazardous to health. The importance of segregation of Hazardous Biomedical Waste and general waste is that only 10% to 25% of waste generated in health facilities is hazardous. Failure of this vital step of segregation turns nonhazardous waste into hazardous. Segregation also enables thosewho handle the containers outside the hospitalwards to identify and treat them appropriately. About 0.5 to 2.0 kg per bed per day hospital waste is generated in India.[2]Hospital waste is a potential health hazard to the health care workers, public, flora and fauna of the area.[3] The BMW invites flies, insects, rodents, cats and dogs that are responsible for the spread of communicable diseases. Rag pickers in the hospital, sorting out the garbage are at a risk of getting various infections. The recycling of disposable syringes, needles, IV sets and other article like

glass bottles without proper sterilization are responsible for Hepatitis, HIV, and other viral diseases. It becomes primary responsibility of Health administrators to manage hospital waste in most safe and eco-friendly manner.[3] Benefits of BMWM results in cleaner and healthier surroundings, reduction in the incidence of hospital acquired and general infectionsand preventsreuse and repackaging of infectious disposables.[3] With this milieu, this study was undertaken to assess and create the awareness with respect to healthcare waste management among the Medical students.

Material and Methods

This interventional study was conducted after getting approval of Institutional Human Ethical Committee and taking informed consent of second MBBS students. The students of second year were administered prevalidated MCQ test of sixteen questions on BMWM.The questions framed were based on Domains- “Existence of Biomedical waste rules”, “Categories of waste”, “Different colour codes used in biomedical waste management” and “Waste disposal methods”

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

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Awareness of BMW Disposal Among Medical Students

A short lecture on Biomedical Waste Management was delivered to the students by an investigator in this study. The same MCQ test was administered after the lecture and after one month. The pre lecture, post lecture and post one month response given was assessed in the overall test and Mean scores were calculated. Also the number of participants were categorised based on performance, both in pre and post test under following headings-a.less than 50%,b.between 50 to 75%,c.more than 75%. Mean scores and the results in the tests were compared. Friedman test and Wilcoxan signed Rank test was used to calculate statistical significance between pre lecture test and post lecture test and between post lecture test and post one month test. P value less than 0.05 was considered as statistically significant.

Results

Total 143 students were present, when this research project was carried out in the class. They volunteered to participated in the research Project.Mean scores were calculated and also the results in pre lecture test,post lecture test and post lecture one month test were categorised into 3 categories-Students scoring less than 50%, 50 to 75% and more than 75%in the pre lecture, post –lecture and post one month assessment. These assessments were carried out for overall results and also separately for each of the above mentioned domains. As shown in Tables 1,2,3,4,5 mean scores in the post lecture test and post one month test were more than the

pre-lecture test.Similar findings are seen in the percentage scored by the students as shown in figures 1,2,3,4,5. P value calculated using Friedman test (Tables 1 to 5) showed that difference in scores of post one month test and post lecture as well as pre lecture test was statistically significant for the entire test and also for individual domains. Mean scores in the post lecture and post one month test were higher than the pre-lecture test. As shown in Table 6, p value calculated using Wilcoxon Signed Ranks test demonstrated that differences between prelecture test and post lecture test was statistically significant for overall test as well as individual domains. However, the mean scores in post one month test were lower as compared to post lecture test, though they were still higher than the baseline level of pre lecture test. P value calculated using Wilcoxan Signed Ranks test for difference between pre lecture test and post one month test was statistically significant for the overall test and also for Domain 2 i.e ‘ Categories of biomedical waste’. However, p value for difference in pre lecture and post one month test was not significant for Domain 1(existence of biomedical rules), Domain 3(Different colour codes used) and Domain 4 (Waste disposal methods). This proves that the knowledge of the students improved significantly immediately after the lecture but did not sustain after one month in all domains of knowledge, though the levels were still higher than the baseline level. This indicates the need for repeated sensitisation.

Table 1: Friedman test for overall test Overall

Pre

Po1

Po2

Mean

6.64

11.92

8.06

Median

Std. Deviation

2.064

2.512

2.948

Minimum

Maximum

Friedman Test

p-value

164.888

<0.0001

Pre=prelecture ,Po 1=post lecture,Po 2=post lecture one month Table 2: Friedman test for Domain(D1)“Existence of biomedical waste management rules” D1

Pre

Po1

Po2

Friedman Test

p-value

Mean

1.99

3.44

2.47

120.766

<0.0001

Median

Std. Deviation

0.915

0.853

0.948

Minimum

Maximum

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Table 3:Friedman test for Domain (D2) “categories of Biomedical waste” D2

Pre

Po1

Po2

Friedman Test

p-value

Mean

1.36

3.32

2.1

137.753

<0.0001

Median

Std. Deviation

0.93

0.969

1.241

Minimum

Maximum

4 Friedman Test

p-value

70.228

<0.0001

Friedman Test

p-value

54.814

<0.0001

Table 4:Friedman test for Domain (D3)“Different colour codes used” D3

Pre

Po1

Po2

Mean

1.66

2.72

1.69

Median

Std. Deviation

0.912

1.051

1.09

Minimum

Maximum

Table 5:Friedman test for Domain(D4)“Waste disposal methods” D4

Pre

Po1

Po2

Mean

1.63

2.44

1.81

Median

Std. Deviation

0.811

0.844

1.007

Minimum

Maximum

Table 6:Wicoxan Signed Ranks test DOM = ALL

Po1 – Pre

Po2_level - Pre

Po2_level - Po1

Wilcoxon Signed Ranks Test

-10.022

-10.361

-10.400

p-value

0.000

DOM = D1

Po1 - Pre

Po2_level - Pre

Po2_level - Po1

Wilcoxon Signed Ranks Test

-9.060

-0.761

-9.808

p-value

0.000

0.446

0.000

DOM = D2

Po1 - Pre

Po2_level - Pre

Po2_level - Po1

Wilcoxon Signed Ranks Test

-9.747

-4.358

-9.010

p-value

0.000

DOM = D3

Po1 - Pre

Po2_level - Pre

Po2_level - Po1

Wilcoxon Signed Ranks Test

-7.030

-1.433

-8.368

p-value

0.000

0.152

0.000

DOM = D4

Po1 - Pre

Po2_level - Pre

Po2_level - Po1

Wilcoxon Signed Ranks Test

-6.719

-0.492

-7.968

p-value

0.000

0.622

0.000

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Fig. 1: Assessment of the Medical students’ overall knowledge in pre lecture, post lecture test and post one month test.

Fig. 2: Assessment of the Medical students’ knowledge in pre lecture, post lecture,and post one month test in Domain(D1) “Existence of Biomedical waste disposal rules”.

Fig. 3: Assessment of the Medical students’ knowledge in pre lecture, post lecture testand post one month test in Domain “Categories of Biomedical waste”

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Fig. 4: Assessment of the Medical students’ knowledge in pre lecture, post lecture test and post one month in Domain(D3) “Different colour codes”.

Fig. 5: Assessment of the Medical students’ knowledge in pre lecture, post lecture and post one month test in Domain(D4) “Waste disposal methods”.

Discussion

Although there is an increased global awareness among health professionals about the hazards and also appropriate management techniques, the level of awareness in India is found to be below par. Adequate knowledge about the health hazard of hospital waste, proper technique, and methods of handling the waste could go a long way toward the safe disposal of hazardous hospital waste and protect the community.[2]

of increasing concern, prompting hospital administration to seek new ways of scientific, safe and cost effective management of the waste, and keeping their personnel informed about the advances in this area.[3,4]

The problem of bio-medical waste disposal in the hospitals and other healthcare establishments has become an issue

Different studies highlight the urgent need for greater awareness and commitments at policy and programme levels for capacity building and resource investments in BMWM.The last decade witnessed a significant increase of public concern regarding Medical Waste disposal. This was fuelled by reports of ‘beach washing” of medical waste on the coasts of Florida and Gulf, and the “recycling” of disposable articles in developing countries.[5] The reports

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and figures available from developed countries indicate that approximately 1-5 kg of waste is generated per bed per day, with substantial inter country and inter specialty differences. The data available from developing countries also indicate that the range is essentially similar but the figures are on a lower side with 1-2 kg per day per bed.[6] In India, it is estimated to be 2.0 kg/ bed/ day.[7]The concern regarding the medical waste is mainly due to the presence of pathogenic organisms and organic substances in hospital solid wastes in significantly high concentrations. The substantial number of organisms of human origin in solid waste suggests the presence of virulent strains of viruses and pathogenic bacteria in undetected numbers.[8] Therefore, improper handling of solid waste in the hospital may increase the airborne pathogenic bacteria, which could adversely affect the hospital environment and community at large.[9] Improper Hospital Waste Management has serious impact on our environment. Apart from risk of water, air & soil pollution, it has considerable impact on human health due to aesthetic effects.[10,11]Government of India reacted towards the global concern and notified the Biomedical waste management rules,1998 (Ministry Of Environment & Forests, Notification, New Delhi 20th July 1998). These rules have been framed in exercise of powers conferred by Sections 6,8 and 25 of Environment (Protection) Act 1986. This is applicable to every hospital and nursing home, veterinary institutions, animal house or slaughterhouses, which generate, Biomedical waste within a time frame.These rules were modified in 2011,2015 and latest in 2016.[12] The Objectives of BMW management are mainly to reduce waste generation,Efficient collection, handling and disposal of waste in such a way that it does not spread infection, Provides safety to employees working in the system andEnsure cost effectiveness by avoiding penalties and fines imposed by regulatory authorities. Accordingly, waste is required to be treated and disposed off in accordance with schedules prescribed. The basic elements is to recognize the waste, identify where waste is generated and determine the cause of generation, plan the disposal of the waste in a scientific manner so as to render it environmentally non-hazardous and eliminate the source of infection.[9] According to a World Health Organization (WHO) report, around 85% of the hospital wasted are actually nonhazardous, 10% are infectious (hence, hazardous), and the remaining 5% are non infectious but hazardous (chemical), pharmaceutical and radioactive.[13] Bio-medical waste differs from hospital waste in the sense that it is â&#x20AC;&#x153;any

solid, fluid or liquid waste, including its container and any intermediate product. These products could be generated during the diagnosis, treatment and immunization of human beings or animals, in research pertaining there to, or in the production or testing of biological and the animal waste from slaughter houses or any other like establishments.[13] According to the WHO, the global life expectancy is

increasing year after year. However, deaths due to infectious disease are increasing.One of major causes for the increase in infectious diseases is improper biomedical waste management.List of infections and diseases documented to have spread through biomedical waste include Tuberculosis, pneumonia, diarrhoeal diseases, tetanus are other common diseases spread due to improper waste management.[14] Occupational health concerns exist for janitorial and laundry workers, nursing, emergency medical personnel, and refuse workers, injuries from sharps and to harmful chemical waste and radioactive waste also cause health hazards to the employees in institutions generating biomedical waste. Proper management can solve the problem of occupational hazards to a large extent.[14] The general publicâ&#x20AC;&#x2122;s health can also be adversely affected by bio-medical waste. Improper practices such as dumping of bio-medical waste in municipal dustbins, or spaces, water bodies etc., leads to the spread of diseases. Emissions from incinerator and open burning also leads to exposure to harmful gases which can cause respiratory diseases and cancer. Plastic waste can choke animals, which scavenge on dumped waste. Injuries from sharps are common feature-affecting animals and rag pickers. Harm chemicals such as dioxins and furans can cause serious health hazards to animal and birds. Certain heavy metals can affect the reproductive health of the animals.[14,15] Bio-Medical Waste Management Rules, 2016, published in the Official Gazette apply to all persons who generate, collect, receive, store, transport, treat, dispose, or handle bio medical waste in any form including hospitals, nursing homes, clinics, dispensaries, veterinary institutions, animal houses, pathological laboratories, blood banks, Ayushhospitals, clinical establishments, research or educational institutions, health camps, medical or surgical camps, vaccination camps, blood donation camps, first aid rooms of schools, forensic laboratories and research labs.[16] A gap between knowledge and actual practice regarding Hospital Waste Management was highlighted in the perception of the hospital staff in a study carried out in

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

kotasthane et al. Ujjain. The participants suggested organisational changes, training and monitoring to address this problem.This is relevant not merely to micro system studied but to other institutions in similar settings.[17] Several studies have been conducted in primary, secondary, tertiary health centres of private and governments institutes in India in urban and rural areas and assessed for the state of BMWM. Multivariate analysis indicated that charts at point of waste generation , availability of designed person , appropriate containers and bags , availability of functional needle destroyers , availability of personal protective gears , segregation of waste at point of generation and log book maintenance were independently associated with better BMWM system in health facilities . This was true for both rural /urban, public/ private facilities.[18] The present study was conducted on Second year MBBS students. The study showed that the baseline knowledge of second MBBS students on Biomedical waste management, which was not adequate, improved significantly after the interventional strategy ,i.e, the lecture delivered by one of the investigators of the study. However the knowledge levels did not sustain after one month, though they were still more than the original baseline levels as was proved by using statistical tests. This indicates that further intermittent sensitization is necessary for sustaining knowledge. After reviewing the literature, not many studies are published regarding knowledge of awareness regarding BMWM among medical students who are one of the stakeholders for BMWM which highlights the importance of this study and need of hour for early sensitization for BMWM among medical students. As present MBBS students are tomorrow’s doctors, early sensitisation and repeated revision of this important topic for MBBS students will be useful in improving the awareness of biomedical waste disposal management in future Doctors and will prove beneficial for the society at large.

Conclusions

Early sensitisation of MBBS students will be useful in improving the awareness of biomedical waste disposal management. Further repetition will be useful for the knowledge levels to sustain As Doctors have to play a role as a leader in a health care team,awareness of this important subject will be beneficial to the society at large.

References 1.

Gopalakrishnan S,.Murali R. Hospital Management Care. Indian J of Community Health 1999;4: 91-4.

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A-201 2.

Shivalli S, Sanklapur V. Healthcare Waste Management:Qualitative and Quantitative Appraisal of Nurses in a Tertiary Care Hospital of India. The Scientific World J. Vol 2014, Article ID 935101, http://dx.doi. org/10.1155/2014/935101.

Mathur P, Patan S, Anand S, Shobhawat A .Need of Biomedical Waste Management System in Hospitals – An Emerging issue – A Review Department of Environmental Science.Curr World environment 2012; 7:117-124.

4. INCLEN Program Evaluation network study group, New Delhi, India Biomedical waste management; situational analysis and predictors of performances in 25 districts across 20 Indian states. Indian J Med Res, 2014 January:139; 141-153. 5.

Ruff GG Jr. Environmental laws in health care. Hosp Mater Manage Q, 1992 Nov; 14 (2) : 28-39.

Report of High Power Committee on Urban- Solid Waste Management, Planning Commission, Govt. of India, Hospital waste management 1995; 35-47.

Archisman M, Gupta MK, Shivali S, Mishra CP,Mohapatra SC. Biomedical Waste Management Practices of Doctors:An Online Snapshot. Nat J of Community Medicine. April-June 2012;3(2):227-231.

8. Wallace LP, Zaltzman R, Burchinal IC, Where solid waste comes from; where it goes, Modem hospitals 1972; 121(3): 92-5. 9.

Li China-shan, Jeng Fu-Tien. Physical and chemical composition of hospital waste. Infect Control Hosp. Epidemiol, 1993; 14:145-150.

10. Rutala WA, Mayhall CG. Medical Waste. Infection control and hospital epidemiology. 1992; 13:38-48. DOI 10.1086/501924. 11. Satpathy S, Pandhi RK, Manual for Hospital waste management at AIIMS Hospital, 1998, New Delhi. 12. Ministry of Environment and Forests, Government of India. Draft Bio-medical Waste ( Management and Handling ) Rules , 2011. 13. Yadav M. Hospital Waste –A Major Problem. JK-Practitioner 2001;8(4):276. 14. Babu R, Parande AK, Rajalakshmi R, Suriyakala P, Volga M. Management of Biomedical Waste in India and Other Countries: A review. J Int Environmental Application and Science. 2009;4(1):65-78. 15. Garg KN, Singh S, Gupta K, Raj N. An Insight on Biomedical Waste Management –A Review. Inter J of Bio and Pharm Res. 2015;6(2):144-9. 16. Government Of India Ministry Of Environment, Forest And Climate. Gazette of India, Extraordinary, Part II, Section 3, Sub-section (i). 2016 March 28.

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17. Bansal M, Mishra A, Gautam P, Changulani R, Srivastava D, Gaur N .Biomedical waste management: Awareness and practices in a district of Madhya Pradesh.Nat J of Community medicine. 2011;2(3):452.

18. Joshi SC, Diwan V, Tamhankar AJ, Joshi R, Shah H, Sharma N et al; Staff perception on biomedical or Health care waste management : a Qualitative study in a rural Tertiary care Hospital in India. PloS one 10950:e0128383. doi 10.1371/ journal.pone.0128383

*Corresponding author: Dr Dhananjay Shrikant Kotasthane, Department of Pathology, Mahatma Gandhi Medical College and Research Institute, Pillaiyarkuppam, Pondicherry, India Phone: +91 9092096244 Email: dskotasthane@gmail.com Date of Submission : 03.01.2017 Date of Acceptance : 04.02.2017 Financial or other Competing Interests: None. Date of Publication : 14.04.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Original Article DOI: 10.21276/APALM.1259

Study of Incidence of Microalbuminuria Among First Diagnosed Diabetic Patients and its Correlation with Body Mass Index and Coexisting Hypertension in A Tertiary Care Hospital 1

Esakki Muthuvel, Vimal Chander R1* and Sowmiyabalu2

Department of Pathology, Saveetha Medical College and Hospital, Thandalam, Chennai, India 2 Tagore Medical College and Hospital, Rathinamangalam, Candour Post, Chennai. India

ABSTRACT Background: Diabetes is raising recently more in the developing countries due to sedentary life style and with increase in duration, complications arise affecting renal, retinal, cardiovascular and nervous systems. Diabetic nephropathy poses a great risk of renal failure and is the cause for performing dialysis. Microalbuminuria is the earliest marker for the renal and cardiovascular involvement. This study aims to study the incidence of microalbuminuria in first diagnosed diabetic patients and to correlate it with the body mass index (BMI), hypertension and waist hip circumference. Methods: This study was conducted on 138 first diagnosed diabetic patients in a tertiary care hospital. Microalbuminuria was analyzed using dipstick method and correlation was made with parameters such as blood pressure, body mass index, and waist-hip circumference. Result: The incidence of microalbuminuria in first diagnosed diabetic patients was 17%. Incidence of microalbuminuria is increased in hypertensive diabetic patients (37%) compared with non-hypertensive diabetic patients (10%), which was statistically significant (p=0.001, r=0.292). Incidence of microalbuminuria among first diagnosed diabetic patients was 17%. The correlation of microalbuminuria with hypertension, BMI and waist-hip ratio suggest that microalbuminuria incidence is significantly associated with hypertension than BMI and waist-hip ratio. Conclusion: Microalbuminuria is the earliest indicator for detecting nephropathy. The incidence of microalbuminuria of 17% during first diagnosis of diabetes indicates the inadequacy in the health care system regarding the screening programs in the rural population. Thus, improvements must be made in the health care system to reduce the incidence rate by effective screening program. Keywords: Microalbuminuria, Diabetes Mellitus, Body Mass Index, Hypertension, Type 2 Diabetes.

Introduction

Diabetes is a rising crisis in India. India leads the world with the largest number of diabetic subjects and this is expected to further rise in the coming years. Hence studies on diabetes related complications are essential to assess the burden of diabetes.[1] The prevalence of diabetes is increasing sharply in the developing countries as people adopt more sedentary life styles. Type 1 and 2 diabetes mellitus have caused a great havoc by increasing the morbidity and mortality by mainly affecting the cardiovascular, renal, retinal system, nervous system and others.[2] Focusing on its effect on the renal system, the main etiology being due to angiopathy of renal capillaries, which leads to chronic renal disease, which is the prime indicator for dialysis. In initial stage, the kidney may leak slightly more albumin than normal amount in urine, which can be sensitively detected by albumin specific dipstick test for albumin, which is known as microalbuminuria. Microalbuminuria is the earliest indicator of diabetic kidney

disease and generalized vascular endothelial dysfunction. [3] The microalbuminuric patients had a significantly higher prevalence of ischemic heart disease compared with normoalbuminuric patients. Retinopathy was also common among the microalbuminuric group. In patients with known diabetes, microalbuminuria is related not only to subsequent proteinuria, but also even more strongly to early death, mainly from cardiovascular disease. Diabetic nephropathy is the leading cause of end stage renal disease worldwide. Diabetic nephropathy is defined by persistent albuminuria (albumin excretion rate > 300 mg/day), declining glomerular filtration rate and rising blood pressure.[4] Microalbuminuria is considered to be an early stage of diabetic nephropathy. Nephropathy is probable cause of the elevated blood pressure found in diabetic patients. It has been suggested that in both Insulindependent diabetes mellitus (IDDM) and Non-insulindependent diabetes mellitus (NIDDM), hypertension increases the chances of development of diabetic

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nephropathy.[5] In NIDDM, microalbuminuria prevalence ranges from 15 to 20% and nephropathy often supervenes after a shorter duration of diabetes than in IDDM.[4] The causal risk factors for microalbuminuria are raised blood pressure and poor glycemic control and achievement of optimal glycemic level reduces the risk of albuminuria and microalbuminuria. This calls for early detection of microalbuminuria and good control of diabetes to reduce the burden of diabetic complication in the future. The present study aims to study the incidence of microalbuminuria in first diagnosed diabetic patients and to correlate it with the body mass index (BMI), hypertension and waist-hip circumference.

Materials and Methods

This cross-sectional study was conducted at Tagore Medical college and hospital in Chennai over a period of 2 months from July to August 2013, wherein suspected patients were diagnosed to have diabetes by random blood sugar test and subsequently confirmed by fasting & postprandial blood sugar values. All first diagnosed diabetic patients are included in this study, while patients with significant proteinuria, urinary tract infection, or heart failure were excluded from this study. In all 138 patients who were included in this study, a complete clinical work up was done including height, weight, and body mass index, waist hip ratio. The body mass index (BMI) was calculated and the patients are classified as normal (BMI <25), overweight (BMI >25 & <30) and obese (BMI >30). The blood pressure was recorded in the right upper arm in the sitting posture, after a five-minute rest. Mean of two blood pressure measurements was used for analysis. Patients were categorized as hypertensive if they were on antihypertensive treatment or in the presence of systolic blood pressure >140 mmHg and/or diastolic blood pressure >90 mmHg. Waist circumference was measured midway between the lower rib and iliac crest while that of the hip at the level of trochanters and the ratio was calculated. The patients are classified as two groups based on the waist hip ratio as group 1 (male <1; female <0.9) and group 2 (male >1; female >0.9). Then spot urine collection is collected from the patient and checked for microalbuminuria. Urine microalbumin concentration was tested using albumin specific dipstick method. In case of dehydrated patients and for those after heavy exercise, urine sample is collected during their next visit to the hospital. Statistical analysis were done using SPSS software version 16. The results were given by median and standard deviation. Correlation was made between microalbuminuria and parameters such as blood pressure, body mass index, and waist hip circumference.

Result

A total of 138 first diagnosed type 2 diabetic patients were included for this study, which included 46 males and 92 females. Overall 24 patients (17%) had microalbuminuria (Figure 1). Among males, microalbuminuria was positive for 6 among 46 (13%) and for females it is 18 among 92 (20%). Hypertensives: Total number of diabetic patients with hypertension was 38 out of 138. Among the 38 hypertensive diabetic patients 14 had Microalbuminuria and among 100 non-hypertensive diabetic patients 10 had microalbuminuria. The incidence is 37% and 10% amongst the Hypertensive diabetic and non-Hypertensive Diabetic patients respectively (Figure 2). The incidence of microalbuminuria is increased in hypertensive patients compared to non-hypertensive patients. The Incidence of Microalbuminuria among Hypertensive Diabetic patients in those with only raised systolic blood pressure is 2 among 13 (15%) or only raised diastolic blood pressure is 2 among 5(40%) or both is 10 among 20 (50%) (Figure 3). The statistical association between microalbuminuria and hypertension is p=0.001, r=0.292. These results suggest that diastolic blood pressure is correlated to microalbuminuria more than systolic blood pressure. Body Mass Index (BMI): Incidence among the patients with normal BMI, overweight, obese are found to be 10 in 63, 8 in 55 and 6 in 20 i.e. 16%, 15%, 30% respectively (Figures 4 and 5). There are no significant changes regarding incidence of microalbuminuria among the overweight patients and those with normal BMI, but it is significantly increased among the obese patients. Statistically association between Body Mass Index and microalbuminuria is p=0.6 and r=0.04, hence BMI is not directly associated with microalbuminuria. Waist hip ratio: Based on the waist hip ratio patients are classified as Group 1 (male <1; female <0.9) and Group 2 (male >1; female >0.9) and incidence among the group 1 and group 2 is 13 among 93 (14%) and 11 among 45 (41%) respectively (Figure 6). Though microalbuminuria appears to be incident more among the person with high waist hip ratio, but statistically the association between waist hip ratio and microalbuminuria is p=0.138, r=0.127 hence this relation was insignificant. Anyway, difference in incidence of microalbuminuria regarding body mass index and waist hip circumference suggests that its incidence differs among different parameters of obesity index. Microalbuminuria was present in 41% of hypertensive diabetic patients with BMI >25, it was present in 37% of hypertensive diabetic patients with BMI <25.

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Fig. 1: Incidence of microalbuminuria among first diagnosed diabetic patients.

Fig. 2: Incidence of microalbuminuria hypertensives and non-hypertensives.

among

Fig. 3: Incidence of microalbuminuria among persons with only high systolic blood pressure (SBP), only high diastolic blood pressure (DBP) and those with both.

Fig. 4: Incidence of microalbuminuria among person with normal BMI (<25), overweight (BMI >25 & <30) and obese (BMI >30).

Fig. 5: Microalbuminuria incidence among person with either body mass index (BMI) >25 or hypertension and those with both.

Fig. 6: Incidence of Microalbuminuria among persons with high waist hip (W/H) ratio and those with normal waist hip (W/H) ratio.

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Discussion

Incidence of microalbuminuria in the initially diagnosed diabetic patients in this study was 17% by dipstick method, whereas the incidence among newly diagnosed diabetic patients using immunoturbidometric test was 27% in a study conducted in South India.[1] The difference in the incidence may be due to large sample size and the sensitivity and specificity of immunoturbidometric assay compared to the albumin specific dipstick method. The prevalence of microalbuminuria increased with the increase in duration of diabetes. Altogether 27.5% of the newly diagnosed diabetic subjects had microalbuminuria by immunoturbidometric assay kits. At the time of diagnosis of diabetes, 24 among 138 patients had microalbuminuria which is a significant and is an opportunity to find the involvement of kidney at the most early stage and taking measures to prevent its progression. Among the other parameters measured, it is probably evident that incidence of microalbuminuria is increased among hypertensive diabetic patients i.e. 14 among 38 having 37% of chances than in non-hypertensive patients i.e. 10 among 100 having a chance of 10%. The result on statistical correlation (p=0.001, r=0.292) between hypertension and microalbuminuria also found to be significant. This also correlates with many studies about the incidence of microalbuminuria among the hypertensive diabetic patients.[6,7,8] In this study, the incidence of microalbuminuria among patients with BMI <25; BMI >25 & <30 and those with BMI >30 is 16%, 15% and 30% respectively. Statistical association of Body mass index and microalbuminuria is p=0.6 and r=0.04, hence Body mass index is not directly associated with microalbuminuria. Though obesity is a general risk factor for cardiovascular problems and many others, there is no statistical significance of incidence of microalbuminuria among the overweight and obese population. In a study conducted among 25 patients with body mass index >25, Blood pressure and urinary albumin excretion rate in hypertensive patients with obesity significantly decreased with weight reduction.[9] Hence there may indirect relevance of microalbuminuria and body mass index. And further studies in this topic may widen our knowledge and fill the gap in the understanding. The incidence of microalbuminuria among those with high waist hip ratio was 24% were as those with moderate waist hip ratio was 14%. Though there was striking incidence of microalbuminuria in patients with abdominal obesity compared to general obesity, there is no statistical correlation (p=0.138, r=0.127) between microalbuminuria and waist-hip ratio in our study. In a multinational, observational study, 20828 hypertensive out-patients

from 26 countries had participated and the suggested that increasing Waist circumference confers an incremental risk for microalbuminuria at any level of BMI, underlining the prognostic importance of abdominal fat accumulation beyond general obesity.[10] This deviation result may be due to small group of people involved in our study or due to short term follow up or only hypertension is strongly correlated to microalbuminuria when compared to diabetes. Testing strategies involving dipstick and laboratory Albumin creatinine ratio (ACR) measurements or dipstick tests had similar accuracy. The costs of using dipstick tests were overall lower than laboratory ACR-based testing. [11] Anyway of the urine screening tests, the dipstick test is inexpensive, easy and rapid to perform, des not delay testing the microalbuminuria, since there is no wait for the screening test. Diabetic patients with micro- or macroalbuminuria should be carefully controlled in order to prevent or to decrease deterioration of renal function due to diabetic nephropathy. Hence steps should take for the early detection of microalbuminuria and to prevent further involvement of the kidney giving more preference to the hypertensive diabetic patients since there are at high risk.

Conclusion

Incidence of microalbuminuria is significantly associated with hypertension than body mass index and waist hip ratio. Microalbuminuria is the earliest detector for the kidney involvement as well as cardiovascular involvement and screening for it in the earliest stage helps to reduce the occurrence of nephropathy. The incidence of microalbuminuria of 17% during first diagnosis of diabetes indicates the inadequacy in the health care system regarding the screening programs in the rural population. Thus improvements must be made in the health care system to reduce the incidence rate by effective screening program.

Reference 1.

Varghese A, Deepa R, Rema M, Mohan V. Prevalence of microalbuminuria in type 2 diabetes mellitus at a diabetes centre in southern India. Postgrad Med J 2001;77:399-402.

Mogensen CE. Preventing end-stage renal disease. Diabet Med 1998;15 Suppl 4:S51-6.

Kong NC, Chia YC, Khalid BA, et al. Microalbuminuria prevalence study in hypertensive type 2 diabetic patients in Malaysia. Med J Malaysia 2006 Oct;61(4):457-65.

Lehmann R, Spinas GA. Diabetic nephropathy: significance of microalbuminuria and proteinuria in Type I and Type II diabetes mellitus. Praxis. 1995;84(44):1265-71.

Valensi P, FerriĂ¨re F, Attali JR, Erault C, Modigliani E, Delrieux C, Sebaoun J. Microalbuminuria in diabetics with moderate hypertension. Arch Mal Ceur Vaiss. 1986;79(6):785-9.

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Zarini GG, Exebio JC, Gundupalli D, Nath S, Huffman FG. Hypertension, poor glycemic control, and microalbuminuria in Cuban Americans with type 2 diabetes. Int J Nephrol Renovasc Dis 2011;4:35-40.

Vasović O, Zamaklar M, Lalić K, Milosević D, Zikić L, Popović L. The prevalence of hypertension and microalbuminuria in diabetes mellitus type 1 and type 2. Srp Arh Celok Lek. 2005;133(5-6):229-32.

Descamps O, Buysschert M, Ketelslegers JM, Hermans M, Lambert E. Microalbuminuria in a population of 653 patients with type 1 and 2 diabetes. Diabete Metab 1991;17(5):469-75.

Ohashi H, Oda H, Ohno M, Watanabe S. Weight reduction improves high blood pressure and microalbuminuria in hypertensive patients with obesity. Nihon Jinzo Gakkai shi 2001; 43(4):333-339.

10. Thoenes M, Reil JC, Khan BV, et al. Abdominal obesity is associated with microalbuminuria and an elevated cardiovascular risk profile in patients with hypertension. Vasc Health Risk Manag. 2009;5(4):577-85. 11. Nagrebetsky A, Jin J, Stevens R, et al. Diagnostic accuracy of urine dipstick testing in screening for microalbuminuria in type 2 diabetes: a cohort study in primary care. Fam Pract. 2013 Apr;30(2):142-52. 12. Mur Martí T, Franch Nadal J, Morató Griera J, Llobera Serentill A, Vilarrubias Calaf M, Ros Espin C. Nephropathy and microalbuminuria in type II diabetes. Aten Primaria. 1995;16(9):516-24.

A-207 13. Weir MR. Albuminuria predicting outcome in diabetes: incidence of microalbuminuria in Asia-Pacific Rim. Kidney Int Suppl. 2004 Nov;(92):S38-9. 14. Gupta DK, Verma LK, Khosla PK, Dash SC. The prevalence of microalbuminuria in diabetes: a study from north India. Diabetes Res Clin Pract 1991 May;12(2):125-8. 15. Lutale JJ, Thordarson H, Abbas ZG, vetvik K. Microalbuminuria among Type 1 and Type 2 diabetic patients of African origin in Dar Es Salaam, Tanzania. Vetvi BMC Nephrol 2007;8:2. 16. Hoffmann IS, Jimenez E, Cubeddu LX. Urinary albumin excretion in lean, overweight and obese glucose tolerant individuals: its relationship with dyslipidaemia, hyperinsulinaemia and blood pressure. J Hum Hypertens 2001 Jun;15(6):407-12. 17. Rossi MC, Nicolucci A, Pellegrini F, et al. Identifying patients with type 2 diabetes at high risk of microalbuminuria: results of the DEMAND (Developing Education on Microalbuminuria for Awareness of reNal and cardiovascular risk in Diabetes) Study. Nephrol Dial Transplant. 2008;23(4):1278-84. 18. Viswanathan M, Snehalatha C, Bhattacharyya PK, Mohan V, Ramachandran A. Microalbuminuria in NIDDM patients in south India. Indian J Med Res 1991;94:125-9. 19. Lum G. How effective are screening tests for microalbuminuria in random urine specimens? Ann Clin Lab Sci 2000;30(4):406-11.

*Corresponding author: Dr. Vimal Chander R, 216, Mahatma Gandhi 2nd Street, Thiruverkadu Cooperative nagar, Chennai – 600077. India Phone: +91 9790645729 Email: rvimalchander@gmail.com Date of Submission : 04.01.2017 Date of Acceptance : 30.01.2017 Financial or other Competing Interests: None. Date of Publication : 15.04.2017

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eISSN: 2349-6983; pISSN: 2394-6466

Original Article DOI: 10.21276/APALM.1269

Blood Transfusion Profile of Beta Thalassemia Major Patients Attending a Tertiary Care Hospital Mallikarjun. A. Pattanashetti1*, Ganga S. Pilli2 and Laxmi A Pattanashetti3 1

Department of Pathology, S. Nijalingappa Medical College and HSK Hospital and Medical Research Centre, Bagalkot, Karnataka, India 2 Department of Pathology, KLE University’s Jawaharlal Nehru Medical College, Belagavi. Karnataka, India 3 Department of Pharmacology, KLE College of Pharmacy, Nehru Nagar, Belagavi, Karnataka, India

ABSTRACT Background: Beta (β) thalassemia major is most common monogenic disorder in the world. Around 1,00,000 children are born each year with the severe homozygous state of the disease in India. The objectives of this study was to assess clinical data and the blood transfusion profile of Beta thalassemia major patients attending tertiary care hospital. This was undertaken as very few studies have been done in this region of the vast country. Methods: The study was done at a tertiary care teaching hospital from January 2014 to December 2014. Universal sampling method was used and 35 β thalassemia major patients who received blood transfusions at 2 to 4 weeks interval in the hospital were included in this study. Clinical details and blood transfusion record was collected on proforma for all patients and data interpreted. Result: Males (80%) outnumbered females (20%) with male to female ratio of 4:1. Nearly half of the study population was aged between 10 to 12 years (51.43). Majority of the patients had one blood transfusion per month (91.43%) and were on chelation therapy (71.43%). Majority of the patients received 100 to 125 blood transfusions during their lifetime (28.57 %). Least number of blood transfusions of more than 250 transfusions was taken by one patient (2.86 %). Conclusion: The present study describes the transfusion profile of β thalassemia major patients attending the tertiary care hospital and emphasizes on maintenance of transfusion record of β thalassemia major patients for better management of these patients. Keywords: Β Thalassemia; Transfusion; Chelation.

Introduction

Beta (β) thalassemia syndromes are a group of hereditary blood disorders characterized by reduced or absent β globin chain synthesis, resulting in reduced hemoglobin (Hb) in red blood cells (RBC), decreased RBC production and anemia. Most thalassemias are inherited as recessive traits. β thalassemia is caused by the reduced (β+) or absent (β0) synthesis of the β globin chains of the hemoglobin tetramer, which is made up of two α globin and two β globin chains (α2 β2). Three clinical and hematological conditions of increasing severity are recognized, i.e., β thalassemia carrier state, thalassemia intermedia, and thalassemia major.[1] Although reliable data are still lacking for many regions of the world, recent data indicate that about 7% of the world’s population is a carrier of a hemoglobin disorder, and that 3,00,000-5,00,000 children are born each year with the severe homozygous state of the disease worldwide. [1] Every year approximately 1,00,000 are born with thalassemia in India.[2] The carrier rate for thalassemia gene varies from 1-3% in southern India to 3-15% in Northern India. [3]

These patients with BTM require regular blood transfusions to survive. Regular transfusion with packed red cells is recommended to maintain a pretransfusion hemoglobin threshold not exceeding 9.5 g/dl, which seems to be associated with adequate marrow inhibition and a relatively low iron burden.[6] Transfusions should generally be given at an interval of three to four weeks. Transfusions should be scheduled in advance and maintained at a fixed schedule. This enables patients and families to establish routines and will improve quality of life. In patients with severe anemia (hemoglobin less than 5 g/dL) or cardiac compromise, the rate of transfusion should be reduced to 2 mL/kg per hour to avoid fluid overload. Diuretics such as furosemide (1 to 2 mg/kg) may be necessary for some patients. If cardiac insufficiency is present, higher pretransfusion hemoglobin levels (10 to 12 g/dL) should be maintained with smaller volume transfusions given every one to two weeks. The only curative treatment for this disease at present is bone marrow transplantation or stem cell transplantation. Management of thalassemia is through lifelong blood transfusion and iron chelation therapy. Even this conventional treatment is often unavailable for patients in remote areas.

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There is lack of studies in this region of country describing blood transfusion profile of β thalassemia major patients. Hence, the present study was undertaken to assess the blood transfusion profile of these patients.

version 20. Categorical data was expressed in terms of rates, ratios and percentage. Continuous data was expressed as Mean ± standard deviation, median and range.

Materials and Methods

A total of 35 patients registered under Blood Bank with β thalassemia major were included in the study. On physical examination splenomegaly and hepatomegaly were present among 65.71% and 14.29% of the patients respectively. In the present study, the mean age was 13.46 ± 3.67 years and median age was 12 years with younger patients being 10 years and oldest being 23 years as shown in Table I. Majority (80%) of the patients were males and the male to female ratio was 4:1.The mean duration of disease was 12.29±3.66 years and the mean total number of transfusions was 151.4±45.65 among all the patients as shown in Table II.

Result

The present cross-sectional study was done at a tertiary care hospital from January 2014 to December 2014. Universal sampling method was used and 35 β thalassemia major patients who received blood transfusions were selected during the study period. Prior to the commencement, ethical clearance for the study was obtained from the Institute ethics committee. The objectives of this study were to assess the clinical data and transfusion record of these patients. All known diagnosed cases of β thalassemia major who were 10 years of age and above, and have received blood transfusions at two to four weeks intervals with or without iron chelation therapy in the tertiary care hospital were included. Patients who are known cases of other types of thalassemias and hemoglobinopathies and patients on transfusion dependent anemia other than β thalassemia were excluded from the study. Written informed consent was obtained from the selected patients. These patients were interviewed for the demographic details and history of disease. The clinical examination was done for all patients. Blood transfusion data was collected in detail. Proforma consisted of data regarding total number of blood transfusions during lifetime, number of transfusions per month and Chelation therapy.

The commonest age group was 10 to 12 years, comprised of 51.43% of the patients followed by 13-15 years age group (25.71 %). Most of the patients were aged between 7 to 12 months (45.71%) and 12 to 18 months (42.86%) at the diagnosis of β thalassemia major . The history of splenectomy was noted in 17.14% of the patients. Majority of the patients received one transfusion per month (91.43%) as shown in Table III. Majority (71.43%) of patients were on chelation therapy. Majority of the patients received one transfusion per month (91.43%) and majority of the patients received 100 to 125 blood transfusions during their lifetime (28.57 %) followed by 126 to 150 blood transfusions (22.86 %) as shown in Table II. Least number of blood transfusions of more than 250 transfusions was taken by one patient (2.86 %) as shown in Table III.

The data obtained was coded and entered into Microsoft Excel Spreadsheet. The data was analysed using SPSS

Table I: Variable

Mean

Age (Years)

13.46

3.67

Age at diagnosis (Months)

8.94

Duration of disease (Years)

Median

Range Min

Max

6.15

12.29

3.66

11.5

Frequency of transfusion (/Month)

1.09

0.28

Total number of blood transfusions

151.40

45.65

139

288

Table II : Gender distribution

Chern et al

Amina adil et al

Khalifa et al

Present study

Male

Female

M: F ratio

0.85

1.21

1.33

4.83

Total

124

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Transfusion Profile of Thalassemia Major Patients

Table III Variables

Age at Diagnosis (Months)

Duration of disease (Years)

Frequency of Transfusion

Number of transfusions

Chelation Therapy

Discussion

Sub-groups

Total No.

6 or less

8.57

7 to 12

45.71

12 to 18

42.86

> 18

2.86

Total

100.00

5 to 10

40.00

11 to 15

40.00

16 to 20

17.14

> 20

2.86

Total

100.00

91.43

8.57

Total

100.00

< 100

5.71

100 to 125

28.57

126 to 150

22.86

151 to 175

17.14

176 to 200

8.57

201to 225

8.57

226 to 250

5.71

> 250

2.86

Total

100.00

Yes

71.43

28.57

Total

100.00

β thalassemia major is a homozygous state which causes hemolytic anemia demanding regular blood transfusions. The availability of safe blood transfusions with adjuvant chelation therapy has facilitated and extended the survival rates of these patients and now their life expectancy has escalated to fourth and fifth decades. The patient’s weight and pre-transfusion hemoglobin and the volume of transfusion should be recorded at each visit. These values should be periodically reviewed to assess the volume of blood required to maintain the desired pre-transfusion hemoglobin level. Alloimmunization is a frequent problem that can be prevented by transfusing blood matched for the patient’s extended red blood cell phenotype. An alloantibody screen should be performed prior to each transfusion. The other complications of blood transfusion include the risk of mismatched transfusion, allergic reactions, and febrile, non-hemolytic reactions.

The mean age observed in the present study was close to that of Chern et al. [4] (14.8 ± 6.9 years) and comparable with the other study from Tehran (15.20 ± 3.1 years) and a study by Khalifa et al [5] (15.9 ± 3.1 years). In a study by Najafipour F et al. [6] in Iran reported mean age was 15.62 ± 4.44 with youngest patient being 10 years and oldest being 27 years. In another study by Khalifa et al [5] showed age range of patients to be 10-30 years as compared to the present study where the age range is 10-23 years. In the present study majority of the patients (80%) were males with male to female ratio of 4:1. Similarly, study done by Khalifa et al [5] showed majority of patients were males. The sex distribution pattern observed in the present study was similar to other studies from Kolkata[7] and Rawalpindi. [8] Considering 10 gm% (in accordance with the moderate transfusion regimen) of pretransfusion hemoglobin as the cut off between adequately transfused and under transfused patients, we found that among those receiving transfusions

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Pattanashetti et al. once a month, 100% were under transfused. Similar percentages for those receiving 1 and 2 transfusions per month were 91.42%, and 8.58 % , respectively The following measures would be optimum for the thalassemia care. Programs that provide acceptable care, including transfusion of safe blood and supportive therapy by chelation, must be established. A careful record of transfused blood should be maintained for each patient, including the volume of the administered units, haematocrit of the donor units and weight of the patient. With this available information, it is possible to calculate transfusion requirements for each patient, which is valuable in early identification of hypersplenism thereby prompting timely splenectomy. Blood transfusion exposes the patient to a variety of risks. Thus, it is vital to continue to improve blood safety and to find ways of reducing transfusion requirements and the number of donor exposures. Adverse events associated with transfusion include: Nonhemolytic febrile transfusion reactions, Allergic reactions, Acute hemolytic reaction, Delayed transfusion reactions, Autoimmune hemolytic anemia, Transfusion-related acute lung injury (TRALI) , Transfusion-associated circulatory overload, Transmission of infectious agents including viruses, bacteria and parasites, are a major risk in blood transfusion. [9]

Conclusion

Overall, the present study describes the blood transfusion profile of Beta thalassemia major patients. These patients need comprehensive care. Transfusion record of all Beta thalassemia major patients needs to be maintained regularly in the hospital blood bank for transfusion requirements of patient and also blood bank. The present study critically evaluated the current transfusion regime.Â Systemic effects of multiple transfusions should be rigorously and meticulously studied.

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Acknowledgement

The authors would like to thank the Department of Pathology and Department of Pediatrics, KLE Universityâ&#x20AC;&#x2122;s Jawaharlal Nehru Medical College , Belgaum for their support. We also thank Mr. S.V. Virgi of KLES Blood Bank and concerned laboratory personnel for all the assistance in this study.

References 1.

Cao A, Galanello R. Beta thalassemia. Genet Med 2010;12:61-76.

Management of birth defects and haemoglobin disorders: Report of a Joint WHO-March of Dimes Meeting. Geneva: World Health Organization; 2006.

Sarnaik, AS. Thalassemia and Related Hemoglobinopathies. Indian J Pediatr 2005;72(4):319-24.

Chern JP, Su S, Lin KH, Chang SH, Lu MY, Jou ST, et al. Survival, mortality and complications in patients with beta thalassaemia major in northern Taiwan. Pediatr Blood Cancer 2006;47:432-7.

Khalifa AS, Salem M, Mounir E, El-Tawil MM, El-Sawy M, Abd Al-Aziz MM. Abnormal glucose tolerance in Egyptian beta-thalassemic patients: possible association with genotyping. Pediatr Diabetes 2004;5:126-32.

Najafipour F, Sorkhabi RS, Aghai NH, Zareizadeh M, Bahrami A. Importance of OGTT for diagnosis of Diabetes in thalassemia major patients J Gorgan Uni Med Sci 2008;10(3):71-6.

Mallik S, Chatterjee C, Mandal PK, Sardar JC, Ghosh P, Manna N. Expenditure to Treat Thalassaemia: An Experience at a Tertiary Care Hospital in India. Iranian J Publ Health 2010;39(1):78-84.

Rai ME, Tanoli ZM, Gandapur ASK. Clinical spectrum of patients of beta thalassemia: a review of fifty four patients. Glomal J Medical sciences 2005;3(2):55-60.

Guidelines for the Clinical Management of Thalassaemia. 2nd Revised ed., No. 9, Nicosia (CY): Thalassaemia International Federation; 2008.

*Corresponding author: Dr. Mallikarjun .A. Pattanashetti , Assistant Professor, Department of Pathology, S. Nijalingappa Medical College and HSK Hospital & MRC Bagalkot, Karnataka, India Email: mallikarjun2030@gmail.com Date of Submission : 08.01.2017 Date of Acceptance : 28.01.2017 Financial or other Competing Interests: None. Date of Publication : 15.04.2017

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Original Article DOI: 10.21276/APALM.1276

A comparison of Effect of Iron Deficiency Anemia on HbA1c Levels in Controlled Diabetics and Non-diabetics: A Cross Sectional Analysis of 300 Cases Lavanya Rajagopal1*, Sundaram Arunachalam1, Shivashekar Ganapathy1, Balaji Ramraj2 and Veena Raja3 2

1 Department of Pathology, SRM Medical College Hospital and Research Centre, Kattankulathur, Chennai India Department of community medicine, SRM Medical College Hospital and Research Centre, Kattankulathur, Chennai India 3 Department of Pathology, SRM Medical College Hospital and Research Centre, Kattankulathur, Chennai India

ABSTRACT Background: Diabetes Mellitus (DM) has become a major health problem worldwide. American Diabetes Association has considered HbA1C levels ≤ 6.5 % as the prime target for glycemic control and as a diagnostic criterion for DM. Anemia is common in DM (8-66%). Studies on alteration of HbA1C in IDA have conflicting results. Objectives: To identify and compare the effect of IDA on HbA1C levels among controlled diabetics (Fasting plasma glucose (FPG) <126mg/ dl since last 6 months) and non-diabetics and its variation according to the degree of anemia. Methods: This cross-sectional study done in SRM Medical College Hospital and Research Centre, Chennai includes 150 Controlled diabetics (75 with IDA and 75 without IDA) and 150 non diabetics (75 with IDA and 75 without IDA). HbA1C, complete hemogram, iron profile and FPG were tested. Medical history was recorded. Results: The mean HbA1C in controlled diabetics with and without IDA were 8.81±0.13 & 5.79±0.01 respectively (P<0.05) and in nondiabetics with and without IDA were 6.84±0.07 & 5.12±0.04 respectively (P<0.05). The difference between no, mild, moderate and severe anemia in both diabetics and non-diabetics was statistically significant (p< 0.05). Mean HbA1C% was highest in groups with severe anemia. Conclusion: IDA falsely elevates HbA1C level independent of blood glucose concentration in both controlled diabetics and non-diabetics. Hence prior to alteration of treatment regimen based on HbA1C for diabetes, IDA should be diagnosed and corrected. Concurrent evaluation for anemia is critical to correctly interpret glycemic status in Indian population with prevalent IDA. Keywords: HbA1C, Iron deficiency Anemia, Diabetics, Non-diabetics

Introduction

Diabetes has become a major health problem worldwide with an estimated 300 million people to be diagnosed with the disease in the next 10 years and 370 million by 2030. [1] In 2010, American Diabetes Association (ADA) has considered HbA1C levels as the prime target for glycemic control and as a diagnostic criterion for Diabetes Mellitus (DM). HbA1C ≥ 6.5% has been established for the diagnosis of DM for its high specificity and certified by the World Health Organization (WHO) in 2011.[1,2,3] Anemia is common among DM and its incidence ranges from 8-66%.[4] Several mechanisms have been discussed about the association of anemia in DM and its complications like nephropathy.[5,6] Initially it was believed that HbA1C was affected only by blood glucose levels.[4,5] However certain study results have proven that HbA1C levels are altered by various other co-existing factors, along with DM, especially that of IDA which is a major public health problem in

developing countries like India. Other factors interfering with HbA1C levels are hemoglobinopathies, renal impairment, pregnancy, hypothyroidism, hemolytic anemia etc. Christy AL et al reported that controlled plasma glucose levels for last 3 months correlates well with controlled HbA1C (< 6.5%).[7] One of the well-known pathological ill effects of IDA in the biological system is the glycosylation of proteins.[8] Although different studies have been carried out to analyze the influence of IDA on HbA1C levels in both diabetic and non-diabetic population individually, only very few studies have been conducted comparing HbA1C variation in both these groups. Due to the variation in the results of multiple studies, we decided to investigate and compare the effects of IDA on HbA1C levels in both controlled diabetic and non-diabetic Indian adults.

Materials and Methods

In this cross-sectional study, we collected data of 1360 individuals aged >18years who consulted our SRM

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Medical College Hospital and Research Centre, Chennai, Tamil Nadu between February 2016 to November 2016.

(TPTZ method), serum ferritin (Bio-Rad Quanimune Ferrin IRMA, Bio-Rad lab)

Of the 1360 persons enrolled in this study, 230 were diabetics and 1130 were non-diabetics. After exclusion of individuals with hypothyroidism, renal insufficiency (elevated serum urea, creatinine), hemoglobinopathies, Pregnancy, Fasting plasma glucose (FPG) >126 mg/dl, hemolytic anemia and those who completed laboratory investigations, 170 controlled diabetics (FPG level is <126 mg/dl since last 6 months) and 365 non diabetics were finalized for our study.

Statistical Analysis: The data are presented as mean ±S.D for continuous variables. Group means were compared by student t-test. Pearson’s co-efficient of correlation was used to determine the correlation between two variables. p value < 0.05 was considered significant.

Among 170 controlled diabetics, 95 were anemic (56%) out of which 75 were diagnosed as IDA and others were excluded. Among 365 non-diabetics, 90 were anemic. Out of 90 nondiabetics with anemia, 75 had IDA and others were excluded. Patients with IDA were chosen by their hemoglobin level (Hb <13gm% in males and <12gm% in females) based on definition of WHO and with predominantly microcytic indices [Mean Corpuscular Volume (MCV) < 76 fl], hypochromic indices [Mean corpuscular Hemoglobin (MCH) < 27 pg/cell] and [Mean Corpuscular Hemoglobin Concentration (MCHC) < 32 g/dl], and microcytic hypochromic picture in peripheral smear. This is later confirmed by low serum iron (< 59 µg/ dl in males and < 37 µg/dl in females) & low serum ferritin (< 15 ng/ml in males and <9 ng/ml in females).[9] Age, Sex and FPG matched controls were selected for each group and the data results were analyzed. Blood samples from both groups were tested for FPG, HbA1C, Hb, RBC count, Hct, MCV, MCH, MCHC, Serum iron and Ferritin. Medical history was recorded On the basis of hemoglobin level, anemic patients were further categorized as Mild anemia (male 12-12.9 gm/dl and female 11-11.9gm/dl), Moderate anemia (male 9-11.9 gm/dl and female 8-10.9 gm/dl) and Severe anemia (male <9 gm/dl and female <8 gm/dl). Measurements: Hb, MCV, MCH, MHC were estimated by SYSMEX XT-1800i analyzer. HbA1C estimated by HPLC method in Bio-Rad D10 analyzer. Glucose oxidase / peroxidase method for plasma glucose and serum iron

Results

The mean HbA1C in controlled diabetics with and without IDA were 8.81±0.13 & 5.79±0.01 respectively and in non-diabetics with and without IDA were 6.84±0.07 & 5.12±0.04 respectively. This result show that the mean HbA1C was higher in those with IDA than in those without IDA in both diabetics and non-diabetics which was statistically significant (p < 0.05) in both groups. [Table1/ Fig 1]. The mean Hb, Hct, MCV, MCH & MCHC in controlled diabetics with and without IDA were statistically significant p (<0.05). [Table 2] Among controlled diabetics, 11 had mild anemia with mean HbA1C % of 7.40±0.70, 39 had moderate anemia with mean HbA1C % of 8.50±0.11 and 25 had severe anemia with mean HbA1C % of 9.15±0.17. Similarly in non-diabetics, 40 had mild anemia with mean HbA1C% of 6.57±0.09%, 28 had moderate anemia with mean HbA1C % of 7.19±0.11 and 7 had severe anemia with mean HbA1C% of 8.31±0.01%. The mean serum iron and ferritin levels in controlled diabetics with and without IDA were 32.68±0.71 µg/ dl,10.06±0.79 µg/l and 75.26±0.79 µg/dl, 45.19±1.11 µg/l respectively which was statistically significant (p < 0.05). The mean serum iron and ferritin levels in non-diabetics with and without IDA were 42±0.61 µg/dl, 12.09±1.21 µg/l and 74±0.32 µg/dl, 41.06±0.43 µg/l respectively which was statistically significant (p < 0.05). The difference was statistically significant (p< 0.05) between no, mild, moderate and severe anemia in both controlled diabetics and non-diabetics and moreover mean HbA1C% was higher in groups with severe anemia in relation to HbA1C levels in patients without IDA. [Table 3/Fig 2]

TABLE 1: COMPARISON OF HbA1C% BETWEEN ANEMIC AND NOT ANEMIC IN CONTROLLED DIABETICS & NONDIABETICS. Parameters

IDA

Not anemic

T test

P value

Controlled diabetics

HbA1C %

8.81 ± 0.13

5.79 ± 0.01

23.974

< 0.05

Non-diabetics

HbA1C %

6.84 ± 0.07

5.12 ± 0.04

22.219

< 0.05

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Effect of IDA on HbA1C Levels

TABLE 2: Comparison of Red Cell Indices Between Anemic and Not Anemic in Controlled Diabetics & Nondiabetics PARAMETERS Hb (g/dl) Hct (%) MCV (fl) MCH (pg/cell) MCHC

SUBJECTS

IDA

NOT ANAEMIC

T TEST

P VALUE

Controlled diabetics

9.22 ± 0.23

13.83 ± 0.13

-17.483

< 0.05

Non diabetics

11.46 ± 0.08

14.31 ± 0.16

-16.078

< 0.05

Controlled diabetics

31.35 ± 0.71

41.61 ± 0.45

-12.221

< 0.05

Non diabetics

37.07 ± 0.29

42.20 ± 0.47

-9.328

< 0.05

Controlled diabetics

66.79 ± 1.53

87.40 ± 0.51

-12.791

< 0.05

Non diabetics

78.56 ± 0.22

86.19 ± 0.59

-3.425

< 0.05

Controlled diabetics

23.88 ± 0.50

29.04 ± 0.14

-9.910

< 0.05

Non diabetics

27.17 ± 0.33

29.75 ± 0.21

-6.511

< 0.05

Controlled diabetics

29.29 ± 0.29

32.72 ± 0.08

-11.409

< 0.05

Non diabetics

29.29 ± 0.29

32.72 ± 0.08

-11.409

< 0.05

TABLE 3: HbA1C Variation According to The Degree of Anemia

CONTROLLED DIABETICS

DEGREE OF ANEMIA

NONDIABETICS

NUMBER

MEAN HBA1C%

NUMBER

MEAN HBA1C%

5.79 ± 0.01

5.16 ± 0.04

MILD

7.40 ± 0.70

6.57 ± 0.09

MODERATE

8.50 ± 0.11

7.19 ± 0.11

SEVERE

9.15 ± 0.17

8.01±0.01

CONTROLLED DIABETICS ANOVA – 202.613, p – 0.0001 No anemia to mild anemia, T test: -7.591, p – 0.0001 Mild anemia to Moderate anemia, T test: -3.306, p – 0.001 Moderate anemia to Severe anemia, T test: -5.805, p – 0.0001 NON DIABETICS ANOVA - 229.815, p – 0.0001. No anemia to mild anemia, T test: -14.323, p – 0.0001 Mild anemia to Moderate anemia, T test: -4.483, p – 0.0001 Moderate anemia to Severe anemia, T test: -3.397, p – 0.0001 TABLE 4: Comparison of Present Study Hba1c Levels with Previous Studies Having Similar Results [Diabetics] Study

Year

Glycemic status

Number screened

IDA

Not-anemic

Significance

1999

10.1±2.7

8.2±3.1

p < 0.05

2010

7.4±0.2

6.9±0.1

p < 0.05

2014

120

6.8±1.4

5.6±0.6

p < 0.05

Present study

2016

150

8.8±0.1

5.7±0.01

p < 0.05

Tarim et al

Ng et al13 Christy et al

TABLE 5: Comparison Of Present Study Hba1c Levels with Previous Studies Having Similar Results [Non-Diabetics] Study

Year

Number screened

IDA

Not-anemic

Significance

El-Agouza et al6

2002

6.1±0.6

5.2±0.4

p < 0.05

2004

100

7.4±0.8

5.9±0.5

p < 0.05

Coban et al

Shanthi et al

2013

100

7.6±0.5

5.5±0.8

p < 0.05

Silva et al14

2015

122

5.6±0.4

5.3±0.4

p < 0.05

Present study

2016

150

6.8± 0.07

5.1± 0.04

p < 0.05

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TABLE 6: Previous Studies with Contradicting Hba1c Results from The Present Study [Non-Diabetics] Study

Year

Glycemic status

Number screened

IDA

Not-anemic

Significance

Ford ES et al17

2011

NDM

8296

5.5±0.1

5.4±0.2

p > 0.05

2012

NDM

100

4.6±0.6

5.5±0.6

p > 0.05

2014

NDM

5.9±0.4

6.5±0.3

p > 0.05

Sinha et al

Kalasker et al

TABLE 7: Comparison of Study Results Showing Hba1c Variation According to The Degree of Anemia Study

Year

Number screened

Silva et al20

2015

Present study

2016

Degree of Anemia

Significance

Mild

Moderate

Severe

122

5.3±0.40

5.5±0.40

5.6±0.40

5.7±0.40

P < 0.05

150

5.1±0.04

6.5±0.09

7.1±0.11

8.0±0.01

P < 0.05

Fig. 1: Comparison of Hba1c% in Study Groups with and Without Anemia.

Fig 2:HbA1C Variation in Mild, Moderate And Severe Anemia.

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Effect of IDA on HbA1C Levels

Discussion

Glycated hemoglobin reflects the glycemic status when monitored over 3 months and predicts the risk of long term complications in diabetics. Glycation of the NH2 terminal valine residue of the β-chain of hemoglobin results in the formation of HbA1C. It also identifies individuals who are at high risk for developing diabetes.[10] According to many study results, anemia is almost twice common in diabetics than non-diabetics.[4,11,12] Apart from blood glucose, HbA1c levels can be affected by factors unrelated to diabetes like IDA.[13] Increased HbA1C levels in IDA were explained by a) Quaternary structure of hemoglobin is altered leading to rapid glycation of globin chain .[14,15] b) Increase in the glycated fraction of hemoglobin due to decrease in total hemoglobin at a constant glucose level occurs because HbA1C is measured as a percentage of total Hemoglobin A.[16] c) higher average age of circulating erythrocytes noticed in IDA due to reduced red cell production lead to increased HbA1C levels.[14] Anemia is a major risk factor for cardiovascular complications and diabetic retinopathy in diabetes. But only very few studies has investigated whether IDA alter the value of HbA1C till now inspite being widely used as a diagnostic tool for DM . Thus leading to over or under diagnosis of DM when diagnosed based on the cutoff value < 6.5% of HbA1C as approved by ADA.[4,5,17] The results of this study show that HbA1C levels are spuriously elevated in the presence of IDA independent of blood glucose concentration in both controlled diabetics and non-diabetics. Our finding confirms the study results of Tarim et al, who reported HbA1C level is elevated in diabetics with IDA than with iron-sufficient controls. This may be explained by iron deficiency related changes in the quaternary structure of hemoglobin molecule increasing the glycation of globin chain.[18] This result also coincides with the study results of Christy et al who also observed that HbA1C levels were significantly higher in IDA patients and decreased after treatment with iron.[7] Ng et al concluded that iron and Erythropoietin stimulating agents in diabetic patients cause a significant fall in HbA1C values without a change in glycemic control.[19] [Table 4] Our results contradicts with the study results of Sharifi et al, who reported that there was no correlation between serum iron , serum ferritin and HbA1c in diabetic patients of either sex. Ferritin levels in patients with DM is high, but

not related to levels of HbA1c and blood glucose control. [20] Ford ES et al concluded that there was no evidence of difference in the relationship between fasting glucose and HbA1c when groups of anemic and non-anemic diabetic individuals with and without iron deficiency when examined individually.[21] Our study results are also consistent with the study done by El-Agouza et al in non-diabetics who reported that a decline in the Hb level might lead to increase in the glycated fraction at a fixed glucose level, because HbA1C is measured as a percentage of total Hb.[16] Our results were also in concordance with the study results of Shanthi et al, Coban et al, and Silva et al.[15,22,23] Coban et al showed a very large difference between HbA1C levels in nondiabetic patients with and without IDA.[22] Shanthi et al conducted study in non –diabetics and reported that iron deficiency was associated with higher proportions of HbA1C and suggested that iron status must be considered during the interpretation of the HbA1c concentrations in Diabetes mellitus.[15] Silva et al reported that IDA affects HbA1c levels and causes spurious increase in their results. Although these upward changes in HbA1c values are statistically significant, they may be not clinically relevant when the overall variability of the HbA1c test is considered. This effect is dependent on anaemia degree and the presence of mild anaemia is likely to have a minor effect on HbA1c levels.[23] [Table 5] Studies by Ford ES et al reported no significant difference in mean HbA1C concentration according to the IDA status as well as before and after iron treatment.[21] Sinha et al and Kalaskar et al contradicts with our results reporting that HbA1C levels are lowered in IDA.[13] [Table 6] Also Saudek et al suggested that red cell age was unlikely to be a significant factor in explaining the changes in HbA1C levels during the treatment of IDA and believed that the reported differences in HbA1C concentrations before and after iron supplementation were due to differences in the laboratory methods used for measuring HbA1C.[24] Ferritin is a storage form of iron, and it reflects the true iron status [WHO]. In our study, serum ferritin as well as serum iron level was indirectly proportional to HbA1C. As explained previously, in IDA, ferritin is decreased with increase in the red cell life span which is associated with increased HbA1C. This goes in hand with other study results of Shanthi et al and Raj et al.[15,25] We also analyzed HbA1c results in different degrees of anemia and found that HbA1C level increases as severity

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Rajagopal et al. of anemia worsens. This result of ours was in accordance with the results of Silva et al.[23] [Table 7] Because the above studies were performed mostly in subjects mostly in individuals without diabetes, they could not conclude whether the presence of IDA affect HbA1C level at the cutoff point of <6.5% vs ≥6.5% the newly recommended diagnostic cutoff point for diabetes by the ADA. This study has few limitations. They are small sample size and the results were obtained from a single centre and with a cross-sectional design, we couldn’t follow up after iron therapy.

Conclusion

Presence of IDA spuriously elevates HbA1C level independent of blood glucose concentration in both diabetics and non-diabetics. Hence prior to alteration of treatment regimen based on HbA1C for diabetes, IDA should be diagnosed and corrected. Also this study suggests concurrent measurement of iron, Hb, HbA1C is critical to correctly interpret glycemic status in Indian population where IDA is highly prevalent.

Acknowledgement

I would like to thank our Dean Dr. A. Sundaram, SRM Medical College Hospital and Research Centre for his support throughout the study. Am thankful to all the participating people for their cooperation.

References 1.

American Diabetes Association. Classification and diagnosis of diabetes. Diabetes care. 2015;38:S8-16

World Health Organization. Use of glycated haemoglobin (HbA1c) in diagnosis of diabetes mellitus: abbreviated report of a WHO consultation.

Cavagnolli G, Comerlato J, Comerlato C, Renz P, Gross JL, Camargo JL. HbA1c measurement for the diagnosis of diabetes: is it enough?. Diabetic Medicine. 2011;28(1):31-5.

Thomas MC, MacIsaac RJ, Tsalamandris C, Power D, Jerums G. Unrecognized Anemia in Patients With Diabetes A cross-sectional survey. Diabetes care. 2003;26(4):1164-9.

Tong PC, Kong AP, So WY, Ng MH, Yang X, Ng MC, et al. Hematocrit, independent of chronic kidney disease, predicts adverse cardiovascular outcomes in Chinese patients with type 2 diabetes. Diabetes care. 2006;29(11):2439-44.

Joss N, Patel R, Paterson K, Simpson K, Perry C, Stirling C. Anaemia is common and predicts mortality in diabetic nephropathy. QJM. 2007;100(10):641-7.

Christy AL, Manjrekar PA, Babu RP, Hegde A. Influence of Iron Deficiency Anemia on Hemoglobin A1C Levels in Diabetic Individuals with Controlled Plasma Glucose Levels. Iran Biomed J. 2014;18(2):88.

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Lapolla A, Traldi P, Fedele D. Importance of measuring products of non-enzymatic glycation of proteins. Clin biochem. 2005;38(2):103-15.

Bain BJ, Lewis SM. Dacie and Lewis Practical Haematology. 11th ed. Edinburgh: Elsevier Churchill Livingstone, 2012. Print.

10. Hong JW, Ku CR, Noh JH, Ko KS, Rhee BD, Kim DJ. Association between the presence of iron deficiency anemia and hemoglobin A1c in Korean adults: the 2011–2012 Korea National Health and Nutrition Examination Survey. Medicine. 2015 May 1;94(20):e825. 11. Sinha N, Mishra TK, Singh T, Gupta N. Effect of iron deficiency anemia on hemoglobin A1c levels. Ann Lab Med. 2012;32(1):17-22. 12. Cawood TJ, Buckley U, Murray A, Corbett M, Dillon D, Goodwin B, et al. Prevalence of anaemia in patients with diabetes mellitus. Irish J Med Sci. 2006;175(2):25-7. 13. Stevens PE, O’Donoghue DJ, Lameire NR. Anaemia in patients with diabetes: unrecognised, undetected and untreated? Current medical research and opinion. 2003;19(5):395-401. 14. Kalasker V, Kodliwadmath MV, Bhat H. Effect of iron deficiency anemia on glycosylated hemoglobin levels in nondiabetic Indian adults. Int J Med Hlth Sci. 2014;3(1):40-3. 15. Shanthi B, Revathy C, Manjula Devi AJ, et al. Effect of iron deficiency on glycation of haemoglobin in nondiabetics. J Clin Diagn Res. 2013;7(1):15–17. 16. El-Agouza I, Abu Shahla A, Sirdah M. The effect of iron deficiency anaemia on the levels of haemoglobin subtypes: possible consequences for clinical diagnosis. Clin Lab Haematol. 2002;24(5):285-9. 17. Zoppini G, Targher G, Chonchol M, Negri C, Stoico V, Pichiri I, et al. Anaemia, independent of chronic kidney disease, predicts all-cause and cardiovascular mortality in type 2 diabetic patients. Atherosclerosis. 2010;210(2):575-80. 18. Tarim ÖM, Küçükerdogan AY, Günay ÜN, Eralp ÖZ, Ercan I. Effects of iron deficiency anemia on hemoglobin A1c in type 1 diabetes mellitus. Pediatr Int. 1999;41(4):357-62. 19. Ng JM, Cooke M, Bhandari S, Atkin SL, Kilpatrick ES.The effect of iron and erythropoietin treatment on the A1C of patients with diabetes and chronic kidney disease. Diabetes Care. 2010;33(11):2310–3. 20. Sharif F, Sazandeh SH. Serum ferritin in type 2 diabetes mellitus and its relationship withHbA1c. Acta Medica iranica. 2004;42(2):142-5. 21. Ford ES, Cowie CC, Li C, Handelsman Y, Bloomgarden ZT. Iron-deficiency anemia, non-iron deficiency anemia and HbA1c among adults in the US. J Diabetes. 2011;3(1):67-73. 22. Coban E, Ozdogan M, Timuragaoglu A. Effect of iron deficiency anemia on the levels of hemoglobin A1C in nondiabetic patients. Acta Haematol. 2004;112(3):126-8.

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23. Silva JF, Pimentel AL, Camargo JL. Effect of iron deficiency anaemia on HbA1c levels is dependent on the degree of anaemia. Clinical Biochemistry. 2016;49(1):117-20. 24. Saudek CD, Herman WH, Sacks DB, Bergenstal RM, Edelman D, Davidson MB. A new look at screening and

diagnosing diabetes mellitus. J Clin Endocrinol Metab. 2008;93(7): 2447-53. 25. Raj S, Rajan GV; Correlation between elevated serum ferritin and HbA1c in type 2 diabetes mellitus. Int J Res Med Sci. 2013; 1(1): 12-15.

*Corresponding author: Dr. Lavanya Rajagopal, Door no. 902, B-Block, SRM Medical Staff Quarters, SRM Medical College Hospital and Research Centre, Kattankulathur, Pincode: 603203, Chennai, INDIA Phone: +91 8754131339 Email: drrlavan@yahoo.co.in Date of Submission : 10.01.2017 Date of Acceptance : 30.01.2017 Financial or other Competing Interests: None. Date of Publication : 15.04.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Original Article DOI: 10.21276/APALM.1278

Cytological and Histomorphological Correlation of Salivary Gland Lesions: An Experience At Rural Tertiary Healthcare Hospital Thangam R1, Vaishali Dhananjay Kotasthane1*, Dhananjay Shrikant Kotasthane1*, Koteeswaran G1 and Kannan N S2 Department of Pathology, Mahatma Gandhi Medical college and Research Institute, Pillaiyarkuppam, Pondicherry, India 2 Department of General Surgery, Sri Manakula Vinayagar Medical College and Hospital, Pondicherry, India

ABSTRACT Background: Salivary gland tumours account for 3-10% of all the head and neck neoplasm. FNAC is being increasingly used in the diagnosis of salivary gland tumours. The objective of this study was to evaluate the age, sex and site distribution and to evaluate the diagnostic accuracy of fine needle aspiration cytology (FNAC) in various salivary gland lesions in correlation with their histopathology, which helps in appropriate management of the patient. Methods: The present study was done at the pathology department, Mahatma Gandhi Medical College and Research Institute, Pondicherry. FNAC was done using 22 gauge needle and 10 ml syringe and smears were stained with H&E and Giemsa stains. Histopathology was assessed on routine H&E stained paraffin sections .Cyto-histo correlation was done and overall diagnostic accuracy, sensitivity and specificity were calculated. Results: The study was conducted on 122 patients having salivary gland lesions .The age ranged from 24-80 years. Benign tumours were common in 3rd decade, whereas malignant tumours in 6th decade of life. There was female preponderance with male to female ratio of 1:1.03.Parotid glands were commonly involved in 74(78.72%).Out of 94 neoplastic cases,78(82.97%)were benign and 14(14.89%) were malignant. Pleomorphic adenoma was the commonest lesion observed accounted for 68.08%. Conclusion: The Overall sensitivity, specificity and diagnostic accuracy were 80%, 94.74% and 91.66% respectively. FNAC of the salivary gland is safe, rapid ,accurate and reliable technique in the primary diagnosis of salivary gland lesions and useful in avoiding surgery (in inflammatory lesions) or limiting surgical procedures(in benign tumours). Keywords: Fine Needle Aspiration Cytology, Histopathology, Salivary Glands

Introduction

FNAC (Fine needle aspiration cytology) has an important role in diagnosis of salivary gland lesions. The salivary glands are subjected to multiple diseases. Hence salivary glands diseases create an inquisitive group of lesions for investigation. The salivary gland tumours accounts for about 6 percentage of tumours in the head and neck region. [1] FNAC of salivary gland tumour is accurate, tolerated and harmless to subjects.[2]Its easy accessibility makes FNAC a popular method for evaluation of tumours of salivary gland. [3] There is widespread acceptance of importance of FNAC in diagnosis of salivary gland lesions.[4] Biopsies or frozen sections of salivary tumours taken for treatment planning carries risk of bleeding, facial nerve injury or inflammation compared to FNAC where complications are very negligible. [4,5] In the present study, the utility of Fine Needle Aspiration Cytology in the diagnosis of salivary gland tumors is studied by correlating the cytological findings with the histopathological features which helps in early diagnosis and effective therapeutic approach.

Materials and Methods

The study was undertaken after approval of Institutional Human Ethical Committte.This is a study of fine needle aspiration of 122 salivary gland lesions. All neoplastic and non neoplastic lesions of salivary gland involving major and minor salivary glands in various sites were studied. The relevant clinical details were noted from the accompanying requisition forms and/or the patient case records. These included age, gender and location and clinical diagnosis. FNAC was done using 22 gauge needle and 10 ml syringe and the smears were wet fixed in isopropyl alcohol for Hematoxylin-Eosin and PAP stains and air-dried for Giemsa stains. Histopathology of the lesions,which were subjected to biopsy, was assessed on routine Haemotoxylin and Eosin stained paraffin sections.Cyto-Histological correlation was done and overall diagnostic accuracy,sensitivity and specificity was calculated.

Results

Fine Needle Aspiration was conducted on a total of 122 cases of lesions of salivary gland. The following observations were made. The patient age ranged from 11

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Cyto-histological Correlation of salivary Gland Lesions

years to 80 years and the maximum number of cases were in the age group of 41-50years(29.5%), followed by 3140yrs(13%) and 51-60yrs(12%). Gender Distribution of Salivary Gland Lesions: In the present study, a female predilection was seen. Among the 122 cases studied, 62 cases (50.82%) were females and 60 cases (49.18%) were males. The female to male ratio was 1.03 Site Distribution of Salivary Gland Lesions: In the present study, the parotid gland (75.40%) was the commonest site involved followed by submandibular gland (22.95%) and minor salivary glands (1.65%) Cytological Findings in Various Salivary Gland Lesions: Out of the 122 cases of salivary gland swellings, 26 (21.3%) were non-neoplastic lesions and 96 (78.7%) were diagnosed as neoplastic lesions. Among the salivary gland lesions, neoplastic lesions 96 (78.7%) were more common and among the neoplastic lesions, benign 80 (83.3%) were more common. Malignant tumours comprised 16.66% (16 cases). Frequency and Site Distribution of Tumours of Salivary Gland: In the present study, the most common site of involvement of salivary gland tumour was parotid gland (78.72%) and second most frequent site was submandibular gland (21.27%). (Table1)

Among the benign tumours, parotid was the most common site (82.5%) followed by submandibular gland (17.5%). Malignant tumours equally involved parotid gland and submandibular gland..(Table 1) Parotid gland was more commonly involved in neoplastic lesions as compared to other salivary glands and this was found to be statistically significant.(p=0.0274) (Table 1) Distribution of Neoplastic Lesions: Among the benign tumours, Pleomorphic Adenoma was the most common; accounting for 66.66% of all tumours and Warthinâ&#x20AC;&#x2122;s tumour was the second most common benign tumour constituting 6.25% of all tumours. (Table 2) Mucoepidermoid Carcinoma was the most common malignant tumour constituting 12.50% of all tumours and Adenoid Cystic Carcinoma and Adenocarcinoma - NOS type were the second most common malignant tumours constituting 2.08% of all tumours .(Table:2) Cyto-Histo Correlation of Salivary Gland Lesions: Out of 122 lesions, Histopathological correlation was available in forty-eight cases, It is shown in Table 3. The parameters calculated showed sensitivity as 80%, specificity as 94.74%, Positive predictive value as 80%, Negative predictive value as 94.74%, Diagnostic accuracy as 91.66%.

Table 1: Percentage frequency and site distribution of tumors of salivary gland. Total no:

Benign

Malignant

Parotid

37(78.72%)

33(82.5%)

4(50%)

submandibular

10(21.27%)

6(17.5%)

4(50%)

Minor salivary gland

1(2.12)

1(2.56)

Total

Chi square(x2 )=6.3752;p=0.0411;p<0.05,s=significant

Table 2: Distribution of neoplastic lesions. Cytological diagnosis

No. of cases

Percentage

Pleomorphic adenoma

66.66

Warthinâ&#x20AC;&#x2122;s tumor

6.25

DD:Pleomorphic adenoma/Myoepithelioma

10.41

Mucoepidermoid carcinoma

12.5

Adenoid cystic carcinoma

2.08

Adenocarcinoma NOS

2.08

TOTAL

100

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Table 3: Cyto-Histo Correlation of Salivary Gland Lesions. Histopathological diagnosis available Cytological diagnosis

Chronic Pleomorphic Basal cell Warthinâ&#x20AC;&#x2122;s Mucoepider- Adenoid cystic Total sialadenitis adenoma adenoma tumor moidcarcinoma carcinoma

Chronic sialadenitis (n=24)

Myoepithelioma/ Pleomorphic adenoma (n=10)

Pleomorhic Adenoma (n=64)

4 4 2

Warthimâ&#x20AC;&#x2122;s tumor (n=6)

Mucoepidermoid carcinoma (n=12)

Total

2 8

10 2

Fig. 1: FNAC of pleomorphic adenoma-Epithelial and myoepithelial cells in a chondromyxoid background (H&E x 10).

Fig. 2: HPE-Pleomorphic adenoma with chondroid differentiation [10x H&E].

Fig. 3: FNAC of Mucoepidermoid carcinoma-clusters of squamous cells in a mucoid background (H&Ex10).

Fig. 4: HPE of Mucoepidermoid cells-intermediate squamoid cells (H&E x10).

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Discussion

Fine needle aspiration gives useful information compared to other diagnostic techniques. The open biopsy gives an accurate diagnosis but it has a disadvantage of contaminating the operative field with tumour cells and difficulty in subsequent surgical management. The main objective of fine needle aspiration is to find if the mass is neoplastic or non neoplastic and in neoplastic whether it is benign or malignant. This preoperative diagnosis about the tumor type can be helpful to plan the best surgical approach. Though, some complications with fine needle aspirations have also been rarely found such as pain, infection, tumour seeding, bleeding and tumour necrosis, they do not usually compromise the accurate diagnosis by histology except sometimes the infarction rarely obscure the diagnosis. [6,7] Another issue encountered is inadequate aspiration and poor cellularity. [8] Ultrasound guided aspiration may be helpful in obtaining better aspirate and decrease the number of unsatisfactory smears. [8] The salivary gland lesions have overlapping morphological patterns in many of the benign and malignant neoplasms of the salivary gland. This makes them one of the difficult areas in cytopathology. In the present study, Pleomorphic Adenoma and Mucoepidermoid Carcinoma were the most common benign and malignant tumours respectively as observed by other authors. These findings were similar to other studies. [7,9] The most common diagnosis made in our study was Pleomorphic Adenoma as observed in other studies. [9] Twenty eight cases were available for histopathological correlation.(Figures 1,2) Out of which twenty four turned out to be pleomorphic adenoma, two as Mucoepidermoid Carcinoma and two to be Adenoid Cystic Carcinoma on histology . Two cases diagnosed as Pleomorphic Adenoma in FNAC turned out to be Adenoid cystic carcinoma in histopathology. Distinction of highly cellular Pleomorphic Adenoma from Adenoid cystic carcinoma in cytology is not always easy. The stroma of adenoid cystic carcinoma was locally fibromyxoid and occasionally hyaline stromal globules seen were mistaken for Pleomorphic Adenoma. With H&E and PAP, the globules are pale, semitranslucent and less conspicous. But specific to pleomorphic adenoma is true chondromyxoid stromal matrix with spindle cells. Accurate diagnosis of adenoid cystic carcinoma may be difficult in poorly differentiated tumors due to lack of characteristic stromal matrix. [10,11,12] The second most common benign tumor after Pleomorphic Adenoma was Warthinâ&#x20AC;&#x2122;s tumour.The three main components for diagnosis are lymphocytes, oncocytes and a dirty background. In our study, two cases were available for histopathological correlation with 100% concordance. [13]

Myoepithelioma (myoepithelial adenoma) may be plasmacytoid, epitheloid or spindle cell type.The cellular pleomorphic adenoma may not be distinguisable from myoepithelioma where selective sampling of solid focus of plasmacytoid cells without specific stroma has been done. In our study, four cases were given differential diagnosis of Pleomorphic Adenoma/Myoepithelioma turned out to be Pleomorphic Adenoma in histology. The plasmacytoid cells of pleomorphic adenoma is difficult to distinguish from Myoepithelioma -plasmacytoid variant. [14] The most common malignant neoplasm is Mucoepidermoid Carcinoma.(Figures 3,4) our study as well as the study conducted by other authors. [11,15,16] Low grade Mucoepidermoid Carcinoma is difficult to diagnose as malignant in FNAC and is one of the most common source of false negative results. The main cause for this is that many of these tumors are predominantly cystic. In our study eight cases were confirmed by histopathology and two which were diagnosed as Mucoepidermoid carcinoma histologically on cytology turned out to be Pleomorphic adenoma on histology. In these two false negative cases, the aspirate was hypocellular with mainly mucoid secretion which was mistakened for myxoid background of Pleomorphic Adenoma.The background should be checked for cohesive epithelial cells that gives clue to the diagnosis of low grade MEC and should lead to assiduous search for more diagnostic components. [11] The high or intermediate grade tumors can be accurately diagnosed by FNAC whereas low grade tumors gives unsatisfactory results as suggested by Klijanienko et al. [11] The second most common malignant tumor was adenoid cystic carcinoma after Mucoepidermoid Carcinoma. FNA smears usually shows epithelial cells and acellular basement membrane material. This material is seen as homogenous spherical structures. The hyaline stromal globules seen in Adenoid cystic carcinoma should be distinguished from that seen in Pleomorphic Adenoma as well as mentioned by other authors. The cytological difference between two tumors can be done if smears are cellular so that distinguising relationship between Pleomorphic Adenoma and extracellular matrix can be recognised. [16] The plasmacytoid cells seen in Pleomorphic Adenoma have abundant cytoplasm compared to the tumor cells of adenoid cystic carcinoma. [17,18] Four cases of Chronic Sialadenitis which were diagnosed by FNAC were operated and turned out to be Warthinâ&#x20AC;&#x2122;s tumor histologically. In predominantly cystic Warthinâ&#x20AC;&#x2122;s tumor, obtaining diagnostic material may be very difficult. Both lymphoid cells and oncocytic cells must be recognised for accurate diagnosis, but either component may be sparse, absent or hidden by mucoid debris. In our case, oncocytic component was absent, which lead to erroneous diagnosis of chronic sialadenitis cytologically

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Thangam R et al. In the present study the Diagnostic accuracy, Sensitivity and specificity was 91.66%,80% and 94.74% This was similar to studies by other authors. [9,19] Despite earlier studies that reported a greater percentage of false-positive results than expected, more recent studies reports greater diagnostic accuracy and lesser sampling errors. The false-positive rates reported in the literature range from 0 to 10% and the accuracy increases with the experience of the pathologist. In our series, the percentages of false positive cases were 4.16%. By using fine needle aspiration as a primary diagnostic tool in salivary gland masses, the number of patients undergoing surgery can be reduced. [20,21]

Conclusion

In patients with salivary gland neoplasm, fine needle aspiration cytology has very useful role in making preoperative diagnosis and management. Most of the benign and malignant neoplasm can be diagnosed with high level of accuracy with adequate sampling and good quality smear preparation. The high accuracy, sensitivity, and specificity of FNAC confirm that preoperative cytology is a useful, quick, reliable diagnostic technique for rapid and early diagnosis and we also conclude that it is simple and costeffective diagnostic tool suitable for developing countries.

References 1.

Stenner M, Klussmann JP. Current update on established and novel biomarkers in salivary gland carcinoma pathology and the molecular pathways involved. Eur Arch Otorhinolaryngol. 2009 Mar. 266(3):333-41.

Zbaren P, Nuyens M, Loosli H, Stauffer E. Diagnostic Accuracy of fine needle aspiration cytology and Frozen section in primary parotid carcinoma. Cancer 2004;9(100):1876-81.

Nanda S KD, Mehta A, Nanda J. Fine needle aspiration cytology: a reliable tool in the diagnosis of salivary gland lesions. Journal of oral pathology & medicine 2012;41(1):106-112.

Wong DSY, Li GKH. The role of fine-needle aspiration cytology in the management of parotid tumors: a critical clinical appraisal. Head Neck 2000; 22:469–473.

Zbaren P, Schar C, Hotz MA, Loosli H. Value of fine-needle aspiration cytology of parotid gland masses. Laryngoscope 2001;111:1989–1992. Rau AR, Pai RR, Nayak S. Infarction of acinic cell carcinoma in a patient infected with HIV: A complication of fine-needle aspiration cytology obscuring definitive diagnosis. Diagn Cytopathol 2001; 24: 301-303.

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Pabuccuoglu HU, Lebe B, Sarioglu S, Lebe E. Infarction of pleomorphic adenoma: A rare complication of fine-needle aspiration obscuring definitive diagnosis. Diagn Cytopathol 2003; 29: 222-224.

Shintani S, Matsuura H, Hasegawa Y. Fine needle aspiration of salivary gland tumors Int J Oral Maxillofac Surg 1997; 26: 284- 286.

Ahmad S, Lateef M, Ahmad R. Clinicopathological study of primary salivary gland tumors in Kashmir. JK- Practitioner 2002; 9: 231- 33

10. Vaidhya S, Sinha. A, Narayan. S, Adhikari. S, Sabira. KC. A comparative study of fine-needle aspiration cytology and histopathology in salivary gland lesions. Journal of pathology of Nepal 2011;108-113. 11. Klijanienko J, Vielh P. Fine needle sampling of salivary gland lesions IV .Review of 50 cases of mucoepidermoid carcinoma with histologic correlation .Diagn cytopathol 1997;17:92-8 12. Yu GH, Caraway NP. Poorly-differentiated adenoid cystic carcinoma:cytologic appearance in fine-needle aspirates of distant metastases. Diagn cytopathol 1996;15:296-300. 13. Veder L, Kerrebijn JD, Smedts FM, den Bakker MA. Diagnostic accuracy of fine-needle aspiration cytology in Warthin tumors. Head Neck. 2010;32(12):1635-40 14. Dodd LG, Caraway NP, Luna MA, Byers PM. Myoepithelioma of the parotid. Report of a case initially examined by fine needle aspiration biopsy. Acta cytol 1994;38:417-21 15. Sousa J, Desa O. Salivary gland tumors : An analysis of 62 cases. Cancer 2001; 38: 38- 45. 16. Khandekar MM, Kavatkar AN, Patankar SA. FNAC of salivary gland lesions with histopathological correlation .J Otolaryngol and Head and Neck Surg 2006; 58: 246- 48. 17. Kapadia SB, Dusenbery D, Dekker A. Fine needle aspiration of pleomorphic adenoma and adenoid cystic carcinoma of salivary gland origin. Acta Cytol 1997; 41: 487- 92. 18. Lee SS, Cho KJ Jang JJ, et al. Differential diagnosis of adenoid cystic carcinoma from pleomorphic adenoma of the salivary gland on fine needle aspiration cytology. Acta Cytol 1996; 40: 1246 19. Haberal I, Golmen H, Safak MA, et al. The value of fine needle aspiration biopsy in salivary gland tumors. International Congress Series 2003; 629- 34 20. Zakowski MF. Fine needle aspiration cytology of tumors: Diagnostic accuracy and potential pitfalls. Cancer Invest 1994; 12: 505-515. 21. Chan MKM, Mc Guire LJ, King W, Li AKC, Lee JCK. Cytodiagnosis of 112 salivary gland lesions, correlation with histologic and frozen section diagnosis. Acta Cytol 1992; 36: 353- 363.

*Corresponding author: Dr Dhananjay Shrikant Kotasthane, Department of Pathology, Mahatma Gandhi Medical college and Research Institute, Pillaiyarkuppam, Pondicherry, India Phone: +91 9092096244 Email: dskotasthane@gmail.com Date of Submission : 11.01.2017 Date of Acceptance : 31.01.2017 Financial or other Competing Interests: None. Date of Publication : 15.04.2017

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Case Report DOI: 10.21276/APALM.1073

Synchronous Primary Carcinoma of Cervix and Endometrium Divya Madhala1, Gouthaman Shanmugasundaram2, Sai Shalini Chinnathambi Narayanan1 and Jaya Vijayaraghavan3 Department of Pathology, Sri Ramachandra Medical College and Research Institute, Chennai, India Department of Surgical Oncology, Sri Ramachandra Medical College and Research Institute, Chennai, India 3 Department of Obstetrics and Gynecology, Sri Ramachandra Medical College and Research Institute, Chennai, India 1

ABSTRACT Introduction: Synchronous primary tumours in the female genital tract are rare. Even rarer are the synchronous tumours involving the cervix and endometrium . We describe a rare case of cervical squamous cell carcinoma and endometrial endometrioid adenocarcinoma in a 62-yearold woman . Case Report: A 62-year-old female presented with post menopausal bleeding for 4 months duration. Per vaginal examination showed a cervical growth . Magnetic resonance imaging revealed a mass in the cervical region and extension into the body and fundus of the uterine cavity. Radical hysterectomy(Type III hysterectomy) was done. The pathology revealed synchronous occurrence of primary neoplasms of squamous cell carcinoma of the cervix and adenocarcinoma of the endometrium. Conclusion: Synchronous malignancies of endometrium and cervix are rare. Surgical treatment is radical surgery- Type 3 hysterectomy with bilateral pelvic lymph node dissection. The knowledge of occurrence of synchronous primaries is needed to plan appropriate adjuvant treatment. Keywords: Cervix, Endometrium, Cancer, Pathology

Introduction

Synchronous tumours are two or more histologically distinct malignancies detected simultaneously. Synchronous primary malignancies of the female genital tract are very rare. The most common combination being ovarian and endometrial primary neoplasms[1] .Very few cases of synchronous malignant tumours of endometrium and cervix have been reported in the world literature[2] . The prognosis of these patients depends on the stage of presentation of these malignant tumours. We present a very rare case of synchronous tumour of endometrial and cervical cancer in a 62 year old female patient.

Case Report

A 62 year old lady presented with complaints of intermittent post menopausal bleeding for 4 months. Patient also had white discharge per vaginum. She is a known diabetic and hypertensive. Pap smear examination revealed malignant squamous cells following which she was referred to our center for further management. Per abdomen examination was within normal limits. Per vaginal examination showed a ulcero-proliferative growth obstructing the cervical os from which cervical biopsy was taken. The histopathology of the cervical biopsy showed features of poorly differentiated squamous cell carcinoma. Magnetic resonance imaging showed a bulky cervix with partially circumferential growth predominantly involving

the anterior and right lateral walls. Tumour extended into body and fundus of uterus with > 50% of myometrial thickness involvement(Figure 1). No serosal involvemt was present. The parametrium was free and pelvic side walls appeared normal. Few bilateral internal iliac nodes were noted. In view of disease confined to the cervix with no parametrial invasion, type 3 hysterectomy with bilateral salphingo - oopherectomy and bilateral pelvic lymph node dissection was planned. Examination under anesthesia was done which showed no parametrial involvement. Maylards incision was made. Exploration of the abdomen was done. There was no significant pelvic or para aortic nodes enlargement. Uterine artery was ligated at the origin. Ureter dissected till bladder base from pelvic brim. Parametrium was divided close to the pelvic side walls. Utero-sacral ligaments were divided close to rectal wall. Upper one third of vagina removed en-bloc with the specimen. Bilateral pelvic lymph node dissection was done. We received total abdominal hysterectomy with bilateral salphingo-oopherectomy specimen along with bilateral pelvic lymph nodes. Uterus with cervix and bilateral tubes and ovaries weighed 30g. Uterus with cervix measures 8.5x5x2 cm. Parametrial length was 6 cm on each side, cervix external surface was unremarkable. Cut surface showed a ill defined , infiltrative, grey white firm lesion measuring 2x2cm and depth of 1.4cm. Tumour is seen

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involving the endo-cervical region. Serosal surface is free of tumour. Distance from the vaginal cuff was 0.7 cm. Endometrium showed a irregular grey white to grey brown proliferative mass measuring 5.5x2.5x0.7cm(Figure 2), and myometrium measures 1 cm. Both ovaries and tubes were unremarkable. Microscopically , cervix showed a FIGO grade 3 poorly differentiated non keratinizing squamous cell carcinoma of cervix with adjacent areas showing cervical dysplasia(Figure 3). Few areas of necrosis was also identified(Figure 4). Endometrium showed a FIGO grade 1 well differentiated, Endometrioid adenocarcinoma not

otherwise characterized( Figure 5 , Figure 6). Margins uninvolved. The bilateral ovaries, tubes, omentum, parametrium and vaginal surgical margins were all free. Surgical vaginal cuff was free of tumor. Also, there was no lower vaginal segment involvement of the endometrial cancer. Bilateral pelvic lymph nodes were free of tumour (27 lymph nodes were examined). Based on pathological examination, a diagnosis of FIGO stage IB1 cervical malignancy and FIGO stage IB endometrial malignancy was made. Following surgery patient was started on adjuvant external beam radiation therapy.

Fig. 1: MRI of cervix and endometrium.

Fig. 2: Cervix showing a ill defined , infiltrative, grey white firm lesion and endometrium with irregular grey white to grey brown proliferative mass.

Fig. 3: Section shows Cervical carcinoma with dysplasia (H&E, 20x).

Fig. 4: Section showing Cervical carcinoma with atypical mitosis and areas of necrosis (H&E, 20x).

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Fig. 5: Section showing Endometrial endometrioid adenocarcinoma (H&E, 4x).

Fig. 6: Section showing endometrial endometrioid adenocarcinoma (H&E, 20x).

Discussion

malignancy is endometrioid adenocarcinoma and in cervix it is squamous cell carcinoma.

Multiple primary tumours can be synchronous or metachronous. Synchronous tumours are two or more histologically distinct simultaneously detected malignancies. The incidence of 1%-6% shows the rarity of these tumours. Out of this the most common synchronous tumour is that of ovarian and endometrium. The etiopathogenesis of these synchronous tumours is very rarely understood. It has been proposed that , embryologically similar tissues when simultaneously subjected to either hormones or carcinogens may develop synchronous neoplasms in genetically susceptible individuals. The concept of field cancerization[1, 3] which was earlier applied for tumours arising from head and neck region has being applied to the synchronous tumours of female genital tract. Here the repeated exposure of the mucosa of the female genital tract to multiple risk factors leads to the development of multicentric disease . It is suggested that cancers developing in different sites originate from histologically similar epithelium. This â&#x20AC;&#x153;secondary mullerian systemâ&#x20AC;? concept attempts to explain the etiology of synchronous primaries in female genital tract. The patients are usually post-menopausal with the median age of presentation being 50-60 years. Most patients present with post menopausal bleeding and vaginal discharge.

The surgical management of these patients include radical hysterectomy(Type III hysterectomy) with bilateral pelvic lymph node dissection in view of cervical involvement. Appropriate surgical staging provides diagnostic, prognostic and therapeutic benefits for women with synchronous primary malignancies. It facilitates appropriate adjuvant treatment as well. In the literature the cases of cervical and endometrial neoplasm occurring at the same time have rarely been reported. A single case study of synchronous cervical and endometrial neoplasm was reported by Cheng-kuo lin et al[2] from national defence center ,Taiwan. They suggested that the second primary cancer in patients having endometrial cancer may offer an opportunity for early detection. In our study , patient was diagnosed to have cervical malignancy and subsequently diagnosed to have carcinoma of endometrium in uterine gross specimen after radical hysterectomy.

Pathologically Synchronous primary endometrial and cervical cancers may have similar or different histologic appearance. The distinction between synchronous primary and metastatic cancers is relatively easy, when they have different histologic types. However, the distinction is relatively difficult, when they have the same histologic type. Most common histopathology seen in endometrium

In a study by Seo-yun tong et al[4] , 20 patients were diagnosed with synchronous tumours , 13 patients had synchronous tumours in the female genital tract and 7 with one primary in the female genital tract and 1 primary in extragenital. The most common was endometrium and ovarian malignancy. Three patients showed primary malignancies of the endometrium and the cervix. All three were associated with adenocarcinoma of the endometrium and two had squamous cell carcinoma of cervix and one had neuroendocrine tumour of cervix. This study proves that synchronous malignancy of endometrium and cervix as seen in our case is very rare.

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A Turkish study by Sultan eser et al[5] was carried out to determine the frequency of occurrence of synchronous female genital tumours from 1993 to 2005. A total of 4185 cases were identified , off which 55 were synchronous tumours. 43 tumours were synchronous tumours of endometrium and ovary whereas just 4 cases of synchronous tumours of endometrium and cervix was present indicating that this combination is very rare. In their study they observed that Endometrioid adenocarcinoma was the most frequent component in a synchronous tumour. Our case also had endometrioid adenocarcinoma. In one study by Axelrod jh et al[6] in 1984 reviewed 78 synchronous or metachronous tumours from 2362 patients. Synchronus tumour pairs of cervix was found in combination with ovary,uterus and kidneys. Also synchronous pairs were found in combinations of endometrium and ovary,endometrium and rectosigmoid, ovary and breast. In this study the synchronous tumours of cervix and endometrium as seen in our study as well was rare when compared to other synchronous tumours. A case of Cervical signet-ring cell carcinoma presenting as a synchronous primary carcinoma with uterine adenocarcinoma was reported by Lower WJ et al[7] in 2009. They found that cervical signet cell carcinoma is very rare with only 11 cases reported in literature and that occurring in combination with uterine neoplasm is even more rarer. But our case was squamous cell carcinoma of cervix along with endometrial endometrial adenocarcinoma. In one study by Derya et al [8] , where triple malignancies of cervical,ovary and endometrial well differentiated endometrioid type carcinoma was reported. The patient underwent radical hysterectomy, bilateral salphingooopherectomy, retroperitoneal lymph node dissection and peritoneal sampling. Post operatively patient received 6 cycles of chemotherapy with cisplatin and paclitaxel. In our study adjuvant external beam radiation was given as a treatment for both FIGO stage IB carcinoma cervix and FIGO stage IB endometrial carcinoma. Chemotherapy was not given in view of early stage of the malignancies. The prognosis of patients with synchronous tumours is more favourable when compared to metastatic lesions of individual tumours. Also the stage at which the patient presents to the health center also determines the outcome.

Another important factor is the lymph node status of these patients. It defines accurately the extent of disease and determines the prognosis of patients.

Conclusion

In conclusion Synchronous primary tumours in the female genital tract are rare. Adjuvant treatment is based on stage of individual primaries. The stage of presentation and extent of disease determines the prognosis of the patients.

Acknowledgements

I would like to thank Professor Dr.Sandhya Sundaram, Head of Department of Pathology for their Guidance and support and my colleagues Dr. Naveen Kumar. S and Dr. Mohan. K.P for their assistance in writing this case report.

Reference 1.

5. 6. 7.

Kambi DP, Mallikarjuna M N, Santosh CS et al; Synchronous malignancies of ovary, fallopian tube and cervix- A rare case. International Journal of Biomedical And Advance Research 2013; 04 [09]. Cheng-Kuo Lin, Mu-Hsien Yu, Ta-Wei Chu1 et al; Synchronus occurrence of primary neoplasms in the uterus with squamous cell carcinoma of the cervix and adenocarcinoma of the endometrium. Taiwanese Journal of Obstetrics and Gynecology. 2006;45: 336–339. Hsu-Dong Sun , Chung-Ru Lai, Ming-Shyen Yen et al; Synchronous occurrence of primary neoplasms of the uterus with mucinous carcinoma of the cervix and endometrioid carcinoma of the endometrium. Taiwanese Journal of Obstetrics & Gynecology 2011;50: 377-378 Seo-Yun Tong, Yong-Sek Lee, Jong-Sup Parket al; Clinical analysis of synchronous neoplasms of female reproductive tract. European Jrnl of Obs and Gyn and Reproductive Biology 2008 ; 136:78-82. Eser S, Gulhan I, Özdemir R et al. Synchronous primary cancers of the female reproductive tract in Turkish Women. Asian Pacific J Cancer Prev 2011;12,857-859. Axelrod JH, Fruchter R, Boyce JG. Multiple primaries among gynecologic malignancies. Gynecol Oncol 1984;18:359–72. Lowery WJ, Difurio MJ, Sundborg MJ, et al. Cervical signetring cell carcinoma presenting as a synchronous primary carcinoma with uterine adenocarcinoma. Mil Med. 2009 Feb;174[2]:212-3. Sakarya DK, Yetimalar MH, Demir M et al; Coexisting triple malignancy of the female genital tract with the same histopathology: An unusual case and review of the literature. Journal of Cases in Obstetrics and Gynecology, 2015;2:10-14.

*Corresponding author: Dr Sai Shalini Chinnathambi Narayanan, Department of Pathology, Sri Ramachandra Medical College and Research Institute, Porur, Chennai, Tamil Nadu, India- 600116 Phone: +91 9940217743 Email: saishalini_cn14@yahoo.com Date of Submission : 01.09.2016 Date of Acceptance : 11.02.2017 Financial or other Competing Interests: None. Date of Publication : 26.03.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Case Report DOI: 10.21276/APALM.1018

Large Hamartomatous Polyp Presenting with Profuse Rectal Bleeding in Colo-colic Intussuception in A Child: A Case Report K N Rattan1 Shruti Bansal2*, Roomi Yadav2, Gurupriya J2 and Neha Singh2 Department of Paediatric Surgery, Pt. B.D. Sharma PGIMS Rohtak, Haryana, India 2 Department of Pathology, Pt. B.D. Sharma PGIMS Rohtak, Haryana, India

ABSTRACT Isolated colocolic intussusception in paediatric age group is quite rare with juvenile polyps being the most important pathological lead points. We are reporting a case of colocolic intussusceptionsecondary to solitary hamartomatous polyp as lead point in a 3.5 year oldmalechild who presented with profuse bleeding per rectum. The patient was successfully managed by reduction and colonic resection withcolo colic anastomosis and is doing well in follow up. Keywords: Colocolic, Intussuception, Children, Solitary Hamartomatous Polyp

Introduction

Intussusceptionis a frequent cause of paediatric intestinal obstruction, seen most commonly in the childrenunder2 years of age with the peak incidence being in between 5 and 10 months of age. Colocolic intussusception (CCI) is an uncommon type of intussusception occurring in paediatric population with majority cases usually associated with a pathologic lead point such as juvenile polyps or tumor mass.[1]To our knowledge, approximately 14 cases of CCI in children have been reported in literature till date with 65% of them associated with juvenile polyps as leading point for this variety of intussusceptions in the pediatric age group. We are reporting a case of colocolic intussusception with solitary hamartomatous polyp as lead point in a three and a half year old male child who presented with profuse rectal bleeding, highlighting the unusual presentation of a rare benign etiology of intussusception at an uncommon site in the child.

Case Report

A 12 kg 3.5 year-old male child was brought to accident and emergency department by his parents with complaints of abdominal pain, profuse rectal bleeding and decreased acceptance. His past medical history was unremarkable. However, the patient was operated outside with the diagnosis of intussusception but it was negative laparotomy as surgeons failed to find intussusception. On physical examination, the child was conscious, irritable, afebrile and severely dehydrated with BP=100/60 mmHg, PR=120/min, RR=24/min and normal breathing. Growth and development was normal. Abdominal examination revealed soft and distended abdomen with no palpable lump. There was diffuse abdominal tenderness. Per rectal

examination was negative. Chest was bilaterally clear and CVS examination was normal. All laboratory investigations including complete haemogram, coagulation studies, renal function test and serum electrolytes were within normal limits. The child was kept NPO and nasogastric aspiration done. Intravenous fluids and antibiotics were given and dehydration was corrected. The child was investigated. X-ray abdomen was normal with no visualization of air fluid levels. On ultrasound, minimal intergut fluid was seen and there was telescoping of one gut loop into another at left hypochondrium and left lumbar region consistent with diagnosis of colo colic intussusception. After adequate resuscitation, child was taken up for surgery. Abdomen was opened through right supraumblical transverse incision. On exploration, colo colic intusucception was seen near the splenic flexure. It was reduced. After reduction, on palpation of large intestine, a firm intramural mass of 3.5Ă&#x2014;3.5cms was palpable. On opening transverse colon, there was large polypoidal growth seen (Figure 1). Hence, resection and colo colic anastomosis was done. Abdomen was closed in layers. Postoperative period was uneventful with no recognizable bleeding. The child was started orally after 3 days of surgery and was discharged on 10thpost operative day. The patient recovered well from surgery and had no further episodes of intussusception. Colonoscopy was performed after 3 months to evaluate for other polypoid lesions but no polyps were found. The resected colonic polyp was sent for histopathological examination. Pathologic examination revealed it to be solitary hamartomatous polyp without atypia (Figure 2&3).

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Profuse Rectal Bleeding in Colo-colic Intussuception in a Child

Fig. 2: Resected polypectomy specimen.

Fig. 1: Peroperative picture showing intussusception and solitary polyp.

Discussion

colo

colic

Intussusception accounts for up to 25% of abdominal emergencies in children up to the age of 5 years.The male-to-female ratio is approximately 3:2.The bowel may simply ‘telescope’ on itself (non-pathological lead point), or some pathology may be the focus of the invagination (pathological lead point).In approximately 95% of children, the intussusception is ileocaecocolic in location with about 90% cases being idiopathic in which no pathologic lead point is evident on barium enema or laparotomy.[2-3]Hyperplasia of paeyers patches is the commonest cause for intussusception in children. Other types of intussusceptions namely jejunojejunal or colocolic are quite rare, usually have a pathological lead point and are seen more commonly in children older than 2 years of age. Possible lead points include intestinal polyps, Meckels diverticulum, Peutz-Jeghers syndrome, mucosal hematoma, Henoch-Schoenlein purpura, neoplasms, lipoma and intestinal duplications.

Fig. 3: Photomicrograph showing histopathology of solitary hamartomatous polyp.

Colo-colic intussusception is an unusual cause of large bowel obstruction in children with no studies documenting the exact prevalence of the disease. Its occurrence is mostly associated with a pathological lead point with majority case reports reporting it to be juvenile polyps. However, the polyps varied in size and clinical presentation. Arthur et al reported a case of colocolic intussusception in a three year old child caused by a colonic polyp.[3]A case of CCI in a four year old male secondary to juvenile polyps was reported by Abrahamsetal in 2012. The child came with complaints of worsening vomiting and diarrhea and on exploration was found to have two large intraluminal pedunculated polyps (measuring 3.3 × 2.5 × 2.0 cm and 2.2 × 2.0 × 1.0 cm) and one small polyp as lead points for CCI.[4] Yamada et al reported CCI in 10 month old boy who presented with bloody stools. Juvenile polyp approximately 15 mm in size was found to be the leading point.[5] However, two cases of CCI without pathologic lead points had been reported in a 7-year-old boy in 2008 by Mahmudloo et al and in

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Rattan et al. a 4 year old boy by Sanchez et al.[6-7] An unusual case of neonatal CCI with intestinal lymphangioma as lead point was reported by Al-Jahdali et al in 2009.[8]Pediatric CCI caused by malignancy has been reported rarely in the literature. A rare case in a 5-year-old child who presented with intermittent CCI with sigmoid colon ganglioneuroma as leading point was reported by Soccorso et al.[9]Although the leading point in our case was colonic polyp similar to that reported in significant number of cases, but its large size of 3.5Ă&#x2014;3.5 cmsand clinical presentation of profuse bleeding per rectum was quite distinct. The clinical manifestations in majority of these cases are paroxysms of colicky abdominal pain and lower intestinal bleeding. Abdominal radiography is the most common initial study used in the workup of abdominal pain and suspected intussusception. Abdominal X-rays may show dilated gas-filled proximal bowel with paucity of gas distally and multiple air fluid levels but they generally have low sensitivity and specificity for the diagnosis of intussusception as a quarter of intussusception cases have a normal abdominal radiograph.[10] The well established imaging modality for the diagnosis of intussusception is an abdominal ultrasonography. It is quite sensitive, specific, reliable and inexpensive tool for diagnosing intussusception and can also help in identification of pathological lead points. Sonographically, the most frequently described appearance of intussusception is a bullâ&#x20AC;&#x2122;s eye or target-like lesion on transverse views which is formed by a loop of bowel (intussusceptum) within another loop of bowel (intussuscepient). Also, the affected bowel segment on longitudinal view resembles the appearance of a kidney which is referred to as pseudo-kidney. CT scan reveals the presence of a target or sausage-shaped lesion and also has high diagnostic accuracy rate. Nevertheless, colonoscopy is a highly useful tool for evaluating patients presenting with intussusception.

Conclusion

Any paediatric patient presenting with fresh rectal bleeding with evidence of intussusception on ultrasonography

C-39 should be subjected to colonoscopy for confirmation of polypoidal lesions. Colonoscopy, under appropriate sedation, is beneficial for both diagnostic and curative purposes. Colonoscopic polypectomy performed by an experienced endoscopist, is generally more useful than open surgical treatment when organic lesions such as juvenile polyps are present.

References 1.

Das A, Ralte L, Chawla AS, Arya SV, Kumar A, Saroha R et al. Colocolic Intussusception in an Older Child: A Rare Case Report and a Literature Review. Case Rep Surg. 2013;2013:1-3.

Dominique TLM. Lower gastro-intestinal bleeding. In: Allan Walker W, Kleinman RE, Sanderson IR, et al (eds). Pediatric Gastrointestinal Disease. 4th ed. Hamilton, Ontario; BC Decker. 2004; p273.

Arthur AL, Garvey R, Vaness DG. Colocolic intussusception in a three-year-old child caused by a colonic polyp. Conn Med. 1990;54:492-4.

Abrahams RB, Franco A, Lewis KN. Pediatric colocolic intussusception with pathologic lead point: a case report. J Med Cases. 2012;3:84-8.

Yamada S, Miura T, Nakamura J, Emura I. Pediatric colocolic intussusception due to juvenile polyp. Gastroenterological Endoscopy. 2010;52:1267-74.

Mahmudloo R, Gheibi S, Vahed SN. Colocolic Intussusception without Lead Point; A Case Report and Literature Review. Iran J Pediatr. 2008;18:373-6.

7. Sanchez S, Javid P, Ricca R, Avansino J. Colocolonic intussusception in a four-yr-old with a heart transplant: A case report and review of the literature. Pediatr Transplant. 2011. 8.

Al-Jahdali A, Lees GM, Gay DP, Al-Sairafi R. Colocolic intussusception in a preterm infant with intestinal malrotation. J Pediatr Surg. 2009;44:e17-18.

Soccorso G, Puls F, Richards C, Pringle H, Nour S. Aganglioneuroma of the sigmoid colon presenting as leading point of intussusception in a child: a case report. JPediatrSurg. 2009;44;e17-e20.

10. Hernandez JA, Swischuk LE, Angel CA. Validity of plain films in intussusception. EmergRadiol. 2004;10:323-6.

*Corresponding author: Dr. Shruti Bansal, Department of Pathology, Pt. B.D. Sharma PGIMS, Rohtak, Haryana, India Phone: +91 07206341546 Email: shruti.b.bansal3@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 11.08.2016 Date of Acceptance : 08.01.2017 Date of Publication : 26.03.2017

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Case Report DOI: 10.21276/APALM.1054

Cavernous Hemangioma of The Testis Mimicking as Torsion of Testis: A Case Report Shreekant Bharti and Narrendran A P* Dept of Pathology, IMS, Banaras Hindu University, India

ABSTRACT Introduction: Cavernous hemangiomas are diagnosed by dilated large vessels with thin walls. Intra-testicular hemangiomas are a rare entity, and often mimic testicular malignancy or may present as testicular torsion. Case Report: 18 year old male presented with a month-long history of lower abdominal pain and clinical examination revealed only a mildly enlarged, non-tender left testis. Ultrasonography of abdomen and scrotum suggested avascular hypoechoic left testis with hydro-hematocoele likely secondary to torsion of testis. A probable diagnosis of torsion of testis was made and the patient underwent left orchiectomy. Microscopic examination of the tumor revealed a cavÂŹernous hemangioma composed of variably sized, clot-filled cavernous spaces separated by fibrous tissue and leading to hemorrhagic necrosis of the testicular parenchyma. Conclusion: Clinicians need to consider the possibility of the rare but benign lesion, testicular hemangioma, in young patients presenting with testicular mass and/or pain, along with normal tumour markers and increased intralesional vascularity on imaging. Keywords: Hemangioma, Cavernous Hemangioma, Testicular Cancer, Benign Testicular Tumor

Introduction

International Society for the Study of Vascular Anomalies (ISSVA) divided vascular anomalies into vascular tumors and vascular malformations. Vascular tumors arise mainly due to endothelial hyperplasia and they may regress spontaneously. Vascular malformations occur by localized defects of vascular morphogenesis, secondary to dysfunction in pathways regulating embryogenesis and they usually persist in adulthood [1]. In 2000, Mazal et al. reviewed the literature and identified four histopathological types of testicular hemangiomas: cavernous, histiocytoid, capillary and papillary endothelial hyperplasia [2]. In the past decade, less than 25 cases of testicular cavernous hemangioma have been reported in literature. Cavernous hemangiomas are diagnosed by dilated large vessels with thin walls. Their walls may be abnormal which may not confirm to arterial or venous morphology. Developmental arrest during the stage of an undifferentiated capillary network has been postulated to result in a cavernous hemangioma. We report a case of an 18-year-old male patient diagnosed with hemangioma of the testis which is a rare entity.

Case Report

An 18 year old male presented with complaints of lower abdominal pain extending till left scrotum since one month. There was no history of trauma to lower abdomen or scrotum. Clinical examination demonstrated unremarkable

abdominal findings with mildly enlarged, non-tender left testis and normal ipsilateral spermatic cord. He underwent an ultrasonogram of abdomen and scrotum which revealed hypoechoic left testicular parenchyma with collection of fluid and epididymis was bulky and heterogenous. Spermatic cord showed normal vasculature. These features suggested avascular hypoechoic left testis with hydro-hematocoele likely secondary to torsion of testis. A probable diagnosis of torsion of testis was made and the patient underwent left orchiectomy. On gross examination, the testis measured 3.5 x 3.5 x 1.5 cm with attached spermatic cord measuring 7.5 cm in length. The orchiectomy specimen showed a testicular tumor of 3 x 3 x 1.5 cm size which on sectioning showed blood clots, areas of necrosis and part of normal parenchyma. Microscopic examination of the tumor revealed a cavÂ ernous hemangioma composed of variably sized, clot-filled cavernous spaces separated by fibrous tissue and leading to hemorrhagic necrosis of the testicular parenchyma.

Discussion

Genital hemangiomas most likely arise from the inner layer of the tunica albuginea, which contains blood vessels and lymphatics and sends septa into the testicular parenchyma. A hemangioma may extend into the testicular parenchyma by way of these septa [3]. Intra-testicular form of hemangiomas is reported rarely, compared with extratesticular genital hemangiomas. Testicular hemangiomas

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Bharti et al.

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Fig. 1: Cavernous hemangioma of testis showing interconnected, thin walled blood vessels (H&E, 40x).

Fig. 2: Cavernous Hemangioma of testis showing endothelium lined, dilated vascular spaces separated by fibrous septae (H&E, 100x).

Fig. 3: Necrosis of the testicular parenchyma associated with cavernous hemangioma of testis (H&E, 100x).

are extremely rare and they occur in diverse age groups appearing in infants to old men [4]. Cavernous hemangioma of the testis primarily presents during childhood [5]. Unilateral testicular enlargement, with or without tenderness, is the chief presenting symptom, which is similar to that of malignant testicular tumors on clinical presentation. Testicular hemangiomas can also present as testicular torsion or associated with testicular infarction [6]. Rarely, it can present with bleeding or ulceration. Spontaneous involution is not reported with this type of hemangioma and long term complications like infertility, hemorrhage and even necrosis can occur as seen in our case. It can be seen extending into penis, anterior abdominal wall, perineum and pelvis [7,8]. An association with hemangiomas in other sites such as skin, liver, rectum and bladder has been reported by other authors [9,10].

. When the tumor markers are negative, presence of a mass with variably sized calcifications is highly suggestive of cavernous hemangioma. However, Serum AFP, beta-hCG and LDH are raised only in half of the patients diagnosed with testicular malignancies. Demonstration of intra-lesional blood flow can be made by Color Doppler studies; however lack of flow does not indicate the absence of cavernous hemangiomas. Treatment options available are surgical excision, laser fulguration, intralesional sclerotherapy and cryotherapy. Testis sparing surgeries are commonly done in pre-pubescent tumors as the proportion of benign tumors is relatively higher than adults [12]. [11]

On ultra-sonogram, testicular hemangiomas are often hypoechoic but sometimes, hyperechoic or even heterogenous

Apart from dilated, endothelium lined vascular spaces separated by fibrous tissue, cavernous hemangiomas also show thrombosis and calcification commonly [5]. The immunohistochemical markers CD31 and CD34 can help in delineating the endothelial lining of the cavernous spaces, while FVIII and vimentin shows variable positivity.

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Differential diagnoses of testicular cavernous hemangioma must include malignant tumors of the testis, epididymis and spermatic cord. The worldwide incidence of testicular cancer is estimated to have doubled in the last 40 years but significant variation exists between races and countries [13]. Hemangiomas and malignant tumors of the testis, especially seminomas have similar findings on ultra-sonogram and magnetic resonance imaging [14]. These findings along with the relatively high incidence of aggressive malignant tumors of testis result in patient undergoing high inguinal orchiectomy. Morphologically, the malignant tumor which forms the main differential diagnosis is well-differentiated angiosarcoma. Angiosarcoma occurs in mid and late adult life and has a broad morphologic spectrum varying from a benign hemangioma like appearance to a spindled or an epithelioid appearance. It shows infiltrative and interanastomosing growth pattern with at least focal endothelial atypia and nuclear hyperchromasia. Genital hemangioma may mimic an inguinal hernia, while subcutaneous location on the scrotum can be confused with a varicocele when it presents on the left side. Infarction associated with torsion of testis is usually analyzed with frozen section and histopathological sections are helpful only in confirming the diagnosis. All the cases of testicular cavernous hemangioma reported in the literature have run a benign course and there were no mentions of metastasis and recurrence following surgical excision [15].

Conclusion

Clinicians and pathologists must be aware of the rare entity of testicular hemangiomas, as clinical examination and imaging studies do not often suffice to arrive at a correct diagnosis. Testicular cancer and torsion of testis are frequent mimickers and considering their high incidence and clinical significance, diagnosis is further delayed. A young patient presenting with testicular mass and/or pain, along with normal tumour markers and increased intralesional vascularity on imaging studies should raise a suspicion of testicular hemangiomas.

Acknowledgements

We acknowledge the technical staff of the histopathology lab and the departmental head for the general support.

Reference 1. 2. 3. 4. 5. 6. 7. 8. 9.

10.

11.

12. 13. 14.

15.

Talmon GA, Stanley SM, Lager DJ. Capillary hemangioma of the testis. Int J Surg Pathol. 2011;19:398–400. Mazal PR, Kratzik C, Kain R, Susani M. Capillary haemangioma of the testis. J Clin Pathol. 2000 Aug; 53 (8):641-2. G. Erdag, E. Kwon, E. Lizza, M. Shevchuk. Cavernous hemangioma of tunica albuginea testis manifesting as testicular pain. Urology. 2006 Sep;68(3):673.e13-5. Numakura K, Tsuchiya N, Inoue T, Yuasa T, Matsuura S, Satoh S, Habuchi T. Case of testicular venous hemangioma. Hinyokika Kiyo. 2007 Jul;53(7):493-5. Chavan D, Javalgi AP. Scrotal Hemangioma: A Case Report. Journal of Clinical and Diagnostic Research : JCDR. 2014;8(12):ND03-ND04. Minagawa T, Murata Y. Testicular cavernous hemangioma associated with intrascrotal testicular torsion: a case report. Hinyokika Kiyo. 2009;55:161–163. R. Rastogi. Diffuse cavernous hemangioma of the penis, scrotum, perineum, and rectum: a rare tumor. Saudi J Kidney Dis Transplant 2008;19(4):614-618. Froehner M, Tsatalpas P, Wirth MP. Giant penile cavernous heman-gioma with intrapelvic extension. Urology. 1999;53:414-5. Keret D, Kam I, Ben-Arieh Y, et al. Scrotal cavernous haemangioma with a family history of cutaneous angiomata. J R Soc Med. 1990;83:402–403. Yanai S, Tsutsumi H, Hotsubo T, et al. Development of a testicular haemangioma after interferon therapy for hepatic haemangiomas: a case report. Eur J Pediatr. 1997;156:784-786. Suriawinata A, Talerman A, Vapnek JM, et al. Hemangioma of the testis: Report of unusual occurrences of cavernous hemangioma in a fetus and capillary hemangioma in an older man. Ann Diagn Pathol. 2001;5:80–3. Atkin G, Miller M, Clarkson KS, et al. Testicular capillary haemangioma in a child. J R Soc Med. 2001;94:638–40. Huyghe E, Matsuda T, Thonneau P. Increasing incidence of testicular cancer worldwide: a review. J Urol 2003:170(1):5-11. Takaoka E, Yamaguchi K, Tominaga T. Cavernous hemangioma of the testis: a case report and review of the literature. Hinyokika Kiyo. 2007 Jun;53(6):405-7. Liu, B., Chen, J., Luo, J., Zhou, F., Wang, C., Xie, L. “Cavernous hemangioma of the testis mimicking a testicular teratoma”. Experimental and Therapeutic Medicine. 2013;6.1:91-92.

*Corresponding author: Narrendran A P, 12B, Fifth Street, North Vinayagapuram, Saravanampatti, Coimbatore – 641035. India Phone: +91 7379141691 Email: drnarrendran@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 14.10.2016 Date of Acceptance : 09.01.2017 Date of Publication : 26.03.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Case Report DOI: 10.21276/APALM.1077

Non-Immune Hydrops Foetalis due to Congenital Toxoplasmosis: A Rare Case Report with Review of Literature. Mallikarjun Adiveppa Pattanashetti1*, Vijayalaxmi V Suranagi2 and Hema B Bannur2 Department of Pathology, S. Nijalingappa Medical College , Bagalkot, India Department of Pathology, Jawaharlal Nehru Medical College, Belagavi, India

1 2

ABSTRACT Non-Immune Hydrops Foetalis (NIHF) implies an excess of total body water which manifests as extracellular accumulation of fluid in serous cavities and soft tissues of foetus without any detectable circulating antibody against RBC antigen. Toxoplasmosis is an important congenitally acquired infectious cause of NIHF. Mother had complaints of fever, reduced foetal movements, USG showed hydrops foetalis and she tested positive for Toxoplasma IgG antibodies. On autopsy, foetus had generalised subcutaneous oedema, bilateral hypoplastic lungs with pleural effusion and oedema of brain with ventriculomegaly suggestive of hydrocephalus. We report a rare case of NIHF due to congenital toxoplasmosis with bilateral hypoplastic lungs, with emphasis on epidemiology and prevention of toxoplasmosis. Keywords: Non-Immune Hydrops Foetalis, Toxoplasmosis, Hypoplastic Lungs

Introduction

Non-Immune Hydrops Foetalis (NIHF) is an excess of total body water evident as extracellular accumulation of fluid in soft tissues and serous cavities of foetus without presence of identifiable circulating antibody against Red Blood Cell (RBC) antigen. Over 80 conditions are known to be associated with hydrops.[1] One of the rare infectious causes of NIHF is Congenital toxoplasmosis. Seroprevalence of toxoplasmosis in reproductive age group Indian women is 2.9 to 42.5 percent.[2] Global burden of congenital toxoplasmosis is 1.5 per 1000 live births.[3] We report a case of NIHF due to congenital toxoplasmosis with hydrocephalus and bilateral hypoplastic lungs for its rarity in India with emphasis on epidemiology and prevention of toxoplasmosis.

Case Report

A 24 year old mother with second gravida delivered a preterm 26 weeks intrauterine still born female foetus by breech extraction. Mother had complaints of fever in the first trimester for a week, reduced foetal movements in 2nd trimester and on USG hydrops foetalis was noted. There was no history of congenital malformation, recurrent spontaneous abortion, bad obstetric outcome or hematological disorder in the family. No history of blood transfusion in the past. The mother was also a known case of Rh negative pregnancy with no Anti-D given after first delivery of healthy male baby weighing 2.5 kg. Serological tests were performed for TORCH panel. Mother was positive for Toxoplasma IgG antibodies - 3.9 IU/L (Negative < 0.8 IU/ L ) and negative for Toxoplasma

IgM antibodies - 0.1 IU/L (Negative < 0.8 IU/L ) both done by ELISA method. She was negative for Rubella 0.7 IU/L (Negative < 0.8 IU/L ), CMV IgM - 0.05 IU/L (Negative < 0.8 IU/L) and HSV 1 & 2 IgM assay - 0.04 IU/L ( Negative <0.8 IU/L). Mother was non- reactive for HBSAg and HIV virus. On autopsy, foetus weighed 600 grams with generalised subcutaneous oedema of skin [Figure 1]. Facial dysmorphism in the form of low set ears and low hairline was seen. Foetus had hypoplasia of lungs with pleural effusion [Figure 2], hepatomegaly and ascites. Congestion and oedema of brain with ventriculomegaly suggestive of hydrocephalus was evident [Figure 3]. This case was diagnosed based on clinical history, autopsy examination of foetus and serological tests for toxoplasma gondii infection in mother.

Discussion

NIHF is the presence of â&#x2030;Ľ 2 abnormal fluid collections of foetus in the absence of red cell alloimmunization. It now accounts for almost 90% of cases of hydrops.[4] The most common causes of NIHF are enlisted [Table 1].[5] Toxoplasma gondii, an opportunistic pathological intracellular parasite which is found in humans and other mammals causes serious zoonoses. In 1948, Sabin and Feldman discovered a serological test for the exploration of toxoplasma. The routes of transmission are ingestion of food contaminated by oocysts in cat faeces or undercooked meat of domestic animals and birds containing toxoplasma cysts, ingestion of raw or undercooked beef, lamb and pork,

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Hydrops Foetalis due to Congenital Toxoplasmosis

Fig. 1 : Generalised subcutaneous oedema of (Arrow).

skin

Fig. 2 : Foetus had hypoplasia of both lungs with pleural effusion ( Arrow).

Fig. 3: Congestion and oedema of brain with ventriculomegaly suggestive of hydrocephalus (Arrow).

blood transfusion, consumption of unpasteurized goatâ&#x20AC;&#x2122;s milk and contaminated drinking water, direct exposure to soil, injured skin and mucosa.[6] Seroprevalence of toxoplasmosis in Indian women of child bearing age has remained a debatable topic as there are no published studies covering all the geographical regions. A seroprevalence study was undertaken by Singh S et al.[7]

on a large population of women of reproductive age from four distinct geographical regions of India : West, East, North and South India. This study showed an estimated total prevalence of 22.4%. Among them, South India had highest prevalence (37.3%) followed by East India (21.2%), North India (19.7%) and West India (8.8%). The possible risk factors of infection in these women in their

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Pattanashetti et al. study included lower socioeconomic status, advanced age, consumption of raw salad, drinking untreated water, residing in mud plastered houses and owning pets. History of contact with animals was found in 29.1% women but pets were significantly more common in South Indian households.[7] Association of sexual transmission of toxoplasmosis with multiple sexual exposures has also been reported in other studies.[8] Several factors which favoured increased seroprevalence of toxoplasmosis in South India included climatic conditions, highly significant number of households with cats, socio-cultural factors wherein South Indians do not wear shoes and most often are barefoot or wear sleepers only, which increases chances of transferring oocysts from soil and water to their food. Dry arid climatic zone and high temperature of Western India was the cause of lower seroprevalence in that region. Water and food-borne outbreaks of toxoplasmosis have been are also reported worldwide and also from India. [9]

C-45 tachyzoites and bradyzoites of toxoplasma. Washing of hands with soap and water after working with soil, after touching raw or undercooked meat or after handling the cat is of utmost importance.[13] Educational programs are a potentially powerful methods due to their low cost, and also because pregnant women are motivated to ensure the health of their babies. Doctors, nurses and health assistants can educate the pregnant women. Health education materials containing information about the prevention of T. gondii infection printed on posters, pamphlets and charts can be explaine to pregnant women during antenatal visits which lead to a decreased rate of seroconversion.

Congenital toxoplasmosis is a preventable infection. Primary prevention is possible by educating the pregnant women about the measures to prevent acquisition of toxoplasma infection. Prevention of primary infection depends upon educating pregnant women on the routes of transmission of Toxoplasma gondii and avoidance of high risk behaviors. Washing of fruits and vegetables should be done before eating. Washing of knives and the sink should be done after preparation of food. Consumption of unpasteurized milk, raw eggs or unfiltered water should be avoided. Measures such as cooking the meat at higher temperatures or keeping it frozen for one day are lethal to

In addition to this, secondary prevention is by serological screening to identify and treat women who acquire toxoplasma infection during pregnancy. Universal toxoplasma screening for is a debatable issue as even very few developed nations have routine antenatal screening for Toxoplasma gondii by serological tests. In developing countries, this is not feasible due to financial burden and hence, only high risk pregnant women must be screened. We comply to the recommendations of high risk category which includes pregnant women with antenatal history of exposure to possible routes of transmission, those who are immunosuppressed or HIV-positive or those with ultrasound findings such as hydrocephalus, microcephaly, intracranial calcifications, ascites, growth restriction or hepatosplenomegaly.[14] Various serological tests are performed to detect the infection by Toxoplasma IgG and IgM assay. Toxoplasma IgG assay done by Sabin Feldman dye test which is the gold standard test to confirm the infection. Other assay methods include Indirect fluorescent antibody test, Latex agglutination test, Direct agglutination test, Enzyme-linked Immunosorbent Assay and Immunosorbent agglutination assay test. Avidity of IgG antibodies test helps in differentiating recently acquired infection and infection from distant past. IgM assay is more sensitive and specific in diagnosing acute congenital infection in the fetus and new born. Amplification of B1 gene of T.gondii DNA by Polymerase Chain Reaction is highly sensitive and specific test for diagnosis of congenital, ocular, cerebral and disseminated toxoplasmosis.[11] Emphasis must be on the early first trimester diagnosis of the mother. According to a study, combination of a sensitive test for Toxoplasma specific IgM antibodies and estimation of the avidity of IgG antibodies for T. gondii has the highest predictive value with regard to the time of infection.[15] Congenital toxoplasmosis can be diagnosed in newborn by isolation of T. gondii from the placental or fetal tissue, the umbilical cord, body fluid or infant blood through various tests. Therapeutic treatment of women who acquire toxoplasma infection can reduce the severity of fetal infection.

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In pregnancy, mother-to-fetal transmission rates rise from 7% in the first trimester, to 24% in the second, to 59% in the third trimester. However, the incidence of severe fetal infection drops from 75% to a negligible risk in late pregnancy.[10] Mother may be asymptomatic even with infection. Around 70â&#x20AC;&#x201C;90% of infants born with congenital infection do not have symptoms at birth. Visual impairment, mental retardation and learning disabilities generally manifest several months to years later. First and second trimester infection has been associated with spontaneous abortion, prematurity and death of the foetus. Clinical features of toxoplasmosis include chorioretinitis, blindness, strabismus, mental retardation, epilepsy, anemia, jaundice, rash, encephalitis, thrombocytopenia, pneumonitis, microcephaly, hypothermia diarrhea, intracranial calcification, hydrocephalus and nonspecific illness. Subclinical infection may be seen in 3rd trimester. [11] The classic triad of chorioretinitis, intracranial calcifications and hydrocephalus is found in less than 10 % of infants infected by toxoplasma.[12]

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Hydrops Foetalis due to Congenital Toxoplasmosis

Conclusion

The diagnosis of primary toxoplasmosis in pregnant women early in the first trimester is of utmost importance in order to provide them early therapy or other interventions to prevent congenital toxoplasmosis of foetus which causes NIHF. Follow-up of children with suspected or confirmed infection from birth to adolescence is necessary in order to avoid the hazard and damage from congenital toxoplasmosis. These children need to be followed up for known clinical spectrum and complications of toxoplasmosis later in life by appropriate clinical examination and pathological, biochemical and radiological investigations. There is a need for more complete and accurate population based data regarding incidence of toxoplasmosis and number of cases by mode of transmission. Foetal death due to congenital toxoplasmosis requires detailed autopsy to understand the pathogenesis, newer associations and clinical spectrum of toxoplasmosis.

Acknowledgements

We thank the patient Department of Obstetrics and Gynaecology, JNMC, Belagavi for the support.

References 1.

Trainor B, Tubman R. The Emerging Pattern of Hydrops Fetalis - Incidence, aetiology and management. Ulster Med J 2006;75(3):185–6.

Nagaraja B, Ramana BV, Murty DS, Naidu K, Reddy Kailasanatha B. Prevalence of

Toxoplasmosis among Antenatal Women with Bad Obstetric History. IJPRBS, 2012;1(3):222–7.

Torgerson PR, Mastroiacovo P. The global burden of congenital toxoplasmosis: a systematic review. Bull World Health Organ 2013;91:501-8.

Norton ME, Chauhan SP, Dashe JS. Society for MaternalFetal Medicine(SMFM) Clinical Guideline #7 : Nonimmune

hydrops fetalis. Am J Obstet Gynecol 2015.;127-133.5) Mascaretti RS, Falcão MS , Silva AM, Costa Vaz FA, Leone CR. Characterization of newborns with Nonimmune Hydrops Fetalis admitted to a Neonatal Intensive Care Unit. Rev. Hosp. Clín. Fac. Med. S. Paulo 2003;58(3):125-132. 6.

Jones JL, Lopez A,Wilson M, Schulkin J, Gibbs R. Congenital toxoplasmosis: a review. Obstet Gynecol Surv 2001;56(5):296-305.

Singh S, Munawwar A, Rao S, Mehta S, Hazarika NK. Serologic Prevalence of Toxoplasma gondii in Indian Women of Child Bearing Age and Effects of Social and Environmental Factors. PLoS Negl Trop Dis 2014;8(3):e2737.

Singh S, Singh N. Toxoplasmosis is sexually transmitted. Proceeding of IX International Conference on AIDS. Berlin, Germany. 1993; 6–11.

Palanisamy M, Madhavan B, Balasundaram MB, Andavar R, Venkatapathy N. Outbreak of ocular toxoplasmosis in Coimbatore,India. Indian J Ophthalmol 2006;54:129-31.

10. Serranti D, Buonsenso D, Valentini P. Congenital toxoplasmosis treatment. Eur Rev for Med Pharmacol Sci 2011;15:193-8. 11. Mittal V, Ichhpujani RL. Toxoplasmosis – An Update. Trop Parasitol 2011; 1(1):9–14. 12. Dubey JP, Beattie CP. Toxoplasmosis of animals and man. Boca Raton, FL: CRC Press, Chapter 1.General Biology: Section V, Epidemiology and Epizootiology. 1988, pp 24. 13. Giannoulis C, Zournatzi B, Giomisi A, Diza E, Tzafettas I. Toxoplasmosis during pregnancy: a case report and review of the literature. Hippokratia 2008;12(3):139–143. 14. Paquet C, Yudin M.H. Toxoplasmosis in Pregnancy: Prevention, Screening and Treatment. J Obstet Gynaecol Can 2013;35:S1–S7. 15. Iqbal J, Khalid N. Detection of acute Toxoplasma gondii infection in early pregnancy by IgG avidity and PCR analysis. J.Med.Microbiol 2007;(56):1495–9.

*Corresponding author: Dr Mallikarjun Adiveppa Pattanashetti, Assistant Professor, Department of Pathology, S. Nijalingappa Medical College , Bagalkot, India Email: mallikarjun2030@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 18.09.2016 Date of Acceptance : 28.12.2016 Date of Publication : 26.03.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Case Report DOI: 10.21276/APALM.1088

Cholesterol Granuloma of Hydrocele Sac Mimicking Testicular Tumour Nehal Ahmad1, Sabina Khan1*, Mohammad Jaseem Hasan1, Sujata Jetley1 and Abhinav Jain2 1 Department of Pathology, Hamdard Institute of Medical Sciences and Research, Jamia Hamdard, New Delhi, India Department of Radiodiagnosis, Hamdard Institute of Medical Sciences and Research, Jamia Hamdard, New Delhi, India

ABSTRACT Cholesterol granuloma is a rare inflammatory granulation tissue that occurs in response to the deposition of cholesterol crystals. It is found most commonly in the paranasal sinuses or temporal bones, but there are also rare reports of their occurrence in other sites also. Here we report a rare case of cholesterol granuloma of hydrocele sac in a 59 year old man who presented with non tender right scrotal swelling. Patient was operated for vaginal hydrocele which on histopathological examination revealed cholesterol granuloma which is very rare in these sites. It can be very difficult to preoperatively distinguish testicular tumours from cholesterol granulomas of the testis or epididymis. Therefore, cholesterol granuloma should be kept in mind in patients with large and non-tender scrotal masses. Keywords: Cholesterol granuloma, Tunica vaginalis, Hydrocele

Introduction

Cholesterol granulomas are a type of non-specific inflammatory reaction to the presence of a foreign body such as cholesterol crystals [1]. They are found most commonly in the paranasal sinuses or temporal bones, but there are also rare reports of their occurrence in the testicular and epididymal sites as well as other sites like peritoneum, parotid gland, lymph nodes, thyroglossal duct, kidney, liver, and spleen [2,3]. These testicular and epididymal lesion may simulate an intrascrotal tumor on physical examination, on ultrasound and at operation. This lesion might be caused by trauma and inflammation. Here we describe a rare incidental case of cholesterol granuloma of hydrocele sac which presented as long standing scrotal swelling simulating a tumour on physical examination and ultrasound.

Case Report

A 59-year-old man came to our surgery OPD with complain of right scrotal swelling that had been present for five years. The patient was apparently asymptomatic with no signs of fever or acute infection. Mild scrotal discomfort was present. His past medical history was irrelevant; the patient was not hypercholesterolemic. The patient denied any history of known scrotal trauma. Laboratory studies including a complete blood count profile, basic biochemical profile, and urinalysis were all within normal ranges. The patientâ&#x20AC;&#x2122;s medical history was unremarkable with respect to tuberculosis, sarcoidosis, syphilis, and fungal infections. Physical examination revealed a 10.5-cm tense cystic,

non-tender scrotal swelling, testes was not palpable separately and transillumination was negative. A scrotal ultrasonography revealed turbid scrotal sac fluid with few echogenic areas and scrotal wall thickening. Surgery was performed for right vaginal hydrocele. At operation yellowish turbid fluid along with the thick sac was found. A partial resection of tunica vaginalis was performed, sparing the testis. A histopathological examination revealed mostly fibrocollagenous tissue, mixed inflammatory infiltrate consisting of lymphocytes, and few histiocytes, eosinophils along with presence of numerous cholesterol crystals, multinucleated foreign body giant cells. Based on these findings, the final diagnosis of a cholesterol granuloma of hydrocele sac was rendered.

Discussion

Cholesterol granuloma is a fibrogranulomatous lesion which forms in response to foreign-body giant cell reaction to cholesterol crystals [2-4]. It can develop in any region of the body where cholesterol crystal deposition may occur, and is a well-recognised lesion affecting the facial skeleton, skull and middle ear [5]. It was first reported in 1894 in middle-ear disease [6]. They have been described with less frequency in the kidneys, breast, peritoneum, mediastinum, parotid gland, lung, liver, spleen and testis. In our case it was seen in the testis [2,3,7]. The clinical presentation is variable and may be encountered incidentally or present due to space occupying effects on surrounding structures [2,5]. The cholesterol crystals stimulate a foreign-body type giant cell reaction that is responsible for the granuloma formation [3,5,7] . Although the exact pathogenesis of cholesterol

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Cholesterol Granuloma of Hydrocele Sac

Fig . 1 : High frequency Ultrasound image obtained with a 12 MHz linear transducer reveals turbid scrotal sac fluid with few echogenic areas and scrotal wall thickening. The testis and epididymis were normal on detailed scan.

Fig . 2 : Photomicrograph showing numerous cholesterol clefts and few epididymal glands (arrows) (H&E stain, 4 x)

Fig. 3 : Photomicrograph showing numerous cholesterol clefts, giant cells (arrows) and inflammatory cells comprising of lymphocytes, histiocytes , plasma cells and occasional eosinophils. (H&E Stain, 40x).

granuloma is unknown, it is believed that a non-infectious local reaction induces ischemic necrosis, granulomatous reaction, and scarring. It has been suggested that the source of cholesterol can be derived from degenerated cells during the inflammatory process and haemolysis or as a transudate from serum [2,3]. The presence of cholesterol crystals results in a foreign body reaction involving inflammatory cells and granulomatous tissue finally develops [8,9]. Histologically cholesterol granuloma consists of extensive granulation tissue with dense masses of cholesterol clefts surrounded by multinucleated giant cells, hemosiderinladen macrophages, lymphocytes and plasma cells [2,3,7].

This characteristic appearance has been described to be diagnostic of cholesterol granuloma which was also seen in our case [7]. There are characteristic features identified on imaging that may reflect a cholesterol granuloma, however surgical excision is required to enable histopathological analysis and diagnostic confirmation. There are very few cases of cholesterol granuloma of hydrocele sac reported in the literature. Farina Perez LAÂ and collegues reported a case of Hydrocele and cholesterol granuloma of the tunica vaginalis simulating a tumor in echography in 1998 [10]. Jain I. Lin and collegues reported

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Ahmad et al.

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a case of cholesterol granuloma of right testis in 1979 and emphasized the difference from lipogranuloma [11]. S. B. Lowenthal and collegues also reported a case of cholesterol granuloma of the tunica vaginalis simulating a testicular tumor [12]. In a study in 2009 it was reported that cholesterol granuloma of the testis is indistinguishable from carcinoma of the testis [1]. They concluded that if in doubt, surgical exploration and histopathologic examination are absolutely necessary. Similarly in our case the excised tissue was sent for histopathological examination. Macroscopically the mass originated from the tunica vaginalis and was a cystic lesion with a thick fibrous capsule of the sac. The cystic lesion was filled with dark yellowish brown material. Microscopically, the sections showed fibrogranulomatous tissue containing innumerable cholesterol clefts and numerous foreign body giant cells. The histological diagnosis was Cholesterol granuloma of hydrocele sac.

2. Luckraz H, Coulston J, Azzu A. Cholesterol granuloma of the superior mediastinum. Ann Thorac Surg 2006; 81: 1509–10.

Management of cholesterol granuloma is largely directed by the location, size and presentation of the lesion [2–5].

Conclusion

In conclusion, cholesterol granuloma of the hydrocele sac is an extremely rare benign condition. They often remain asymptomatic and are discovered incidentally on imaging or intra-operatively. Given that it is a benign and curable lesion, early recognition and management is important, and because it can mimic a malignant neoplasm, surgical resection should be considered.

Reference 1.

Lam CY, Lin CM, Tsai SW, et al. Cholesterol granuloma of the epididymis mimicking a paratesticular tumor. JTUA 2009; 20: 89-91.

Fujimoto K, Takamori S, Yano H, Sadohara J, Matsuo T, Terazaki Y, et al. Focal cholesterol granuloma in the anterior mediastinum. J Thorac Oncol 2007; 2: 1054–6.

Gore M, Zanation A, Ebert C, Senior B. Cholesterol granuloma of the petrous apex. Otolaryngol Clin North Am 2011; 44: 1043–58.

Nikolaidis V, Malliari H, Psifidis D, Metaxas S. Cholesterol granuloma presenting as a mass obstructing the external ear canal. BMC Ear Nose Throat Disord 2010; 10: 4.

Manasse P. Ueber Granulationsgeshwulst mit Fremdkoerriesenzellen. Virchows Arch 1894; 136: 245

Leon M, Chavez C, Fyfe B, Nagorsky M, Garcia F. Cholesterol granuloma of the maxillary sinus. Arch Pathol Lab Med. 2002; 126: 217–9.

Nistal M, Mate A, Paniagua R. Granulomatous epididymal lesion of possible ischemic origin. Am J Surg Pathol 1997; 21: 951-6.

Spajic B, Cupic H, Stimac G, et al. Cholesterol granuloma of the right epididymis mimicking an acute scrotum. Asian J Androl 2006; 8: 749-50

10. Fariña Pérez LA, Menéndez P, Macho V. Hydrocele and cholesterol granuloma of the tunica vaginalis simulating a tumor in echography. Actas Urol Esp. 1998 Jan; 22(1): 70-3. 11. Lin JI, Tseng CH, Marsidi PJ, Bais VC. Cholesterol granuloma of right testis; Urology 1979 ; 14(5): 522–523 12. Lowenthal SB, Goldstein AMB, Terry R. Cholesterol granuloma of tunica vaginalis simulating testicular tumor. Urology July 1981; 18(1): 89–90

*Corresponding author: Dr Sabina Khan. Associate Professor, Department of Pathology, Hamdard Institute of Medical Sciences and Research, Jamia Hamdard , New Delhi 110062, India Phone: +91 9540248289 Email: drsabina1@gmail.com Date of Submission : 29.09.2016 Date of Acceptance : 31.12.2016 Financial or other Competing Interests: None. Date of Publication : 28.03.2017

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Case Report DOI: 10.21276/APALM.1094

Primary Neuroendocrine Carcinoma of Breast: An Uncommon Variant Sunil V Jagtap1*, Vidya Chandrashekhar Aher1, Suresh Jaywantrao Bhosale2, Atul Hulwan1 and Anup Nanagir Gosavi1 Department of Pathology, Krishna Institute of Medical Sciences Deemed University (KIMSDU). Maharashtra, India 2 Department of Surgery, Krishna Institute of Medical Sciences Deemed University (KIMSDU). Maharashtra, India

ABSTRACT Neuroendocrine carcinoma of breast ( NECB) is an aggressive variant of invasive mammary carcinoma. Very few cases of Primary NECB have been reported in the literature. Here we report a 50 year female presented with left breast lump measuring 15 x 12 cm. of gradual onset with left axillary lymphadenopathy. On fine needle aspiration cytology of breast and lymphnode were positive for malignant cells. On ultrasonography of abdomino- pelvis showed metastasis to liver, peri pancreatic and para aortic lymph nodes with minimal ascites. On histopathology reported as Primary NECB-Large cell type-left breast. We are presenting this case for its extreme rarity and aggressive clinical behaviour. Keywords: â&#x20AC;&#x201C; Neuroendocrine Carcinoma of Breast, Endocrine Tumor, Breast Cancer, Histopathology

Introduction

Neuroendocrine carcinoma of breast ( NECB) is an aggressive variant of invasive mammary carcinoma. It is extremely rare tumor accounting for 0.27 to 0.50% of histopathologically proven breast cancers [1,2]. Most neuroendocrine carcinomas are located in gastrointestinal tract and lung [3]. Only a limited number of studies on neuroendocrine breast have been reported in the literature and most of them are case studies.

Case Report

A 50 year old postmenopausal female presented with a single, large, hard, non-tender, gradually increasing, lump in the left breast of 8 months duration. At that time there was no other systemic disease was evident on clinical and other investigations. Patient delayed the initial treatment and came later with retraction of nipple and left axillary lymphadenopathy. She had no history of significant systemic disease. Her obstetrics history was G4P4D0L4 with menopause six years back. Her past and family history was non contributory. Radiological examination by ultrasonography of abdomen pelvis at second visit revealed metastasis to liver, peri pancreatic and para aortic lymph nodes with minimal ascites. Mammography showed heteroechoeic lesions with hypoechoic rims. Fine needle aspiration cytology left breast was positive for malignant cells. Modified radical mastectomy was performed .Histopathological examination revealed gross specimen of left breast measuring 20x18x9 cm and weighing 900gms. It was covered with skin measuring 17x15cm (Fig.1). Tumour was fixed to skin with nipple areola retraction. Peau-d-orange was also noted. On cut section

revealed a tumor measuring 15x12x6 cm (Fig.2). Tumor was grey white, firm to hard in consistency with pushing peripheral margins. On microscopy, multiple sections showed tumor composed of neoplastic cells, arranged in large sheets, alveolar pattern, solid nests separated by thin fibrous septae(Fig.3). Individual tumor cells were large, round, pleomorphic having salt and pepper chromatin and moderate eosinophilic granular cytoplasm which constituted more than 60% of tumor morphology(Fig.4). Focally invasive breast carcinoma is noted. Intervening stroma showed diffuse mononuclear cell infiltration and fibrosis. On histopathological examination diagnosed as NECB-Large cell type â&#x20AC;&#x201C; left breast. On Immunohistochemistry tumor showed > 50 % tumor population showing positivity for Synaptophysin and chromogranin. Tumor was positive for ER and PR receptors. Our patient underwent left modified radical mastectomy and received chemotherapy and radiotherapy. On follow up, the patient is doing well.

Discussion

Primary NECB is an extremely rare specific subtype of breast malignancy . Most of the neuroendocrine tumors are located in the gastrointestinal tract and lung .The morphological features of primary NECB are similar to that of other sites. Many benign and malignant lesions of various organs show neuroendocrine differentiation [4]. Neuroendocrine differentiation in breast carcinoma shows morphological similarity to carcinoma of gastrointestinal tract, lung and other nonendocrine organs with endocrine differentiation and carcinoid tumor [5]. In breast carcinoma,

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Jagtap et al.

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Fig. 1: Gross specimen of left breast- modified radical mastectomy.

Fig. 2: left breast on cut open showing grey white, homogenous, firm to hard tumor with pushing borders m. 15x12x6cm.

Fig. 3: photomicrograph showing breast tissue with tumor composed of neoplastic cells arranged in large sheets, alveolar pattern and solid nests separated by thin fibrovascular septa. H&E stain 100X

Fig. 4: Individual tumor cells are medium to large size, round to pleomorphic having salt and paper chromatin and moderate amount of eosinophilic granular cytoplasm. . H&E stain 400X

not otherwise specified (NOS) focal neuroendocrine differentiation can be seen scattered singly or in groups [6].

more than 50% of the population of tumor expressing immunohistochemical staining for neuroendocrine markers for diagnosing primary NECB and the extra mammary sites should be excluded [7].

NECB used to be known as argyrophilic breast carcinoma, Carcinoid tumor or Endocrine carcinoma and now classified as breast carcinoma with neuroendocrine differentiation or Primary Neuroendocrine carcinoma [7,8]. In 50% of breast tumors, scattered neuroendocrine cells can be detected. Neuroendocrine differentiation has been reported in both in situ and invasine breast carcinoma. It is observed that mucinous carcinoma of breast has the greatest association with neuroendocrine differentiation [8] . WHO in 2003 defined NECB as breast carcinoma with

Our patient was a 50 year old woman who presented with breast lump of 8 months duration which was gradually increasing in size with axillary node enlargement .Initially patient was not ready for surgical management but came at the clinic in late stage of disease. The mean age at diagnosis of the patient with NEC is 64 years. The mean tumor size is 3.2cm for NEC [6].. Where as in our case tumor was very large size as patient presented in late stage of disease. It

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Primary Neuroendocrine Carcinoma of Breast

is reported that about 43% of NEC cases presented with lymph node metastasis at the time of diagnosis [9]. In our case patient had significant axillary node involvement. On histology, neuroendocrine carcinoma usually shows neoplastic cells arranged in alveolar pattern, solid sheets with tendency to produce peripheral palisidation. Depending on cell type and differentiation it is subtyped into- Solid neuroendocrine carcinoma ,Oat cell carcinoma , Large cell neuroendocrine carcinoma [7, 9, 10]. In large series by Sapino et al, described tumors in 5 subtypes; solid cohesive, alveolar, small cell, solid papillary, cellular mucinous [6] .Our case shows predominant solid, alveolar and nest pattern having large cells with focal areas of mucin production. Few cases reported in literature of primary breast NEC of large cell type by Kim JW et al [2], Stita W et al [10], Hanna MY et al [11], Wei B et al. [12]. Overall survival of patients with NEC is determined significantly by tumour size, lymph node status, and proliferation rate. Clinical outcomes reported in literature showed 15% of local recurrence by 5 years, with median recurrence free time of 177 months and 34% risk for distant recurrence within 5 years [12].

Kim JW, Woo OH, Cho KR, Seo BK, Yong HS, Kim A, et al. Primary large cell neuroendocrine carcinoma of the breast: radiologic and pathologic findings. J Korean Med Sci 2008;23:1118-20.

Modlin IM, Lye K D, Kidd M. A five decade analysis of 13,715 carcinoid tumors. Cancer.2003;97:934-959.

David O, Bhattacharjee M. Diffuse neuroendocrine differentiation in a morphologically composite mammary infiltrating ductal carcinoma:a case report and review of literature. Arch Pathol Lab Med.2003;127(3):e131-4.

Ozbilim G, Kilicarslan B, Tezer E, Buyukkece A, Ustum M, Karaveli S et al. Breast carcinoma showing neuroendocrine differentiation characterized with ectopic hormone production (2 case report). Turk J Med Sci 2000;30:609-13.

6. Sapino A, Righi F, Cassoni P, Papitti M, Gugliotta P, Bussolati G. Expression of the neuroendocrine phenomenon in carcinoma of the breast. SeminDiagnPathol 2000;17:127-37. 7.

Ellis IO, Schnitt SJ, Saste-Garau X. Invasive breast carcinoma. In: Tavassoli FA, Devilee P, editors. World Health Organization classification of tumours. Pathology and genetics of the tumours of breast and female genital organs. Lyon: IARC press;2003:13-59.

Tovosoli FA, Pathology of the breast. 1st ed. Norwalk: Connecticut. Appleton and Lange;2000.

Primary NECB is an unusual and aggressive carcinoma of breast. We are presenting this case for its extreme rarity and with advanced clinical behaviour. The treatment modality is not different from those conventional breast malignancies, however chromogranin production in neuroendocrine carcinoma has clinical and genetic implications owing to the biochemical analogy between granin and breast cancer (BRCA) protein. Hence, targeted gene therapy may be a future treatment strategy.

Wang Jun, Bing Wei, Constance TA, et al. Invasive neuroendocrine carcinoma of breast: a population based study from the surveillance, epidemiology and end result (SEER) database, BMC cancer.2014;14:147.

References

12. Wei B, Ding T, Xing Y, Wei W, Tian Z, Tang F, et al. Invasive neuroendocrine carcinoma of the breast: A distinctive subtype of aggressive mammary carcinoma. Cancer. 2010;116:4463â&#x20AC;&#x201C;73.

Conclusion

Zhang Y, Chen Z, Bao Y, Du Z, Li Q, Zhao Y, et al. Invasive neuroendocrine carcinoma of the breast: a prognostic research of 107 Chinese patients. Neoplasma 2013;60:215-22.

10. Stita W, Trabelsi A, Gharbi O, Mokni M, Korbi S. Primary solid neuroendocrine carcinoma of the breast Can J Surg 2009;52:E289-E290. 11. Hanna MY, Leung E, Rogers C, Pilgrim S. Primary large-cell neuroendocrine tumor of the breast. Breast J. 2013;19:204â&#x20AC;&#x201C;206.

*Corresponding author: Dr Sunil Vitthalrao Jagtap, Gadkari-mala, Vadgaon haveli-415110, Maharashtra, India. Email: drsvjagtap@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 10.06.2016 Date of Acceptance : 19.01.2017 Date of Publication : 28.03.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Case Report DOI: 10.21276/APALM.1129

Inflammatory Myofibroblastic Tumor of Urinary Bladder Ronica Baruah1, Abhijit Kalita1* and Phanindra Mohan Deka2 Department of Pathology, Ekopath Metropolis Lab Services Pvt. Ltd. Guwahati, Assam, India 2 Department of Urology, Dispur Hospitals Pvt. Ltd. Guwahati, Assam, India

ABSTRACT Inflammatory myofibroblastic tumor (IMT) is an uncommon benign tumor of intermediate neoplastic potential, characterised by spindle cell proliferation with characteristic fibroinflammatory and pseudosarcomatous appearance. A subset of IMT with histologic atypia and/or clinical aggressiveness is also known. IMTs occur in the mesentery, omentum, retroperitoneum, pelvis, and abdominal soft tissues. However, the occurrence of IMT in urinary bladder is unusual. IMT exhibits morphologic and immunophenotypic overlap with malignant spindle cell tumors of the urinary bladder and is diagnostically challenging. In the case presented, ALK-1 and SMA immunostains helped to identify IMT. Anaplastic Lymphoma Kinase (ALK) gene translocation or ALK gene expression can further confirm IMT. Complete surgical excision with follow-up is the treatment of choice for IMT. Keywords: Inflammatory Myofibroblastic Tumor, ALK-1 Immunostain, Spindle Cell Proliferation.

Introduction

Inflammatory myofibroblastic tumor (IMT) is a distinctive neoplasm composed of myofibroblastic and fibroblastic spindle cells accompanied by inflammatory infiltration of plasma cells, lymphocytes, and eosinophils [1,2]. IMT is also known as pseudosarcoma, atypical myofibroblastic tumor, atypical fibromyxoid tumor and pseudosarcomatous appearance. IMT is a neoplasm of intermediate biologic potential[3]. A subset of IMT is identified with histologic atypia and clinical aggressiveness, and may be difficult to distinguish from other sarcomatous proliferations. It may affect any age group, but it is more common in children and young adults with slight female preponderance . The origin of IMT is controversial, but a recent report suggests that it is neoplastic because of aggressive behaviour, involvement of chromosome 2p23, and congenital clonality. IMTs occur in the mesentery, omentum, retroperitoneum, pelvis, and abdominal soft tissues. However, the occurrence of IMT in urinary bladder is unusual[1]. IMT exhibits morphologic and immunophenotypic overlap with malignant spindle cell tumors of the urinary bladder and diagnostic distinction from these tumors can be problematic.Â Both epithelial and myogenic markers can be expressed in IMT and may lead to a misdiagnosis of sarcomatoid carcinoma, leiomyosarcoma, and rhabdomyosarcoma [4].The treatment of choice is total excision of the tumor [5].

Case Report

A 45 year old male presented with painless hematuria, clots in urine and burning micturition for one month. No past history or family history of any specific disease was elicited. Urine microscopic examination showed plenty

of red cells as the only abnormal finding. Cystoscopic examination was done which showed a large number of blood clots which were subsequently evacuated. Following evacuation of the clot, a large broad based solid tumor was found in the posterior wall of the urinary bladder for which transurethral resection of the tumor was done and biopsy sent for histopathological examination. The tissue received consisted of multiple bits aggregating to 3x3x1 cm. Sections from the formalin fixed paraffin embedded bladder mass stained with Hematoxylin and Eosin showed spindle cell proliferation with moderate nuclear atypia, occasional mitotic figures and few scattered lymphocytes. Similar spindle cells were also seen infiltrating the muscularis mucosae and involving the base of tumor (Figure 1). It presented as a malignant spindle cell tumor. However, there was absence of atypical mitotic figures and areas of necrosis, presence of delicate abundant vascular network, scattered oedematous / myxoid areas and inflammatory cells in deeper layers of the tumor. These features prompted a possible benign nature of the tumor. A provisional diagnosis of Sarcomatoid Transitional cell carcinoma was given based on the location of the tumor, invasion of muscular layer and nuclear atypia. Immunohistochemistry was recommended for final diagnosis. Considering various possibilities, a battery of immunostains were done on the tumor section, to come to a final diagnosis. The immunostains included Ki 67, CK 7, CK 20, Uroplakin, S-100, Chromogranin, Synaptophysin,

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Inflammatory Myofibroblastic Tumor

EMA, desmin, SMA, and ALK-1. The tumor showed a high proliferative index (high Ki 67 - Figure 2). Transitional carcinoma was ruled out following negative staining for CK20, CK7 and Uroplakin. S-100, EMA and desmin were also negative, ruling out the possibility of the tumor having neural or muscular origin. Neuroendocrine lesion was ruled out based on negativity for synaptophysin and chromogranin. However, SMA showed focal positivity and ALK-1 showed strong positivity (Figure 2).

The immunohistochemistry results were compiled and most sarcomatous lesions were ruled out. Consequently, a benign spindle cell lesion , either IMT or Post-operative Spindle Cell Nodule (PSCN) were considered in the differential diagnosis. PSCN was excluded as there was no past history of any surgical intervention or biopsy. So, a final diagnosis of IMT was given. However, translocation study of ALK gene was recommended (by cytogenetics or Fluorescent-in-situ Hybridisation) for the confirmation.

Fig. 1: Fascicles of spindle cells (A) with nuclear atypia (B) and involvement of muscular layer (C).

Fig. 2: Immunohistochemistry showing high Ki67 (A) and strong positivity for ALK-1.

Discussion

Inflammatory myofibroblastic tumor (IMT) of the urinary bladder is an unusual spindle cell lesion that exhibits cytologic atypia, infiltrative growth, and mitotic activity mimicking malignant tumors, such as leiomyosarcoma, rhabdomyosarcoma, and sarcomatoid carcinoma [4]. It is idiopathic and no known predisposing condition exist for myofibroblastic tumor of the bladder [5]. However, trauma is considered as a strong possible cause in many cases. Essential criterion for the diagnosis of IMT are : spindle myoepithelial cell proliferation and lymphocyte infiltrate [7].

Studies have demonstrated clonality, suggesting that they are neoplastic rather than reactive in nature; a possible exception is those that occur immediately following instrumentation[8] (post-operative spindle cell nodule). Post-operative spindle cell nodule (PSCN) is a rare nonneoplastic lesion of the bladder consisting of a reactive proliferation of spindle cells, occurring between several weeks or months following surgical intervention, such as transurethral resection or biopsy [9]. Recently, anaplastic lymphoma kinase (ALK) gene translocation or ALK protein expression in IMT has been reported, in relatively

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younger age group. The detection of ALK protein and ALK gene rearrangements are useful in distinguishing IMT from other spindle cell malignancies in the urinary bladder [4,10]. Coffin CM et al [3] concluded from his study that ALK reactivity was associated with local recurrences, but not distant metastasis, which was confined to ALKnegative lesions.

immunohistochemistry panel is needed for a final diagnosis. Identification of spindle cell proliferative lesions like IMT is important to avoid radical resections. 1.

Etani T, Naiki T, Nagai T, Lida K, Ando R, Naiki-Ito A et al. Inflammatory Myofibroblastic tumor of urinary bladder; A case report. Case Rep Oncol 2016; 9(2) : 464-469

The case under discussion is an aggressive spindle cell tumor on account of its invasion of muscular layer of the bladder and involvement of the base of the tumor, coupled with an atypical cellular morphology which also showed high proliferative index with Ki67 on immunohistochemistry. Similar aggressive histological features were also identified by case studies of Etani T et al [1] , Yagnik V et al [7] and Tanny SPT et al [11]. However, benign nature of the tumor was also considered by the histological features discussed above.

Fletcher CDM, Bridge JA, Hogendoorn P, Mertens F. WHO Classification of Tumours of Soft Tissue and Bone. Vol. 5. Lyon : IARC Press ; 2013 ; 83–84.

Coffin CM, Hornick JL, Flethcher CD. Inflammatory myofibroblastic tumor: Comparison of clinicopathologic, histologic, and immunohistochemical features including ALK expression in atypicall and aggressive cases. Am J Sur Pathol 2007; 31(4): 509-520

Rao RN, Ranjan P, Singla N, Pandey R. Inflammatory myofibroblastic tumor of the urinary bladder diagnosed by anaplastic lymphoma kinase immunostaining. Urol Ann 2012; 4(2): 115-118

Dobrosz Z, Rys J, Palen P, Wlaszczuk P, Ciepiela M. Inflammatory myofibroblastic tumor of the bladder – an unexpected case co-existing with an ovarian teratoma. Diagnost Pathol 2014:9;138

Pettinano G, Manivel JL, De Rosa N, Dehner LP. IMT (plasma cell granuloma): Clinico-pathologic study of 20 cases with immunohistochemical and ultrastructural observations. Am J Clin Pathol 1990; 94: 538-546

Yagnik V, Chadha A, Chaudhari S, Patel K. Inflammatory myofibroblastic tumor of the urinary bladder. Urol Ann 2010; 2(2): 78-79

Mills SE. Editor. Sternberg’s Diagnostic Surgical Pathology. 5th ed.; Lippincott Williams & Wilkins; 2010

Zhao J, Ping H, Xing N. Post-operative spindle cell nodule of the bladder : a case report and review of literature. Oncol Lett. 2014; 7(5): 1507-1510

The immunostains done for the tumor in our case also assisted in clinching the final diagnosis. A strong positivity for ALK with a high proliferative index (Ki 67), and focal positivity for SMA, projected the tumor as an atypical form of IMT. Positivity for SMA and ALK-1 was also seen in the case report of Yagnik V et al [7]. However, desmin was additionally expressed in that case study. The case report of Etani T et al [1] showed positivity for ALK, vimentin and SMA, but was negative for desmin. Study of the immunostains also ruled out the malignant sarcomatous proliferations, as discussed above. It is important to rule out IMT and PSCN from malignant spindle cell proliferations of the bladder, in order to avoid radical surgical interventions. IMTs and PSCNs can be treated with total excision alone, followed by a close surveillance.

Conclusion

IMT needs to be suspected in case of spindle cell proliferation with/without atypia and chronic inflammatory infiltrate in urinary bladder, particularly with no history of instrumentation (which rules out PSCN). A thorough histopathological examination along with a suitable

References

10. Toyonori T, Christina M, Jonathan E. ALK-1 Expression in Inflammatory Myofibroblastic Tumor of the Urinary bladder. Am J Sur Pathol 2004; 28(12): 1609-1614 11. Tanny SPT, Wang LL, Liddell HA, Longano A, Appu S, Shahbaz S. Inflammatory myofibroblastic tumor of the urinary bladder : a case report. Urol Case Rep 2016 ; 6: 58-59

*Corresponding author: Dr. Abhijit Kalita, House no 66, Happyvilla, Ujanbazar, Guwahati, Assam, India. Pin: 781003. Email: abhighy1985@gmail.com

Financial or other Competing Interests: None.

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Date of Submission : 01.11.2016 Date of Acceptance : 01.02.2017 Date of Publication : 28.03.2017

eISSN: 2349-6983; pISSN: 2394-6466

Case Report DOI: 10.21276/APALM.1146

A Case Series of Congenital Ovarian Cyst in A Tertiary Care Hospital in Mumbai, India Bhavana Madhukar Bharambe*, Saroj Ashok Bolde, Sushma Chandeshwar Bharti and Neha Satyanarayan Somani Department of Pathology, Grant Government Medical College, Mumbai, India

ABSTRACT The congenital ovarian cysts are one of the most frequent lesions to be diagnosed antenatally. The manifestations postnatally determine the line of management of the cysts. These can manifest as abdominal lump, torsion or may be asymptomatic. We present a case series of four cases of congenital ovarian cyst presenting to our tertiary care hospital. Keywords: Ovarian Cyst, Congenital Cyst, Antenatal Ovarian Cyst, Childhood Ovarian Cyst

Introduction

Congenital ovarian cyst is a rare clinical finding seen in infants. This entity was first described by Valenti et al[1] in 1975. These cysts are present prenatally and are increasingly being diagnosed in fetal or postnatal period by various diagnostic modalities of the modern era, especially antenatal ultrasonography (USG) and magnetic resonance imaging (MRI). The ovarian cysts can be symptomatic or asymptomatic. They are classified into simple and complex radiologically by Nussbaum et al.[2]These are usually unilocular and unilateral. We present four cases of congenital ovarian cysts with varied manifestations and histomorphology.

Case Report(S)

Case 1: A 14 day female child born to 26 year old mother was diagnosed with left ovarian cyst in the antenatal scan. The cyst was sized 2.8x2.2x1.5 cm and was unilocular. The remaining investigations were normal. Case 2: A 27 day old child was bought to pediatric surgery department with complaints of crying spells and vomiting. The patient had left lumbar and iliac tenderness and guarding of abdomen. On USG, a twisted complex ovarian cyst sized 5.5x3.7x4.2 cm was seen on left side with tiny cystic spaces within. The child was immediately operated upon for derotation of ovarian cyst with deroofing done. Bilateral oophoropexy was done. Postoperatively, patient is doing well and is healthy. Case 3: A one month and 6 day old female child was bought by her 25 year old mother with complaints of constant crying. On examination, tenderness was noted in abdomen on left side. USG abdomen showed well defined cystic lesion in left lumbar region suggestive of mesenteric

or ovarian cyst. Contrast enhancing computed tomography (CECT) abdomen showed a well defined cystic lesion suggestive of ovarian teratoma or enteric duplication cyst. Diagnostic laparoscopy revealed twisted ovarian cyst which was removed and sent for histopathology. The child was stable after surgery. Case 4: A full term, one day old female child, born to a primigravida was admitted with complaints of a pelvic cyst. The cyst was diagnosed prenatally by USG. It was a well defined cyst sized 4x4.8x4.9 cm on left side showing septations. The ovarian cystectomy was performed with oophorectomy. None of the above babies had any associated congenital malformation as described in literature. The gross and histopathological examination of the received ovarian cysts confirmed the ovarian origin with focally preserved flattened to cuboidal lining epithelium. There was evidence of dystrophic calcification in all the cases along with stromal hemorrhages (Fig. 1 -3).

Discussion

The term congenital ovarian cyst encompasses all the ovarian cysts detected around neonatal period. Though exact age limit hasnâ&#x20AC;&#x2122;t been proposed in the literature. The retrospective evaluation of still borns and newborns with death within 4 weeks of birth suggests that the overall incidence of congenital ovarian cyst is approximately 30%.[3]The other intra-abdominal cysts which are common include ureterocele, urachal cyst, hydrocolpos, renal cysts, enteric duplication cyst, meconium cyst, lymphangioma and fetus in fetu. These ovarian cysts are believed to arise from the persistent effect of maternal gonadotrophin levels on the fetal ovarian

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Fig. 1: A unilocular cyst with smooth external surface and hemorrhagic inner lining.

Fig. 2: Shows dystrophic calcification (red arrow) and focally ulcerated flattened epithelial lining (blue arrow). (H&E, X 400).

Fig. 3: Shows haemorrhages in the cyst wall. (H&E, X 400).

tissue resulting in the stimulation of a naive ovarian follicle causing a cystic transformation.[4] These cysts are earliest detected on prenatal imaging done as routine ultrasonography evaluation and can be picked up as early as 28th week of gestation. However, all cysts identified on prenatal ultrasound do not need an intervention. The need to actively intervene depends on serial documentation of features which act as predictive markers for development of complications significant to affect the life of neonate. The most common complication to arise in congenital ovarian cyst is development of torsion of the ovarian pedicle which forms 50 to 70% of all complications.[4,5] The remaining complications being haemorrhage, rupture, peritionitis secondary to inflammation, urinary tract obstruction,

bowel obstruction, autoamputation and in rare case of sterile necrosis of the ovary. These congenital ovarian cysts are known to undergo spontaneous regression.

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Several studies have tried to evaluate the predictive markers in prenatal ultrasonography which can predict an increased risk of developing complication and planning an active intervention. Such predictive markers on prenatal USG include length of the ovarian pedicle, size of the cyst, laxity of the ovarian and infundibular ligament, solitary or multiple cyst in the same ovary, unilateral or bilateral involvement, simple versus complex nature of cyst, presence /absence of increased vascularity or congestion of ovarian pedicle on colour doppler.[5] Torsion of ovarian pedicle does result in inflammatory process reflected

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Congenital Ovarian Cyst

by subtle rise in alpha foeto protein/carcinoembryonic antigen levels but they have not been found to be predictive in identifying development of complications in congenital ovarian cysts. The most important markers on prenatal ultrasonogram to predict a complication include size and eco pattern of the cyst. A simple uniform solitary cyst of size less than 4 cm is unlikely to give rise to any complication, however such cyst have a potential for undergoing a reduction with progression of pregnancy and spontaneous resolution. This advocates a serial ultrasonographic evaluation in prenatal phase, at weekly interval to document the progress of cyst with pregnancy. Bagolan et al. managed 34 simple ovarian cysts< 5 cm with wait and see policy and have observed spontaneous regression in 26 cases, 1 persistent at birth and 7 cases of torsion.[6,7]Certain studies report conservative management for simple cyst upto size of 8cm. Intrauterine ovarian cyst aspiration remains another promising option for management of congenital ovarian cyst. However, the risk associated with invasive procedure, the potential to cause haemorrhage, rupture/ spillage with inflammation, or infection does merit a careful selection of patents to be considered for aspiration. Bagolan et al in their study performed in utero aspiration of fourteen simple cysts measuring 5 cm observing resolution in twelve cases and torsion in two concluding that “in utero” aspiration of ovarian cyst is a safe procedure.[6,7] Cases of congenital ovarian cysts which present with torsion or rupture warrant a surgical exploration either open or minimal invasive approach. In cases where complex ovarian cyst appears likely, ultrasonography may not be reliable and CT scan or MRI is more informative to define the nature of cyst. The time to consider for surgical intervention is also influenced by the extent of fetal lung maturation to salvage the cystic ovary. Most of the cases present as unilocular cysts which histologically may be follicular cyst, simple cyst, theca lutein cyst or corpus luteum cyst with characteristic presence of dystrophic calcification and haemorrhages in the cyst wall. The lining epithelium may or may not be seen.

In our study, all cases were symptomatic and all presented in post natal period under the age of 2 yrs with symptoms of abdominal pain. All the patients underwent surgical procedure in view of ultrasonography findings, symptomatic presentation and development of complications. The retrospective evaluation of clinical spectrum and histopathology reveals presence of cyst in prenatal ultrasonography which subsequently underwent complication requiring surgical intervention.

Conclusion

Congenital ovarian cyst is a significantly prevalent finding on prenatal ultrasonography, which usually requires a regular sonographic monitoring. Intervention is only required in patients with complications or those who are at potential risk for developing complications as evaluated on serial monitoring. Overall prognosis for all congenital ovarian cysts remains excellent.

Reference 1.

Valenti C, Kassner EG, Yermakow V, Comb E. Antenatal diagnosis of a fetal ovarian cyst. Am J Obstet Gynecol 1975;15:216–219.

Nussbaum AR, Sanders RC, Hartmann DS. Neonatal ovarian cysts: sonographic-pathologic correlation. Radiology 1988;168:817–821.

Anna Dera-szymanowska, Mariolaropacka-lesiak, Michałbłaszczyński, Marta Szymankiewicz, Grzegorz H. Bręborowicz. Torsion of adnexal cyst in utero – case report. Archives of Perinatal Medicine 2012;18,229-232.

Mudholkar V, Acharya A, Kulkarni A, Hirgude S. Antenatally diagnosed neonatal ovarian cyst with torsion. Indian Journal of Pathology and Microbiology 2011;54:228- 229.

Ibrahim H, Lewis D, Harrison GK, Tice H, Sangster G. Journal of Perinatology 2007;27,523–526.

Bagolan P, Rivosecchi M, Giorlandino C, Bilancioni E, Nahom A, Zaccara A, et al. Prenatal diagnosis and clinical outcome of ovarian cysts . J Pediatr Surg 1992;27:879-881.

Bagolan P, Giorlandino C, Nahom A, Bilancioni E, Trucchi A, Gatti C, et al. The management of fetal ovarian cysts. J Pediatr Surg 2002;37:25-30.

*Corresponding author: Dr. Bhavana Madhukar Bharambe, Flat 32, Trimurti Bldg, JJ Hospital Campus, Byculla, Mumbai-400008, India Phone: +91 9892906747 Email: bhavanab.136@gmail.com

Financial or other Competing Interests: None.

Date of Submission : 07.11.2016 Date of Acceptance : 03.02.2017 Date of Publication : 28.03.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Case Report DOI: 10.21276/APALM.1149

Occult Primary Thyroid Carcinoma Presenting as Lateral Cervical Mass: Report of Two Cases Shalini Bahadur , Priyanka Anand*, Kamlesh Prajapati and Namrata Nargotra Dept. of Pathology; NDMC and Hindu Rao hospital. New Delhi, India

ABSTRACT Background: Thyroid cancer presenting with cervical lymphadenopathy as a sole occurrence is uncommon. It usually presents as a palpable thyroid mass or nodule. Isolated cervical lymphadenopathy is rare and hence poses a diagnostic challenge. Fine needle aspiration- cytology (FNAC) or biopsy are important modalities which help establish the origin. Case Report: Two cases of lateral cervical lymphadenopathy with no other specific clinical findings were encountered within a span of two weeks with different clinical backgrounds. First to present was a 28 year old young male with a months’ history of lateral cervical swelling. Later a 58 year old male also presented without any history of overt primary thyroid carcinoma. FNAC in both the cases confirmed presence of metastasis from occult primary papillary cancer thyroid. Following this ultrasonography neck was performed which revealed a solid-cystic mass in both the cases. In the younger male an ill-defined solid nodule with microcacifications was identified in the left thyroid lobe while in the case of elderly male; thyroid gland was normal. Subsequently both underwent total thyroidectomy with left neck dissection and histologically proven primary papillary thyroid cancer was found. Conclusion: Posterior cervical lymphadenopathy occurring primarily as a result of papillary carcinoma thyroid is rare. Keywords: Cervical Lymphadenopathy, Papillary Thyroid Cancer, Fine Needle Aspiration- Cytology, Histologic Examination.

Introduction

Thyroid cancer presenting with cervical lymphadenopathy as sole occurrence is uncommon.[1,2,3] Cervical lymphadenopathy is attributable to a variety of nonneoplastic and neoplastic causes with likelihood of a benign process in younger patients. Metastatic cervical lymphnodes potentially have primary source in head and neck as well as elsewhere and need investigations to guide further management. Fine needle aspiration cytology (FNAC) or biopsy are important modalities which help establish this origin. We hereby report two cases of papillary carcinoma thyroid presenting as lateral cervical masses.

Case Reports

Case 1: A 28-year old male patient presented with brief history of a months’ duration of an enlarging left neck mass. There were no associated symptoms. On examination a soft to firm non- tender posterior cervical swelling measuring 2 x 2.5cm with restricted mobility was noted. Thyroid gland was not palpable. Baseline investigations including complete blood counts (CBC), electrolytes and thyroid function tests were within normal limits. A family history of head and neck cancer was denied. Previous exposure to radiation was not found. A FNAC performed showed cells in syncytial aggregates, balls and sheets with anatomical borders. Nuclear crowding and

overlapping was noted with relatively abundant cytoplasm. Several of the nuclei showed nuclear grooving as well as occasionally presence of intranuclear cytoplasmic inclusions. Possibility of metastatic papillary carcinoma thyroid was suggested on FNAC. Following this Ultrasonography of neck was done. A 2.5cm solid–cystic mass with thick irregular walls was

found. An ill-defined hypoechoic solid nodule with micro-calcifications was identified in left thyroid lobe. A Technetium perfusion scan showed evidence of increased perfusion in cold nodule involving left lobe of thyroid gland with homogenous uptake in right thyroid lobe. Thyroidectomy with left neck dissection was performed. Case 2: A 58-year old male presented with a left sided neck mass of three months’ duration. No positive contributory clinical history was noted. On examination a firm, nontender 3x2cm mass was noted on left side of neck. FNAC was performed that showed syncytial aggregates and papillary fragments without apparent fibrovascular cores. Nuclei had a powdery chromatin with many showing longitudinal cytoplasmic grooves. Occasional intranuclear cytoplasmic inclusions were evident along with thick streaks of colloid-like material. Again, possibility of metastatic carcinoma thyroid was suggested. USG on follow up revealed a well-defined solid- cystic mass in left side of neck of size 2cms and both lobes of thyroid showed apparently normal size and echotexture.

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Thyroid Carcinoma Presenting as a Cervical Mass.

Fig. 1(Case 1): Metastatic Papillary Thyroid Carcinoma in cervical lymph node: (A)- Papillary cytoarchitecture with anatomical edges. (MGG; 10x) (B)- Cells having round to oval nuclei with pale powdery chromatin and many of them showing intranuclear grooves. (Papanicolaou; 100x) (C)- Ultrasonography- A 2.5cm solid â&#x20AC;&#x201C; cystic mass with thick irregular walls. (D)- Cells with intra-nuclear cytoplasmic inclusion (arrow) with a foamy macrophage seen on the right side of the image. (MGG; 40x).

Fig. 2 (Case 2): Metastatic Papillary Thyroid Carcinoma in cervical lymph node: (A)- Cells with intranuclear grooves. (PAP; 40x) (B)- Cells with crowded nuclei and one intra-nuclear cytoplasmic inclusion seen as shown by an arrow. (MGG; 40x) (C)Cells with dense metaplastic squamoid cytoplasm. (MGG; 40x) (D)- Papillae with anatomical borders. (PAP; 10x).

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Discussion

cytoplasmic positivity of antibodies against CK19 and CK903 while the benign thyroid follicles stain only weakly and focally for it.[15]

Thyroid neoplasms commonly present in unsuspecting patients as a thyroid mass.[1,2,4] Less commonly instead of a dominant thyroid lesion, lateral neck mass occurs heralding an occult thyroid primary. Evaluation of a lateral neck mass is diagnostically difficult since a variety of differential diagnosis need to be excluded in both adults and young for which a variety of evaluation options are available. A multitude of inflammatory or congenital lesions need to be considered in younger patients while malignant process considered more likely in elderly.[5] FNAC in a country like India is an easy, simple, uncomplicated and cost-effective diagnostic tool to help clinch diagnosis even when clinicians are least suspecting a malignancy.[6,7,8] Radiological techniques can be a useful adjunct in arriving at a conclusive diagnosis when reviewing cytology smears. Lateral neck mass as an initial manifestation in thyroid cancer has been reported as 10-21% [1,2,4] but are uncommon particularly to be reported on FNAC alone. Occult thyroid carcinoma presenting with cervical metastasis has been reported in autopsy studies on histopathology between 10-26% .[1,2,9-12] Most of these discovered cervical masses have been located in mid jugular (51%) and low jugular (33%) lymph nodes followed by supraclavicular (2%), posterior cervical (1.7%) and high jugular (10%) lymph nodes.[2,4] In our cases, posterior cervical lymph nodes were involved with clinically normal thyroid gland without any palpable lumps. Role of FNAC is very vital in arriving at a presumptive diagnosis with subsequent cyto-histologic co-relation and histologic examination being gold standard.[13]

Thyroglobulin measurement in FNA material is a useful ancillary test that improves the detection of cystic papillary thyroid cancer metastases. Particulary in cytologically non- diagnostic cases, the measurement of Tg- FNA helps to distinguish benign from malignant cystic lesions.[16] Thyroglobulin levels in 28 yr old patient was borderline and hence inconclusive. It could not be done in the second case. Multiple criteria of papillary structure without adherent blood vessels, intra-nuclear cytoplasmic inclusions, dense metaplastic cytoplasm, nuclear grooves and thick ropy or blobs of colloid help arrive at correct diagnosis. In cystic degenerations of lymph nodes afflicted by papillary carcinoma metastasis, macrophages have tendency to form cohesive clusters, a feature that if present should raise suspicion of papillary neoplasm and must be sought.

Conclusion

Posterior cervical lymphadenopathy occurring primarily as a result of papillary carcinoma thyroid is rare. A confident correct diagnosis can be made on cytopathology in combination with clinical evaluation and radiological investigations. This is particularly fruitful in occult nonpalpable lesions that are undetected prior to FNAC. Diagnosis of metastatic papillary carcinoma thyroid warrants follow up by total thyroidectomy with lymph node dissection.

References 1.

Maceri DR, Babyak J, Ossakow SJ. Lateral neck mass. Sole presenting sign of metastatic thyroid cancer. Arch Otolaryngol Head Neck Surg. 1986;112:47–9.

Park CS, Min JS. Lateral neck mass as the initial manifestation of thyroid carcinoma. Head Neck. 1989;11:410–13.

Machado NO, Chopra PJ, AI Hamdani A. Papillary Carcinoma of the thyroid presenting primarily as cervical lymphadenopathy: An approach to management. Sultan Qaboos Univ Med J. 2009;9(3):328-32.

De Jong SA, Demeter JG, Jarosz H, Lawrence AM, Paloyan E. Primary papillary thyroid carcinoma presenting as cervical lymphadenopathy: the operative approach to the lateral aberrant thyroid: Am Surg. 1993;59:172–7.

McGuirt FW. Differential diagnosis of Neck Masses. In: CummingC, et al., eds. Otolaryngology Head Neck Surgery. St. Louis: Mosby-Year Book, 1993: 1543–1553.

Bagwan IN, Kane SV, Chinoy RF. Cytologic evaluaton of the enlarged neck node: FNAC utility in metastatic neck disease. Int J Pathol 2007;6:2.

Cytologic evaluation results can be improved by several variables; pathologists expertise and minute observation of

cytological details being of utmost importance with equal emphasis on clinical findings. In present cases, both lateral cervical lymph nodes were cystic. It is imperative to re-assert importance of second aspiration of residual solid masses left after primary fluid aspiration of solid-cystic lumps. This helps to substantially reduce possibility of missing cystic carcinomas.[13] This protocol was strictly adhered to in both cases. Branchial cleft cysts are common lateral cystic neck masses. Ectopic thyroid tissue within a branchial cleft cyst is a rare phenomenon. Papillary thyroid carcinoma arising from this tissue is an extremely rare possibility and should be kept in mind.[14] Immunohistochemistry helps in the diagnosis of papillary thyroid carcinoma with diffuse www.pacificejournals.com/apalm

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Thyroid Carcinoma Presenting as a Cervical Mass.

Alam K, Khan A, Siddiqui F, Jain A, Haider N, Maheshwari V. Fine needle aspiration cytology (FNAC): A handy tool for metastatic lymphadenopathy. Int J Pathol 2010;10:2.

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

Sanders LE, Rossi RL. Occult well differentiated thyroid carcinoma presenting as cervical node disease. World J Surg 1995;19:642–647.

10. Vassilopoulou-Sellin R, Weber RS. Metastatic cancer as an incidental finding during neck dissection: significance and management. Head Neck 1992;14:459–463. 11. Nussbaum M, Bukachevsky R. Thyroid carcinoma presenting as a regional neck mass. Head Neck Surg 1990;12:114–117. 12. Cady B, Myssiorek D, Thompson N, et al. Management of papillary carcinoma of the thyroid, metastatic to the lateral

neck, without apparent primary carcinoma in the thyroid gland. Collected Lett Surg 1993;16:1. 13. Ravetto C, Colombo L, Dottorini ME. Usefulness of fineneedle aspiration in the diagnosis of thyroid carcinoma. Cancer Cytopathol 2000;90:357-363. 14. Mehmood RK, Basha SL, Ghareeb E. A case of papillary carcinoma arising in ectopic thyroid tissue within a branchial cleft cyst with neck node metastasis. Ear, Nose and Throat Journal 2006;85:675-676. 15. Wang Z, Qui S, Mahmoud A. Histopathologic and immunohistochemical characterization of a primary papillary thyroid carcinoma in the lateral cervical lymph node. Experimental and Molecular Pathol 2007;82:91-94. 16. Holmes BJ, Sokoll LJ. Measurement of fine needle aspiration thyroglobulin level increases the detection of metastatic papillary thyroid carcinoma in cystic neck lesions. Cancer Cytopathol 2014;122:521-526.

*Corresponding author: Dr. Priyanka Anand, Hindu Rao Hospital, NDMC, Malkaganj, New Delhi- 110007. India Phone: +91 9990331813 Email: priyankaanand19@yahoo.com

Financial or other Competing Interests: None.

Date of Submission : 07.11.2016 Date of Acceptance : 27.12.2016 Date of Publication : 28.03.2017

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Case Report DOI: 10.21276/APALM.1152

Cytodiagnosis of a Testicular Epidermoid cyst in a Young Male: A Rare Entity Dilip Kumar, Anupama Arya, Shruti Mahawar, Poonam Das and Nitin Dayal Institute of Lab Medicine, Max Super Speciality Hospital, Saket, New Delhi, India

ABSTRACT Intratesticular epidermoid cyst is a rare benign lesion of testis. It is important to distinguish this benign lesion from malignant testicular tumor. This is a case report of a 18yr old patient who presented with a mass in the left testis. His ultrasound showed typical onion skin appearance and tumour markers were within normal limits. Subsequently FNAC was performed which showed anucleated clumps of squamous epithelial cells admixed with the native testicular cells, thus diagnosis of testicular epidermoid cyst was made. Cytology along with sonographic findings provide rapid diagnosis and patient can be relieved of unnecessary anxiety. Keywords: Epidermoid Cyst, Testis, Anucleated Squamous Epithelial Cells

IntroductIon

Testicular epidermoid cyst is an infrequent tumor, accounting for 1% of all testicular masses occurring in mid adulthood (2 nd to 4thdecade). Such lesion is benign entity without any malignant potential. Teratoma is one of the important differential diagnosis, however cytology along with classical sonographic appearance rules out the same. An 18 year old boy presented to the OPD with complaint of swelling in the scrotum. Serum Beta HCG and AFP were found to be within normal limits. USG revealed classical onion skin appearance. Cytology showed presence of anucleated clumps of squamous epithelial cells along with normal cellular component of testis. Cytology along with sonographic findings provides early diagnosis of the lesion and thus unnecessary Orchidectomy can be avoided.

Case Report

A 18 year old boy presented to the out patient department with the complain of pain and swelling in the scrotal region. On physical examination, the swelling was palpated in the left upper part of the scrotum .The swelling was 1x1 cm, firm, tender and not fixed to the skin. The skin over the swelling appeared normal. There was no history of trauma or fever. The right testis appeared normal. On sonographic examination, the left testis measured approximately 4.4x2.1x2 cm . A lesion was seen in left testis measuring approximately 1.7 x1.1x 1.9 cm in size giving rise to a characteristic onion skin appearance. A subsequent Doppler showed mild intralesional vascularity in the lesion. The right testis and both the epididymus was found to be normal in size and echotexture. USG abdomen was normal. A Contrast enhanced CT scan of the testis

revealed a focally enhancing left testicular lesion without any significant retroperitoneal/ inguinal lymphadenopathy. Serum tumour marker, Beta-HCG was less than 0.2 mIU/ ml (normal- 0.5 – 2.67mIU/ml) and serum AFP was 1.20ng/ ml (normal- 0-9 ng/ml)., which ruled out the possibility of it being a teratoma. FNAC was performed with 22G needle after giving cord block with 2% lignocaine. Two passes were made and on each occasion, the needle was manipulated in different directions. A clear aspirate was obtained on both the occasions. The slides were air dried and wet fixed in 95% alcohol. The air dried slides were stained with May Grunwald Giemsa stain and the wet fixed slides were stained with Papanicolaou stain. On microscopic examination, anucleated clumps of squamous epithelial cells were seen along with normal spermatogenetic cells including immature germ cells, sertoli cells and spermatid heads. No cells of mesodermal or endodermal origin were seen. No evidence of parasite or malignancy was observed. Thus cytological diagnosis of “Testicular Epidermoid Cyst” was suggested in correlation with radiological findings.

Discussion

FNAC has become a preliminary preoperative diagnostic tool for diagnosis of any tumour, whether benign or malignant. It has an inevitable role in preoperative diagnosis of a testicular epidermoid cyst as it surpasses an unnecessary orchidectomy in a patient . Testicular epidermoid cyst is an uncommon entity, constituting 1-2% of testicular mass lesion. [1] These are keratin-filled intratesticular cysts,which generally present as solid appearing testicular masses. These are benign and

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Cytodiagnosis of A Testicular Epidermoid Cyst

Fig. 1: Photomicrograph showing anucleate squamous epithelial cells admixed with testicular spermatogenetic cells.(MGG,1000X).

Fig. 2: Sonogram showing a round mass with concentric rings of alternating echogenecity.

Fig. 3: Color Doppler Sonogram showing minimal color doppler from within the mass.

do not have a risk of recurrence or metastasis. [2] It poses a clinical dilemma, as such testicular mass lesion gives an impression of it being a testicular tumour. [3] The most important of which is a teratoma ,which has a strong malignant potential. But the absence of mesodermal and endodermal component in cytology and serum tumour markers being within normal range, rules out the possibility of it being a teratoma.. Malek RS et al(1986)[4] described that testicular epidermoid cyst presenting as painless swelling, however, in the present case the patient complained of pain in the swelling . Macroscopically, these are cystic and smooth. Cytologically it shows anucleated clumps of

squamous epithelial cells admixed with the native testicular cells. Cytology along with various other tools help us to reach a diagnosis. The include (1) absence of elevated tumour markers; (2) ultrasound comprises of hyperechoic heterogenous sonographic pattern which gives “onion skin appearance; (3) MRI typically showing “ bull’s eye” appearance i.e. a high intensity mid-zone which consist of scaly squamous cells and periphery zone of low intensity in T1 and T2 due to compact keratin fibres; (4) no Doppler vascularization. [5] Although the sonographic findings are strongly suggestive but they are not completely diagnostic of an epidermoid cyst. The surgeon has to take the help of an intra-operative frozen section to plan the further course of treatment.

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Kumar et al. The treatment of choice is excision of the cyst along with surrounding testicular parenchyma to exclude any accompanying teratomatous component or malignant germ cell neoplasia. Postoperative monitoring is always recommended. [6] However, in our case the patient refused the surgical option and a diagnosis was rendered on the classical cytological features along with sonographic â&#x20AC;&#x153; onion skinâ&#x20AC;? appearance .The case is presented because its diagnosis on FNAC is a rare entity and it provided a rapid and conclusive diagnosis to the surgeon, thus, saving a young patient from undergoing an orchidectomy.

References 1.

Loberant N, Bhatt S, Messing E et al. Bilateral Testicular Epidermoid Cysts :A rare Tumor. Journal of Clinical Imaging Science. 2011A;1:4.

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Somuncu S, Cakmak M, Atasoy P et al. Testicular epidermoid cyst and organ preserving surger y. Journal of Indian Association of Pediatric Surgery .2006;11:99-100

3. Maizlin ZV, Belenky A, Baniel J etc al. Epidermoid cyst and Teratoma of the testis: sonographic and histologic similarities. Journal of Ultrasound Medicine .2005;24:1403-9 4.

Malek RS, Rosen JS, Farrow GM. Epidermoid cyst of testis: a critical analysis . British Journal of Urology.1986;58;55-59.

Fernandez JA, Santiago SA, Diez BC etc al.Intratesticular Epidermoid Cyst:A rare Tumor. Journal of Clinical Medicine Research.2010;2: 281-283.

Heidenreich A, Egelmann UH, Vietsch HV et al. Organ preserving surgery in testicular epidermoid cysts. Journal of Urology.1995; 153: 1147-1150.

*Corresponding author: Dr Dilip Kumar, Flat no.- G3, Plot no.- 617,Sector, Vaishali, Ghaziabad (U.P.)- 201010 India Phone: +91 9810758472 Email: drkum ardil ip@gma il.c om

Financial or other Competing Interests: None.

www.pacificejournals.com/apalm

Date of Submission : 09.01.2017 Date of Acceptance : 12.02.2017 Date of Publication : 28.03.2017

eISSN: 2349-6983; pISSN: 2394-6466

Letter to Editor DOI: 10.21276/APALM.1171

Trichilemmal Cyst at Wrist: A Rare Site of Occurence Ruchi Sinha1, Iffat Jamal1*, Shashikant Kumar1 and Purushottam Kumar2 1

Department Of Pathology , All India Institute Of Medical Sciences, Patna, India 2 Department Of Surgery, All India Institute Of Medical Sciences, Patna, India

Dear Sir,

A 30 year old lady presented with a painless swelling on the dorsum of right wrist for one year which was gradually increasing in size .There was no associated pain or difficulty in movement of the wrist joint. Clinical examination revealed a well-defined, 3x2 cm ,firm,nontender subcutaneous lesion over the dorsum of right wrist which was free from underlying structures (Figure 1). There was no impairment of function and sensation of the right wrist,hand and digits.Fine needle aspiration of the lesion was performed and cytological diagnosis of epidermal inclusion cyst was made .

The possible differential diagnoses that should be considered at this location are epidermal inclusion cysts,lipoma,ganglion and hibernoma. [5] Histopathology is needed for a definitive diagnosis. Knowledge about morphology of different cystic lesion and its behavior is essential to come to a correct diagnosis.Unusual presentation of benign cystic lesion can lead to clinical misdiagnosis. This case not only highlights the unusual location of trichilemmal cyst but also the importance of doing histopathological examination to avoid misdiagnosis. Treatment consists of surgical excision.

Considering the progressive increase in size, the lesion was surgically excised and sent for histopathological examination. Grossly a globular ,smooth ,well encapsulated and pearly white mass of size 2x1.5x1 cm was received ,cut section of which revealed a solid and homogenous mass with lamellated appearance (Figure 2) . Histopatholgical examination confirmed it to be a trichilemmal cyst (Figure 3). Trichilemmal cyst also called pilar cyst,isthmus-catagen cyst or wen are commonly present in areas with dense hair follicle concentrations.About 90 % of trichilemmal cysts occur in scalp. Other sites are face,neck, trunk,buttock and elbow .[1] A trichilemmal cyst over the wrist is quite rare and very few cases have been reported so far in literature. This makes our case worth reporting. Histopathology of trichilemmal cyst shows epithelial cells possessing no clear visible intercellular bridges and is characterized by sudden keratinisation without the formation of a granular layer with an uneven interphase between the keratinized and the nonkeratinised cells.[2 ] The keratin in the cyst is non- lamellated with retention of some nuclei. Focal calcification and foreign body giant cell reaction is also present.[3 ] Trichilemmal cysts are almost always benign, although malignant transformation can be seen in 2% cases.A spectrum of transformation from benign pilar cyst to a proliferating tumor to malignant proliferating trichilemmal tumor can be encountered .[4]

Fig. 1: Clinical photograph of the patient showing a swelling on the dorsum of right wrist.

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Trichlemmal Cyst at Wrist

Fig. 2: a) Gross image of the resected specimen from wrist showing a globular and pearly white lamellated mass. b) FNA aspirate of the lesion showing anucleate squames in keratinous background. ( MGG;400X)

3c Fig. 3: a) Microphotograph showing absence of granular layer with abrupt interphase between keratinized and nonkeratinised cells.( H & E;400X). b): microphotograph showing a cyst lined with squmaous epithelium undergoing sudden keratinization ( H&E;100 X) and c): focal calcification.( H & E ;400X).

Annals of Pathology and Laboratory Medicine, Vol. 04, No. 02, March - April, 2017

Sinha et al.

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Reference 1. 2. 3.

Mcgavran MH, Bennington B. Keratinous cysts of the skin.Identification and differentiation of Pilar cysts from Epidermal cysts. Arch Dermatol 1996;94:499-508. Adya KA, Inamdar AC. Multiple firm swellings over the scalp. Int.J Trichology 2012;4:98-9. Ikegami T, Kameyama M, Orikasa H, Yamazaki K. Trichilemmal cyst in the pulp of index finger.A case report. Hand Surg 2003;8:253-5.

Sadath HN, Ramachandra S, Kumar MA, Harithka K. Multicentric calcified trichilemmal cysts with alopecia universalis affecting siblings.Indian Journal Deramtol Venereol Leprol 2013;79:88-91.

Anolik R, Firoz B, Walters RF, Meehan SA, Tsou HC, Whitlow M , et al.Proliferating trichilemmal cyst with focal calcification.Dematol Online J 2008;14-25.

*Corresponding author: Dr. Iffat Jamal, Flat No-D/2, Phase-1, Sapna Apartment, Nayatola, Patna-800004, Bihar, India Phone: +91 - 09835498843 Email: Iffatjamal111@Gmail.Com

Financial or other Competing Interests: None.

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Date of Submission : 18.11.2016 Date of Acceptance : 12.01.2017 Date of Publication : 31.03.2017

eISSN: 2349-6983; pISSN: 2394-6466

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