Ramji Rai

Real Time-PCR HBV-DNA Analysis: Significance and First Experience in Armed Forces.

BACKGROUND HBV DNA quantitation is used extensively world wide for the diagnosis and monitoring of treatment of Hepatitis B virus (HBV) infection. However, it has still to be popular in India. The aim of this study was to quantitate HBV - DNA by Real time - PCR method in Hepatitis B and in immuno-compromised patients, to compare the results with HBeAg detection and to monitor the response to therapy of chronic Hepatitis B patients to antivirals. METHODS Ninety one serum samples of Hepatitis group of patients (all HBsAg positive), 41 samples from immuno-compromised patients (all HBsAg negative) and 49 patients of Chronic Hepatitis B group (all HBsAg positive) were the subjects of this first ever study in Armed Forces. Twenty serum samples from healthy volunteers and non-hepatitis B patients served as negative controls. The amplification detection was carried out in a Rotor-Gene 2000-sequence detector. RESULTS Amongst Hepatitis B group, 33% (30/91) of the samples were positive for HBV-DNA and 26% (24/91) of samples were positive for HBeAg. In the immuno-compromised group of patients 14.6% (6/11) of samples were positive for HIV-DNA and 9.7% (4/41) were positive for HBeAg. Of the Chronic Hepatitis B patients on treatment, all (100%) were positive by HBV-DNA, whereas 29/49 (59.2%) were positive by HBeAg before treatment. After treatment with antivirals, 06/49 (12.2%) were positive by both tests and 11/49 (22.5%) were positive only by HBV-DNA. 32/49 (65.3%) patients became negative serologically after therapy. CONCLUSION HBeAg status did not necessarily reflect HBV-DNA level in the serum, as 10/91 (11%) in the Hepatitis B group, 2/41 (4.9%) in the immuno compromised group and 20/49 (40.8%) patients in the Chronic Hepatitis B group were positive for HBV-DNA but negative for HBeAg. HBV-DNA was not found to be positive amongst any of the negative controls. Real time - PCR is a sensitive and reproducible assay for HBV-DNA quantitation and may be started in Armed Forces referral centers in the near future.

SPECTRUM OF NEPHROPATHIES WITH SPECIAL REFERENCE TO PRIMARY GLOMERULOPATHIES

A retrospective study was carried out where histopathology records of Department of Pathology, Armed Forces Medical College, Pune were scrutinised covering a period of 10 years. We came across 325 cases of nephropathies. The sample comprised of 92.6% adults and 7.4% children. 79.7% were males and 20.3% were females. The patient population comprised of 35.1% serving soldiers, 37.8% their family members and 27.1% not related to Armed Forces. The study is based on only light microscopy findings. It was observed that primary glomerular disease was the commonest entity and comprised of 61.5% of all the nephropathies. Acute diffuse proliferative glomerulonephritis was not only the commonest primary glomerulopathy (23.5%) but the commonest nephropathy as well. The other primary glomerulopathies in descending order of frequency were, membranoproliferative glomerulonephritis (22.5%), mesangioproliferative glomerulonephritis (21.5%), chronic glomerulonephritis (9%), minimal change disease (7.5%), membranous glomerulopathy (7%), focal segmental glomerulosclerosis (5%) and crescentic glomerulonephritis (4%). We had only 14 cases (4.3% of all nephropathies) of secondary glomerulopathies, amongst which amyloidosis was the commonest cause. We came across 2 cases (0.6% of all nephropathies) of Alports syndrome. In the other nephropathies, 22.2% were tubulointerstitial diseases and 5.5% were malignant kidney tumors. In 5.8% renal biopsies, no significant pathology was seen on light microscopy.

Value of Silver Binding Nucleolar Organiser Regions (AgNOR) in Squamous Cell Carcinomas of Upper Aero-digestive Tract

BACKGROUND Study of Silver-binding nucleolar organizing regions (AgNORs) in 50 cases of squamous cell carcinomas (SCC) of upper Aero-digestive tract was carried out with an aim of correlating AgNOR positivity with histological grade of tumour and evaluating value of AgNOR staining in predicting the progress of disease. METHODS Tissue sections of the cases were stained with 50 percent silver nitrate solution for AgNOR evaluation and the data analysed. All the sections were also stained with haematoxylin and eosin stain for tumour typing and grading. RESULTS In this study, the mean AgNOR (mAgNOR) score of normal squamous epithelium (50 cases) was 1.56 (range1.00-2.80), that of well differentiated squamous carcinoma (27 cases) was 3.29 (range 2.4-4.6), moderately differentiated squamous carcinoma (21 cases) was 4.29 (range 2.7-5.6) and of poorly differentiated squamous cell carcinoma (2 cases) was 5.21. The mAgNOR scores were statistically significant. Comparison of the percentage AgNOR (pAgNOR) scores between well differentiated and moderately differentiated carcinomas showed that all the pAgNOR values (1-9) were significant. However, pAgNOR 3 and pAgNOR 4 were most significant. Cut off values for pAgNOR score to differentiate between the various grades of squamous cell carcinomas could not be calculated due to lack of data on survival rates. CONCLUSION AgNOR technique can definitely be used as a supportive tool to routinely performed Hemotoxylin and Eosin staining and may help in prognosis as well as therapeutic decision-making in squamous cell carcinomas. Studies of larger number of cases in a prospective study are needed to arrive at more substantial conclusions.

Platelet Functions in Cardiopulmonary Bypass Surgery

BACKGROUND Haemorrhage after Cardio Pulmonary Bypass (CPB) Surgery is a well recognised complication that leads to significant morbidity and mortality. The incidence varies between 5-25% depending upon the clinical situation. Several factors are implicated as causative but none have been precisely proved. METHODS Our study was an attempt to evaluate the haemostatic defect with particular reference to platelet function abnormalities during cardio pulmonary bypass surgery, in order to reduce the morbidity and mortality associated with post CPB haemorrhage. Flow cytometric evaluation of different platelet glycoproteins like GPIb/IX, GPIIb/IIIa and GMP-140 was done. RESULTS The marker expression showed deregulation during surgery which returned to base after bypass was terminated. In contrast, the cases with bleeding showed significant variation. P-Selectin (GMP 140) expression decreased progressively till 3(rd) post-operative day showing lack of activation of platelets in cases of severe bleeding. CONCLUSION Longer duration of CPB initiates plasmin generation through heparin, which raises the PAI-1-tPA complex and thereby down regulating the functions of platelets. This suggests a link between duration of CPB, bleeding, platelet dysfunction and fibrinolysis. Hence serial estimations of the levels of GMP-140 and tPA can predict severe bleeding.

Study of p53 and bcl-2 Oncoproteins in Ulcerative Colitis with Dysplasia

BACKGROUND Dysplasia in ulcerative colitis has been graded on haematoxylin and eosin stain using Riddles criteria. This system was formed to nullify the inter-observer variation. Few cases of early dysplasia were missed when purely screened on morphology. This study was carried out to detect early dysplasia using p53 and bcl-2 oncoproteins. METHODS A retrospective study was carried out on paraffin blocks of 100 histologically diagnosed cases of ulcerative colitis at a large service hospital. Haematoxylin and Eosin stained (H &E) slides of these cases were re-examined as per standard techniques. RESULTS On correlating histological grades with p53 immunoscore it was found that 90.76% of cases graded as negative for dysplasia on H & E, got an immunoscore of 0 and other 9.24% cases which were graded as negative for dysplasia got a score 1+. This shows that the immunohistochemistry was able to pick up 6 cases, which were missed by routine histology. Nine out of 11 cases in which the pathologists could not rule out a dysplasia and graded them as indefinite (probably negative for dysplasia) got a score of 0. In these cases possibly the histological features may be construed as an acute inflammation or repair induced dysplasia which were suspicious for neoplastic dysplasia on routine histology sections. On analyzing our findings on bcl-2 immunohistochemistry it was seen that there was no significant concordance (p>0.05) of immunoscore with the grades of dysplasia estimated morphologically. CONCLUSION Our study recommends that p53 should be used as regular immunohistochemical marker while grading the dysplasia of ulcerative colitis, especially in indefinite cases as it brings objectivity in grading. Our study also came to a conclusion that use of bcl-2 for grading dysplasia of ulcerative colitis is not of any significant help.

Carcinosarcoma of the Breast With An Unusual Secretory Carcinoma as the Carcinomatous Component

Carcinosarcoma (CaSa), as defined by the World Health Organization, is a tumour composed of intimately admixed malignant epithelial and stromal components [1]. It is considered a distinct form of metaplastic carcinoma of the breast. Secretory carcinomas of the breast are uncommon tumours, generally encountered in children and are therefore called by some authors as juvenile carcinomas. However, they have also been reported in older patients including post-menopausal women [2]. We report a case of CaSa of the breast with an unusual secretory carcinoma as the malignant epithelial component.

Signet Ring Carcinoma of the Breast : An Uncommon Type of Breast Carcinoma

Signet ring cell carcinoma (SRCC) is a unique subtype of adenocarcinoma characterized by abundant intracellular mucin accumulation. SRCC of the breast, originally grouped as a variant of mucinous carcinoma, was later realized to be a pathologically distinct group [1]. We report a case of SRCC of the breast that is ductal in origin.

Retroperitoneal Teratoma in an Adult With Carcinoid as the Main Component

Teratomas are uncommon in adults. A case report in an adult is described. The histopathology revealed a teratoma with a main component of the tumour to be a Carcinoid. This combination has not been found in the literature till date. Most teratomas arise in infancy and are seen in the sacrococcygeal region [1, 2]. They are also seen in the retroperitoneum, mediastinum and other parts of the body. The presentation in adults is uncommon. The frequency of malignancy increases with age [3]. The incidence of endocrine carcinoma in the retroperitoneum is extremely low [4]. Carcinoid tumours usually involve the gastrointestinal tract or the bronchial tree [5, 6]. Retroperitoneum is a very rare site [6]. In searching literature we could not find any case of a retroperitoneal teratoma in an adult with Carcinoid as the main component.

ASTROCYTOMAS AND PROGNOSIS-FROM MORPHOLOGY TO TUMOR BIOLOGY

Twenty-seven cases of Astrocytoma were studied to assess the role of a newly introduced proliferation marker-Proliferating Cell Nuclear Antigen (PCNA) in improving prognostic accuracy in comparison to traditional histologic methods like grading. The study revealed a direct correlation between grading and PCNA expression as determined by labelling indices (LI). A 25% PCNA LI separated low and high grade tumors. The difference between PCNA LIs of patients who were alive and those who were dead at the end of the study was statistically significant. However, in this study with limited follow-up, statistically significant relation to survival and recurrence could not be established. The study introduces a new method of assessing tumor biology that enables objectivity in prediction of tumor behaviour.

CORRELATIONSHIP OF HORMONE RECEPTOR STATUS, p53 MUTATION AND c-erb B-2 OVEREXPRESSION WITH NUCLEAR GRADING IN BREAST CANCERS

Thirty cases of infiltrating duct carcinoma of the breast were studied for the expression of estrogen receptor (ER) and progesterone receptor (PR) status; p 53 protein mutation and c-erb B2 overexpression. The results were correlated with the morphological differentiation, as determined by the Nottinghams modification of the Bloom-Richardson system. Hormone receptor positivity was seen in 46.67% cases, whereas p 53 mutation and c-erb B2 overexpression were seen in 50.00% and 60.00% cases respectively. In grade II tumours receptor positivity, p53 mutation and c-erb B-2 overexpression were 57.15%, 42.85% and 52.38% respectively. The corresponding figures for grade III tumours were 33.33%, 83.33% and 66.67% respectively. As grade 1 comprised only 3 cases no statistical correlation could be observed. Thus we conclude that receptor positivity declined, whereas p 53 mutation and c-erb B-2 overexpression increased, with increase in tumour grade.