A COMPARATIVE STUDY BETWEEN IMMUNOHISTOCHEMISTRY, HEMATOXYLIN & EOSIN AND GEIMSA STAIN FOR HELICOBACTER PYLORI DETECTION IN CHRONIC GASTRITIS

Transcription

1 Original Research Article Pathology International Journal of Pharma and Bio Sciences ISSN A COMPARATIVE STUDY BETWEEN IMMUNOHISTOCHEMISTRY, HEMATOXYLIN & EOSIN AND GEIMSA STAIN FOR HELICOBACTER PYLORI DETECTION IN CHRONIC GASTRITIS DR.PRIYADHARSHINI.M *1, DR.SINDU.V 2 1 Department of Pathology, Madras medical college, Tamil Nadu, India 2 Department of Pathology, Coimbatore medical college, Tamil Nadu, India ABSTRACT Helicobacter pylorus (H.pylori) is now accepted as a major cause of chronic active antral gastritis. There is accumulating evidence to incriminate this microbe in the aetiology of duodenal ulcer and gastric carcinoma. The aim of this study was to ascertain the reliability of hematoxylin & eosin (H&E) and Geimsa stains in comparison with immunohistochemical (IHC) technique for the diagnosis of H.pylori.50 gastric biopsies were analysed in the present study. Sensitivity and specificity of Geimsa was 94% and 100% respectively. Sensitivity and specificity of H&E was found to be 70.59% and 100% respectively. Immunohistochemistry was positive in all cases in which H.pylori was detected by other methods. When the density of organism is low immunohistochemistry can be recommended for detection of H.pylori where the other two stains have low detection rate. It was concluded that Geimsa stain is less time consuming, relaible and readily available when compared to IHC technique. KEYWORDS: Antral biopsy, Geimsa, H.pylori, H&E, Immunohistochemistry, chronic gastritis. DR.PRIYADHARSHINI.M * Department of Pathology, Madras medical college, Tamil Nadu, India *Corresponding Author Received on: Revised and Accepted on: DOI: B-727

2 INTRODUCTION Helicobacter pylorus plays a significant role in the genesis of several gastric diseases like acute and chronic gastritis, gastric adenocarcinoma and gastric mucosa associated lymphoma. At least half the world's population are infected by the bacterium, making it the most widespread infection in the world. 1 Most of the cases reported were from low socio-economic status. 2 The human stomach is the primary reservoir for the organism and it is transmitted via oral-oral, gastric-oral and faecal-oral routes. 3 Inflammation of the pyloric antrum leads to duodenal ulcers, while inflammation of the corpus (body of the stomach) leads to gastric ulcers and gastric carcinoma. 4 It is therefore of paramount importance to determine the presence of the organism in surgical pathology specimens in order to manage these common diseases of the upper gastrointestinal tract. Antral biopsy specimens processed for histology would provide an easier and more cost-effective means of diagnosing H.pylori infection. Intraepithelial neutrophils and subepithelial plasma cells are 5 characteristic of H.pylori infection. Various special stains have been devised to detect H.pylori in histological sections but their specificity and sensitivity vary greatly. The haematoxylin and eosin stain, the most frequently used stain in histology, has been found to be the most unreliabl,because the detection rate was only 66% and there were few false cases reported. 6 Modified Giemsa stain described by Gray et al (1986) has been favoured by many researchers because of its easiness to perform and availability in most histopathology. 7 Immunohistochemical techniques using anti H.pylori antibody which reacts with somatic antigens of the whole bacteria have been found to correlate well with the presence of the bacteria. The aim of this study is to analyse the role of IHC versus Hematoxylin & Eosin and Geimsa in detection of H.pylori. MATERIALS AND METHODS The study is a longitudinal prospective study undertaken in the department of pathology over a period of 3 years. Plan The patients who were included in this study were screened with predetermined inclusion and exclusion criteria. Selected patients underwent through consent protocols. Brief clinical history regarding risk factors for gastritis and examination findings was collected with predetermined proforma. This study was approved by our institutional ethical committee. This study was done in Stanley Medical College, Chennai, Tamilnadu Inclusion criteria All the cases proved to be of chronic gastritis by histopathology irrespective of age were included for study. Exclusion criteria Those with poor clinical data were excluded from the study. Method of data collection Out of the 120 cases 100 cases had adequate clinical data. Of these 100 cases 50 cases were selected randomly. Those biopsy materials were processed and sections were cut at 5 micron thickness. H&E staining of sections was done. Histopathological examination of these sections was done. Necessary microphotographs were taken. The Sydney system is a novel system for grading gastritis that was devised by a group of experts at the 9th World Congress of Gastroenterology in Sydney, Australia in In 1994 in Houston, Texas, experts devised the new updated Sydney system. 8 H&E Section from gastric biopsy had been categorized using Sydney grading system (Table.1) based on activity, extent of chronic inflammation, metaplasia, atrophy and Helicobacter pylori colonisation and the results were tabulated. Special stain (Geimsa stain) and Immunohistochemical study using Helicobacter pylori polyclonal antibody were done in 50 cases and H.pylori colonisation in antral biopsy was graded. RESULTS In 100 patients presented with gastritis 55 were female, 45 were male. 58% were in the age group of Average age of the person included in the study range from % of patients were from lower socio economic status. Table 1 Sydney grading of gastritis in gastric biopsy Features Grade Chronic inflammation Mild Moderate Marked Activity <1/3 of pits mild 1/3 to 2/3 moderate >2/3 marked Atrophy mild moderate marked Intestinal metaplasia mild moderate marked Helicobacter pylori colonization <1/3 of surface mild 1/3 to 2/3 moderate >2/3 marked Table 2 Dietary habits 3days in <3days in a Food habits Absent present week week Spices Fries Cola Peppermint Garlic B-728

3 Ginger Most common dietary habits associated with gastritis are intake of spices, fries, garlic, and ginger(table no:2). Of this p value obtained for intake of spices by using chi square test was found to be 0.005, which is statistically significant. Table 3 Sydney grading in gastric biopsy of 100 cases Sydney grade Activity Chronic inflamation Intestinal metaplasia Atrophy H.pylori This table (Table3) indicates that most of the cases show associated chronic inflamation.(fig 1). Intestinal metaplasia (Fig 2) seen in only few cases. Most of the cases which shows H.pylori positivity where low grade (Fig 3). Figure 1 H&E (100X) - Lamina propria showing dense lymphocytic infiltration, atrophy of glands and Intestinal metaplasia (grade 3) Figure 2 H&E(450X)-Antral mucosa showing Intestinal metaplasia B-729

4 Figure 3 H&E (1000X) - Helicobacter pylori present in luminal surface Figure 4 Geimsa (1000X) - Mucosal surface epithelium showing grade 2 H.pylori colonisation Figure 5 Geimsa (1000X) - Grade 3 Helicobacter pylori B-730

5 Figure 6 IHC-(100X) Grade 1 H.pylori colonisation Figure 7 IHC-(100X) - Grade 2 H.pylori colonisation Figure 8 IHC-(100X) - Grade 3 H.pylori colonisation Table 4 Grading of H.pylori infection in 50 gastric biopsies using Sydney scoring system. Staining method H.pylori Positive Grade 1 Grade 2 Grade 3 H.pylori negative IHC 34(68%) 20(40%) 9(18%) 5(10%) 16(32%) GIEMSA 32(64%) 18(36%) 9(18%) 5(10%) 18(36%) H&E 24(48%) 11(22%) 8(16%) 5(10%) 26(52%) From this table (table no :4) it was evident that H.pylori detection using H&E, Geimsa and IHC were similar in grade 2 & grade 3 H.pylori colonisation(fig no4,5,7,8). Discrepancies in the result were seen when the H.pylori colonisation was low (grade1, Fig no:6). B-731

6 Table 5 Comparison of H&E and IHC IHC H&E Positive Negative Positive 24 0 Negative Among the H&E negative cases H.pylori was detected in 10 cases using IHC which were of grades 1 and 2. Table 6 Comparison between IHC and Geimsa IHC Geimsa Positive Negative Positive 32 0 Negative 2 16 Out of 50 cases, IHC detected H.pylori in 34 cases whereas Geimsa were positive in 32 cases only. Table 7 H&E & Geimsa in comparison with immunohistochemistry H&E Geimsa Sensitivity 70.59% 94.1% Specificity 100% 100% Positive predictive value 100% 100% Negative predictive value 61.54% 88.89% % of false negative % of false positive 0 0 Sensitivity and specificity of Geimsa was 94% and 100% respectively. Sensitivity and specificity of H&E was found to be 70.59% and 100% respectively. Hence Giemsa is more sensitive than H&E in H.pylori detection. DISCUSSION Infection with H. Pylori occurs worldwide, but the prevalence varies greatly among countries. It is more common in developing countries where the prevalence is generally over 80% in middle aged adults as compared to 20-50% in industrialized countries. 9 According to Javed et al prevalence of H.Pylori infection increases with age. 10 Adisa et al in his retrospective study observed that the prevalence of H.pylori associated gastritis was maximum between the age group of 41 to 50 years. 9 In the present study the positive cases fall between the age group of years. According to Javed et al 80% of H.pylori infection was fond in the low and middle class. 10 In our present study H.Pylori infection was found to be more prevalent in low socio economic group. Sensitivity of H&E stain is low due to lack of contrast between the bacteria and the surrounding tissue. The specificity of H&E is also low due to its non specific staining of non H.pylori bacteria in the stomach. Modified Geimsa is a cheap, easily applicable stain that can be performed in 15minutes. The results are reliable. The sensitivity and specificity values are acceptable. Lack of contrast is the disadvantage of the Geimsa technique but careful observation allows identification of the organisms correctly. Immunohistochemistry is an expensive and time consuming technique with procedure duration ranging from 1 hour to 24 hours. Sensitivity and specificity are high for the detection of H.pylori using IHC.In the current study, the histochemical methods such as H&E, Geimsa and IHC were analysed and compared for the sensitivity and specificity in detecting the H.pylori (Table no:5,6). Kacar N et al observed that the sensitivity and specificity for H&E, Geimsa and IHC in detection of H.Pylori was 97%/80%; 97%/90%; 100%/100% respectively. 11 HR. Wabinga et al in his study evaluated the staining ability of Geimsa and IHC in gastric biopsies and inferred that the sensitivity of Geimsa stain was 85%, specificity was 89%, positive predictive value was 93% and negative predictive value was 74%. 12 Sensitivity of detection of H.pylori in gastric biopsies and resected specimens using modified Geimsa and IHC were compared by Babic et al which revealed the sensitivity of Geimsa to be 73.3% and 90% for IHC. 13 In the present study sensitivity and specificity of geimsa was 94% and 100% respectively. Sensitivity and specificity of H&E was 70.59% and 100% respectively (Table no:7). CONCLUSION In our present study the Giemsa was found to be more sensitive with low false negative rates than H&E but Immunohistochemistry is the most sensitive and reliable method for detection of H.pylori. Considering the cost of Immunohistochemistry it can be recommended only when the density of organism appears to be low. The cost effectiveness, applicability and the reliability of the Geimsa stain make it an ideal technique in detecting H.pylori infection in gastric biopsies. Geimsa stain is also less time consuming and readily available when compared to Immunohistochemistry technique. CONFLICT OF INTEREST Conflict of interest declared none. B-732

7 REFERENCES 1. Pounder RE, Ng D. The prevalence of Helicobacter pylori infection in different countries. Alimentary pharmacology & therapeutics Dec;9: Mohankumar Pandurangan. Studies on Helicobacter pylori in asymptomatic children. Int J Pharm Bio Sci April; 6(2):(B) Parsonnet J, Shmuely H, Haggerty T. Fecal and oral shedding of Helicobacter pylori from healthy infected adults. Jama Dec 15;282(23): Suerbaum S, Michetti P. Helicobacter pylori infection. New England Journal of Medicine Oct 10;347(15): Jerold R.Turner. Robbins and cotran Pathologic basic of disease. 8 th edition. New Delhi: Elsevier publishers; 2010; p Molyneux AJ, Harris MD. Helicobacter pylori in gastric biopsies--should you trust the pathology report?. Journal of the Royal College of Physicians of London Apr;27(2): Madan E, Kemp J, Westblom TU, Subik M, Sexton S, Cook J. Evaluation of staining methods for identifying Campylobacter pylori. American journal of clinical pathology Oct 1;90(4): Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis: the updated Sydney system. The American journal of surgical pathology Oct 1;20(10): Adisa J.O.1, Musa A.B.2, Yima U.I.2, Egbujo E.C.3. Helicobacter Pylori Associated Gastritis In North-Eastern Nigeria: A Histopathological Study. EISRJC; 2011: 3: Javed M, Amin K, Muhammad D, Husain A, Mahmood N. Prevalence of H. Pylori. Professional med. Sep 2010;17(3): Kacar F, Culhaci N, Yükselen V, Meteoglu I, Dikicioglu E, Levi E. Histologic demonstration of Helicobacter pylori in gastric biopsies: which is the best staining method. Internet J Pathol. 2004;3(1): Wabinga HR. Comparison of immunohistochemical and modified Giemsa stains for demonstration of Helicobacter pylori infection in an African population. African health sciences Aug 1;2(2): Babić T, Bašić H, Selimović-Miljković B, Kocić B, Tasić GD. Detection of Helicobacter pylori in gastric biopsy and resection specimens. Vojnosanitetski pregled. 2005;62(1): B-733