Comparison of Haematoxylin and Eosin and Calretinin Detection of Ganglion Cells in Colorectal Biopsies Suspected of Hirschsprung’s Disease

Abstract

Background: Hirschsprung’s disease is a functional obstruction of the gastrointestinal tract due to the congenital absence of ganglion cells. The diagnosis and extent of resection for the management of Hirschsprung’s disease depend on specific identification of ganglion cells. Recently, calretinin immunohistochemistry has been introduced as a new, simple and reliable tool for the diagnosis of Hirschsprung’s disease compared to haematoxylin & eosin. This study compared detection of ganglion cells by the haematoxylin & eosin and calretinin immunohistochemistry, at Muhimbili National Hospital. Methodology: The study was a retrospective hospital based, of all suspected Hirschsprung’s disease cases diagnosed either by clinical, histopathological or both methods in a 1 year period from 1 stJanuary 2018 to 31 stDecember 2018. All 99 cases were stained by haematoxylin & eosin, and all cases which were negative for ganglion cells by haematoxylin & eosin (36 cases) and those positive with adequate submucosa or muscular layers (23 cases) were stained by calretinin immunohistochemistry. Data was recorded and analysed by Statistical Product and Service Solutions version 23. Statistical methods used were descriptive and inferential (p-value and Cohen’s Kappa value). Concordance of haematoxylin & eosin and calretinin immunohistochemistry in ganglion cells’ detection was calculated. Results: A total of 99 cases were studied, 69 males and 30 females. The median age was 3 years with range of 1 month to 19 years. Using the routine haematoxylin & eosin stain, ganglion cells were seen in 63 cases and no ganglion cells were seen in 36 cases. Calretinin immunohistochemical stain found ganglion cells in all 23 cases that showed ganglion cells by haematoxylin & eosin and 6 cases that did not show ganglion cells by haematoxylin & eosin, making 30 confirmed cases by calretinin immunohistochemistry. There was a substantial agreement between haematoxylin & eosin and calretinin immunohistochemistry with concordance of 90% (Cohen’s kappa=0.790). There was a Positive predictive value of 100%, negative predictive value of 83%, sensitivity of 79% and specificity of 100% in detection of ganglion cells. Most cases (50.5%) presented with constipation. The sites of biopsies submitted were mostly from the rectum (64.6%) and most (69.7%) of the submitted biopsies were full thickness. Location of the ganglion cells as seen by the haematoxylin & eosin for majority of the biopsies 31/63(49.2%) were on both submucosa and muscularis propria nerve plexuses. The proportion of Hirschsprung’s disease for the year 2018 among the submitted biopsies was 36/99 (36.4%). Male to female ratio of positive cases was 2:1 (p=0.62) and 39% were between 1 to 3 years of age, which was the age group with the highest frequency (p=0.37). Conclusion: Comparison of detection of ganglion cells by the routine haematoxylin & eosin and calretinin immunohistochemistry has shown a very high concordance rate with a slightly higher detection rate by calretinin for the cases that were missed by the routine haematoxylin & eosin. The overall capability of ganglion cells’ detection by calretinin immunohistochemistry has been accompanied by a significant shorter time and simplicity in sighting out the ganglion cells.