Objective: To determine the frequency of nerve bundle hypertrophy in aganglionic segments in relation to the site of biopsy, along with the assessment of value of Calretinin immunostaining in the diagnosis of suspected cases of Hirschsprung disease. Study Design: Cross Sectional study. Setting: Department of Pathology, Pakistan Institute of Medical Sciences (PIMS) Islamabad. Period: September 2018 to March 2019. Material & Methods: After routine tissue processing colonic biopsies were examined for presence of ganglion cells and hypertrophic nerve presence or absence. Diagnosis of Hirschsprung disease was based on the absence of ganglion cells in submucosal and myenteric plexus, the presence or absence of hypertrophic nerves (more than 4 nerves >30 µm thick/×200 field or more than 2 nerves >40 µm thick/×200 field) was also noted in all cases of Hirschsprung disease (aganglionic segments). Calretinin immunostaining was applied to all the cases and controls and findings were recorded as positive or negative staining. Statistical Analysis: Data was analyzed using SPSS version 23. Qualitative data was calculated as frequencies and percentages. Pearson Chi square test was used to establish the association of nerve bundle hypertrophy with the site of biopsy. Results: Total biopsies were 60; 30 each from ganglionic and aganglionic segments. Calretinin sensitivity in our study was 90%, specificity 83.3%. In 30 cases of aganglionosis hypertrophic nerves were present in 13(21.7%) and they were absent in 17 (28.3%). No significant association (p value= 0.447) was seen in nerve bundle hypertrophy and site of biopsy. Conclusion: Calretinin immunohistochemistry can be used as a reliable ancillary technique in the diagnosis of HD. Aganglionosis may not always be associated with submucosal nerve hypertrophy which alone should not be used as a criteria for HD diagnosis but instead adjunct methods like Calretinin immunostaining must be utilized to confirm presence or absence of ganglion cells. There is no association of nerve hypertrophy with site of biopsy.

Background: Tissue margin marking with India ink is important in decision making for surgeons. The present study was conducted to examine the reliability of different shades of locally available poster colours in tissue marking and to evaluate the colour perceptibility microscopically in comparison with similar tissues marked by India ink. Methodology: This experimental study was conducted at Department of Pathology, HBS Medical & Dental College & Hospital, Islamabad from 27th February 2021 to 29th April 2021. Sample size was fifty, collected through convenient sampling technique. Five types of formalin fixed tissue specimens were selected for the study so as to evaluate the effectiveness of poster ink marking on different tissue surfaces. From each specimen, four sections were taken from the margins. Three shades of poster colour (black, blue and green) were used to ink three sections while one section from each tissue type was marked with India ink. After complete tissue processing and routine haematoxylin and eosin (H&E) staining, slides were examined microscopically. Scoring was done on a scale 0 to 3 on the basis of visibility. Results: The present study showed that poster colours inking of the tissues was quite reliable as compared to India ink. Most consistent results were achieved with black and blue colours as compared to green colour. Conclusion: Poster colours are reliable tool for tissue marking when India ink is not available. Their availability in a variety of colours provides them an edge over India ink. Key words: Eosin, India ink, Resection Margins