Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5

Summary

This protocol describes the process of immunostaining rectal suction biopsies for calretinin, S100 protein, and protein gene product 9.5. This novel adjuvant diagnostic method for Hirschsprung's disease has preferable sensitivity and specificity rates.

Abstract

Hirschsprung's disease (HD) is a congenital intestinal disease that is clinically manifested as an inability to pass meconium in infants or as long-term constipation in children. Rectal suction biopsy (RSB) to determine the absence of ganglion cells and neural hypertrophy is the most accurate test for the diagnosis of HD at present. Traditional hematoxylin-eosin staining lacks sensitivity and specificity. Acetylcholinesterase staining cannot be widely used due to its complex process. Our novel protocol of immunostaining for calretinin, S100 protein, and protein gene product 9.5 (PGP9.5), which we conducted on RSBs, exhibits high sensitivity and specificity rates of 96.49% (95% confidence interval, 0.88-0.99) and 100% (95% confidence interval, 0.97-1.00), respectively. The HD-affected segments often present as the absence of the expression of calretinin, S100 protein, and PGP9.5, which are markers of neural hypertrophy in the submucosal tissue. This protocol describes the detailed operating process of this new diagnostic method.

Introduction

Hirschsprung's disease (HD) is a common congenital gut intestinal disorder characterized by a lack of ganglion cells in different segments of the distal intestinal tract¹. The human enteric nervous system is formed when the invasion of the embryonic neural cells is completed. If there is a disturbance of the process and the invasion fails to complete, the distal intestine of the newborn becomes aganglionic². This potentially fatal condition is called Hirschsprung's disease. Proliferation, motility, and gut growth are the three main components of successful colonization.

Traditional hem....

Protocol

This protocol was approved by the Research Ethics Board of the Union Hospital of Huazhong University of Science and Technology.

1. Rectal Suction Biopsy

1. Perform the rectal suction biopsy by a well-trained pediatric surgeon and an assistant using a Rbi2 suction rectal biopsy system after obtaining informed consent from the guardian.
   1. Perform RSB on patients that have the following indications: inability to pass meconium in infants or long-term bloating with or without constipation in children; children with long-term constipation who need paraffin oil to complete defecation; intestinal obstruction or intestinal perfora....

Results

In total, 318 patients were enrolled in our study. All of the patients underwent RSB, and the tissues were stained for calretinin, S100, and PGP9.5. The diagnosis based on our novel protocol was HD in 97 cases, non-HD in 213 cases, and suspected HD in 8 cases. Among the 132 surgical patients, 99 patients were diagnosed with HD by immunostaining of full-thickness specimens after surgery. S100 and PGP9.5 staining showed that 92% and 93% of the 99 patients, respectively, had hypertrophied ne.......

Discussion

Here we described a procedure using three different immunohistochemical antibodies to stain RBS sections for the diagnosis of HD. The sensitivity of our diagnostic protocol was 96.49% (95% CI, 0.88-0.99), and the specificity was 100% (95% CI, 0.97-1.00).

The most critical steps of the protocol are RSB and antigen-antibody reaction. The size of the biopsy determines the accuracy of the staining. A small biopsy will not provide enough tissue to make a precise diagnosis, while a large biopsy lead.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
calretinin antibody	MXB Biotechnologies	MAB-0716 170416405c	antibody: primary antibody
S-100 antibody	MXB Biotechnologies	Kit-0007	antibody: primary antibody
PGP9.5 antibody	Shanghai long island antibody Co. Ltd	R-0457-03	antibody: primary antibody
enhancer reagent	MXB Biotechnologies	KIT-9902-A 170416405a	antibody: secondary antibody A
Goat anti-Rabbit/Mouse IgG Secondary Antibody	MXB Biotechnologies	KIT-9902-B	antibody: secondary antibody B
DAB staining kit (containing reagent A B and C)	MXB Biotechnologies	DAB-0031	staining kit
Heat incubator	Shanghai yiheng instrument Co. Ltd	DHP-9082	instrument
Rbi2 suction rectal biopsy system	Aus Systems Pty Ltd, South Australia, Australia	CP1200 HP1000 SS1000	instrument
microtome	Leica	leica RM2016	instrument
citric acid sodium citrate buffer(100X)	MXB Biotechnologies	MVS-0101	antigen retrieval buffer
pathological tissue dehydrator	wuhan junjie electronic Co. Ltd	JT-12F	instrument