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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 con.......

Representative 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.......

Acknowledgements

The authors thank Weibing Tang for his great help in providing the filming lab. This article is supported by the Public Welfare Research, and special funds were received from the National Health and Family Planning of China (Grant No.201402007).

....

Materials

NameCompanyCatalog NumberComments
calretinin antibodyMXB BiotechnologiesMAB-0716 170416405cantibody: primary antibody
S-100 antibodyMXB BiotechnologiesKit-0007antibody: primary antibody
PGP9.5 antibodyShanghai long island antibody Co. LtdR-0457-03antibody: primary antibody
enhancer reagentMXB BiotechnologiesKIT-9902-A 170416405aantibody: secondary antibody A
Goat anti-Rabbit/Mouse IgG Secondary AntibodyMXB BiotechnologiesKIT-9902-Bantibody: secondary antibody B
DAB staining kit (containing reagent A B and C)MXB BiotechnologiesDAB-0031staining kit
Heat incubatorShanghai yiheng instrument Co. LtdDHP-9082instrument
Rbi2 suction rectal biopsy systemAus Systems Pty Ltd, South Australia, AustraliaCP1200 HP1000 SS1000instrument
microtomeLeicaleica RM2016instrument
citric acid sodium citrate buffer(100X)MXB BiotechnologiesMVS-0101antigen retrieval buffer
pathological tissue dehydratorwuhan junjie electronic Co. LtdJT-12Finstrument

References

  1. Tam, P. K. Hirschsprung's disease: A bridge for science and surgery. Journal of Pediatric Surgery. 51 (1), 18-22 (2016).
  2. Heanue, T. A., Pachnis, V. Enteric nervous system development and Hirs....

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