Name: diagnosis of hirschsprung's disease by immunostaining rectal suction biopsies for calretinin, s100 protein and protein gene product 9.5
Uploaded: 2019-04-26T12:30:00
Description: Watch this Scientific Journal Video about Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5 at JoVE.com

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).

This protocol was approved by the Research Ethics Board of the Union Hospital of Huazhong University of Science and Technology.
1. Rectal Suction Biopsy
<ol>
	<li>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.
	<ol>
		<li>Perform RSB on patients that have the following indications: inability to pass meconium in infants or long-term bloating with or without con...

<ol>
	<li>Tam, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hirschsprung's+disease:+A+bridge+for+science+and+surgery.">Hirschsprung's disease: A bridge for science and surgery.</a> Journal of Pediatric Surgery. 51 (1), 18-22 (2016).</li><li>Heanue, T. A., Pachnis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+nervous+system+development+and+Hirschsprung's+disease:+advances+in+genetic+and+stem+cell+studies.">Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies.</a> Nature Reviews Neuroscience. 8 (6), 466-479 (2007).</li><li>Setiadi, J. A., Dwihantoro, A., Iskandar, K., Heriyanto, D. 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N., Ekberli Agirbas, G., Tiryaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calretinin+immunohistochemistry+in+Hirschsprung's+disease:+An+adjunct+to+formalin-based+diagnosis.">Calretinin immunohistochemistry in Hirschsprung's disease: An adjunct to formalin-based diagnosis.</a> The Turkish Journal of Gastroenterology. 23 (3), 226-233 (2012).</li><li>Bachmann, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+panel+for+the+diagnosis+of+Hirschsprung&#39;s+disease+using+antibodies+to+MAP2,+calretinin,+GLUT1+and+S100.">Immunohistochemical panel for the diagnosis of Hirschsprung&#39;s disease using antibodies to MAP2, calretinin, GLUT1 and S100.</a> Histopathology. 66 (6), 824-835 (2015).</li><li>Jiang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S100+and+protein+gene+product+9.5+immunostaining+of+rectal+suction+biopsies+in+the+diagnosis+of+Hirschsprung'+disease.">S100 and protein gene product 9.5 immunostaining of rectal suction biopsies in the diagnosis of Hirschsprung' disease.</a> American Journal of Translational Research. 8 (7), 3159 (2016).</li><li>Takawira, C., D'Agostini, S., Shenouda, S., Persad, R., Sergi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+procedures+update+on+Hirschsprung+disease.">Laboratory procedures update on Hirschsprung disease.</a> Journal of Pediatric Gastroenterology &amp; Nutrition. 60 (5), 598 (2015).</li><li>Meier-Ruge, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholinesterase+activity+in+suction+biopsies+of+the+rectum+in+the+diagnosis+of+Hirschsprung's+disease.">Acetylcholinesterase activity in suction biopsies of the rectum in the diagnosis of Hirschsprung's disease.</a> Journal of Pediatric Surgery. 7 (1), 11-17 (1972).</li><li>Barshack, I., Fridman, E., Goldberg, I., Chowers, Y., Kopolovic, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+loss+of+calretinin+expression+indicates+aganglionosis+in+Hirschsprung's+disease.">The loss of calretinin expression indicates aganglionosis in Hirschsprung's disease.</a> Journal of Clinical Pathology. 57 (7), 712-716 (2004).</li><li>Kapur, R. 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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...

Hirschsprung&#39;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&#39;s disease. Proliferation, motility, and gut growth are the three main components of successful colonization.
Traditional hematoxylin and eosin (H&#38;E) staining of a limited submucosal biopsy cannot attain as satisfactory of a result as H&#38;E staining of a full-thickness tissue obtained from surgery. Additionally, acetylcholinesterase (AChE) staining of rectal suction tissue is theoretically challenging due to its inadequate sensitivity, which is 91%, and complex processing of frozen sections3,4. Several other immunohistochemical markers of ganglion cells and nerve fibers that can be stained in formalin-fixed and paraffin-embedded specimens are gradually becoming the mainstream HD diagnostics. Calretinin is a vitamin D-dependent calcium-binding protein that is not expressed in the myenteric and submucosal plexus of HD-affected segments5. S100 protein is expressed in cells derived from the neural crest, such as nerve fibers and glial cells, which often present neural hypertrophy in the submucosal tissue of HD-affected segments6. Protein gene product 9.5 (PGP9.5) reliably stains nerve fibers and ganglion cells; PGP9.5 staining acts as a supplement to calretinin staining, especially in cases of isolated hypoganglionosis. Double staining with S100 and PGP9.5 can decrease the false-negative rate and increase the sensitivity. As a prerequisite, the current study aims to ensure adequate specificity and high sensitivity of this novel diagnostic method. Our novel protocol used all three markers for the discrimination of aganglionic intestine and hypertrophic nerve fibers. A prospective study of 318 children was performed by our lab and previously published without a detailed protocol7. The detailed protocol and precautions are discussed in this article. Any neonates who suffered from a severe defecation problem since birth or children with chronic constipation excluding other common diseases are potential candidates for rectal suction biopsy (RSB). Our novel protocol is suitable for staining not only RSBs but also full-thickness biopsies or surgical specimens to make a final diagnosis.

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

diagnosis of hirschsprung's disease by immunostaining rectal suction biopsies for calretinin, s100 protein and protein gene product 9.5

Hirschsprung&#39;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.

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...

Watch this Scientific Journal Video about Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5 at JoVE.com

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

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&#39;s disease has preferable sensitivity and specificity rates.

Hirschsprung&#39;s disease (HD) is a congenital intestinal disease that is clinically manifested as an inability to pass meconium in infants or as ...

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