Immunostaining in Hirschsprung's Disease: Al-Khobar Experience
From the Departments of Pathology (Drs. Al-Sohaibani and Anim) and Surgery (Dr. Khawaja), King Fahd University Hospital, Al-Khobar.
MO Al-Sohaibani, JT Anim, S Khawaja, Immunostaining in Hirschsprung's Disease: Al-Khobar Experience. 1990; 10(4): 434-438
Abstract
The role of immunoperoxidase stains in the diagnosis of Hirschsprung's disease was evaluated in a total of 60 sections from 40 cases originally diagnosed based on the findings from hematoxylin-eosin staining done in our laboratory. Neuron-specific enolase was very effective in highlighting rare scanty ganglion cells in two cases that had been classified as aganglionic. S-100 protein was effective in illustrating nerve fiber proliferation in submucosa and lamina propria, which may be difficult to demonstrate using hematoxylin-eosin alone. We recommend the use of immunoperoxidase stains as an important adjunct to hematoxylin-eosin for the diagnosis of Hirschsprung's disease when using paraffin sections.
Hirschsprung's disease is a congenital disorder in which the ganglion cells are absent from the walls of the rectum and colon, resulting in abnormal function and movement.1,2 Severe constipation is seen early in life, with abdominal distention and associated complications, including growth retardation. Other conditions (i.e., hypoganglionosis, immaturity of ganglia and hypogenesis of ganglia, zonal aganglionosis, and neuronal colonic dysplasia) have been found to mimic Hirschsprung's disease clinically.3-11 In these disorders ganglion cells are present but they show various abnormal distribution patterns as well as abnormal proliferation of nerve fibers.
Traditionally, the histological demonstration of the absence of ganglion cells using conventional stains such as hematoxylin-eosin (H & E) has been used to diagnose Hirschsprung's disease. The introduction of acetyl Cholinesterase, which can demonstrate ganglion cells and the distribution of cholinergic fibers in the submucosal and myenteric plexuses,12-22 has made it possible to define more clearly the abnormalities in patients with Hirschsprung's disease. However, the use of this technique has remained limited, not only because it uses fresh frozen tissue but also because it requires considerable experience in the interpretation of results.21
In recent years, the use of immunoperoxidase techniques as part of routine histopathological examinations has been extended to the demonstration of ganglion cells and nerve fibers in the colonic walls.23-26 Antisera useful in the study of Hirschsprung's disease include neuron-specific enolase (NSE) which stains the perikaryon of ganglion cells and S-100 protein which stains Schwann cells and nerve fibers. This technique is preferable to the acetyl Cholinesterase method because it can be performed easily on routinely fixed paraffin-embedded tissue. The presence or absence of ganglion cells as well as the size and distribution of nerve fibers within the mucosa and submucosa can be easily detected. However, there are few studies confirming the usefulness of the technique in the diagnosis of Hirschsprung's disease and most pathologists are not familiar with the interpretation of these stains. The purpose of this study is to review a series of cases of Hirschsprung's disease diagnosed in our laboratory during the past eight years and assess the usefulness of immunoperoxidase staining for S-100 protein and NSE in its diagnosis.
Methods
A total of 60 sections from 40 cases of clinically suspected Hirschsprung's disease were retrieved from the histopathology laboratory files at King Fahad University Hospital, Al-Khobar, Saudi Arabia. These 40 cases included 32 male and 8 female children aged between 2 weeks and 5 years (average, 1.5 years). Sections from paraffin blocks were cut at 4 μm and stained with H & E. Additional sections were also cut for immunoperoxidase staining.
Immunoperoxidase Method
Immunoperoxidase kits (Cambridge Research Laboratories, Cambridge, Massachusetts, USA) for NSE and S-100 protein were used according to manufacturer's instructions. These kits apply the peroxidase antiperoxidase method and use amino-9-ethyl carbazole as the substrate. Negative controls were included. Each section was examined thoroughly for ganglion cells in the muscularis mucosae layer and in the submucosa. Hyperplastic nerve fibers in the lamina propria, submucosa, and muscularis mucosae were evaluated and graded on a scale of + to + + +.
Results
Findings are compared in Table 1. Two sections that were previously considered aganglionic were found to contain ganglion cells in sections stained for NSE or S-100 protein (Figure 1). NSE defined the ganglions well by staining their perikaryon and cytoplasm (Figure 2). S-100 protein, on the other hand, did not stain the body of the ganglion cells but stained Schwann cells, nerve fibers, and other supportive elements around the ganglion cells, producing a "negative image" pattern (Figure 3) (Figure 3. S-100 protein stain for ganglion cells showing "negative image pattern."). Nerve fibers in the muscularis mucosae, submucosa, and muscular layer were found to be strongly positive and prominent with S-100 protein (Figure 4). However, the nerve endings in the lamina propria mucosal surface were not shown by either NSE or S-100 protein.
Table 1. Summary of immunoperoxidase stains for ganglion cells compared to hematoxylin-eosin.
|
Staining method |
Aganglionic sections |
Ganglionic sections |
|
Hematoxylin-eosin |
27 |
33 |
|
Neuron-specific enolase |
25 |
35 |
|
S-100 protein |
25 |
33 |
Table 2. Summary of nerve fiber proliferation in ganglionic and aganglionic sections using S-100 protein.
|
Sections |
Grade of nerve fiber proliferation |
||
|
I |
II |
III |
|
|
Aganglionic sections (N = 25) |
2 |
5 |
18 |
|
Ganglionic sections (N = 35) |
18 |
10 |
7 |
The degree of nerve fiber proliferation was graded on the basis of I to III and the results tabulated according to the findings in ganglionic and aganglionic segments (Table 2). There was considerably more proliferation in the aganglionic compared to the ganglionic segments. However, there was some overlap between the two groups.
Discussion
The immunoperoxidase technique was originally found to be useful in demonstrating gastrointestinal innervation.27,28 Recently its effectiveness in the diagnosis of Hirschsprung's disease has been investigated in several studies,23,26 specifically for superficial biopsies. These studies have shown that immunoperoxidase staining for NSE and S-100 protein easily demonstrates the presence or absence of ganglion cells.
The ability to demonstrate ganglion cells in the mucosa and submucosa using this technique has helped to highlight its specificity. The common practice of taking superficial biopsies as an outpatient procedure means the amount and depth of tissue can sometimes be unpredictable and this can hamper evaluation of tissue for the presence or absence of ganglion cells. Detection of ganglion cells has been made easier by the use of NSE because it stains ganglion cell cytoplasm in a distinctive manner. S-100 protein staining has proved excellent for demonstrating the supportive elements around the ganglion cells, producing a "negative image" pattern in Schwann cells and nerve fibers.29-31 These methods are useful in the quantitative and qualitative analysis of ganglion cells in cases of inflammation, crushing artifact, and heat artifact. They are also of value in distinguishing between small ganglion cells and other cells, including Schwann cells, because of the intensity of staining and morphological acuteness. These methods can also differentiate vacuolated ganglion cells and immature ganglion cells from, for example, macrophages which are often seen in young patients.
Besides the absence of ganglion cells, there is prominent proliferation of cholinergic nerve fibers in the lamina propria and muscularis mucosae in bowel biopsy specimens from patients with Hirschsprung's disease. This is best demonstrated by acetyl Cholinesterase staining. Previous studies have shown that NSE and S-100 protein staining fail to reveal the nerve fibers of the lamina propria in a consistent manner. However, the nerve fibers in the muscularis mucosae and submucosa can be easily demonstrated. Thus, although acetyl Cholinesterase staining is superior to the immunoperoxidase staining for S-100 protein and NSE, it is more practical.23,26,32
Our findings revealed that S-100 protein and NSE staining of cholinergic nerve fibers within the lamina propria is erratic, but staining of the fibers within the muscularis mucosae and submucosa was quite consistent. Comparison of the ganglionic and aganglionic segments revealed that the size and number of the nerves in the muscularis mucosae and submucosa were considerably higher in the aganglionic portion, though there was some overlap in the findings between ganglion and aganglionic segments (Table 1). This finding may also be used in arriving at a diagnosis of Hirschsprung's disease.
Acknowledgment
We wish to thank Mr. Mohamoud Fakeer, Mr. A. Rahim Othman, and Ms. Sarla Patel for their technical assistance, and Ms. Tess Pasqual for typing the manuscript.
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