Diagnosis

Hirschsprung’s Disease: Using Monoclonal Antibody

171B5

By A. Yamataka, T. Miyano, M. Urao, and H. Nishiye

Tokyo, Japan l A new reliable immunohistochemical method for diagnosing Hirschsprung’s disease (HD) using our unique monoclonal antibody (MAb) 17185 against synaptic vesicles is described. Fresh frozen sections of rectal tissues were used from 13 patients with HD aged 2 weeks to 13 months; g had rectosigmoid HD and 4 had total colonic aganglionosis (TCA). Comparable normal colonic and rectal specimens were also obtained from 13 age-matched controls. All specimens were labeled with MAb 171B5, to demonstrate neuronal innervation patterns of both mucosa and submucosa. In all control specimens, many synapses arranged in variciform plexuses were seen in the lamina propria, a moderate number in the muscularis mucosae, and dense clusters in the submucosal plexus. In all aganglionic specimens, only scanty numbers of synapses which were not organized in variciform plexuses were seen in the lamina propria, none in the muscularis mucosae, and a few in the submucosa. These findings suggest that MAb 171B5 immunohistochemistry on the lamina propria alone can differentiate between normal and aganglionic bowel and appears to be a reliable and useful method for detecting HD on suction rectal biopsy.

Copyright o 1992 by W.B. Saunders Company INDEX body.

WORDS:

Hirschsprung’s

disease;

monoclonal

anti-

I

N THE

DIAGNOSIS of Hirschsprung’s disease (HD), the demonstration of acetylcholinesterase (AChE)-positive nerve fibres in the lamina propria is used to circumvent the problem related to inadequate specimens obtained by suction rectal biopsy.’ Falsenegative results are occasionally obtained in the case of neonates and total colonic aganglionosis (TCA)2,3 because AChE histochemistry looks at a secondary increase in extrinsic nerve fibres in the lamina propria occurring around the time of birth4 and there may be no or only a slight increase in extrinsic nerve fibres in TCA.5 Wakely and McAdam@ also showed that examination of the lamina propria alone is insufficient for diagnosis and its use alone lead to errors; an adequate segment of the muscularis mucosae is necessary.

Furness and Costa7 reported dense networks of fine interconnecting nerve fibres arranged in variciform plexuses in the lamina propria of the normal bowel. In HD these mucosal plexuses are congenitally absent, in addition to the absence of myenteric and submucosal plexuses .8 Therefore, a technique that could confirm the absence of the neuronal innervation in the mucosa (lamina propria and muscularis mucosae) would demonstrate a primary pathological process. This study evaluates the effectiveness of MAb171B5 immunohistochemistry for diagnosing HD and recommends its application on suction rectal biopsy specimens. MATERIALS AND METHODS Resected specimens of rectum were obtained at pull-through operation from one neonate and seven infants with HD and four patients with TCA. Their ages ranged from 4 weeks to 13 months (mean, 5.7 months). One specimen was obtained at autopsy from a neonate with HD, Down’s syndrome, and congenital heart disease who died at the age of 2 weeks. Sixty full-thickness tissue blocks, each measuring 0.7 cm x 0.7 cm, were prepared from the 13 rectal specimens. For the control group, 11 colonic and 2 rectal specimens were obtained from age-matched patients without gastrointestinal disease. Twenty comparable control tissue blocks were then prepared from these 13 specimens. Part of each tissue block was taken for paraffin sections for hematoxylin-eosin (H&E) staining and the remainer snap frozen for study as follows. Alternate cryostat sections, 5 urn thick, were stained with H&E and examined by conventional light microscopy. Immunohistochemical studies were carried out on the remaining sections with the indirect immunofluorescent method9J0 using MAb 171B510-r2(diluted 1:SOO)as a primary antiserum that labels synapses, to demonstrate the distribution of synapses in the lamina propria, muscularis mucosae, and suhmucosa. A normal control section was also processed simultaneously with each tissue block. The number of synapses seen were graded from no synapses visible (-) to many synapses (+ + +) as outlined in Table 1. RESULTS

The results are summarized in Table 1. From the Departments of Pediatric Surgery and Physiology, Juntendo University School of Medicine, Tokyo, Japan. Presented at the 38th Annual International Congress of the British Association of Paediatric Surgeons, Budapest, Hungary, Juiy 24-26, 1991. Address reprint requests to A. Yamataka, MD, Department of Pediatric Surgery, Juntendo University School of Medicine, 2-I-1, Hongo, Bunkyo-ku, Tokyo 113, Japan. Copyright 8 I992 by W.B. Saunders Company 0022-3468/92/2707-0007$03.00/0 820

Colon and Rectum in Control Group H&E staining. Ganglia were present in all frozen and paraffin sections. In all gangliM4b 171B5 immunohistochemistry. onic specimens, many synapses arranged in variciform plexuses were seen in the lamina propria and a moderate number of synapses in the muscularis JournalofPediarric Surgery,

Vol27, No 7 (July). 1992:

pp 820-822

HIRSCHSPRUNG’S

821

DISEASE

Table 1. Distribution of Synapses in the Mucosa and Submucosa in the Aganglionic Rectum and Control Groups VeSSd Walls in the

Lamina

Muscularis

Propria

Mucosee

Submucosa

+++

++

+++

+

It

_

+

++

Submucosa

Controls

(n = 13, t = 60) Aganglionic rectum (n = 13, t = 20)

Abbreviations: n, number of patients; t, number of tissue blocks; -, no synapses visible; 2, occasional synapses; +, a few synapses; ++. a moderate number of synapses; + + +, many synapses.

mucosae (Fig 1A). In the submucosa, dense clusters of synapses were observed in the plexuses, with a few synapses around the vessel walls (Fig 1B). Rectum in Patients with HD H&E staining. Ganglia were absent in all frozen and paraffin sections. A4Ab 171B5 immunohistochemistry. In all aganglionic specimens of patients with HD (including TCA), occasional synapses were seen in the lamina propria with no organization of variciform plexuses and none in the muscularis mucosae (Fig 2A). In the submucosa, a few synapses were detected but there were no plexuses (Fig 2A). A moderate number of synapses were observed around the vessel walls (Fig 2B).

DISCUSSION MAb 171B5 identifies the synaptic vesicle-specific 38,000-d protein that is widely distributed in synapses of both the central and peripheral nervous systems.‘@‘* It selectively labels the synaptic vesicles but not other structures.” In the present study, using this MAb, a marked decrease in the number of synapses in both mucosa and submucosa was demonstrated in patients with HD, suggesting that the proliferative extrinsic nerve fibres do not have sufficient synapses in these layers of the aganglionic rectum. Thus, the h4Ab 171B5 immunohistochemical technique identifies the primary pathology of HD, which is an absent or poor mucosal neuronal innervation. Its use would avoid the false-negative problem encountered with AChE histochemistry on suction rectal biopsy. Our results also suggest that normal and aganglionic bowel can be accurately distinguished by MAb 171B5 immunohistochemical technique from the examination of the lamina propria alone. The present study shows a larger number of synapses around the vessels in the aganglionic rectum than in controls. The basis for this phenomenon remains unexplained. It is possible that the extrinsic nerve fibres in the aganglionic rectum have synaptic contacts with vessel walls. However, this finding, which is limited to vessel walls, does not influence our

Fig 1. MAb 171S54abeled synapses (arrows) in the rectal (A) mucosa and (6) submucosa from the control group. Note the many synapses arranged in variciform plexuses in the lamina propria (Ip), a moderate number in the muscularis mucosae (mm), dense clusters in the eubmucosal plexus (SP), and the relatively few synapses around the vessel walls (V).

YAMATAKA

ET AL

Fig 2. MAb 171654sbeled synapses (arrows) in the rectal (A) mucosa and (B) submucosa in a patient with HD. Note only a few synapses in the lamina propria (Ip), none in the muscularis mucosae (mm), and a few synapses without formation of plexuses in the submucosa. Around the vessel walls (V) the synaptic distribution is increased compared with the control group (Fig 16).

criteria for diagnosis of HD using the MAb 171B5 immunohistochemical technique. We conclude that MAb 171B5 immunohistochemistry can distinguish between normal and aganglionic bowel and should be a reliable and useful diagnostic method for detecting HD. ACKNOWLEDGMENT The authors thank Dr Daan den Hollander and Dr P.M.R. Carneiro for advice in preparing the manuscript and K. Sato for his immense contribution.

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the acetylcholinesterase-activity in rectal suction biopsy from neonates with Hirschsprung’s disease. Z Kinderchir 39:246-249, 1984 5. Lake BD: Acetylcholinesterase in the diagnosis of Hirschsprung’s disease and other gastro-intestinal disorders, in eds Filipe I, Lake BD (eds): Histochemistry in Pathology. New York, NY, Churchill Livingstone, 1983, pp 145-149 6. Wakely PE, McAdams AJ: Acetylcholinesterase histochemistry and the diagnosis of Hirschsprung’s disease: A 3%year experience. Pediatr Path01 2:35-46,1984 7. Furness JB, Costa M: The Enteric Nervous System (ed 1). New York, NY, Churchill Livingstone, 1987, pp 6-25 8. Weinberg AG: Hirschsprung’s disease-A pathologist’s view. Perspect Pediatr Path01 2:207-239,1975 9. Coons AH: Histochemistry with labeled antibody. Int Rev Cytol5:1-23,1956 10. Yamataka A, Miyano T, Nishiye H, et al: Innervation of aganglionic intestines of Hirschsprung’s disease examined by monoclonal antibody 171B5. Lancet 2:1038-1039, 1989 11. Obata K, Nishiye H, Fujita SC, et al: Identification of synaptic vesicle-specific 38,000-dalton protein by monoclonal antibodies. Brain Res 375:37-48, 1986 12. Obata K, Kojima N, Nishiye H, et al: Four synaptic vesiclespecific proteins; identification by monoclonal antibodies and distribution in the nervous tissue and the adrenal medulla. Brain Res 404:169-179,1987