Hirschsprung's disease: diagnosis using monoclonal antibody 171B5.

A new reliable immunohistochemical method for diagnosing Hirschsprung's disease (HD) using our unique monoclonal antibody (MAb) 171B5 against synaptic vesicles is described. Fresh frozen sections of rectal tissues were used from 13 patients with HD aged 2 weeks to 13 months; 9 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.     

Histochemical acetylcholinesterase reactions in total colonic aganglionosis

Reliability of the histochemical diagnosis in total colonic aganglionosis is controversial. We studied histochemical acetylcholinesterase (AChE) reactions in three children with total colonic aganglionosis. In all three, there was an increase in AChE positive nerve fibers in the rectal mucosa, obtained when the patients were over the neonatal period. However, a proximal extension of the increase in AChE positive nerve fibers was noted in the distal end of the resected bowel obtained at the definitive operation in two cases out of three. In one, there was no proximal extension in the resected bowel, and in another, there was an increase in AChE positive nerve fibers in the mucosa of the ganglionic ileum. Our findings indicate that the histochemical study of AChE activity in rectal suction biopsy is useful, even in cases of total colonic aganglionosis.     

Histochemical diagnosis of Hirschsprung's disease and 2 point rectal mucosal biopsies for selection of surgical treatment

In order to establish the diagnostic criteria of Hirschsprung's disease by Ach. E. staining of rectal mucosal biopsy, we analysed the specimen obtained from 110 infants with abdominal distension and constipation. We found the pattern of nerve fiber proliferation was not uniform even in patients with Hirschsprung's disease. The pattern was classified into 4 types. I. total layer type (Typical type), II. lamina propria mucosal type, III. lamina submucosal type, and IV. lamina propria mucosal and lamina submucosal type. In case of types II, III, IV which do not show a typical finding, detection of Meissner's ganglion cells is the most important factor for the diagnosis of Hirschsprung's disease. Since we confirmed that the Meissner's ganglion cells were always present at the level of just above to the dentate line of the rectum in the specimen of normal controls, we concluded that 5mm oral to the dentate line was the most appropriate position of the rectal mucosal biopsy for the diagnosis of Hirschsprung's disease including patients with ultrashort segment aganglionosis. Two points rectal mucosal biopsies, namely, the biopsies from the site of 5mm and 5 cm oral to the dentate line, was useful for selection of surgical treatment in patients with short segment aganglionosis.     

Histochemical criteria for the diagnosis of Hirschsprung's disease in rectal suction biopsies by acetylcholinesterase activity

Histochemistry to demonstrate cholinergic fibers in rectal suction biopsies is a reliable method to diagnose or exclude Hirschsprung's disease. The histochemical criterion of a positive reaction consists of the presence of many coarse discrete cholinergic fibers in the muscularis mucosae and in the immediately subjacent submucosa, best assessed under medium power with the condenser lowered. A marked increase in cholinergic fibers in the lamina propria is seen only in some cases of Hirschsprung's disease. At least some cases of Hirschsprung's disease may be the result of destruction of ganglian cells by a viral infection in the intrauterine and early postnatal life.     

Intestinal neuronal dysplasia: results of treatment in 33 patients

PURPOSE: Intestinal neuronal dysplasia (IND) is a disease of the enteric nervous system, which clinically resembles Hirschsprung's disease. The authors reviewed their experience of IND over an 8-year period. METHODS: Between 1992 and 1999, 418 patients underwent rectal suction biopsy for persistent constipation. Thirty-three (7.8%) patients had histologic evidence of IND. There were 26 boys and 7 girls (age range, 1 week to 10 years). The diagnosis of IND was based on the presence of hyperganglionosis of the submucous plexus and giant ganglia and at least one of the following features in rectal biopsies: (1) ectopic ganglia, (2) increased acetylcholinesterase (AChE) activity in the lamina propria, and (3) increased AChE nerve fibers around the submucosal blood vessels. All patients were started on laxatives with or without enemas after the diagnosis was made. Patients have been followed up from 1 to 8 years (mean, 2.4 years). RESULTS: Twenty-one (64%) patients had a good response to conservative management and currently have normal bowel habits. Twelve patients (36%) underwent internal sphincter myectomy after failed conservative management. Seven of these patients now have normal bowel habits. Two patients are able to stay clean with regular enemas. Three patients who continued to have persistent constipation after myectomy and underwent resection of redundant and dilated sigmoid colon now have normal bowel habits. CONCLUSION: The majority of patients with IND can be treated successfully with conservative treatment.     

Erythrocyte acetylcholinesterase activity in Hirschsprung's disease in Israel

Hirschsprung's disease is a relatively prevalent disorder in newborn babies and young children. Acetylcholinesterase (AChE) activity is augmented locally, in the neural plexus of the affected gut segment. It has been suggested that this enzyme will also be increased in red blood cells (RBCs) of affected children. Thus, we studied AChE activity in newborn babies and children, as well as age-adjusted data. This Israeli study consisted of healthy and affected Bedouins as well as Ashkenazi and Sephardic Jews. This study supports the Japanese study in that the RBC AChE is not augmented in Hirschsprung's disease. However, final conclusions cannot yet be drawn, because of the small sample.     

Problems and advantages of acetylcholinesterase histochemistry of rectal suction biopsies in the diagnosis of Hirschsprung's disease

One hundred fifty-seven children, aged 2 days to 15 years, had rectal biopsies for suspected Hirschsprung's disease from 1980 to 1987. Sixty had adequate suction rectal biopsies stained for acetylcholinesterase (ACE); 41 of these were reported as normal and eight showed atypical innervation in children who progressed satisfactorily without surgery. Two showed hyperganglionosis and nine were from children with Hirschsprung's disease. Four of the latter nine biopsies (44%) gave false-negative results in infants aged 9, 10, 13 days, and 6 weeks. The last had total colonic aganglionosis. There were two false-positive results, one in a normal child with atypical innervation and the other in a child with hyperganglionosis. Thus, six of 60 (10%) of ACE-stained suction rectal biopsies gave misleading results.     

Intestinal neuronal dysplasia: association with digestive malformations

Isolated Intestinal neuronal dysplasia is a disease characterized by symptoms of diarrhea or intestinal obstruction along with histopathologic diagnosis based on hyperplasia of submucous plexus with giant ganglia containing more than seven nerve cells, increased acetylcholinesterase activity in the lamina propria and around the submucosal blood vessels and heterotopic ganglia in the lamina propria. The aims of this study have been to determine the incidence of the congenital malformations associated with the isolated intestinal dysplasia type B (not associated to another anomaly of the enteric nervous system) and to correlate them with the severity of the symptoms and their possible familial involvement. We have retrospectively reviewed all the patients diagnosed with IND type B nonassociated to Hirschsprung's disease in our hospital from 1981 to 2002. Our serie consists of 44 cases, 1 for every 7500 newborn. 35% of the patients presented associated congenital anomalies, with digestive malformations being the most commonly found (20% of the total). 40% of the patients studied had previous history of constipation in the family. Onset of symptoms was detected in 75% of children during newborn period.     

Aberrant colonic expression of MHC class II antigens in Hirschsprung's disease.

The major histocompatibility complex (MHC) Class I and II cell surface antigens responsible for the recognition of self vs non-self were studied in patients with documented Hirschsprung's disease. Monoclonal antibodies reactive with monomorphic determinants of human lymphocyte antigen (HLA)-A,B,C (Class I) and HLA-DR (Class II) were used to demonstrate immunohistochemically the expression of MHC antigens in 27 biopsy specimens from a variety of colorectal disorders. The rectal specimens examined from patients with Hirschsprung's disease showed an unexpected, marked elevation of Class II antigens with abnormal localization in the mucosa and lamina propria. This ectopic expression was not seen in any portion of the small or large bowel of patients who did not have Hirschsprung's disease. Furthermore, proximal normal colon of children with Hirschsprung's disease failed to show increased expression of Class II antigen. In an attempt to better define the effector arm at a cellular level, the distribution of helper T cells (CD4+), cytotoxic/suppressor T cells (CD8+) and natural killer cells (NK; CD16+) was examined in 5 cases. In Hirschsprung's disease, rectal infiltration of CD8+ and CD16+ cells was found, but not CD4+ cells. Ectopic expression of Class II antigen with increased numbers of rectal T cells and NK cells suggested that an early immunologic event may be causal in Hirschsprung's disease.     

Intestinal neuronal dysplasia is a possible cause of persistent bowel symptoms after pull-through operation for Hirschsprung's disease

The proximal margin of the resected bowel specimens from 33 consecutively treated patients undergoing a definitive pull-through operation for Hirschsprung's disease (HD) and control specimens consisting of suction rectal biopsy specimens obtained from 24 age-matched patients evaluated for constipation (and proven not to have HD) were examined using conventional H&E staining and acetylcholinesterase (AChE) histochemistry. Complete resection of the aganglionic segment was confirmed in 31 patients. In one patient, the proximal margin was found to be aganglionic; in another, the proximal margin was in a transitional zone. In both patients, frozen sections at the time of surgery were interpreted as having ganglion cells. In 10 of 31 patients, intestinal neuronal dysplasia was demonstrated in the proximal margin of the resected bowel. The abnormalities included hyperplasia of the submucous plexus, giant ganglia (with > 7 ganglion cells), and ectopic ganglion cells (all 10 patients) and increased AChE activity in the lamina propria (5 patients). All ten patients with IND had persistent bowel problems after the definitive operation for HD, such as enterocolitis, soiling, or constipation. Only four of the other 21 patients had persistent bowel symptoms. This study suggests that IND is commonly associated with HD. It also emphasizes the importance of histochemical examination of the resected segment to predict postoperative bowel function in patients with HD.     

Ectopic class II major histocompatibility antigens in Hirschsprung's disease and neuronal intestinal dysplasia.

Although the etiology of Hirschsprung's disease and neuronal intestinal dysplasia remains obscure, both have histological abnormalities involving ganglion cells and neuronal elements. Searching for a common pathway that may inhibit normal maturation of neurogenic precursors, we examined the possible role of an immune mechanism in the maldevelopment of the enteric neural network. Six patients with Hirschsprung's disease were studied by comparing biopsy specimens from diseased colon with ones taken from proximal ganglionic colon in the same patients. These were similarly compared with colonic biopsy specimens from patients studied with chronic constipation or bowel removed at the time of operation for other disorders. Biopsies were taken from four other patients with neuronal intestinal dysplasia. Each was examined by hematoxylin & eosin staining, acetylcholinesterase histochemistry, and immunohistochemistry of major histocompatibility complex (MHC) class I and class II antigens. All rectal samples from Hirschsprung's disease patients exhibited elevated acetylcholinesterase histochemistry and absent ganglia to confirm the diagnosis. These findings were correlated with marked elevation of class II MHC in the aganglionic area, whereas the proximal normal ganglionic segments showed no elevation. Rectal biopsy specimens from patients with chronic constipation exhibited no such elevation. A similar elevation of class II MHC was detected in the mucosa and submucosa of all four patients with the rare neuronal intestinal dysplasia disorder whose diagnosis was confirmed by giant ganglia in Auerbach's plexuses, aberrant Meissner's ganglia in the lamina propria mucosa, and giant neurofibrils in the mucosa and submucosa. The correlation of elevated class II MHC in these two neuronal dysfunction disorders may indicate an underlying autoimmune mechanism as is seen in thyroiditis and insulin dependent diabetes mellitus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphohistochemical study of the large intestine in Hirschsprung's disease in children

The morphohistochemical study of the biopsy specimens of the large intestine and operative material, obtained in 41 children with Hirschsprung's disease, in 34 children with combination of the Hirschsprung's disease and dolichosigmoid, and in 25--with dolichosigmoid, was carried out. It is shown, that the presence of a thick network of neural fibers with positive response to acetylcholinesterase (ACE) in the biopsy specimens of the large intestine and positive, or sharply positive response to ACE in the lamina propria and lamina muscularis of the mucosal layer is a precise morphologic marker of the Hirschsprung's disease.     

Short segment Hirschsprung's disease as a cause of discrepancy between histologic, histochemical, and clinical features

Diagnostic difficulty may be encountered in Hirschsprung's disease when discrepancy is noted between the histochemical pattern, the presence or absence of ganglion cells, and clinical features. This is mainly a problem in neonates, and the cause of the discrepancy is probably due to biopsy specimens being taken above a short aganglionic segment. When short segment Hirschsprung's disease is suspected, a low suction biopsy should be taken for the demonstration of acetylcholinesterase activity.     

Fourteen-year experience of acetylcholinesterase staining for rectal mucosal biopsy in neonatal Hirschsprung's disease

BACKGROUND/PURPOSE: Acetylcholinesterase (AChE) staining of rectal mucosal biopsy specimens is the most important and popular examination for making a definite diagnosis of Hirschsprung's disease. This examination often is performed for patients with constipation in the daily clinic. The results of this examination are reflected immediately in the treatment. However, the authors sometimes encountered difficult cases to diagnose, especially in neonates. Therefore, a retrospective investigation was conducted on the benefits and problems of AChE staining of rectal mucosal biopsy specimens in neonates. METHODS: The authors encountered 459 cases (91 neonates) of suspected Hirschsprung's disease, clinically, from April 1986 to March 2000. Mucosal specimens were taken by punch biopsies. Samples were stained by the modified Karnovsky Roots method using rubeanic acid as an amplifier and immediately examined with a light microscope. These results were collected and assessed mainly on neonatal cases. The authors also analyzed the 104 cases of Hirschsprung's disease diagnosed in patients less than 1 year of age to evaluate the relationship between the grade of proliferation of AChE positive fiber and age. RESULTS: Forty-one neonatal cases of Hirschsprung's disease were diagnosed based on the findings of AChE staining. A definite diagnosis of Hirschsprung's disease was confirmed based on the pathologic findings of operative samples. Forty-eight cases that were diagnosed as normal included 4 cases that turned out to be false-negative (3 Hirschspurung's disease cases and 1 case of an allied disorder of Hirschsprung's disease). There were no major complications in mucosal punch biopsy. In the cases of Hirschsprung's disease diagnosed in a patient less than 1 year of age, the grade of AChE-positive fiber tended to increase with the aging of patients. CONCLUSIONS: The specificity of AChE staining was high (100%), but its sensitivity was slightly low (91%). Careful long-term follow-up is required for any cases diagnosed as normal. Mucosal biopsies should be repeated in cases of persistent clinical symptoms.     

Intensity and proximal extension of acetylcholinesterase activity in the mucosa of the rectosigmoid in Hirschsprung's disease

By staining for AChE activity, 51 consecutive rectal biopsies were investigated for the presence of mucosal nerve fibers in order to evaluate the possibilities of suction biopsies for the diagnosis of Hirschsprung's disease. Of nine patients suffering from Hirschsprung's disease, AChE activity was increased in eight. This study suggest that the intensity and proximal extension of AChE activity increase with age, and that mucosal activity does not extend as far proximally as in the submucous and intramuscular plexuses. From the study of resected bowel segments of eight patients it is concluded that false-negative findings in the rectal mucosa of patients with Hirschsprung's disease are avoidable by taking the biopsy immediately beyond the pectinate line. There were no false-positive findings.     

A study of mucosal gut immunity in infants who develop Hirschsprung's-associated enterocolitis.

The aim of this study was twofold. First, to establish quantitatively the distribution of the immunoglobulin-containing (plasma) cells, T and B lymphocytes in the lamina propria of the rectal mucosa of normal neonates and neonates with Hirschsprung's disease (HD). Second, to review the neonates with HD to determine any differences in these cell populations between those who subsequently developed Hirschsprung's enterocolitis (HEC) and those who did not. Two conclusions can be drawn from the results of our study of rectal mucosal immune defenses. First, neonates with HD have no deficiencies in these defenses when compared with normal neonates. Second, neonates with HD who subsequently develop HEC have no premorbid deficiency in these defenses. It was noted that the pan-T cell count in the infants who went on to develop HEC appeared to be increased, although this did not reach statistical significance. The use of fresh or frozen material would permit a more detailed analysis of the separate T cell subsets.     

Clinical and histologic studies of abnormal intramural plexus with special reference to hypoganglionosis

Since April 1973, 230 patients, who were suspected of Hirschsprung's disease, have been examined by rectal mucosal biopsy, barium enema and ano-rectal manometry. Their rectal mucosal specimens have been taken both from the regions 2cm upwards and just above the dentate line, and have been studied by means of acetylcholinesterase (AChE)-staining and hematoxylin-eosin (H-E)-staining. Specimens obtained by rectal full-thickness biopsies or operations were examined by means of AChE-staining, H-E-staining and silver-impregnation. Eleven out of the 230 patients were histologically diagnosed as hypoganglionosis. All rectal mucosas of hypoganglionosis showed a few small submucous plexus. According to the findings of nerve fibers, however, cases of hypoganglionosis were divided into three groups. The recognition of nerve fibers partially proliferated in rectal mucosa at the newborn age leads the diagnosis to type A. The type B is diagnosed as Hirschsprung's disease only through the recognition of proliferations of nerve fibers in rectal mucosa after the suckling age. In type C, no proliferation of nerve fibers can be recognized even after the suckling age. An accurate diagnosis of type C can be, therefore, made only through the examinations of myenteric plexus in full-thickness rectal specimen. On barium enema, a narrow segment was definite in 7 cases of hypoganglionosis, but was indefinite in the remaining 4 cases. The diseased segments of intestines were limited to the rectum or the rectosigmoid colon in 10 cases. Recto-anal sphincteric reflex showed an atypical reflex in 6 cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

An improved staining technique for acetylcholinesterase activity using rubeanic acid in the diagnosis of Hirschsprung's disease

An improved staining technique for acetylcholinesterase (AChE) activity using rubeanic acid was used to make a clinical diagnosis in 54 children with constipation. Nineteen were thus confirmed to have Hirschsprung's disease. False positive or negative reactions were nil. The sites of AChE activities were in the form of black deposits and the contrast was sharp. This approach should find a wide application for the diagnosis of Hirschsprung's disease.     

Two-point rectal mucosal biopsy for selection of surgical treatment of Hirschsprung's disease

A two-point rectal mucosal biopsy (utilizing a histochemical study), namely biopsies at sites 5 to 10 mm and 30 to 50 mm oral to the dentate line, was developed to differentiate patients who can be treated adequately by rectal myectomy without colostomy and those who require other definitive surgery for Hirschsprung's disease. The examinations were performed in 28 patients suspected having a short aganglionic rectum. Ganglion cells were demonstrated by upper biopsy in four of 13 neonates, six of nine infants, and three of six children. These cases were successfully treated by rectal myectomy. The two-point rectal mucosal biopsy is useful not only for making definitive diagnosis but also for the selection of the surgical treatment for patients with Hirschsprung's disease.     

Rectal suction biopsy for the diagnosis of Hirschsprung's disease.

The diagnosis of Hirschsprung's disease is at times difficult, particularly in the young patient. Since 1972 we have used rectal suction biopsy as a screening technique in neonates and infants with failure to pass meconium or evidence of obstruction. In addition, it is used to confirm the diagnosis of Hirschsprung's disease when suspected by barium enema study. This technique has been used in 444 patients, 302 of whom were less than one year of age. No anesthesia is necessary, and there have been no associated complications. Only one patient early in the study had an initial misdiagnosis. There have been no false-positive or false-negative specimens since this initial problem, and no patients have undergone inappropriate pull-through procedures for suspected Hirschsprung's disease. It is recommended that all neonates who do not pass meconium in the first 48 hours of life undergo rectal suction biopsy to establish the diagnosis of congenital megacolon.