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Fourteen-year experience of acetylcholinesterase staining for rectal mucosal biopsy in neonatal Hirschsprung's disease
Fourteen-Year Experience of Acetylcholinesterase Staining for Rectal Mucosal Biopsy in Neonatal Hirschsprung’s Disease By Makoto Nakao, Sachiyo Suita, Tomoaki Taguchi, Ryuichiro Hirose, and Yuichi Shima Fukuoka, Japan
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.
B
ECAUSE OF THE RECENT advances in perinatal medicine, almost all patients with Hirschsprung’s disease are now referred to our department in the neonatal period. It is generally accepted that constipation that is present from birth or that begins in the neonatal period is most likely to be congenital in origin, such as Hirschsprung’s disease.1 A barium enema and manometric study usually are the first examinations performed. In a barium enema, however, the caliber change sometimes is unclear, especially in total colonic aganglionosis and even in short segmental type of neonatal cases.2,3 In a manometric study, a significantly lower anal resting pressure and pain threshold, as well as a decreased frequency of spontaneous rhythmic oscillations usually, are observed in patients with Hirschsprung’s disease.4 However, the rectoanal inhibitory reflex in the manometric study findings often is negative even in normal neonates.5 Therefore, it is accepted generally that the combination of barium enema and anorectal manometry allows for sufficient definition in those infants and children who do not need biopsy or surgery for Hirschsprung’s disease.5,6 However, these 2 examinations often
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. J Pediatr Surg 36:1357-1363. Copyright © 2001 by W.B. Saunders Company. INDEX WORDS: Hirschsprung’s disease, neonate, acetylcholinesterase staining, rectal mucosal biopsy, punch biopsy.
are insufficient for a definite diagnosis of Hirschsprung’s disease. Because AChE staining of a rectal mucosal biopsy is essential for a definite diagnosis of Hirschsprung’s disease, we usually perform this examination for patients ranging from neonates to adults who are suspected to have Hirschsprung’s disease. However, sometimes we come across the diagnostic difficulties, especially in neonates. We, therefore, retrospectively evaluated the benefits and the problems of AChE staining of rectal mucosal biopsy specimens in the neonate. From the Departments of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. This work was supported in part by a Grant-in-Aid for Scientific Research (B2) from the Ministry of Education. Address reprint requests to Makoto Nakao, MD, Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. Copyright © 2001 by W.B. Saunders Company 0022-3468/01/3609-0008$35.00/0 doi:10.1053/jpsu.2001.26369
Journal of Pediatric Surgery, Vol 36, No 9 (September), 2001: pp 1357-1363
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MATERIALS AND METHODS We established the current method of acetylcholinesterase staining using a rubeanic acid amplifying technique in April 19867 and have since encountered 459 cases of AChE staining of rectal mucosal biopsy specimens, in which 91 cases were neonates, until March 2000. Here, we review all these cases. In the 1980s, a suction biopsy was the procedure of choice, and 17 samples were taken by this method. Three samples were taken by full-thickness biopsies during surgery. In the last 10 years, a punch biopsy has become the most popular method, and it was performed for 71 of 91 neonatal cases. In this procedure, mucosal specimens were obtained from the rectum 2 cm above the anal verge using foreign body forceps and a plastic test tube with a small hole on its tip.8 Samples were frozen immediately by liquid nitrogen and stored in a refrigerator at ⫺80°C. AChE staining was done according to the modified method of Karnovsky and Roots.9 AChE staining solution was made just before staining by mixing 6.5 mL of 0.1 mol/L sodium hydrogen maleate buffer, 0.5 mL of 0.1 mol/L sodium citrate, 1 mL of 30 mmol/L copper sulfate, 1 mL of 5 mmol/L potassium ferricyanide, 1 mL of distilled water, and 10 mg of acetylcholine iodide. Rubeanic acid solution was made by mixing 10 mg of rubeanic acid, 10 mL of 100% ethanol, 6.55 g of sodium acetate, and 40 mL of distilled water.7 Frozen samples were sliced into 10-m sections by a cryostat (SAKURA Precision Instrument Co, Tokyo, Japan). The sections then were placed on microscope slides and put into the AChE staining solution for 20 minutes and the rubeanic acid solution for 10 minutes. After staining, the samples were dehydrated and embedded in Canada balsam. We thereafter observed the samples under light microscopy and made a diagnosis. To evaluate the AChE staining results, we categorized the grade of the proliferation of AChE positive fiber as follows: (⫺), no proliferation of AChE-positive fibers in the lamina propria mucosa (Fig 1); (⫾), a slight proliferation of thin fiber only at the bottom of the lamina propria mucosa; (⫹), obvious proliferation in the lower part of the lamina propria mucosa; (⫹⫹), AChE-positive fibers extend to the tip of the lamina propria mucosa (Fig 2); (⫹⫹⫹), AChE-positive fibers proliferate diffusely, run transversely, and make a network. We diagnosed Hirschsprung’s disease when both the proliferation of AChEpositive fiber([⫾]⬃[⫹⫹⫹]) and a lack of ganglion cells were observed. To evaluate the relationship between the grade of proliferation of AChE-positive fiber and age, we also analyzed 104 cases
Fig 2. AChE staining of Hirschsprung’s disease (original magnification ⴛ 40). The proliferation of AChE-positive fibers is seen in lamina propria, and thick fiber bundles are also seen in muscularis mucosa (grade [ⴙⴙ]). No ganglion cells are found in this section.
of Hirschsprung’s disease diagnosed in patients less than 1 year of age.
RESULTS
Analysis of 91 Neonatal Cases We diagnosed 41 of 91 neonatal Hirschsprung’s disease by AChE staining findings. There were 2 cases of (⫾, 4.9%), 22 cases of (⫹; 53.6%), 15 cases of (⫹⫹; 36.6%), and 2 cases of (⫹⫹⫹; 4.9%). All of the patients underwent radical operations and were confirmed to have Hirschsprung’s disease pathologically. There were 36 short segment cases (80.5%), 6 long segment cases (14.6%), and 2 cases of total colonic aganglionosis (TCA; 4.9%). There was no statistical relationship between the grade of AChE-positive fiber and the extent of the aganglionic segment (Table 1). However, 4 of the 48 cases diagnosed to be normal were found to have been false-negative. Three cases were proved to be Hirschsprung’s disease after the second rectal mucosal biopsy using AChE staining in patients at 6 months, 29 days, and 3 months of age, respectively. All of them were short segment cases. The other patient had an allied disorder of Hirschsprung’s disease (immaturity of ganglion cells), which was confirmed by histologic examination in a fullTable 1. Analysis of 41 of Initially Diagnosed Hirschsprung’s Disease: Relationship Between the Grade of AChE-Positive Fiber and the Extent of the Aganglionic Segment
Fig 1. AChE staining of normal case (original magnification ⴛ 40). The proliferation of AChE-positive fiber is not seen in lamina propria at all, and several ganglion cells are found in submucosal layer (grade [ⴚ]).
Short segment Long segment TCA Total
(⫾)
(⫹)
(⫹⫹)
(⫹⫹⫹)
Total
2 0 0 2
20 1 1 22
10 4 1 15
1 1 0 2
33 6 2 41
NOTE. There was no statistical relationship between the grade of AChE-positive fiber and the extent of the aganglionic segment.
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Table 2. Summary of the Analysis of AChE Staining of Neonates Initial Diagnosis by AChE Staining
Definite Diagnosis
Hirschsprung’s disease
41
3
Normal
48
Unknown Total
2 91
3 3 3 3
Hirschsprung’s disease short segment long segment TCA Normal Hirschsprung’s disease (short segment) Allied disorder of Hirschsprung’s disease Unknown Total
41 33 6 2 44 3 1 2 91
NOTE. We diagnosed 41 of 91 neonatal cases of Hirschsprung’s disease by AChE staining findings. Forty-eight cases were diagnosed as normal, which included 4 false-negative cases. The diagnostic specificity of AChE staining was 100%, and its sensitivity was 91%.
thickness biopsy sample at creating an ileostomy. Two cases were impossible to diagnose because of poor sample quality, such as a shortage of sample and insufficient freezing. The diagnostic specificity of AChE staining was 100% (41 of 41 cases), and its sensitivity was 91% (44 of 48 cases; Table 2). False-negative cases (3 of Hirschsprung’s disease and 1 of allied disorder of Hirschsprung’s disease) are shown as case reports.
Case Reports We would like to report 4 false-negative cases (case 1, 2, 3, and 4) and 1 typical case of Hirschsprung’s disease that was followed chronologically from the neonatal period (case 5). Case 1: False negative case of Hirschsprung’s disease 1. The patient was an 8-day-old girl whose chief problem was constipation. A rectal mucosal biopsy was
Fig 3. AChE staining of false-negative Hirschsprung’s disease (original magnification ⴛ 40). There is no proliferation of AChE-positive fibers in mucosal layer (grade [ⴚ]; left) But a few thick fiber bundles exist in lamina propria showed by arrow (right).
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performed immediately. There was no proliferation of AChE-positive fibers in the lamina propria mucosa (grade [⫺]). However, no typical ganglion cells were seen either (Fig 3A). We diagnosed normal rather than Hirschsprung’s disease. Because of the persistence of the symptoms, an additional biopsy was performed 6 months later, which showed aganglionosis and AChE staining positive fibers in the mucosal layer. We looked at the first section again and found a localized AChE staining positive fiber bundle (Fig 3B). This finding is thought to be an early phase of Hirschsprung’s disease. But it was difficult to make a definite diagnosis in an 8-day-old patient. Case 2: False-negative case of Hirschsprung’s disease 2. The patient was an 8-day-old boy. Because of dyschezia, he underwent a rectal mucosal biopsy. Although no typical ganglion cells were detected, proliferation of AChE-positive fibers in the lamina propria mucosa was not seen at all. Therefore, our diagnosis was normal at that point. An additional biopsy was performed at 29 days of age because of persistent constipation. The
NAKAO ET AL
result was grade (⫹⫹) and the definite diagnosis of Hirschsprung’s disease was obtained, and he underwent a Z-shaped anastomosis at 10 months of age. Case 3: False-negative case of Hirschsprung’s disease 3. The patient was a girl who underwent a rectal mucosal biopsy at 15 days of age. Although the ganglion cells were not found at all, the result was grade (⫺). However, a conservative treatment such as enema and laxatives was not effective, and the second biopsy was done at 3 months of age. The results indicated grade (⫹⫹⫹). A definite diagnosis of Hirschsprung’s disease, thus, was made at that time. Case 4: An allied disorder of Hirschsprung’s disease. The patient was a 2-day-old boy born at 38 weeks gestation, and his birth weight was 2,830 g. His chief problems were abdominal distension and vomiting. An emergency rectal mucosal biopsy was performed with a suspicion of Hirschsprung’s disease. There was no proliferation of AChE-positive fibers in the mucosal layer, and several ganglion cells were seen in the submucosal layer (Fig 4). Because of the progress of abdominal
Fig 4. AChE and HE staining of Allied disorder of Hirschsprung’s disease. There is no proliferation of the AChE-positive fibers in mucosal layer, and several ganglion cells are seen in submucosal layer (Grade [ⴚ]; left; original magnification ⴛ 40). H&E staining of ileum shows the definite diagnosis as immaturity of ganglion cells (right, original magnification ⴛ 100).
AChE STAIN IN NEONATAL HD
distension, an ileostomy was made on the same day. The pathologic findings of the ileum showed a definite diagnosis to be an immaturity of ganglion cells. Case 5: Typical case of neonatal short segment aganglionosis that was followed chronologically. The patient was 2-day-old boy who showed abdominal distension and delayed passage of meconium. He underwent rectal mucosal biopsy 3 times at 4 days, 1 month, and 4 months of age, respectively. Only a slight proliferation of AChE-positive fibers was seen in mucosal layer at 4 days (grade: [⫹]), and no ganglion cells were detected. The diagnosis of Hirschsprung’s disease was made at that point. The findings at 1 month of age did not change remarkably. He received the conservative therapy, such as bowel lavage. However, the proliferation became significant at 4 months of age (grade [⫹⫹]; Fig 5). He underwent a Z-shaped anastomosis at 5 months of age and showed good progress. As shown in this case, the AChE-positive fiber was thought to proliferate after the aging.
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Analysis of 104 Cases of Hirschsprung’s Disease Diagnosed in Patients Less Than 1 Year of Age The relationship between the age and grade of AChEpositive fiber on 104 Hirschsprung’s disease cases in our institute is shown in Fig 6. Almost all (⫾) cases existed only in neonatal period and no (⫹) cases existed after 100 days of age. As a result, the grade of AChE-positive fiber tended to increase after the aging of patients. Furthermore, the proliferation of AChE-positive fibers in the mucosal layer often is insufficient, especially in the neonatal period, which makes it hard to make a definite diagnosis. DISCUSSION
The standard technique of AChE staining is based on the enzyme histochemical method of Karnovsky and Roots.9 We modified this method by using rubeanic acid as an amplifier, which increased the contrast and reduced the staining time to 30 minutes.7,8 The technique of
Fig 5. Chronological change of AChE staining of Hirschsprung’s disease (original magnification ⴛ 40). Only a little proliferation of AChE-positive fibers is seen in mucosal layer at both 4 days and 1 month of age (grade [ⴙ]). However, the proliferation becomes significant at 4 months of age (grade [ⴙⴙ]).
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Fig 6. Age-related distribution of the grade of AChE-positive fiber of Hirschsprung’s disease cases. The relationship between age and grade of AChE-positive fiber on 104 Hirschsprung’s disease patients less than 1 year old. Grade (ⴚ) and almost all (ⴞ) cases exist only in neonatal period and no (ⴙ) cases existed after 100days of age.
introducing supplemental oxidation also is reported to reduce the staining time to less than 10 minutes.10 However, our technique is simple and highly reproducibile, and a 30-minute staining time is short enough to start the adequate therapy. The immunostaining for other substances such as neuropeptide Y, protein gene product 9.5, tyrosine hydroxylase, calcitonin gene-related peptide, and substance P also has been reported to be a useful examination for the diagnosis of Hirschsprung’s disease instead of AChE staining.11-14 However, AChE staining is superior to the other examinations because of its simplicity, fastness, and accuracy. Ghosh and Griffiths15 reported that all the children with Hirschsprung’s disease had an onset of symptoms before 4 weeks of age. In addition, the rectoanal inhibitory reflex of a manometric study often is inaccurate in the neonatal period.5 Therefore, AChE staining of a rectal mucosal biopsy in the neonatal period is becoming more important following the progress of neonatal care. We have encountered 91 neonatal cases of AChE staining after a rectal mucosal biopsy since 1986. All of the cases diagnosed as Hirschsprung’s disease by AChE staining were confirmed based on the pathologic findings, and its specificity was 100%. We had 2 cases of TCA, which we could easily diagnose by AChE staining in the neonatal period. Generally, the barium enema and manometric study findings are often obscure in TCA cases.2 Therefore, AChE staining is invaluable for evaluating TCA cases. We had 4 false-negative cases among the 48 cases diagnosed normal by AChE staining. These 4 cases contained 3 cases of Hirschsprung’s disease and 1 case of allied disorder of Hirschspurung’s disease. These 3 falsenegative cases of Hirschsprung’s disease resulted from the insufficiency of the proliferation of AChE-positive fibers in mucosal layer. After settlement of the definite diagnosis, we looked at the samples again. All the sam-
ples had a very little proliferation of AChE-positive fibers or a few thick AChE-positive fiber bundles not in mucosal layer but in the submucosal layer. Furthermore, no ganglion cells were recognized at all. In a case with such a findings, we have to consider the second biopsy after neonatal period. It is generally considered that AChE-positive fiber grows up after the age.7 According to the analysis of our Hirschsprung’s disease cases of patients less than 1 year of age, false-negative and almost all grade (⫾) cases exist only in neonatal period. After 100 days of age, all cases belong to grade (⫹⫹) or (⫹⫹⫹). Taking these results into consideration, the second biopsy should be performed after the neonatal period, and the biopsy must be repeated until obtaining the definite diagnosis if the clinical symptoms are persisted. In case of allied disorder of Hirschsprung’s disease, the proliferation of AChE-positive fibers is not seen, and normal appearance of ganglion cells were recognized in AChE staining, and H&E staining of operative samples showed the definite diagnosis. Because the sample volume is limited in AChE staining of rectal mucosal biopsy, it is impossible to decide the abnormality of ganglion cells both qualitatively and quantitatively. We have to take this fact into consideration and correspond to the patient accurately according to the symptoms. AChE staining of rectal mucosal biopsy is an effective examination in specificity, but its sensitivity is slightly low. We are required careful clinical follow-up for the case diagnosed as normal in the neonatal period, and the repeated mucosal biopsy is recommended in case of persistent symptoms. ACKNOWLEDGMENT The authors thank Brian Quinn for reading the manuscript.
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REFERENCES 1. Leung AK, Chan PY, Cho HY: Constipation in children. Am Family Physician 54:611-618, 1996 2. Masuyama H, Watanabe K, Ikawa H, et al: Motor activity of refashioned colorectoanus in patients with total colonic aganglionosis. J Pediatr Surg 31:283-290, 1996 3. Rosenfield NS, Ablow RC, Markowitz RI, et al: Hirschspurung’s disease: Accuracy of the barium enema examination. Radiology 150: 393-400, 1984 4. Lanfranchi GA, Bazzocchi G, Federici S, et al: Anorectal manometry in the diagnosis of Hirschsprung’s disease—Comparison with clinical and radiological criteria. Am J Gastroenterol 79:270-275, 1984 5. Emir H, Akman M, Sarimurat N, et al: Anorectal manometry during the neonatal period: Its specificity in the diagnosis of Hirschsprung’s disease. Eur J Pediatr Surg 9:101-103, 1999 6. Fotter R: Imaging of constipation in infants and children. Eur Radiol 8:248-258, 1998 7. Goto S, Ikeda K, Toyohara T: An improved staining technique for acetylcholinesterase activity using rubeanic acid in the diagnosis of Hirschsprung’s disease. Jpn J Surg 14:135-138, 1984 8. Hirose R, Hirata Y, Yamada T, et al: The simple technique of rectal mucosal biopsy for the diagnosis of Hirschsprung’s disease. J Pediatr Surg 28:499-506, 1993
9. Karnovsky MJ, Roots L: A “direct-coloring” thiocholine method for cholinesterase. J Histochem Cytochem 12:219-221, 1964 10. Kobayashi H, O’Briain DS, Hirakawa H, et al: A rapid technique of acetylcholinesterase staining. Arch Pathol Lab Med 118:11271129, 1994 11. Galvis DA, Yunis EJ: Comparison of neuropeptide Y, protein gene product 9.5, and acetylcholinesterase in the diagnosis of Hirschsprung’s disease. Pediatr Pathol Lab Med 17:413-425, 1997 12. Wiedenmann B, Riedel C, John M, et al: Qualitative and quantitative analysis of synapses in Hirschsprung’s disease. Pediatr Surg Int 13:468-473, 1998 13. Watanabe Y, Ito F, Ando H, et al: Morphological investigation of the enteric nervous system in Hirschsprung’s disease and hypoganglionosis using whole-mount colon preparation. J Pediatr Surg 34:445449, 1999 14. Robey SS, Kuhajda FP, Yardley JH: Immunoperoxidase stains of ganglion cells and abnormal mucosal nerve proliferations in Hirschsprung’s disease. Human Pathol 19:432-437, 1988 15. Ghosh A, Griffiths DM: Rectal biopsy in the investigation of constipation. Arch Dis Child 79:266-268, 1998
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.
B
ECAUSE OF THE RECENT advances in perinatal medicine, almost all patients with Hirschsprung’s disease are now referred to our department in the neonatal period. It is generally accepted that constipation that is present from birth or that begins in the neonatal period is most likely to be congenital in origin, such as Hirschsprung’s disease.1 A barium enema and manometric study usually are the first examinations performed. In a barium enema, however, the caliber change sometimes is unclear, especially in total colonic aganglionosis and even in short segmental type of neonatal cases.2,3 In a manometric study, a significantly lower anal resting pressure and pain threshold, as well as a decreased frequency of spontaneous rhythmic oscillations usually, are observed in patients with Hirschsprung’s disease.4 However, the rectoanal inhibitory reflex in the manometric study findings often is negative even in normal neonates.5 Therefore, it is accepted generally that the combination of barium enema and anorectal manometry allows for sufficient definition in those infants and children who do not need biopsy or surgery for Hirschsprung’s disease.5,6 However, these 2 examinations often
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. J Pediatr Surg 36:1357-1363. Copyright © 2001 by W.B. Saunders Company. INDEX WORDS: Hirschsprung’s disease, neonate, acetylcholinesterase staining, rectal mucosal biopsy, punch biopsy.
are insufficient for a definite diagnosis of Hirschsprung’s disease. Because AChE staining of a rectal mucosal biopsy is essential for a definite diagnosis of Hirschsprung’s disease, we usually perform this examination for patients ranging from neonates to adults who are suspected to have Hirschsprung’s disease. However, sometimes we come across the diagnostic difficulties, especially in neonates. We, therefore, retrospectively evaluated the benefits and the problems of AChE staining of rectal mucosal biopsy specimens in the neonate. From the Departments of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. This work was supported in part by a Grant-in-Aid for Scientific Research (B2) from the Ministry of Education. Address reprint requests to Makoto Nakao, MD, Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. Copyright © 2001 by W.B. Saunders Company 0022-3468/01/3609-0008$35.00/0 doi:10.1053/jpsu.2001.26369
Journal of Pediatric Surgery, Vol 36, No 9 (September), 2001: pp 1357-1363
1357
1358
NAKAO ET AL
MATERIALS AND METHODS We established the current method of acetylcholinesterase staining using a rubeanic acid amplifying technique in April 19867 and have since encountered 459 cases of AChE staining of rectal mucosal biopsy specimens, in which 91 cases were neonates, until March 2000. Here, we review all these cases. In the 1980s, a suction biopsy was the procedure of choice, and 17 samples were taken by this method. Three samples were taken by full-thickness biopsies during surgery. In the last 10 years, a punch biopsy has become the most popular method, and it was performed for 71 of 91 neonatal cases. In this procedure, mucosal specimens were obtained from the rectum 2 cm above the anal verge using foreign body forceps and a plastic test tube with a small hole on its tip.8 Samples were frozen immediately by liquid nitrogen and stored in a refrigerator at ⫺80°C. AChE staining was done according to the modified method of Karnovsky and Roots.9 AChE staining solution was made just before staining by mixing 6.5 mL of 0.1 mol/L sodium hydrogen maleate buffer, 0.5 mL of 0.1 mol/L sodium citrate, 1 mL of 30 mmol/L copper sulfate, 1 mL of 5 mmol/L potassium ferricyanide, 1 mL of distilled water, and 10 mg of acetylcholine iodide. Rubeanic acid solution was made by mixing 10 mg of rubeanic acid, 10 mL of 100% ethanol, 6.55 g of sodium acetate, and 40 mL of distilled water.7 Frozen samples were sliced into 10-m sections by a cryostat (SAKURA Precision Instrument Co, Tokyo, Japan). The sections then were placed on microscope slides and put into the AChE staining solution for 20 minutes and the rubeanic acid solution for 10 minutes. After staining, the samples were dehydrated and embedded in Canada balsam. We thereafter observed the samples under light microscopy and made a diagnosis. To evaluate the AChE staining results, we categorized the grade of the proliferation of AChE positive fiber as follows: (⫺), no proliferation of AChE-positive fibers in the lamina propria mucosa (Fig 1); (⫾), a slight proliferation of thin fiber only at the bottom of the lamina propria mucosa; (⫹), obvious proliferation in the lower part of the lamina propria mucosa; (⫹⫹), AChE-positive fibers extend to the tip of the lamina propria mucosa (Fig 2); (⫹⫹⫹), AChE-positive fibers proliferate diffusely, run transversely, and make a network. We diagnosed Hirschsprung’s disease when both the proliferation of AChEpositive fiber([⫾]⬃[⫹⫹⫹]) and a lack of ganglion cells were observed. To evaluate the relationship between the grade of proliferation of AChE-positive fiber and age, we also analyzed 104 cases
Fig 2. AChE staining of Hirschsprung’s disease (original magnification ⴛ 40). The proliferation of AChE-positive fibers is seen in lamina propria, and thick fiber bundles are also seen in muscularis mucosa (grade [ⴙⴙ]). No ganglion cells are found in this section.
of Hirschsprung’s disease diagnosed in patients less than 1 year of age.
RESULTS
Analysis of 91 Neonatal Cases We diagnosed 41 of 91 neonatal Hirschsprung’s disease by AChE staining findings. There were 2 cases of (⫾, 4.9%), 22 cases of (⫹; 53.6%), 15 cases of (⫹⫹; 36.6%), and 2 cases of (⫹⫹⫹; 4.9%). All of the patients underwent radical operations and were confirmed to have Hirschsprung’s disease pathologically. There were 36 short segment cases (80.5%), 6 long segment cases (14.6%), and 2 cases of total colonic aganglionosis (TCA; 4.9%). There was no statistical relationship between the grade of AChE-positive fiber and the extent of the aganglionic segment (Table 1). However, 4 of the 48 cases diagnosed to be normal were found to have been false-negative. Three cases were proved to be Hirschsprung’s disease after the second rectal mucosal biopsy using AChE staining in patients at 6 months, 29 days, and 3 months of age, respectively. All of them were short segment cases. The other patient had an allied disorder of Hirschsprung’s disease (immaturity of ganglion cells), which was confirmed by histologic examination in a fullTable 1. Analysis of 41 of Initially Diagnosed Hirschsprung’s Disease: Relationship Between the Grade of AChE-Positive Fiber and the Extent of the Aganglionic Segment
Fig 1. AChE staining of normal case (original magnification ⴛ 40). The proliferation of AChE-positive fiber is not seen in lamina propria at all, and several ganglion cells are found in submucosal layer (grade [ⴚ]).
Short segment Long segment TCA Total
(⫾)
(⫹)
(⫹⫹)
(⫹⫹⫹)
Total
2 0 0 2
20 1 1 22
10 4 1 15
1 1 0 2
33 6 2 41
NOTE. There was no statistical relationship between the grade of AChE-positive fiber and the extent of the aganglionic segment.
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Table 2. Summary of the Analysis of AChE Staining of Neonates Initial Diagnosis by AChE Staining
Definite Diagnosis
Hirschsprung’s disease
41
3
Normal
48
Unknown Total
2 91
3 3 3 3
Hirschsprung’s disease short segment long segment TCA Normal Hirschsprung’s disease (short segment) Allied disorder of Hirschsprung’s disease Unknown Total
41 33 6 2 44 3 1 2 91
NOTE. We diagnosed 41 of 91 neonatal cases of Hirschsprung’s disease by AChE staining findings. Forty-eight cases were diagnosed as normal, which included 4 false-negative cases. The diagnostic specificity of AChE staining was 100%, and its sensitivity was 91%.
thickness biopsy sample at creating an ileostomy. Two cases were impossible to diagnose because of poor sample quality, such as a shortage of sample and insufficient freezing. The diagnostic specificity of AChE staining was 100% (41 of 41 cases), and its sensitivity was 91% (44 of 48 cases; Table 2). False-negative cases (3 of Hirschsprung’s disease and 1 of allied disorder of Hirschsprung’s disease) are shown as case reports.
Case Reports We would like to report 4 false-negative cases (case 1, 2, 3, and 4) and 1 typical case of Hirschsprung’s disease that was followed chronologically from the neonatal period (case 5). Case 1: False negative case of Hirschsprung’s disease 1. The patient was an 8-day-old girl whose chief problem was constipation. A rectal mucosal biopsy was
Fig 3. AChE staining of false-negative Hirschsprung’s disease (original magnification ⴛ 40). There is no proliferation of AChE-positive fibers in mucosal layer (grade [ⴚ]; left) But a few thick fiber bundles exist in lamina propria showed by arrow (right).
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performed immediately. There was no proliferation of AChE-positive fibers in the lamina propria mucosa (grade [⫺]). However, no typical ganglion cells were seen either (Fig 3A). We diagnosed normal rather than Hirschsprung’s disease. Because of the persistence of the symptoms, an additional biopsy was performed 6 months later, which showed aganglionosis and AChE staining positive fibers in the mucosal layer. We looked at the first section again and found a localized AChE staining positive fiber bundle (Fig 3B). This finding is thought to be an early phase of Hirschsprung’s disease. But it was difficult to make a definite diagnosis in an 8-day-old patient. Case 2: False-negative case of Hirschsprung’s disease 2. The patient was an 8-day-old boy. Because of dyschezia, he underwent a rectal mucosal biopsy. Although no typical ganglion cells were detected, proliferation of AChE-positive fibers in the lamina propria mucosa was not seen at all. Therefore, our diagnosis was normal at that point. An additional biopsy was performed at 29 days of age because of persistent constipation. The
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result was grade (⫹⫹) and the definite diagnosis of Hirschsprung’s disease was obtained, and he underwent a Z-shaped anastomosis at 10 months of age. Case 3: False-negative case of Hirschsprung’s disease 3. The patient was a girl who underwent a rectal mucosal biopsy at 15 days of age. Although the ganglion cells were not found at all, the result was grade (⫺). However, a conservative treatment such as enema and laxatives was not effective, and the second biopsy was done at 3 months of age. The results indicated grade (⫹⫹⫹). A definite diagnosis of Hirschsprung’s disease, thus, was made at that time. Case 4: An allied disorder of Hirschsprung’s disease. The patient was a 2-day-old boy born at 38 weeks gestation, and his birth weight was 2,830 g. His chief problems were abdominal distension and vomiting. An emergency rectal mucosal biopsy was performed with a suspicion of Hirschsprung’s disease. There was no proliferation of AChE-positive fibers in the mucosal layer, and several ganglion cells were seen in the submucosal layer (Fig 4). Because of the progress of abdominal
Fig 4. AChE and HE staining of Allied disorder of Hirschsprung’s disease. There is no proliferation of the AChE-positive fibers in mucosal layer, and several ganglion cells are seen in submucosal layer (Grade [ⴚ]; left; original magnification ⴛ 40). H&E staining of ileum shows the definite diagnosis as immaturity of ganglion cells (right, original magnification ⴛ 100).
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distension, an ileostomy was made on the same day. The pathologic findings of the ileum showed a definite diagnosis to be an immaturity of ganglion cells. Case 5: Typical case of neonatal short segment aganglionosis that was followed chronologically. The patient was 2-day-old boy who showed abdominal distension and delayed passage of meconium. He underwent rectal mucosal biopsy 3 times at 4 days, 1 month, and 4 months of age, respectively. Only a slight proliferation of AChE-positive fibers was seen in mucosal layer at 4 days (grade: [⫹]), and no ganglion cells were detected. The diagnosis of Hirschsprung’s disease was made at that point. The findings at 1 month of age did not change remarkably. He received the conservative therapy, such as bowel lavage. However, the proliferation became significant at 4 months of age (grade [⫹⫹]; Fig 5). He underwent a Z-shaped anastomosis at 5 months of age and showed good progress. As shown in this case, the AChE-positive fiber was thought to proliferate after the aging.
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Analysis of 104 Cases of Hirschsprung’s Disease Diagnosed in Patients Less Than 1 Year of Age The relationship between the age and grade of AChEpositive fiber on 104 Hirschsprung’s disease cases in our institute is shown in Fig 6. Almost all (⫾) cases existed only in neonatal period and no (⫹) cases existed after 100 days of age. As a result, the grade of AChE-positive fiber tended to increase after the aging of patients. Furthermore, the proliferation of AChE-positive fibers in the mucosal layer often is insufficient, especially in the neonatal period, which makes it hard to make a definite diagnosis. DISCUSSION
The standard technique of AChE staining is based on the enzyme histochemical method of Karnovsky and Roots.9 We modified this method by using rubeanic acid as an amplifier, which increased the contrast and reduced the staining time to 30 minutes.7,8 The technique of
Fig 5. Chronological change of AChE staining of Hirschsprung’s disease (original magnification ⴛ 40). Only a little proliferation of AChE-positive fibers is seen in mucosal layer at both 4 days and 1 month of age (grade [ⴙ]). However, the proliferation becomes significant at 4 months of age (grade [ⴙⴙ]).
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Fig 6. Age-related distribution of the grade of AChE-positive fiber of Hirschsprung’s disease cases. The relationship between age and grade of AChE-positive fiber on 104 Hirschsprung’s disease patients less than 1 year old. Grade (ⴚ) and almost all (ⴞ) cases exist only in neonatal period and no (ⴙ) cases existed after 100days of age.
introducing supplemental oxidation also is reported to reduce the staining time to less than 10 minutes.10 However, our technique is simple and highly reproducibile, and a 30-minute staining time is short enough to start the adequate therapy. The immunostaining for other substances such as neuropeptide Y, protein gene product 9.5, tyrosine hydroxylase, calcitonin gene-related peptide, and substance P also has been reported to be a useful examination for the diagnosis of Hirschsprung’s disease instead of AChE staining.11-14 However, AChE staining is superior to the other examinations because of its simplicity, fastness, and accuracy. Ghosh and Griffiths15 reported that all the children with Hirschsprung’s disease had an onset of symptoms before 4 weeks of age. In addition, the rectoanal inhibitory reflex of a manometric study often is inaccurate in the neonatal period.5 Therefore, AChE staining of a rectal mucosal biopsy in the neonatal period is becoming more important following the progress of neonatal care. We have encountered 91 neonatal cases of AChE staining after a rectal mucosal biopsy since 1986. All of the cases diagnosed as Hirschsprung’s disease by AChE staining were confirmed based on the pathologic findings, and its specificity was 100%. We had 2 cases of TCA, which we could easily diagnose by AChE staining in the neonatal period. Generally, the barium enema and manometric study findings are often obscure in TCA cases.2 Therefore, AChE staining is invaluable for evaluating TCA cases. We had 4 false-negative cases among the 48 cases diagnosed normal by AChE staining. These 4 cases contained 3 cases of Hirschsprung’s disease and 1 case of allied disorder of Hirschspurung’s disease. These 3 falsenegative cases of Hirschsprung’s disease resulted from the insufficiency of the proliferation of AChE-positive fibers in mucosal layer. After settlement of the definite diagnosis, we looked at the samples again. All the sam-
ples had a very little proliferation of AChE-positive fibers or a few thick AChE-positive fiber bundles not in mucosal layer but in the submucosal layer. Furthermore, no ganglion cells were recognized at all. In a case with such a findings, we have to consider the second biopsy after neonatal period. It is generally considered that AChE-positive fiber grows up after the age.7 According to the analysis of our Hirschsprung’s disease cases of patients less than 1 year of age, false-negative and almost all grade (⫾) cases exist only in neonatal period. After 100 days of age, all cases belong to grade (⫹⫹) or (⫹⫹⫹). Taking these results into consideration, the second biopsy should be performed after the neonatal period, and the biopsy must be repeated until obtaining the definite diagnosis if the clinical symptoms are persisted. In case of allied disorder of Hirschsprung’s disease, the proliferation of AChE-positive fibers is not seen, and normal appearance of ganglion cells were recognized in AChE staining, and H&E staining of operative samples showed the definite diagnosis. Because the sample volume is limited in AChE staining of rectal mucosal biopsy, it is impossible to decide the abnormality of ganglion cells both qualitatively and quantitatively. We have to take this fact into consideration and correspond to the patient accurately according to the symptoms. AChE staining of rectal mucosal biopsy is an effective examination in specificity, but its sensitivity is slightly low. We are required careful clinical follow-up for the case diagnosed as normal in the neonatal period, and the repeated mucosal biopsy is recommended in case of persistent symptoms. ACKNOWLEDGMENT The authors thank Brian Quinn for reading the manuscript.
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REFERENCES 1. Leung AK, Chan PY, Cho HY: Constipation in children. Am Family Physician 54:611-618, 1996 2. Masuyama H, Watanabe K, Ikawa H, et al: Motor activity of refashioned colorectoanus in patients with total colonic aganglionosis. J Pediatr Surg 31:283-290, 1996 3. Rosenfield NS, Ablow RC, Markowitz RI, et al: Hirschspurung’s disease: Accuracy of the barium enema examination. Radiology 150: 393-400, 1984 4. Lanfranchi GA, Bazzocchi G, Federici S, et al: Anorectal manometry in the diagnosis of Hirschsprung’s disease—Comparison with clinical and radiological criteria. Am J Gastroenterol 79:270-275, 1984 5. Emir H, Akman M, Sarimurat N, et al: Anorectal manometry during the neonatal period: Its specificity in the diagnosis of Hirschsprung’s disease. Eur J Pediatr Surg 9:101-103, 1999 6. Fotter R: Imaging of constipation in infants and children. Eur Radiol 8:248-258, 1998 7. Goto S, Ikeda K, Toyohara T: An improved staining technique for acetylcholinesterase activity using rubeanic acid in the diagnosis of Hirschsprung’s disease. Jpn J Surg 14:135-138, 1984 8. Hirose R, Hirata Y, Yamada T, et al: The simple technique of rectal mucosal biopsy for the diagnosis of Hirschsprung’s disease. J Pediatr Surg 28:499-506, 1993
9. Karnovsky MJ, Roots L: A “direct-coloring” thiocholine method for cholinesterase. J Histochem Cytochem 12:219-221, 1964 10. Kobayashi H, O’Briain DS, Hirakawa H, et al: A rapid technique of acetylcholinesterase staining. Arch Pathol Lab Med 118:11271129, 1994 11. Galvis DA, Yunis EJ: Comparison of neuropeptide Y, protein gene product 9.5, and acetylcholinesterase in the diagnosis of Hirschsprung’s disease. Pediatr Pathol Lab Med 17:413-425, 1997 12. Wiedenmann B, Riedel C, John M, et al: Qualitative and quantitative analysis of synapses in Hirschsprung’s disease. Pediatr Surg Int 13:468-473, 1998 13. Watanabe Y, Ito F, Ando H, et al: Morphological investigation of the enteric nervous system in Hirschsprung’s disease and hypoganglionosis using whole-mount colon preparation. J Pediatr Surg 34:445449, 1999 14. Robey SS, Kuhajda FP, Yardley JH: Immunoperoxidase stains of ganglion cells and abnormal mucosal nerve proliferations in Hirschsprung’s disease. Human Pathol 19:432-437, 1988 15. Ghosh A, Griffiths DM: Rectal biopsy in the investigation of constipation. Arch Dis Child 79:266-268, 1998