NSE和S-100蛋白在巨结肠同源病的表达

目的探讨巨结肠同源病的病理形态学特征，提高其诊断符合率。方法对131例行巨结肠根治术患儿的病变肠段进行常规HE染色，观察肠神经元及神经节细胞形态和数量的变化，与正常组进行比较，并对21例标本采用NSE和S-100蛋白进行免疫组织化学染色。结果巨结肠同源病（HAD）标本的神经元和神经节细胞数与正常组及巨结肠症（HD）比较，差异有显著统计学意义（P〈0．01）。结论巨结肠同源病的诊断主要根据病理特征而定，在HE染色基础上结合NSE及S-100蛋白免疫组织化学染色能提高确诊率。     

NSE和S-100蛋自在巨结肠同源病的表达

目的 探讨巨结肠同源病的病理形态学特征,提高其诊断符合率.方法 对131例行巨结肠根治术患儿的病变肠段进行常规HE染色,观察肠神经元及神经节细胞形态和数量的变化,与正常组进行比较,并对21例标本采用NSE和S-100蛋白进行免疫组织化学染色.结果 巨结肠同源病(HAD)标本的神经元和神经节细胞数与正常组及巨结肠症(HD)比较,差异有显著统计学意义(P＜0.01).结论 巨结肠同源病的诊断主要根据病理特征而定,在HE染色基础上结合NSE及S-100蛋白免疫组织化学染色能提高确诊率.     

快速乳酸脱氢酶染色在先天性巨结肠及肠神经元发育异常术中诊断的应用

目的 探讨快速乳酸脱氢酶(LDH)染色在术中诊断先天性巨结肠(HD)及肠神经元发育异常(IND)的可行性及临床意义.方法 20例术前诊断为HD或巨结肠同源病(HAD)患儿术中分别在直肠及近端结肠取全层标本行快速LDH染色,通过预热孵育液和提高氧化型辅酶Ⅰ(NAD)水平将反应时间缩短在20 min内,同时行快速乙酰胆碱酯酶(AChE)染色,并与术后病检结果 进行Kappa诊断一致性检验.结果 LDH染色孵育20 min后出现蓝紫色沉淀,可识别为神经节细胞,增加染色时间并不增加强染色程度;20例患儿中HD 8例,IND 6例,HD并IND 3例,均与术后病理检查结果 相一致;1例直肠LDH染色有少量表达阳性,但AChE染色阳性,术后病检为神经节细胞减少症;2例直肠LDH和AChE染色均阴性,术后病检为HD、神经节细胞减少各1 例.Kappa检验HD、IND、HD并IND的患儿检验结果 与术后病理检查结果 有较强的吻合性(K≥0.7).结论 快速LDH染色联合快速AChE染色术中诊断HD和IND结果 可靠,可帮助术中判断病变肠管的范围.     

新生儿肠动力影响因素分析

目的从病理角度分析新生儿期非巨结肠性动力性肠梗阻的发病机制,以指导临床,避免不必要的手术。方法收集本院2005年1月至2010年1月新生儿期非巨结肠动力性肠梗阻病例32例,均经保守治疗无效后行手术探查,其中30例行回肠造瘘术,2例行横结肠造瘘术,取造瘘处肠管石蜡切片为实验第一组;以上32例患儿均于3个月后返回本院行封瘘术,取封瘘术中同段肠管石蜡切片为实验第二组;取10例同日龄同段正常肠管石蜡切片做对照组。三组石蜡切片分别行HE染色,分别对CD3、CD117行免疫组化检测及凋亡（Tunnel）检测。结果HE染色光镜下观察第二组较第一组黏膜及肌问神经节细胞数量增多,体积增大,光镜下观察肠壁肌纤维未见异常;CD3于实验组及对照组神经节周均未见表达;CD117第二组较第一组及对照组表达增多,实验组及对照组Tunnel凋亡实验见神经节周无表达。结论肠神经节周围炎症反应及肠神经节凋亡这两个影响年长儿肠动力的因素指标在新生儿动力性肠梗阻中并不常见;Cajal细胞发育异常及神经节细胞成熟度可能是新生儿肠动力异常的重要原因。     

The expression and significance of calretinin in the colon of patients with Hirschsprung's disease

目的 本研究应用免疫组织化学染色方法,观察对照组肠壁、先天性巨结肠症(Hirschsprung's disease,HD)患儿有神经节细胞段和无神经节细胞段肠壁神经组织中钙视网膜蛋白(Calretinin,CR)的表达结果,目的在于了解HD的病理生理改变以及寻找诊断先天性巨结肠的简便有效的方法.方法 收集苏州大学附属儿童医院小儿外科2005至2008年手术切除HD标本54例,包括HD扩张段与痉挛段.以15例年龄与之相符的无HD患儿的手术切除结肠标本作为对照组.分别对HD痉挛段、扩张段、对照组肠壁组织切片进行CR的免疫组织化学染色和HE染色,计算机成像系统照相存盘,用图像分析软件(Image-Pro-Plus)分别判定CR在HD扩张段与痉挛段神经丛中阳性染色面积百分比.所得数据用SPSS 12.0统计软件包进行处理分析.结果 ①无论是HE或免疫组化染色HD的扩张段神经节细胞皆存在,肌层神经纤维有不同程度的排列改变,神经节细胞大小不等;痉挛段均未见神经节细胞;②在正常结肠及HD扩张段肠壁免疫组化染色可见钙视网膜蛋白在肌间及黏膜下神经丛中对神经节细胞呈强阳性表达,神经纤维也呈阳性反应;而HD痉挛段肠壁的免疫组化染色则可见钙视网膜蛋白在肌间神经丛及黏膜下神经丛大多表达阴性(90.7％),仅少量呈弱阳性表达(9.3％);③定量分析:CR分别在HD痉挛段之间神经丛中阳性染色面积百分率(0.00665±().00387)与其在HD扩张段神经丛中阳性染色面积百分率(0.26483±0.14626)存在差异,有显著统计学意义(P＜0.01).结论 钙视网膜蛋白免疫组化染色可很好的显示正常结肠及HD扩张段肠壁的神经节细胞及神经纤维,而在HD痉挛段该指标免疫组化染色结果呈阴性或弱阳性表达.钙视网膜蛋白可能作为诊断HD的神经标志物之一.     

疑似全结肠巨结肠回肠造瘘术后的二期手术策略

目的：探讨疑似全结肠巨结肠病例行急诊回肠造瘘术后二期手术的诊疗流程及手术方案。方法对本院2012年1月至2013年6月收治的22例回肠造瘘术后的疑似全结肠巨结肠患儿进行回顾性分析。患儿手术年龄3～13个月,平均年龄5．8个月。经下消化道造影后24 h复查腹平片及直肠测压检查。均行术中回结肠多处活检,冰冻病理检查。根据检查结果决定下一步手术方案。结果11例诊断为全结肠巨结肠,行经腹部切口辅助经肛门巨结肠根治术（全结肠切除回肠肛门吻合术）。2例确诊为长段型巨结肠,1例确诊为常见型巨结肠,行巨结肠根治术。8例排除巨结肠及巨结肠同源病,行关瘘术。术后患儿恢复可。结论对于因疑全结肠巨结肠而行回肠造瘘的患儿,二期手术前可行下消化道造影,造影后24 h复查腹平片以评估远端肠管排钡功能。术中行回结肠多处浆肌层活检快速冰冻病理检查可以明确诊断及病变范围,从而指导二期手术方案的选择,是一种可靠、可行、简便快捷的方法。     

巨结肠类缘病病理形态学诊断的探讨

目的采用NSE和S-100蛋白免疫组化染色方法,探讨巨结肠类缘病的病理形态学特征,提高其诊断率。方法对97例行巨结肠根治术后的病变肠段进行常规HE染色,并对其中21例采用NSE和S-100蛋白免疫组化,观察肠神经元及神经节细胞形态和数量的变化并与正常组进行比较。结果巨结肠类缘病(alliedHD)标本的神经元与神经节细胞数与正常组及巨结肠症(HD)比较差异有显著性(P<0.01)。结论巨结肠类缘病的诊断主要根据病理特征而定,在HE染色基础上结合NSE及S-100蛋白免疫组织化学染色能提高确诊率。     

神经元特异烯醇化酶和S100蛋白在巨结肠及其类缘病中的表达

目的探讨神经元特异烯醇化酶(NSE)和S100蛋白(S100)在巨结肠及其类缘病的表达和临床意义。方法对120例拟诊为先天性巨结肠的病变肠段行HE染色,结合光镜下肠壁神经节细胞的形态特征,并对29例应用免疫组织化学技术行NSE、S100蛋白染色,比较其染色结果和分布特点。结果巨结肠类缘病标本的神经丛及神经节细胞数与正常组比较差异有显著性(P<0.01)。结论NSE、S100可作为巨结肠与其类缘病鉴别诊断的标记物,能提高临床巨结肠类缘病的确诊率。     

S-100蛋白与Cathepsin D在先天性巨结肠诊断中的应用

目的观察S-100蛋白(S-100)和组织蛋白酶D(cathepsin D，CAD)在先天性巨结肠(Hirschspnmg’s disease，HD)中的表达。方法应用免疫组织化学染色观察25例HD患儿及10例对照组儿童结肠。结果(1)在正常对照组与HD扩张段组肠壁肌间和黏膜下层可见CAD只对神经丛中神经节细胞阳性表达，对神经纤维与神经胶质细胞均不表达。S-100染色与CAD染色相反，神经纤维与神经胶质细胞均阳性表达，神经节细胞阴性表达，表现为阳性神经丛中细胞状“空白区”。(2)在HD狭窄段组肠壁神经丛中缺乏CAD阳性表达。S-100染色神经丛中细胞状“空白区”未观察到；S-100强阳性表达的神经纤维束显著增生，扭曲呈波浪状。结论S-100和CAD的免疫组化染色对诊断HD具有重要意义。     

结肠次全切除术治疗巨结肠同源病23例

目的探讨小儿巨结肠同源病(HAD)的诊断和治疗方法。方法回顾性分析23例巨结肠同源病的病例资料,男8例,女15例,年龄5个月～16岁,其中5个月～3岁4例,3～16岁19例。患儿均以便秘、腹胀为主诉,术前常规行钡灌肠、直肠肛管测压检查。其中11例行直肠粘膜活检术;5例因肠梗阻行结肠造瘘术;3例为先天性巨结肠术后复发便秘。结果全部病例均行腹会阴Soave法结肠次全切除术。23例中,钡剂灌肠均未见明显狭窄段、移行段,24h延迟拍片提示钡剂滞留;部分病例有结肠扩张和结肠冗长表现;直肠肛管测压均有抑制反射,11例直肠粘膜活检AchE阴性。23例术后病理诊断与术前诊断一致。术后均有不同程度腹泻,1例出现伤口裂开,2例直肠粘膜脱垂,2例粘连性肠梗阻;无吻合口瘘、肌鞘感染等并发症。结论腹会阴Soave法结肠次全切除术是治疗HAD较为彻底的术式。     

A new rapid acetylcholinesterase histochemical method for the intraoperative diagnosis of Hirschsprung's disease and intestinal neuronal dysplasia.

The most commonly used acetylcholinesterase (AChE) method for the diagnosis of Hirschsprung's disease (HD) and intestinal neuronal dysplasia (IND) was first introduced in 1964 by Morris Karnovsky and Logan Roots. This technique requires about 80 - 120 minutes incubation time and cannot be used for the intraoperative diagnosis of HD and IND. To avoid these limitations, in 1994 Kobayashi et al first proposed an accelerated modified method in two different versions, the first using diaminobenzydine (DAB) reagent, the second using 4-chloro-1-naphthol as final reagent. In the present study, we propose a new rapid variation of AChE staining which avoids the use of DAB and naphthol, notably toxic reagents, but follows the same acceleration principle of Kobayashi's technique. Our modified rapid AChE requires a total incubation time of only 8 minutes, which is compatible with intraoperative histochemical examination purposes. Intraoperative seromuscular or full-thickness intestinal biopsies were obtained from 92 children affected by intestinal dysganglionoses. The biopsies were frozen and cut in 15 microm cryostatic sections. Rapid AChE was performed with a special incubation medium using 3-amino-9 ethylcarbazole (AEC) as chromogenic substance. The two complementary histochemical techniques alpha-naphthylesterase (ANE) and lactate-dehydrogenase (LDH) were also used intraoperatively for the staining of ganglion cells. The diagnosis was confirmed postoperatively with conventional AChE Karnovsky technique, comparing the extensions of hyperganglionic, hypoganglionic and aganglionic segments in each studied case. The new rapid AChE modified method can identify ganglion cells and fibers using a dark brown precipitate. In all the cases studied, the intestinal innervation pattern identified with this modified technique was similar to that obtained with Karnovsky AchE. Seventy-eight HD, 8 isolated IND and 6 HD associated with an evident IND segment were diagnosed. This new rapid AChE histochemical technique avoids the use of DAB and naphthol, and can thus be considered safe for operators. Rapid AChE is a valid tool for both the evaluation of aganglionosis extension and for the identification of IND pattern during surgery. We recommend this very reliable method for the intraoperative diagnosis of HD and IND, in association with other enzymatic markers of ganglion cells (ANE or LDH). We propose the following diagnostic protocols: a) for preoperative histochemical study: conventional AChE plus LDH and NADPH-diaphorase; b) for intraoperative study: rapid AChE plus ANE.     

Acetylcholinesterase-stained suction rectal biopsies in the diagnosis of Hirschsprung's disease.

Conflicting reports in the literature regarding the sensitivity and specificity of the acetylcholinesterase (AChE) stain in establishing or excluding the diagnosis of Hirschsprung's disease (HD) prompted this review of 497 rectal biopsies performed on 455 children. Using hematoxylin and eosin (H&E) to stain formalin-fixed, paraffin-embedded tissue sections is our preferred method of identifying ganglion cells. In this series, however, there were eight children with HD, and nine without HD in whom the AChE-stained portion of the sample provided invaluable diagnostic information not obtained from the concomitant, formalin-fixed, H&E-stained portion of the sample. The AChE stain also provided at least suggestive evidence of HD in some of the anal or anorectal biopsy specimens.     

The use of rapid assessment of enteric ICC and neuronal morphology may improve patient management in pediatric surgery: a new clinical pathological protocol

Alterations in interstitial cells of Cajal (ICC) distribution and density may seriously influence gut motility. We and others have documented a disturbance of ICC and neurons in Hirschsprung’s disease (HD), intestinal ischemia, and inflammation. The ability to remove intestine with permanent dysmotility improves significantly the prognosis. This may be important in the developing intestine especially in HD. More complete pathological information increases the accuracy of surgical decisions. Therefore we sought to develop a rapid, intraoperative immunohistochemical protocol for ICC and neurons in surgical specimens for routine diagnostic and therapeutic assessment of pediatric patients. To date, cKit is the only reliable immunohistochemistry for ICC identification. There are multiple antibodies in use for neuronal identification. A comparison of fixation methods and immunostaining using cKit and multiple intestinal neuronal antibodies was done. Fresh segments of surgically resected intestine were sectioned and stained using antibodies for ICC cell identification (anti-cKit) and for neuronal characterization. By carefully changing tissue fixation methods, different neuronal antibodies were tested to determine an optimal rapid protocol. Each suggested protocol was tested on normal and pathological intestinal tissue and compared to the previous overnight immunostaining of the same tissue. A new rapid tissue fixation and immunostaining protocol using cKit for ICC identification and NF 68 was developed. By employing this protocol, we could obtain ICC and neuro-immunohistochemistry in unfixed frozen sections within 1 h with tissue vibration to diminish the time for immunostaining. Without vibration the protocol takes 3 h. ICC and enteric neuronal changes could be readily observed and the quality of staining was comparable to standard immunohistochemistry. Each gut pathology displayed characteristic changes in ICC and neuronal density/distribution in the affected bowel. The time-scale of the 1-h immunoprocessing is still longer than the standard clinical pathological “quick” sections using H&E staining; however, the protocol duration is within the surgery timescale. Standard H&E stain used in combination with our rapid neuronal and ICC immunohistochemistry protocol enables a fast, comprehensive, and accurate assessment of the pathophysiology of signaling networks controlling gut motility while the patient is still in the operating room. We propose that the addition of this simple and rapid immunohistochemical assessment in the pathologist evaluation of surgical specimens would result in a more complete characterization for diagnosis and prognosis of the pediatric patient. Specifically, we propose that this test will differentiate good versus poor prognosis HD patients based on their neuron/ICC ratio and present a rapid, standardized method for use in general pathology.     

Study of acetylcholinesterase activity in rectal suction biopsy for diagnosis of intestinal dysganglionoses: 17-year experience of a single center

Although the utility of the acetylcholinesterase (AChE) histochemistry on rectal suction biopsy in diagnosing Hirschsprung's disease (HD) has been documented, few reports address a great number of biopsies and patients. Our aim is to present a 17-year experience on the method of rectal suction biopsy and AChE histochemical staining for diagnosis of intestinal dysganglionoses. Between August 1989 and July 2006, 297 children suspected of having HD were submitted to rectal suction biopsies that were evaluated by the same two surgeons. There were 18 complications (6.0%), namely one self-limited rectal bleeding and 17 (5.7%) inadequate procedures that were repeated. A total of 157 patients (52.8%) showed no increased AChE activity and the remaining patients (140-47.2.0%) presented patterns of increased AChE activity confirming the diagnosis of HD or neuronal intestinal dysplasia. Among the 140 cases suspected as having HD, in 131 children the diagnosis of HD was confirmed and they were operated on. The histological studies showed that 111 children presented the classic form of HD or a long spastic segment. Sixteen children presented total colonic aganglionosis and four children proved to have intestinal neuronal dysplasia, according to histological and radiological criteria. Nine (6.6%) newborns were identified as false-positives and no false-negative results were verified. The rectal suction biopsy combined with AChE staining is advantageous for the differentiation between normal bowel and intestinal dysganglionoses. The rectal suction method is simple and can easily be performed by experienced surgeons. The histological evaluation is very objective and can be performed by a non-pathologist.     

Histochemical diagnosis of Hirschsprung disease

A histochemical staining technique for detection of acetylcholinesterase (AChE) in rectal suction biopsies was compared with the presence or absence of ganglion cells in full-thickness or suction biopsies for the diagnosis of Hirschsprung disease (HD) in infants and children. Biopsies from 55 of 58 children were adequate for both the AChE assay and routine pathologic examination for ganglion cells. Two patterns of AChE staining were noted. With pattern A, prominent nerve fibers staining for AChE were seen throughout the muscularis mucosa and the lamina propria. With pattern B, similar fibers were seen only in the muscularis mucosa and the areas of lamina propria that were immediately adjacent. No "false-negative" AChE staining reactions were found in patients with HD. No "false-positive" reactions showing pattern A were found. This pattern was diagnostic for HD. Three false-positive reactions were found showing pattern B in patients with conditions other than HD. Among 22 patients with HD, 19 were males and three were females. Pattern A occurred in all age groups and in both sexes. Pattern B in patients with HD was seen exclusively in male infants 1 month of age or less. Experience suggests that the AChE staining of rectal suction biopsies is an excellent screening test for HD in infants and children. If pattern B is encountered, however, the specimen should be examined by routine pathologic techniques for the presence of submucosal ganglion cells.     

先天性巨结肠RET蛋白免疫组化研究

目的 了解RET蛋白与先天性巨结肠(HD)组织改变之间的关系,以探讨HD的发病机理.方法 利用抗RET蛋白的三种抗体,以及抗磷酸酪氨酸抗体采用免疫组织化学方法,对42例散发性HD肠组织中RET蛋白表达情况进行研究,10例正常结肠作对照。结果 抗RET蛋白的三种抗体在HD扩张段、移行段、对照组均显强阳性染色,P>O.05;在狭窄段大多数表现阴性染色,少数可见强阳性或弱阳性染色,与前三者比较PO.05;HD移行段与狭窄段大多数呈现阴性或弱阳性染色,P>O.05。前两者与后两者比较P相似文献   

A new rapid acetylcholinesterase staining kit for diagnosing Hirschsprung’s disease

Conventional acetylcholinesterase (AChE) histochemistry is both time consuming and complicated and requires the mixing of reagents that are toxic to the human body. We developed a rapid technique for performing AChE histochemistry, which has already been published, and now present a kit for performing AChE histochemistry that is a further improvement. Rectal suction biopsy specimens taken from 20 constipated patients and three full thickness biopsy specimens taken from 4 Hirschsprung’s disease (HD) patients during pull-through surgery from aganglionic, transitional, and ganglionic bowel segments were tested using our rapid technique and the new kit. Each specimen was incubated for only 6 min. All ganglion cells stained clearly for AchE in just 6 min using both techniques. However, the kit was able to stain AchE positive nerve fibers more clearly and did not detect endogenous peroxidase-containing histiocytes, as did the earlier rapid technique. The kit could also detect AchE positive nerve fibers in the circular and longitudinal muscle layers, unlike the earlier rapid technique. The kit allows AChE histochemistry to be performed rapidly with complete accuracy, without any risk for toxicity. Moreover, the kit provides more focused information on AchE distribution in the bowel itself without any extraneous staining and can be used for diagnosing HD and allied disorders as well as establishing the exact level of innervation for pull-through resection.     

Acetylcholinesterase histochemistry and the diagnosis of Hirschsprung's disease: a 31/2-year experience

Using a modification of the histochemical acetylcholinesterase (ACE) reaction of Karnovsky and Roots, 154 suction rectal biopsies performed during the clinical investigation for Hirschsprung's disease were examined and compared to simultaneously obtained paraffin-embedded, hematoxylin-eosin (H&E)-stained tissue. Serial paraffin sections were adequate to identify normal ganglia or to raise suspicion of Hirschsprung disease at initial biopsy in 129 of 154 (83%) cases. Interpretation of the ACE-stained tissue correlated exactly with this interpretation in 123 instances; 6 cases had uninterpretable ACE frozen sections. From this group of adequate paraffin sections, 18 patients with aganglionic megacolon were identified with the ACE preparation, relying primarily on a pattern of unusually thick and numerous nerve fibers within the muscularis mucosa. Twenty-five paraffin-embedded specimens were deemed uninterpretable because of absent or insufficient submucosa or because of location. The ACE-stained section, however, correctly predicted the presence or absence of ganglia in 23 of these additional cases where the diagnosis could subsequently be confirmed either by repeat biopsy or repeated clinical follow-up examination of the patient. Using the ACE method alone, therefore, a diagnosis was obtainable at initial biopsy in 146 of 154 specimens (95%). This percentage was increased to 99% (152 of 154 specimens) if both ACE and paraffin sections were utilized. Recognition of the normal and abnormal patterns of nerve fiber staining in ACE preparations permits reliable, decisive interpretation of rectal suction biopsies in the evaluation of Hirschsprung's disease.     

Studies of <Emphasis Type="Italic">RET</Emphasis> gene expression and acetylcholinesterase activity in a series of sporadic Hirschsprung’s disease

The purpose is to present the studies of RET gene expression and acetylcholinesterase activity in 23 patients operated for Hirschsprung's disease (HD). The patients underwent either transanal endorectal pull-through or Duhamell's procedure. Full-thickness intestinal samples from the three different segments (ganglionic, intermediate and aganglionic) were collected. Each tissue sample was divided in two portions, one for AChE histochemical staining and the other for examination of RET mRNA expression level. All patients had an uneventful postoperative recovery. In all patients, the AChE stainings demonstrated the absence of activity in the ganglionic area, the marked increase of positive fibers in the aganglionic area, and little increase of positive fibers in the intermediate area. In the ganglionic and intermediate areas, all patients (100%) showed significant RET gene expression. In the aganglionic area, 18 patients (78.3%) did not present gene expression and the other five patients (21.7%) presented gene expression that was similar to the ganglionic and intermediate areas. The results reinforce the conclusion that the method of AChE staining is effective for the diagnosis of intestinal aganglionosis and confirm the knowledge that genes beyond RET may be implicated in the genesis of sporadic cases of HD.     

Whole-mount NADPH-diaphorase histochemistry is a reliable technique for the intraoperative evaluation of extent of aganglionosis

Multiple seromuscular biopsies at three levels (narrow segment, transitional zone, and dilated segment) were taken and investigated intraoperatively to determine the extent of aganglionosis. Using the whole-mount preparation technique, circular muscle fibers were separated from the specimens. After a short prefixation, the muscle fibers were stained by the NADPH-diaphorase technique and were examined within 20–25 min. A fine and dense neuronal meshwork was observed between circular muscle fibers in the normal and ganglionic part of the bowel. In contrast, there was a complete lack of NADPH-diaphorase-positive fibers in the circular muscle of aganglionic colon. In the transitional zone, NADPH-diaphorase-positive fibers were markedly reduced compared to the ganglionic region. The density of these fibers increased and attained normal levels in the proximal bowel above the transition zone. These results suggest that whole-mount NADPH-diaphorase histochemistry is a three-dimensional technique suitable for the intraoperative evaluation of extend of aganglionosis. The technique is sufficiently rapid to be used in conjunction with routine frozen sections to assist in the diagnosis and in selecting the optimal level of resection at the time of pull-through operation.