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 (HID) and intestinal neuronal dysplasia (IND) wa s first introduced in 1964 by Morris Karnovsky and Logan Roots. This techni que requires about 80-120 minutes incubation time and cannot be used for th e intraoperative diagnosis of HD and IND. To avoid these limitations, in 19 94 Kobayashi et al first proposed an accelerated modified method in two dif ferent versions, the first using diaminobenzydine (DAB) reagent, the second using 4-chloro-1-naphthol as final reagent. In the present study, we propo se a new rapid variation of AChE staining which avoids the use of DAB and n aphthol, notably toxic reagents, but follows the same acceleration principl e of Kobayashi's technique. our modified rapid AChE requires a total incuba tion time of only 8 minutes, which is compatible with intraoperative histoc hemical examination purposes. Intraoperative seromuscular or full-thickness intestinal biopsies were obta ined from 92 children affected by intestinal dysganglionoses. The biopsies were frozen and cut in 15 mum cryostatic sections. Rapid AChE was performed with a special incubation medium using 3-amino-9 ethylcarbazole (AEC) as c hromogenic substance. The two complementary histochemical techniques alpha- naphthylesterase (ANE) and lactate-dehydrogenase (LDH) were also used intra operatively 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 e ach studied case. The new rapid AChE modified method can identify ganglion cells and fibers u sing a dark brown precipitate. In all the cases studied, the intestinal inn ervation pattern identified with this modified technique was similar to tha t obtained with Karnovsky AchE. Seventy-eight HID, 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 napht hol. and can thus be considered safe for operators. Rapid AChE is a valid t ool for both the evaluation of aganglionosis extension and for the identifi cation of IND pattern during surgery. We recommend this very reliable metho d 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: conventiona l AChE plus LDH and NADPH-diaphorase; b) for intraoperative study: rapid AC hE plus ANE.