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.