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

Abstract

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.