Improvised AChE assay on homogenized rectal biopsy: An Alibi to AChE histochemistry in the diagnosis of Hirschsprung's disease?

The surgical treatment of Hirschsprungs disease (HD) is reliant on the accurate tissue diagnosis based on increased acetylcholine esterase enzyme (AChE) activity in frozen rectal biopsy sections demonstrated by histochemical staining techniques. The latter techniques used for ascertaining the presence of AChE mandates the use of inaccessible sophisticated instruments thereby limiting the performance of this technique to urban resource rich settings. This bottleneck translates into delayed treatment for patients who live in suburban, resource limited regions. Given the indispensable nature of the surgical treatment, scope for improving accessibility is restricted to the diagnostic modality of the disease. In our study, we attempted to quantitatively estimate AChE in homogenized rectal mucosal biopsy samples of paediatric population (n=68) using an in-house modified AChE (mACHE) assay. The assay procedure for homogenization of the tissue was modified to selectively extract the mucosal portion of the rectal mucosal biopsy sample to be used for the assay. The efficiency of specific mucosal extraction was confirmed by the absence of neurotrophic AChE nerve fibres in remnant tissue obtained post homogenization by histochemical AChE staining. The mAChE assay was carried out in a blinded manner and the results were compared with the gold standard histochemical AChE (hAChE) assay. Samples with activity lesser than 1 x 10-6 mole/min/g wet weight of tissue were diagnosed as Non Hirschsprungs Disease (NHD) and those above 1 x 10-6 mole/min/g wet weight of tissue were diagnosed as HD. mAChE assay was found to have a specificity, sensitivity of 81.08 % and 92.85%; positive, negative predictive values of 78.78% and 93.75 % respectively. The mAChE assay was able to diagnose three cases which were deemed inconclusive as per hAChE. The improvised in-house mAChE assay had a mutually exclusive range to differentiate between control and test, thus crediting its use as a diagnostic tool for Hirschsprungs Disease.