Preservation of acetylcholinesterase enzyme activity in non-frozen rectal biopsy specimens for Hirschsprung disease

Abstract

The acetylcholinesterase (AChE) stain has been a commonly accepted adjunctive study for rectal biopsy specimens in patients being evaluated for Hirschsprung disease (HD). However, the requirement for immediately frozen rectal biopsy specimens (to preserve the enzyme activity) has proved to be an obstacle to its use in some centers. In this study, we developed a microwave procedure for the AChE stain, which we then used to evaluate a special tissue transport medium, Michel’s medium, for its utility in preserving AChE enzyme activity without immediate freezing in fresh rectal specimens collected from known HD patients. When compared with similar rectal biopsy specimens that were immediately snap-frozen, the specimens stored in Michel’s medium at room temperature or with refrigeration (for up to 108 hours) followed by sucrose cryoprotection showed preservation of AChE enzyme activity, with comparable nerve fiber staining and no significant background staining. This technique provides a convenient alternative method to immediate snap-freezing for the demonstration of AChE enzyme activity and allows the courier transport or mailing of specimens without freezing to laboratories where this special stain is performed routinely.

Keywords:

• Acetylcholinesterase
• Hirschsprung disease
• Michel’s medium
• Microwave
• N-ethylmaleimide

The authors express their deep appreciation to Walter Scott Cain, MD, pediatric surgeon at the Children's Hospital of Alabama, who operated on many of these patients.