Single Muscle Fiber Calcium Wave Frequency Assay for Malignant Hyperthermia Diagnosis: An Exploratory Validation Study.

Abstract

The current accepted standard for diagnosing malignant hyperthermia (MH) is an in vitro contracture test (IVCT), which has many limitations. Here the authors evaluate a newly developed test, the calcium wave frequency assay (CaWFa), which aimed to directly measure calcium release from the ryanodine receptor 1 (RyR1) of single muscle fibers. A small segment of muscle (50 mg) was sectioned from 30 patients undergoing routine IVCT muscle biopsies, and mechanically skinned single muscle fibers were isolated. Using calcium-dependent fluorescence and confocal microscopy, the authors were able to examine RyR1 sensitivity of single fibers challenged with graded concentrations of halothane and caffeine. The induction of regenerative calcium waves and wave frequencies were compared with IVCT results to assess diagnostic sensitivity and specificity. The proportion of muscle fibers that responded with regenerative calcium waves on exposure to 0.5 mM and 1 mM halothane was higher in muscle from MH susceptible (MHS) patients compared with MH normal (MHN) patients (36.5% vs. 0% and 77.5% vs. 23.1%, respectively). Calcium wave frequency was also elevated in the MHS fibers compared to MHN in halothane at all tested concentrations. No difference in calcium wave onset or frequency was demonstrated between groups exposed to caffeine. Using CaWFa, an onset concentration of 1 mM halothane in combination with a wave frequency threshold of 1.57 waves/min achieved 92% sensitivity and 88% specificity. The CaWFa effectively discriminates MHS from MHN muscle in response to halothane, offering a comparable sensitivity and specificity to the IVCT. The CaWFa shows promising potential as a minimally invasive alternative to IVCT for diagnosing MH susceptibility.