Malignant hyperthermia (MH) is a rare, life-threatening pharmacogenetic disorder triggered by volatile anesthetics or certain neuromuscular blocking agents. It causes uncontrolled skeletal muscle hypermetabolism due to excessive calcium release through the ryanodine receptor type 1 (RyR1), resulting in clinical features such as hyperthermia, muscle rigidity, and tachycardia. The gold standard to diagnose MH susceptibility (MHS) is the in-vitro contracture test (IVCT), which needs invasive muscle biopsy and has limitations in availability and specificity. Genetic testing offers a non-invasive alternative, but it does not detect all pathogenic variants. A recent pilot study introduces the calcium wave frequency assay (CaWFa), which measures RyR1-mediated calcium release in single muscle fibers and may represent a potential alternative to IVCT.
The published study in Anesthesiology aimed to determine the sensitivity, specificity, and feasibility of CaWFa in distinguishing MHS from MH-normal (MHN) individuals and to compare its diagnostic performance with the IVCT. The assay was designed to improve upon previous functional tests by directly quantifying intracellular calcium dynamics under physiologic conditions instead of relying on indirect measures like muscle contractile force.
Ethical approval was obtained from relevant institutional committees in Australia, and patients scheduled for IVCT from February 2020 to July 2023 were recruited. Muscle biopsies from the vastus lateralis were collected. A small portion of each biopsy, comparable to a needle biopsy sample, was transported to the laboratory for CaWFa testing. Of the 43 transported samples, 13 were excluded because of protocol refinement, which leaves 30 samples (13 MHS and 17 MHN) for analysis. A total of 181 single muscle fibers were isolated using a mechanically skinned fiber technique, which preserves calcium transport mechanisms and RyR1-dihydropyridine receptor coupling.
Fibers were equilibrated in a physiologic solution containing controlled concentrations of calcium, magnesium, ATP, and other intracellular components. The calcium-sensitive dye rhod-2-enabled real-time fluorescence imaging by confocal microscopy. Fibers were exposed stepwise to increasing concentrations of halothane (0.5 to 3 mM) or caffeine (1 to 4 mM), and regenerative calcium waves were recorded. The primary outcome was the proportion of fibers demonstrating calcium waves, and secondary outcomes included wave frequency, onset concentration, and the lowest halothane concentration that induced a response. Statistical analysis was conducted using GraphPad Prism.
Results showed clear differentiation between MHS and MHN individuals in response to halothane but not caffeine. At 0.5 mM halothane, 36.5% of MHS fibers exhibited calcium waves as compared to 0% of MHN fibers. At 1 mM, 77.5% of MHS fibers responded vs 23.1% of MHN fibers. Higher concentrations (2 to 3 mM) produced plateau responses in both groups. Wave frequency was consistently higher in MHS fibers; at 1 mM halothane, mean wave frequency was 1.91 ± 0.49 waves/min in MHS as compared with 1.25 ± 0.53 waves/min in MHN.
A positive correlation between CaWFa wave frequency and IVCT force was observed with Spearman coefficients of r = 0.74 at 0.5 mM, r = 0.59 at 2 mM, and r = 0.49 at 3 mM. ROC analysis revealed that wave frequency at 1 mM halothane had the strongest discriminatory capacity (AUC = 0.921). Onset concentration analysis showed that MHS individuals responded at significantly lower halothane concentrations (mean 0.69 ± 0.25 mM) as compared with MHN individuals (1.59 ± 0.51 mM).
A two-step diagnostic model was developed: individuals responding at 0.5 mM were classified as MHS, those first responding at 2 mM as MHN, and those responding at 1 mM were further classified by using a wave frequency threshold of 1.57 waves/min. This combined approach yielded 92% sensitivity and 88% specificity, correctly diagnosing 27 of 30 individuals, with 2 false positives and 1 false negative.
Overall, CaWFa showed strong diagnostic performance compared with IVCT while offering significant practical advantages, which include reduced invasiveness and compatibility with smaller muscle samples suitable for needle biopsy. The assay overcomes limitations of prior functional tests that relied on contractile force measurements by directly measuring RyR1-mediated calcium release under physiological conditions. Although caffeine did not provide discriminatory value in this cohort, halothane-based parameters showed robust sensitivity and specificity. Blinded validation studies are needed to refine diagnostic thresholds and confirm reproducibility. This pilot study provides compelling evidence that CaWFa represents a promising, less invasive alternative to IVCT to diagnose MH susceptibility.
Reference: Singh DP, Perry MJ, Bjorksten AR, et al. Single muscle fiber calcium wave frequency assay for malignant hyperthermia diagnosis: an exploratory validation study. Anesthesiology. 2026;144(3):570-581. doi:10.1097/ALN.0000000000005882
