Split-Sample Quality Assurance for the (1,3)-β-D-Glucan Assay

Background: (1,3)--D-Glucan (BDG) is an adjunctive biomarker for invasive fungal infections, but its analytical variability and lack of external quality assessment (EQA) programs limit assay reliability.This study evaluated BDG stability under practical storage conditions and explored the feasibility of a split-sample procedure as an alternative quality-assurance approach.Methods: The reliability of BDG assay results was assessed through three experiments addressing pre-analytical handling, storage stability, and interlaboratory comparability.To examine the effect of freeze-thaw stress, samples with low, moderate, and high BDG concentrations were repeatedly frozen and thawed.Short-term stability was then evaluated using pooled samples at near-cutoff, weak-positive, and strong-positive levels stored at different temperatures for up to 72 hours, with triplicate testing at predefined intervals.In addition, seven serum samples were aliquoted and analyzed in parallel at two institutions to assess interlaboratory agreement.Results: Repeated freeze-thaw cycles resulted in a marked reduction in BDG concentrations, whereas values remained largely stable for up to 72 hours under both refrigerated and frozen storage.Variability was the greatest near the assay cutoff.Interlaboratory comparison showed acceptable agreement in six of seven samples, with one discrepant result likely attributable to specimen-specific or handling-related factors.Conclusions: BDG assays remain stable under short-term refrigerated or frozen storage but are susceptible to repeated freeze-thaw cycles and variability near the cutoff range.The split-sample procedure offers a practical interim approach when EQA is unavailable and may serve as a foundation for standardized quality-assurance frameworks for BDG testing.