A flow cytometric assay for HLA‐DR expression on monocytes validated as a biomarker for enrollment in sepsis clinical trials

PURPOSE: Decreased expression of HLA-DR on monocytes (mHLA-DR) is a reliable indicator of immunosuppression in patients with sepsis and is correlated with increased risk of secondary infection and mortality. A flow cytometry-based laboratory developed test for the measurement of mHLA-DR in whole blood was validated for clinical trial enrollment, which is considered medical decision-making, for patients with severe sepsis or septic shock. METHODS: The BD Quantibrite™ anti-HLA-DR/anti-monocyte reagent measures antibodies bound per cell of HLA-DR on CD14+ monocytes. The mHLA-DR assay was planned to support inclusion/exclusion of patients for a clinical trial and was validated according to New York State Department of Health (NYSDOH) requirements for a new non-malignant leukocyte immunophenotyping assay. RESULTS: Normal, healthy donor and sepsis patient samples were stable up to 72 h post-collection in Cyto-Chex BCT phlebotomy tubes. Pre-determined acceptance criteria were met for precision parameters (average %CV ≤ 20%) and global laboratory-to-laboratory comparisons (average %Δ ≤ 20%). The approaches taken to evaluate and report accuracy, analytical specificity and sensitivity, reportable range, reference interval, and the proposed multi-level quality control were accepted by NYSDOH. CONCLUSIONS: In this study, the validation strategy necessary when the intended use of assay results changes from exploratory to medical decision making (patient enrollment), which successfully resulted in regulatory approval, is described.