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

Abstract

PurposeDecreased 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.MethodsThe 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.ResultsNormal, 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.ConclusionsIn 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.

Bicentric evaluation of stabilizing sampling tubes for assessment of monocyte HLA-DR expression in clinical samples

“…Results, obtained with standardized staining protocol (cited above) and Cyto-Chex® BCT tubes demonstrated (i) the stability of mHLA-DR values at room temperature (until 72 h after sampling) and (ii) the adequacy of this approach (e.g., precision, accuracy, lab-to-lab comparison) to fulfill requirements for regulatory approval (5). Nevertheless, the strict comparison of mHLA-DR values between blood sampled in EDTA versus Cyto-Chex® BCT tubes was not performed in this validation study.…”

“…Cell Preparation and Flow Cytometric Analysis mHLA-DR determination was performed on both types of tube using standardized protocol as already described (5,10,11). Briefly, whole blood (25 µL) was stained with 10 µL of QuantiBrite HLA-DR/Monocyte mixture (QuantiBrite anti-HLA-DR PE (clone L243)/Anti-monocytes (CD14) PerCP-Cy5.5 (clone MUP9), Becton Dickinson San Jose, CA, USA) at room temperature for 30 min in a dark chamber.…”

“…This paved the way for tailored immunostimulation for patients presenting with the most altered or persistent immune functions (3). To date, the diminished expression of the human leucocyte antigen-DR on circulating monocytes (mHLA-DR) is consensually accepted as a reliable marker of immunosuppression in sepsis and severe injuries (4,5). It has been used to guide therapy in randomized clinical trials or complicated clinical cases (6)(7)(8)(9).…”

“…In this intended use, a strict reproducibility of values is essential to meet regulatory requirements. To this end, an extensive characterization of assay performance has been conducted according to the New York State Department of Health (NYSDOH) requirements (5).…”

“…In order to complete the work of Quadrini and colleagues (5), the objective of the present work was to compare mHLA-DR values from blood simultaneously collected in EDTA and Cyto-Chex® BCT tubes and to confirm over time stability of mHLA-DR expression with Cyto-Chex® BCT tubes.…”