A-135 Characterization of refrigerator-stable erythrocyte and platelet product with utility for clotting assay external control and experimental applications

Abstract

Background Shelf-stable material to accurately replicate whole blood clotting for use as proficiency/external control, validation samples, and drug/biomarker discovery tools is not widely available. During clot formation, activated platelets change shape and aggregate, and erythrocytes (RBCs) are deformed by contractile forces; therefore, functional RBCs and platelets responsive to clotting pathway signaling are both fundamental to a representative clotting control. Standard storage methods preserve RBCs for up to 42 days and platelets up to 7 days, with functional deterioration during that period. Cryopreservation preserves clotting function for longer periods, but lot-to-lot performance is not well-characterized and cryopreserved material presents challenges for use as point-of-care external control. Lyophilization causes extensive membrane damage. Other membrane stabilization methods successfully prevent hemolysis but interfere with clotting. Here, we show that increasing RBC resistance to shear in conjunction with preserving RBC deformability and platelet responsiveness generates a refrigerator-stable product with acceptable clotting time precision and stability for 75 days post-draw, suitable as a proxy whole blood clotting sample. Methods Two lots of RBC/platelet material produced under ISO13485 were stored at 2–8°C during characterization, with periodic equilibration to room temperature, mimicking typical assay external control handling. The ClotChip System (IUO), intended for point-of-care use and utilizing dielectric spectroscopy to determine whole blood clotting time, was employed to assess clotting function. Both lots underwent 20-day precision assessment of clotting performance in the presence of normal plasma or clotting-factor deficient plasma (CLSI EP05-03). Material was also assessed for long-term and in-use stability assessments of clotting function (CLSI EP25). RBC deformability was assessed weekly via ektacytometry (LorrcaMaxSis) under normoxia and hypoxia. Complete blood counts (CBCs) were periodically measured (Abacus3CP). Responsiveness of RBCs and platelets to clotting factors was evaluated on ClotChip after combination with plasmas whose clotting factor levels varied. Results Clotting time repeatability was 3.3%-13.8% for both lots and total imprecision was 4.6%-15.0%. Mean clotting time for material combined with normal plasma remained within 20% of initial values for up to 75 days after blood draw; in-use stability at room temperature remained within 10% of initial values for the full period tested (5.5 hrs). RBC deformability (EImax) under normoxia or hypoxia fell within the fresh whole blood reference range and exhibited no significant change over 75 days (p>0.05) (see Figure). Half-maximal shear stress required to elongate cells was ∼30% elevated vs fresh blood at the start of storage, increased over time, and plateaued at 3-4x fresh blood levels. CBCs at baseline and 10 weeks indicated minimal changes in RBC or platelet counts. Conclusion These data characterize refrigerator-stable blood-derived material exhibiting consistent clotting activity for up to 75 days post draw. Our results indicate clotting function can be preserved for months by conditions that stiffen the RBC membrane (hypothesized to enhance cell viability and resilience) but maintain RBC deformability and platelet activation. These findings suggest a novel approach to creating stable whole blood clotting control materials. The ability of material thus prepared to respond dose-dependently to plasma factor levels demonstrates utility in experimental studies of patient-specific clotting factors.