Evaluation of the functional properties of cryopreserved buffy coat-derived monocytes for monocyte monolayer assay.

Abstract

Monocyte monolayer assay (MMA) is a compatibility testing method for evaluating the clinical significance of red blood cell (RBC) alloantibodies. Time-consuming monocyte isolation procedures and requirement for fresh monocytes have limited application of the MMA. The aim of this study was to develop and assess the utility and efficacy of cryopreserved buffy coat (BC)-derived monocytes for MMA application. Peripheral blood mononuclear cells (PBMNCs) were isolated from BC or peripheral blood (PB) and pooled and BC PBMNCs were cryopreserved. Monocytes from pooled PBMNCs were incubated with anti-D-sensitized, anti-Scianna2 (Sc2)-sensitized, anti-AnWj-sensitized, or anti-Jra -sensitized RBCs or lipopolysaccharide (LPS). MMA phagocytic index (PI) and membrane integrity were determined microscopically, and cytokine release was measured by Luminex technology. PBMNC isolation rates from fresh BC and PB were not comparable (67.4±6.3 and 75.8±7.7% respectively, p=0.024). There was no significant difference in PBMNC membrane integrity (fresh PB, 100%; fresh BC, 100%; cryopreserved BC, 95.2±1.2%), postwash recovery (fresh PB, 85.9±3.1; fresh BC, 86.9±6.7; cryopreserved BC, 84.8±5.1), or monocyte PI (fresh PB, 82±10; fresh BC, 77±11; cryopreserved BC=80±6). Monocytes from pooled cryopreserved BC PBMNCs reacted with RBCs sensitized with anti-D and RBC alloantibodies, including anti-Sc2, anti-Jra , and anti-AnWj. Monocytes from pooled cryopreserved BC PBMNCs can be used reliably to evaluate phagocytic responses of sensitized RBCs and to assess clinical significance of RBC alloantibodies.