Comparison of established and novel methods for the detection and enumeration of microparticles in canine stored erythrocyte concentrates for transfusion.

McEntire, Meredeth C; Wardrop, K Jane; Davis, William C

BACKGROUND Microparticles (MP) are submicron, phosphatidylserine (PS)-bearing lipid vesicles with physiologic and pathologic roles in coagulation and inflammation. Microparticles accumulate in packed RBC (pRBC) stored for transfusion, potentially increasing recipient morbidity. Historically, canine MP have been detected with the PS label annexin V in supernatant samples. Other detection methods are available but have not been evaluated in dogs. OBJECTIVES The purpose of the study was to detect and enumerate MP in canine pRBC using annexin V, lactadherin, and bio-maleimide to compare label performance and assess microparticle accumulation under standard storage conditions. METHODS Microparticles (0.5-1.0 μm) in canine dog erythrocyte antigen 1.1 positive, nonleukoreduced pRBC were labeled with FITC-annexin V, FITC-lactadherin, and the fluorescent dye bio-maleimide, and were counted using flow cytometry at 3 time points (days 7, 21, and 35) of storage. Unprocessed pRBC, rather than supernatant, were used. RESULTS Annexin V and bio-maleimide labeling produced comparable microparticle counts (P = .16), while lactadherin labeling resulted in higher microparticle counts than annexin V (P = .002) and bio-maleimide (P = .006), particularly on day 7. Bio-maleimide- and annexin V-based microparticle counts increased significantly from day 7 to 35 (P = .04), and increases from day 21 to 35 approached statistical significance (P = .05). CONCLUSIONS Bio-maleimide- and annexin V-mediated microparticle counts were comparable in unprocessed canine pRBC using flow cytometry. Whether the increased microparticle counts with lactadherin were due to increased sensitivity for small, PS-bearing MP or due to labeling of membrane fragments and debris requires further investigation.