A Characterization of Microparticles in Stored Packed Red Blood Cell Units.

Microparticles are cell membrane derived vesicles that are released from apoptotic and activated cells. Recent studies have identified microparticles as the main source of circulating functionally active tissue factor, and increased numbers of tissue factor positive plasma microparticles have been associated with hypercoagulable states. Although the subject is controversial, some authors have suggested that red blood cell transfusions may be associated with increased risk of myocardial infarction in patients with acute coronary syndromes. This association could be due to prothrombotic elements in packed red blood cell units. We decided to characterize the microparticle content of packed red blood cell units, examining the microparticles for expression of annexin V, tissue factor, glycophorin-A, and CD41. We chose units near their expiration date because we hypothesized that the generation of microparticles would be time dependent and that units at this time would have the maximum number of microparticles. We isolated microparticles from 7 packed red blood cell units and from 4 samples of normal control plasma, and then used flow cytometry to count the number of annexin V positive microparticles with forward scatter characteristics that were similiar to 1 um beads. Next we used flow cytometry to examine these microparticles for expression of tissue factor, CD41, and Glycophorin A. We found that packed red blood cell units contain annexin V positive microparticles that segregate into two subsets- one group with a high level of annexin V expression and one group with a low level of annexin V expression. These two subsets appear to be distinct, with the high annexin V group expressing significantly more glycophorin-A (81.8% vs 8.9%, p<0.01) and more CD41 (71.4% vs 2.0%, p<0.01) while the low annexin V group showed more tissue factor reactivity (20.3% vs 4.8%, p<0.01). The significance of the tissue factor reactivity is not clear, and we plan to confirm the presence of active tissue factor in future samples with assays for tissue factor activity. We found that microparticles from normal plasma also appear to segregate into high annexin V and low annexin V subsets, although the percentage of microparticles with high annexin V expression was significantly lower than that found in packed red blood cell units (17.8% vs 26.0%, p<0.01). Relative to normal control plasma, packed red blood cells units appear to contain greater numbers of microparticles (3.05E+07/mL vs 4.64E+5/mL, p=0.07), greater numbers of annexin V positive microparticles (1.80E+7/mL vs 2.83E+5/mL, p=0.06), greater numbers of low annexin V microparticles (9.11E+6/mL vs 2.55E+05/mL, p=0.06), greater numbers of high annexin V microparticles (8.77E+6/mL vs 2.83E+4/mL, p=0.06), and a higher level of microparticles with tissue factor reactivity (2.06E+6/mL vs 9.66E+3/mL, p=0.06). We plan to run additional samples to see if these differences will reach statistical significance. We also examined one leukoreduced packed red blood cell unit and found it to have lower levels of microparticles, lower levels of high annexin V microparticles, and lower levels of tissue factor expression as compared to non-leukoreduced units. We plan to run additional leukoreduced units to see if these differences are significant. Overall our study has partially characterized the microparticle content of packed red blood cell units, which to our knowledge has not been reported in the literature.