High Counts of Potentially Pathogenic Cell Membrane Microparticles in Apheresis Platelets.
Authors: Gelderman Monique P. , Carter Laura B. , Simak Jan 
Published: October 2019

Abstract
Moderate to severe reactions to platelet transfusion have been documented in more than 20% of recipients, and a significant portion of the adverse effects is likely not antibody mediated. Cell membrane microparticles (MP), 0.2–1.0 μm in size, are released into the circulation from stimulated platelets, blood or endothelial cells, and the elevated MP in blood are associated with various vascular pathologies. Moreover, MP potential for different pathogenic activities has been documented. We analyzed MP in apheresis platelet units (APU) (n=26) sent to our laboratory from blood centers nationwide. The analysis was performed on day 6 of the platelet storage period. The median platelet count and pH in APU were 1386x103/μL (range: 697x103–1612x103), and 7.38 (6.82–7.51), respectively. A three-color flow cytometry assay (Simak et al, British J Haematol125, 804–813, 2004) was used for analyzing MP in the non-frozen platelet supernatants. Plasma from healthy volunteers (HVP) (n=12) was used for comparison. To identify MP of platelet, white blood cell (WBC), red blood cell (RBC), and endothelial cell (EC) origin, the antibodies to CD41, CD45, CD235a, and CD105 or CD144, respectively, were used. We also analyzed the expression of CD54 (ICAM 1), a likely marker of proinflammatory status. To study procoagulant phenotypes of MP, phosphatidylserine-positive MP were assayed using the annexin V binding (AVB), and the tissue factor (CD142) was detected with the VIC7 Mab clone. As expected, we found a nine-fold higher platelet CD41+MP in APU (median: 16830/μL; range: 5860–65210) vs. HVP (1910/μL; 1020–2200; p<0.001). Surprisingly, the RBC CD235a+MP were two-fold increased in APU (14650/μL; 10390–21410) vs. HVP (7350/μL; 6160–8960; p<0.001), whereas WBC CD45+MP were about five times higher in APU (4310/μL; 2620–10960) vs. HVP (880/μL; 670–1330; p=0.018). In addition, EC MP (CD105+CD45-MP) were about three times higher in APU (2790/μL; 840–9870) vs. HVP (930/μL; 710–1930; p=0.002). Moreover, the tissue factor specific Mab VIC7 detected in APU about a three times higher count of CD142+MP (4670/μL; 1830–11150) than in HVP (1580/μL; 1070–2510; p<0.001). Counts of CD142+AVB+MP were 2080/μL (730–8040) in APU, and 1330/μL (720–1700; p=0.042) in HVP. Unexpectedly, only 20% of the CD142+MP were CD142+CD45+MP in both, APU and HVP samples, while over 50% were CD142+CD41+MP, and about 20% were CD142+CD144+MP. Although counts of CD54+MP in the APU group (1760/μL; 700–4760) were not significantly different from the HVP group (1350/μL; 970–1560), several APU samples exceeded nearly four times the HVP median. In a functional assay, the overnight incubation of washed APU MP with cultured EC (HUVEC) induced a significant increase in EC apoptosis (TUNEL), and an increase in the cell expression of CD54 and CD142, as compared to control. In conclusion, we found in APU significantly elevated counts of MP derived from platelets, WBC, RBC, and EC, and also MP of procoagulant phenotypes, when compared to HVP. Moreover, APU MP exhibited potentially pathogenic activities on cultured EC. Our results warrant further studies to investigate if the outlying high counts of MP populations with procoagulant or proinflammatory phenotypes, observed in some APU samples, could be related to the clinical adverse effects of APU transfusions. In addition, the potential impact of collection, processing, and storage steps on MP generation in APU should be investigated. The views of the authors represent a scientific opinion and should not be construed as FDA policy.