Activation during preparation of therapeutic platelets affects deterioration during storage: a comparative flow cytometric study of different production methods

Abstract

Summary. Three different separation methods, all using centrifugation, are routinely used to prepare therapeutic platelet concentrates from human donor blood. Platelet concentrates derived from platelet-rich plasma (PRP-PC), buffy coat (BC-PC) and apheresis (AP-PC) were investigated at the end of production, and over an 8 d storage period. Change in platelet surface markers were measured by flow cytometry, using fluorescein-conjugated antibodies to fibrinogen, P-selectin (CD62P), GPIIb-IIIa (CD41), GPIba (CD42b) and GPV (CD42d), and fluorescein-conjugated Annexin V was used to measure expression of anionic phospholipid.All concentrates showed some changes during preparation but PRP-PC underwent the greatest changes with significantly higher levels of P-selectin (P < 0 . 001) and bound Annexin V (P ¼ 0 . 001) than AP-PC or BC-PC, and lower levels of GPIba (P ¼ 0 . 002) and GPV (P < 0 . 001). These changes were attributable to component separation rather than venesection. These markers all continued to change on storage with a strong positive correlation between the changes seen during production and those after 5 d storage. PRP-PC continued to show the greatest changes whereas BC-PC showed the least. Fibrinogen was bound to 40-50% of platelets in all preparations and this did not alter significantly on storage whereas total expression of GPIIb-IIIa remained unchanged throughout.There was no evidence that the platelet surface changes were thrombin-mediated and leucocyte depletion of BP-PC by filtration had no effect on the changes. It is proposed that the deterioration of platelet concentrates during storage may be related to activation occurring during preparation. 'Whole blood' flow cytometry using a panel of fluoresceinlabelled reagents provides an informative method for evaluating platelet concentrates.

BCL2/BCL-XL inhibition induces apoptosis, disrupts cellular calcium homeostasis, and prevents platelet activation

“…Cytoplasmic condensation and budding, to yield microparticles, have been associated with apoptosis in aging platelets in stored concentrates, 18 but these platelets have also undergone a degree of activation during their preparation, which increased during storage in parallel with their decline into an apoptotic-like state. 38 However, morphologic changes similar to those observed in our study were also reported during apoptosis in platelets aged in vitro, which was also accompanied by a reduced aggregatory response. 39 Exposure of patients to ABT-263 results in thrombocytopenia during the first 4 weeks of treatment.…”

Platelet adhesion changes during storage studied with a novel method using flow cytometry and protein-coated beads

“…The GPIIb/IIIa receptor is responsible for platelet binding to fibrinogen and aggregation. Several studies have shown a well preserved GPIIb/IIIa expression during storage [6,21,22] but GPIIb/IIIa have also been reported to increase [23] and the receptor can exist in an activated or non-activated form, thus further complicating interpretations. Analysis of the active conformation of GPIIb/IIIa was not assessed in our study, which would have been of interest since there are studies reporting that the GPIIb/IIIa receptor undergoes changes with storage [2,24], which might be one explanation to the increased platelet binding to fibrinogen beads observed in our study.…”

Methods for evaluation of platelet function

“…Surface expression of GPIIb/IIIa, Pselectin and GPIb can be measured by flow cytometry. Increased P-selectin expression during storage has been reported by several authors [15][16][17] whereas GPIb has been shown to decrease during storage [15,17]. It is however unclear whether the level of in vitro platelet activation in stored PCs correlates with in vivo survival and haemostatic function of platelets after transfusion [18].…”