Effects of Liquid Storage and Cryopreservation on Platelet Surface Glycoproteins, Light Scatter, and Procoagulant Activity

This study was designed to examine the effects of standard 22°C platelet storage and platelet cryopreservation ;pn surface glycoproteins, light scattering properties and surface-bound factor V using flow cytometry. Single donor platelet units were collected in ACD (formula A) using a^Haemonetics Mobile Collection System. The units were either liquid-preserved at 22°C with rotation for up to 7 days or were cryopreserved using 6% DMSO as the cryoprotectant at -80°C. Storage at 22°C for 7 days resulted in an increased platelet-derived microparticle (PDMP) content which actively bound coagulation factor V in the absence of added agonist. Addition of 2 U/mL thrombin or a combination of 20 uM ADP and 20 uM epinephrine in the presence of 3 mM calcium resulted in an increased production of PDMP On days 5 and 7 compared to the day of collection. Plasma factor V activity of both the platelet unit and autologous ACD plasma decreased with 22°C storage, although the decrease was greater in the platelet product. Platelet surface P-selectin increased progressively during the 7 day 22°C storage period with a parallel decrease in responsiveness to the in vitro addition of 2 U/mL thrombin or 0.5 uM U46619 (a thromboxane A2 analog). Platelet surface glyco-protein (GP) lb decreased during 22°C storage, but GPIX, which is complexed with GPIb, did not change. Platelet surface GPIV increased slightly and the GPIa-lla complex decreased slightly with 22°C storage. While constitutive expression of the platelet surface GPIIb-llla complex (measured by antibody 7E3) doubled over 7 days of 22°C storage, the exposure of the functional fibrinogen binding site on GPIIb-llla with the addition of agonist (measured by antibody PAC1) decreased dramatically during this same time period. This decreased reactivity to agonist was largely the result of an emerging sub-population of unresponsive platelets which could be readily observed by the bimodal distribution of the flow cytometry histograms of PAC1 specific fluorescence. Platelet cryopreservation resulted in the formation of PDMP post-thaw similar to the 7 day 22°C storage. A portion of these PDMP were lost after washing the thawed cryopreserved platelet unit, a step required to remove DMSO from the transfusion product. In contrast, the freeze-thaw process did not stimulate the formation of platelet .microaggregates. Platelet surface P-selectin decreased slightly during the initial 22°C 24 hr testing period prior to freezing and increased significantly post-thaw. The platelet responsiveness to thrombin as measured by surface P-selectin decreased after the 'cryopreservation procedure. Cryopreservation also resulted in a decreased surface expression of GPIb, which was at least partially due to the development of a subpopulation of platelets with reduced surface GPIb. This subpopulation could be separately analyzed from the GPIb normal platelets, and, using 3 color flow cytometry, was shown to possess relatively higher amounts of surface bound factor V after the platelets were thawed. The GPIb reduced subpopulation was also less responsive to thrombin, as ifeported by surface P-selectin, than the GPIb normal platelets in the same sample. In conclusion, flow cytometry based assays coupling light-scattering properties and multiple immunologic probes directed at functional sites on the platelet surface is a promising technology for the determination of pre-transfusion plateletpheresis product quality.