Composition of plasma in apheresis-derived platelet concentrates under cold storage

Abstract

Background: Compared to room temperature (RT, 22-24°C) storage, refrigeration of platelet concentrates (PC) may provide advantages due to lower risks of bacterial growth and increased responsiveness of platelets. However, storage at cold temperature (CT, 2-6°C) may also strongly influence the plasmatic composition of PC. This study analysed the content of plasma in apheresis-derived platelet concentrates (APC).

Materials and methods: APC were stored under blood bank conditions at CT or RT. On days 0 and 6, samples were drawn for analysis. Coagulation parameters comprised global coagulation assays, single factors or inhibitors. The distribution pattern of von Willebrand multimers was investigated by immunoblotting. Thrombin generation was assessed with a fluorescence assay. Immunological and clinical chemistry parameters were determined on automated analysers.

Results: After storage at CT, coagulation factors V, VII, IX or protein S activity are partially reduced, but less compromised than under RT. There was a large reduction in Factor VIII levels and this was similar at both temperatures. In contrast to RT, von Willebrand Factor (vWF) activity was remarkably decreased at CT, and this was accompanied by the shift from high molecular to low molecular weight multimers. Thrombin generation showed improved preservation at CT. Other plasma proteins like immunoglobulins were stable at both conditions.

Discussion: Refrigeration mediates a bivalent effect on plasmatic coagulation in APC. At CT, the partial reduction of labile coagulation factors is less emphasised. However, CT does not prevent Factor VIII depletion, but induces an additional loss of vWF activity by multimer cleavage. Preserved thrombin generation may indicate a higher hemostatic capacity for cold storage.

Figure 1

von Willebrand Factor (vWF) multimer analysis in apheresis-derived platelet concentrates (APC) stored under room temperature (RT) and cold temperature (CT) Samples of APC from day 0 or day 6, either stored at RT or CT, were separated on an SDS-agarose gel and blotted on a nitrocellulose membrane. von Willebrand multimers were stained with a primary polyclonal rabbit anti-human von Willebrand factor antibody and a secondary alkaline phosphatase-conjugated donkey anti-rabbit IgG antibody. A representative immunoblot is shown (A) with indicated multimer sizes (HMW: high molecular weight; IMW: intermediate molecular weight; LMW: low molecular weight). The densitometric pattern, given in arbitrary units (AU; Rγ: lane position), shows the distribution of different multimer sizes (B). Cold storage induces a relative shift from HMW to LMW multimers, indicated by blue arrows. The shift is also calculated in %, as relative fraction of multimer band intensities (C). Comparison of band intensities for multimer fractions was performed with Kolmogorov-Smirnov test and rank analysis with the two-tailed Mann-Whitney test. n=4; *p<0.05, compared as indicated.

Figure 2

Thrombin generation in apheresis-derived platelet concentrates (APC) induced with low phospholipid concentrations Samples from APC on day 0 or day 6, either stored at RT or CT, were analysed for thrombin generation with the RCL thrombin generation reagent containing low concentrations of phospholipids. The mean trace of thrombin generation is shown for each sample type (A). The box-and-whisker plots illustrate the values for thrombin generation as area under the curve (AU: nmol thrombin×min) (B), for thrombin peak height (nM) (C), and for lag phase time (min) (D). n=9; mean±Standard Deviation; #p<0.1 (as tendency), compared as indicated.

Figure 3

Thrombin generation in apheresis-derived platelet concentrates (APC) induced with high phospholipid concentrations Samples from APC on day 0 or day 6, either stored at room temperature (RT) or cold temperature (CT), were analysed for thrombin generation with the RCH thrombin generation reagent containing high concentrations of phospholipids. The mean trace of thrombin generation is shown for each sample type (A). The box-and-whisker plots illustrate the values for thrombin generation as area under the curve (AU: nmol thrombin x minute) (B), for thrombin peak height (nM) (C), and for lag phase time (minutes) (D). n=9; mean±Standard Deviation; *p<0.05, #p<0.1 (as tendency), compared as indicated.