Characterization of factors affecting the stability of frozen heparinized plasma

The use of heparin rather than citrate as primary anticoagulant has been shown to significantly improve the initial activity, stability and recovery of factor VIII:C from human plasma, cryoprecipitates or factor VIII concentrates if the plasma was initially frozen at -80 degrees C and subsequently stored at this temperature. If frozen and stored at progressively warmer temperatures however, increasing amounts of insoluble protein aggregates, termed storage precipitates (SPs), were recovered in the thawed plasma and cryoprecipitate fractions. Plasma recovery by centrifugation at 7,000 g for 7 min [Method I (MI)], 2 x 10 min (MII) or 15 min (MIII) had little effect on SP formation after 1 month at any storage temperature. After 4 months at -20 degrees C, more SP was recovered from MIII plasma whereas at -40 degrees C, more SP was recovered from MI plasma. Also, the preparation method had little or no effect on factor VIII:C activity at equivalent storage times or temperatures. A trend towards improved factor VIII recoveries was noted at lower freezing and storage temperatures however. SP formation was associated with reduced fibrinogen levels in the recovered plasma without loss of antithrombin-III or increased fibrinopeptide-A. Western blots showed polymerization of A alpha or gamma-chains of fibrinogen. SP formation was reduced or eliminated with factor XIII inhibitors, antibody to the active factor XIII a subunit or adjustment of heparinized plasma to 5-10 mM sodium citrate before initial freezing and storage. Although plasma factor VIII:C recoveries were only slightly affected at these citrate concentrations under most conditions, its recovery in cryoprecipitates was substantially improved owing to the reduction or absence of SPs.