CHARACTERIZATION OF FACTORS AFFECTING THE STABILITY OF FROZEN HEPARINIZED PLASMA

The use of heparin rather than citrate as primary anticoagulant has be en shown to significantly improve the initial activity, stability and recovery of factor VIII:C from human plasma, cryoprecipitates or facto r 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 inso luble protein aggregates, termed storage precipitates (SPs), were reco vered in the thawed plasma and cryoprecipitate fractions. Plasma recov ery 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 mont h 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 n o effect on factor VIII:C activity at equivalent storage times or temp eratures. A trend towards improved factor VIII recoveries was noted at lower freezing and storage temperatures however. SP formation was ass ociated with reduced fibrinogen levels in the recovered plasma without loss of antithrombin-III or increased fibrinopeptide-A. Western blots showed polymerization of Aalpha or gamma-chains of fibrinogen. SP for mation was reduced or eliminated with factor XIII inhibitors, antibody to the active factor XIII a subunit or adjustment of heparinized plas ma to 5-10 mM sodium citrate before initial freezing and storage. Alth ough plasma factor VIII:C recoveries were only slightly affected at th ese citrate concentrations under most conditions, its recovery in cryo precipitates was substantially improved owing to the reduction or abse nce of SPs.