The Factor VIII procoagulant activity in plasma, cryoprecipitate, and their polyethylene glycol (PEG) precipitates is markedly increased if blood is collected into heparin rather than into citrate phosphate dextrose (CPD). There is a 34% increase in the initial level of the Factor VIII activity in the heparinized plasma with 78% of this initial activity (184 U) recovered in the cryoprecipitate. As well, the stability of the Factor VIII activity is improved: after 24 h of incubation at 22°C, 99% of the initial activity is retained in heparinized plasma whereas only 64% remains in CPD plasma. The cryoprecipitate prepared from heparinized plasma is equally stable after 24 h. The PEG concentrate prepared from the cryoprecipitate of heparinized plasma is increased to 128 U compared to only 54 U from CPD plasma. Relative recoveries were 531 U/l for heparinized plasma versus 215 U/l for CPD plasma. This represents a 147% increase in yield.

DDAVP (l-desaminocysteine-(8-D-arginine)-vasopressin) produces a marked increase in plasma factor VIII procoagulant (F VIII :C) levels. Previously, we have reported that blood collected into heparin rather than into CPD anticoagulant results in higher starting levels of plasma F VIII:C activity. We therefore wished to determine whether the effects of these two agents were accumulative and whether they would result in any difference in the relative molecular distribution of F VIII:C. Blood was collected into CPD or heparin immediately before and 15 min after an intravenous dose of 0.2 μg/kg body weight of DDAVP. Pre-stimulation factor VIII levels were approximately 36% higher in heparinized plasma than in CPD plasma. Following DDAVP stimulation, the final factor VIII activity was increased 3.9-fold when either of the anticoagulants was used, with the heparin sample maintaining a 37% increase over the CPD sample. Column chromatography on Sepharose CL-6B of pre- and post-DDAVP plasma samples collected into either heparin or CPD indicated that there was no change in the relative distribution of the high and low molecular weight forms of F VIII:C. The heparinized sample showed the typical distribution of approximately 60% F VIII:C at void volume (V(0)) and 40% at 2.3 V(0), suggesting that DDAVP-stimulated increases of plasma F VIII : C are equally distributed between the carrier and non-carrier associated F VIII:C activities.

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°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°C, more SP was recovered from MI plasma whereas at -40°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 Aa or y-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.