Preparation of Cryoprecipitated Factor VIII Concentrates

Abstract

Factors affecting the yield of factor VIII in cryoprecipitate have been investigated in the context of a blood component program. Both in vitro and in vivo measurements were used to assess the effects of critical variables on the yield of factor VIII activity. Variables such as anticoagulant, plastic bag, mixing during collection, and platelet contamination had no significant effect on yield of factor VIII activity in cryoprecipitate. Among the most critical factors affecting yield were storage time of whole blood and procedures for freezing, thawing, and reconstitution. The following procedures were found to assure a 60 per cent recovery of factor VIII in cryoprecipitate: 1)processing of whole blood within six hours of collection; 2)use of a technique to freeze plasma within 30 minutes either in a -70 C ethanol bath or -85 C freezer; 3)rapid thawing (1 1/2 hour or less) in a 4 C circulating water bath; 4)centrifugation at 4,500 X g for 10 minutes at 4 C followed by draining of the supernatant in a 4 C cold room; 5) storage of the precipitate at -20 C until ready for use; 6) thawing in a 37 C water bath for at least 15 minutes followed by addition of 20 ml of 0.15 M saline for a 20 minute period at room temperature, and gentle mixing before pooling units for transfusion. The recovery of factor VIII in cryoprecipitate appears to be limited to about 65 per cent by its solubility in plasma at 4 C. Therefore, further effort to increase the amount available for treatment should involve improving the supply of plasma for its preparation and decreasing the cost of processing.

Disulfide Bonds and the Quaternary Structure of Factor VIII/von Willebrand Factor

“…At the same times that samples were taken for activity assays, 25-1.l samples were also removed for electrophoresis. 25 ,ul of 0.1 M iodoacetamide was added to each sample and the mixture reacted for 3 min at room temperature. 25 ,lA of 1% SDS was then added.…”

“…25 ,ul of 0.1 M iodoacetamide was added to each sample and the mixture reacted for 3 min at room temperature. 25 ,lA of 1% SDS was then added. Electrophoresis was carried out in the large-pore gel system.…”

Influence of the Primary Anticoagulant on the Recovery of Factor VIII in Cryoprecipitate

“…On the contrary, Vermeer et al [5,6] noted that the choice of anticoagulant should be dependent on the time between bloodletting and the freezing of plasma, as the loss of factor VIII in CPD during storage is substantially greater than in ACD. Several authors do not find a significant difference between ACD, CPD and CPD-A [7][8][9][10]. The beneficial effect of heparin is debated: Preston [11] does not find a higher yield, whereas Rock and Palmer [12], Rock et al [13], Smit Sibinga et al [14] and Krachmalnicoff and Thomas [15] described less inactivation of factor VIII with sometimes much higher yields.…”

Segregation of Proteins and Sodium in Human Plasma upon Freezing

“…Factor VIII (antihemo philic factor) is recovered from the source plasma by cryoprecipitation. The recovery of factor VIII from thawed plasma is re ported to be strongly dependent on the freez ing rate of the plasma [8,11]-the yield and ing plasma is an interesting aspect to inves tigate the segregation.…”