Mixing study

Abstract: In a previous paper, we showed that the abnormality of ristocetin-induced platelet aggregation in platelet-rich plasma in 10 patients with von Willebrand's disease could be corrected by a factor in normal plasma that was present in the same fractions as factor VIII procoagulant activity (antihemophilic factor, AHF, VIII(AHF)) when prepared by chromatography on Bio-Gel 5 M (Bio-Rad Laboratories, Richmond, Calif.). This observation suggests that patients with this disorder are deficient in a plasma factor, associated with the factor VIII molecule, that is necessary for normal platelet function. In the present paper, we describe, an assay for this factor, the von Willebrand factor (VIII(VWF)), based on the observation that a log-log relationship exists between the amount of ristocetin-induced aggregation of washed, normal platelets and the concentration of normal plasma present in the test system. We assayed the activity of VIII(VWF) as well as antihemophilic factor procoagulant activity (VIII(AHF)) and factor VIII antigen (VIII(AGN)) in 15 patients with von Willebrand's disease and 20 normal subjects. A highly significant correlation (r approximately 0.80) between VIII(VWF) and both VIII(AHF) was found in normal subjects and in patients with von Willebrand's disease. This finding, in addition to the observation that agarose gel chromatography fractions that have VIII(AHF) procoagulant activity also have VIII(VWF) activity, strongly suggests that the von Willebrand factor is associated with the factor VIII molecule. VIII(VWF) in normal plasma was not inhibited by human anti-VIII, and VIII(VWF) levels were normal in hemophilic plasma. Thus, the VIII(VWF) site on the factor VIII molecule appears to be different from that determining VIII(AHF). Finally, the activity of VIII(VWF) appeared to correlate better with the bleeding time than either VIII(AHF) or VIII(AGN). This suggests that VIII(VWF) assayed in this study may be the "anti-bleeding factor" that is deficient in von Willebrand's disease. These findings are consistent with a decreased synthesis of the factor VIII molecule in von Willebrand's disease and suggest the possibility of additional abnormalities of the site on the molecule that determines the activity of VIII(VWF).

Abstract: Lupus anticoagulants (LAs) are antibodies that interfere with phospholipid dependent coagulation reactions in vitro. This workshop was designed to provide the participants with an experience in identification of LAs, to evaluate different criteria for mixing studies, to assess the performance of recently introduced confirmatory studies and to assess the performance of two potential surrogate LA control plasmas. The results demonstrate that there continues to be significant variation in the sensitivity and responsiveness of APTT reagents to the presence of LAs, confirming the need for more than one screening assay before the presence of a LA can be ruled out. In this workshop, the best distinction between factor deficiency and inhibitors was obtained using a 1:1 mix of normal plasma with patient plasma and the criterion defining correction as shortening of the APTT to within 5 s of the APTT of pooled normal plasma. A 4:1 mix of patient to normal plasma did not work well in distinguishing factor deficiency from inhibitors. The platelet neutralization procedure, DVVconfirm® and StaClot LA® all gave positive results with the LA samples. False positive platelet neutralization procedures were seen with the samples from patients on oral anticoagulants and a factor V inhibitor. False positive StaClot LA results were obtained with high titer factor VIII inhibitors. Both of the potential surrogate plasmas gave variable results with multiple assays; they can not be recommended for routine use at present.