Sources of variation in factor VIII, von Willebrand factor and fibrinogen measurements: Implications for detecting deficiencies and increased plasma levels

Abstract

Objectives

Differences in pre-analytical and assay conditions, inappropriate reference ranges, or inflammation may have the potential to impair clinical decisions based on measurements of factor VIII (FVIII), von Willebrand factor (VWF) and fibrinogen (Fg). This study examined the impact on FVIII, VWF and Fg in plasma of freezing and thawing, different citrate anticoagulant concentrations, and inflammation, as determined by high-sensitivity C-reactive protein (hsCRP).

Materials and Methods

FVIII was determined prior to freezing and after thawing using a one-stage clotting assay (FVIII:C), an amidolytic assay (FVIII:AM) and an enzyme immunoassay (FVIII:Ag). Samples were anticoagulated with 106 or 129 mmol/L of citrate. FVIII, VWF and Fg were quantified in 300 individuals to establish reference ranges and to investigate associations with hsCRP.

Results

Freezing and thawing reduced FVIII:C and FVIII:AM markedly. FVIII coagulant activities were not significantly different between samples anticoagulated with 106 or 129 mmol/L of citrate, respectively. FVIII, VWF and Fg were significantly associated with hsCRP. FVIII:C was greater than FVIII:AM and FVIII:Ag in all experiments, indicating that the presence of activated FVIII may lead to overestimation of FVIII:C.

Conclusions

Standardized freezing and thawing of plasma samples appears to be indispensable if reliable FVIII results are to be obtained. Because inflammation can potentially mask deficiency states or mimic an increased risk of thrombosis, FVIII, VWF and Fg determinations should be supplemented by measurements of hsCRP.

Introduction

Measurements of factor VIII (FVIII), von Willebrand factor (VWF) and fibrinogen (Fg) are an integral element of diagnostic strategies for bleeding disorders [1], [2] and for identifying patients with an increased risk of venous thromboembolism (VTE) [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]. Increased plasma levels of these hemostatic proteins have also been associated with inflammation and arterial cardiovascular events [13], [14], [15], [16], [17]. Factors including insufficiently standardized pre-test and assay conditions, inappropriate reference ranges, or disregard of inflammation as a significant cause of variability continue to hamper clinical decisions based on lower and upper reference limits for these proteins. Inflammation can mask decreased plasma levels that potentially may be associated with bleeding or mimic persistently elevated levels, which can increase the risk of first or recurrent VTE and arterial cardiovascular events. The results of these shortcomings can be seen in substantial differences in results between studies, for example FVIII cut-off values to define an increased risk of VTE range between 150 to 298 IU/dL [4], [5], [6], [7], [10], [18], [19], or lower reference limits for VWF between 37 and 65 IU/dL [20], [21]. Although previous findings in therapeutic plasma preparations have indicated that FVIII coagulant activity decreases markedly after freezing and thawing [22], or with increasing final citrate concentrations [23], similar examinations in diagnostic plasma samples have not yet been performed. There is also a lack of data on the interrelationship between FVIII, VWF and Fg as determined using different assays and the degree to which they are associated in normal individuals with the inflammatory marker C-reactive protein (CRP).

We therefore examined FVIII, VWF, Fg and high-sensitivity CRP (hsCRP) in 300 healthy subjects. FVIII coagulant activity and antigen were determined using a one-stage clotting assay (FVIII:C), a two-stage amidolytic (chromogenic) assay (FVIII:AM), and an enzyme immunoassay (FVIII:Ag). VWF antigen (VWF:Ag) was measured turbidimetrically, Fg by the Clauss´ method (clottable Fg, Fg:C) and nephelometrically (Fg antigen, Fg:Ag). The influence on FVIII levels of freezing and thawing plasma samples and of different citrate anticoagulant concentrations was also analyzed.