Influence of blood handling on determination of plasma resistance against activated protein C

Determination of activated protein C resistance ratio (APC-R) by a modified activated partial thromboplastin time test may be used as a screening assay to identify carriers of the Factor V Leiden mutation. However, blood handling procedures may influence the test result. Thus, we have examined influence of preanalytical procedures such as freezing and centrifugation on determination of APC-R. Blood samples were collected from 28 healthy volunteers and from citrate-stabilized blood three different plasma preparations were obtained: 1) the blood was immediately centrifuged at 2,000 g for 20 minutes and plasma was kept at room temperature for one hour until analysis; 2) the blood was immediately centrifuged at 2,000 g for 20 minutes and plasma was frozen for eight days at -70°C and thawed at 37°C just before analysis; 3) the blood was immediately centrifuged at 2,000 g for 20 minutes, followed by a new centrifugation for 10 minutes at 10,000 g and plasma was kept at room temperature for one hour until analysis. APC-R decreased significantly in plasma samples frozen for eight days compared with APC-R determined in unfrozen plasma samples 3.26 (1.88-3.81) vs. 3.30 (1.87-4.50), respectively, P < 0.004. In contrast, APC-R increased significantly in samples plasma centrifuged two times compared with plasma samples centrifuged one time, 3.37 (1.88-4.74) vs. 3.30 (1.87-4.50), respectively, P < 0.008. We conclude that simple blood handling procedures influence significantly the determination of APC-R. In order to reduce preanalytical variation and to increase accuracy of the assay result we suggest to determine APC-R on unfrozen plasma samples centrifuged twice.