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Thromb Haemost 2002; 87(03): 483-492
DOI: 10.1055/s-0037-1613029
Review Article
Schattauer GmbH

Effects of (Pre-)analytical Variables on Activated Protein C Resistance Determined Via a Thrombin Generation-based Assay

Joyce Curvers
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
,
M. Christella
,
G. D. L. Thomassen
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
,
Hans de Ronde
2   Hemostasis and Thrombosis Research Center
,
Rogier M. Bertina
2   Hemostasis and Thrombosis Research Center
,
Frits R. Rosendaal
3   Department of Clinical Epidemiology, Leiden University Medical Center, The Netherlands
,
Guido Tans
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
,
Jan Rosing
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 12 June 2001

Accepted after resubmission 17 December 2001

Publication Date:
14 December 2017 (online)

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Summary

The normalized activated protein C sensitivity ratio (nAPC-sr) determined with an assay that quantifies the effect of APC on thrombin formation initiated via the extrinsic coagulation pathway identifies hereditary and acquired defects of the protein C system. We investigated the influence of assay conditions (analytical variables) and plasma handling (pre-analytical variables) on nAPC-sr obtained with this APC resistance test. The effect of the analytical variables (CaCl2, phospholipid and APC concentrations and the concentration and source of tissue factor) was determined in pooled normal plasma. Inhibition of thrombin formation by APC was dependent on the APC concentration and was also affected by the tissue factor, Ca2+ and phospholipid concentrations. Thus, strict standardization of reactant concentrations is required to obtain reproducible nAPC-sr. Three different tissue factor preparations were compared by determining nAPCsr in plasma samples obtained from 90 healthy individuals. nAPC-sr were similar for all three tissue factor preparations although, compared with the noncommercially available tissue factor used in earlier studies, values determined with commercial tissue factor preparations showed larger variation. Pre-analytical variables, investigated in plasma of nine volunteers (3 normal individuals and 6 individuals with an APCresistant phenotype) were: concentration of anticoagulant (3.2% vs. 3.8% trisodiumcitrate), time before processing of blood (0, 4 and 24 h), centrifugation speed, storage temperature of plasma (–20° C vs. –80° C) and sample thawing. Multiple linear regression analysis showed that only the citrate concentration affected the nAPC-sr, which was higher in samples collected in 3.2% trisodiumcitrate than in samples collected in 3.8% trisodiumcitrate.

Keywords

APC Resistance - Factor VLeiden
 

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