Laboratory Diagnosis of APC-Resistance: A Critical Evaluation of the Test and the Development of Diagnostic Criteria

Summary

The APC-resistance test consists of two APTT’s, one in the presence and one in the absence of a fixed amount of Activated Protein C (APC), and is a simple and reliable method to detect a reduced sensitivity to the anticoagulant action of APC (APC-resistance). At a fixed concentration of APC the prolongation of the APTT is dependent on the activator, the CaCl2 concentration, the citrate concentration in the sample, and on sample handling. The effect of sample handling can be reduced by calculating the APC-Sensitivity Ratio (APC-SR). The actual prolongation of the APTT is also influenced by low protein S levels (reduction of APC-SR) and by reduced levels of factors V, VIII and IX (increase of APC-SR). The APC-SR is most dramaticly effected by reduced levels of factors II and X, which result often in “unmeasurable” APC-SR’s in plasmas of patients on oral anticoagulant treatment. So at present no reliable APC-SR’s can be measured in these patients. Patients treated with heparin can be tested after treatment of their plasma with Hepzym®. The inter- and intra-assay variation in the APC-SR is 4% and 2%, respectively, when using the same batches of activator and APC. The variation which is introduced in the APC-SR by use of different batches of activator or APC, or by the use of different APC or CaCl2 concentrations, can effectively be avoided by expressing the result of the test in normalized-APC-SR (n-APC-SR).

The diagnosis of APC-resistance is defined by a n-APC-SR < 0.84. Patients who are heterozygous for the Factor V Leiden mutation have a n-APC-SR of 0.45-0.70, while patients who are homozygous for the mutation have a n-APC-SR <0.45.

• References

• 1 Dahlbäck B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognised mechanism characterized by poor anticoagulant response to activated protein C: prediction ofa cofactor to activated protein C. Proc Natl Acad Sci USA 1993; 90: 1004-8
• 2 Dahlbäck B, Hildebrand B. Inherited resistance to activated protein C is corrected by anticoagulant cofactor activity found to be a property of factor V. Proc Natl Acad Sci USA 1994; 91: 1396-400
• 3 Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA, Reitsma PH. Mutation in blood coagulation factorV associated with resistance to activated protein C. Nature 1994; 369: 64-79
• 4 Jenny RJ, Pittman DD, Toole JJ, Kriz RW, Aldape RA, Hewich RM, Kaufman RJ, Mann KG. Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci USA 1987; 84: 4846-50
• 5 Kalafatis M, Haley PE, Mann K. Membrane bound human factor Va is inactivated by activated protein C after cleavage of the heavy chain at Arg 506 and Arg 306. Blood 1993; 82: 58 a (abstract)
• 6 Koster T, Rosendaal FR, de Ronde H, Briët E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden Thrombophilia Study. Lancet 1993; 342: 1503-6
• 7 Svensson PJ, Dahlbäck B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994; 330: 517-22
• 8 Griffin JH, Evatt B, Wideman C, Fernandez JA. Anticoagulant protein C pathway defective in majority of thrombophilic patients. Blood 1993; 82: 1989-93
• 9 Faioni EM, Franchi F, Asti D, Sacchi E, Bernardi F, Mannucci PM. Resistance to activated protein C in nine thrombophilic families: interference in a protein S functional assay. Thromb Haemost 1993; 70: 1067-71
• 10 Halbmayer WM, Haushofer A, Schön R, Fischer M. The prevalence of poor anticoagulant response to activated protein C (APC-resistance) among patients suffering from stroke or venous thrombosis and among healthy subjects. Blood Coag Fibrinol 1994; 5: 51-7
• 11 Legnani C, Palareti G, Giagi R, Coccheri S. Activated protein C resistance in deep-vein thrombosis. Lancet 1994; 343: 541-2 (letter)
• 12 van Hinsbergh VWM, Bertina RM, van Wijngaarden A, van Tilburg NH, Emeis JJ, Haverkate F. Activated protein C decreases plasminogen activa-tor-inhibitor activity in endothelial cell-conditioned medium. Blood 1985; 65: 444-51
• 13 Laurell CB. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 1966; 15: 45-52
• 14 Boyer C, Rothchild C, Wolf M, Amiral J, Meyer D, Larrieu MJ. A new method for the estimation of protein C by ELISA. Thromb Res 1984; 36: 579-89
• 15 Deutz-Terlouw PP, Ballering L, van Wijngaarden A, Bertina RM. Two ELISA’s for measurement of protein S and their use in the laboratory diagnosis of protein S deficiency. Clin Chem Acta 1989; 186: 321-34
• 16 Rosendaal RF, BertinaE RM, Reitsma PH. Evaluation of activated protein C resistance in stored plasma. Lancet 1994; 343: 1289-90 (Letter)
• 17 Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH, Evatt B. Activated protein C resistance caused by Arg506Gln mutation in factor Va. Lancet 1994; 343: 1361-2 (Letter)
• 18 Cadroy Y, Sié P, Boneu B. Frequency of a defective response to activated protein C in patients with a history of venous thrombosis. Blood 1994; 83: 2008-9 (Letter)