Poor Comparability of Prothrombin Fragment 1 + 2 Values Measured by Two Commercial ELISA Methods: Influence of Different Anticoagulants and Standards

Summary

We compared F 1 + 2 results obtained with two commercial ELISA methods (Behring and Baxter) assaying the same plasma samples. There was little correlation between the results of the two methods, as shown by the low correlation coefficient (r = 0.50) and by low percentage of concordant classification (normal or abnormal) of the samples (24%). Such poor correlation is probably due to the different anticoagulants suggested, because correlation improved when both methods were carried out in plasmas collected with the same anticoagulant. However, the Baxter method still gave significantly lower F 1 + 2 values than the Behring method. Assuming that this difference is due to the use of standards with different F 1 +2 concentrations, the standards from Behring and Baxter were evaluated by both methods. Parallel dose-response curves were obtained when the standards were run by the Behring method but not by the Baxter method, indicating that the two standards are qualitatively different. This study demonstrates that the two F 1 + 2 methods give different values for the same samples and that these values are poorly correlated. Standardization of the F 1 + 2 assays cannot be achieved easily simply by using a common standard and the use of different anticoagulants appears to be the main reason for poor standardization.

• References

• 1 Aronson DL, Stevan L, Ball AP, Franza BR Jr, Finlayson JS. Generation of the combined prothrombin activation peptide (FI.2) during the clotting of blood and plasma. J Clin Invest 1977; 60: 1410-1418
• 2 Bauer KA, Rosenberg RD. The pathophysiology of the prethrombotic state in humans: insights gained from studies using markers of the hemostatic system activation. Blood 1987; 70: 343-350
• 3 Teitel JM, Bauer KA, Lau HK, Rosenberg RD. Studies of the prothrombin activation pathway utilizing radioimmunoassays for the F2/ F 1 + 2 fragment and thrombin-antithrombin complex. Blood 1982; 59: 1086-97
• 4 Pelzer H, Schwarz A, Stuber W. Determination of human prothrombin activation fragment 1 + 2 in plasma with an antibody against a synthetic peptide. Thromb Haemost 1991; 65: 153-9
• 5 Shi D, Ruiz JA, Perez LM, Denis RF, Sio R, Alvarez D, Tang SM, Mills R, Arbuthnott K, Gaur P. Detection of prothrombin activation with a two-site enzyme immunoassay for the fragment F 1 + 2. Thromb Haemost 1989; 62: 165
• 6 Hursting M, Butman B, Steiner J, Moore B, Szewczvk K, Bell M, Dom- brose F. A quantitative ELISA for plasma prothrombin fragment 1.2 (F1.2). Blood 1989; 65-153
• 7 Boisclair MD, Lane DA, Philippou M, Sheikh S, Hunt B. Thrombin production. Inactivation and expression during open heart surgery measured by assays for activation fragments including a new ELISA for prothrombin fragment F 1 + 2. Thromb Haemost 1993; 70: 253-8
• 8 Bauer KA, Goodman TL, Kass BL, Rosenberg RD. Elevated factor Xa activity in the blood of asymptomatic patients with congenital antithrombin deficiency. J Clin Invest 1985; 76: 826-36
• 9 Conway EM, Bauer KA, Barzegar S, Rosenberg RD. Suppression of hemostatic system activation by oral anticoagulants in the blood of patients with thrombotic diathesis. J Clin Invest 1987; 80: 1535-44
• 10 Bauer KA, Broekmans AW, Bertina RM, Conard J, Horellou ML, Samama MM, Rosenberg RD. Hemostatic enzyme generation in the blood of patients with hereditary protein C deficiency. Blood 1988; 71: 1418-1426
• 11 Mannucci PM, Bauer KA, Gringeri A, Barzegar S, Bottasso B, Simoni L, Rosenberg RD. Thrombin generation is not increased in the blood of hemophilia B patients after infusion of a purified factor IX concentrate. Blood 1990; 76: 2540-5
• 12 Mannucci PM, Bauer KA, Gringeri A, Barzegar S, Santagostino E, Tradati FC, Rosenberg RD. No activation of the common pathway of the coagulation cascade after a highly purified factor IX concentrate. Brit J Haematol 1991; 79: 606-11
• 13 Wilkes HC, Meade TW, Barzegar S, Foley AJ, Hughes LO, Bauer KA, Rosenberg RD, Miller GJ. Gemfibrozil reduces plasma prothrombin fragment F 1 + 2 concentration a marker of coagulability in patients with coronary heart disease. Thromb Haemost 1992; 67: 503-506
• 14 Bauer KA, Mannucci PM, Gringeri A, Tradati F, Barzegar S, Kass BL, ten Cate H, Kestin AS, Brettler DB, Rosenberg RD. Factor IXa-lactor Vlla-ccll surface complex does not contribute to the basal activation of the coagulation mechanism in vivo. Blood 1992; 79: 2039-47
• 15 Milenson MM, Bauer KA, Kistler JP, Barzegar S, Tulin L, Rosenberg RD. Monitoring “mini-intensity” anticoagulation with warfarin: comparison of the prothrombin time using a sensitive thromboplastin with prothrombin fragment F 1 + 2 levels. Blood 1992; 79: 2034-8
• 16 Mannucci PM, Tripodi A, Bottasso B, Baudo F, Finazzi G, De Stefano V, Palareti G, Manotti C, Mazzucconi MG, Castaman G. Markers of procoagulant imbalance in patients with inherited thrombophilic syndromes. Thromb Haemost 1992; 67: 200-2
• 17 Demers C, Ginsberg JS, Henderson P, Ofosu FA, Weitz JI, Blajchman MA. Measurement of markers of activated coagulation in antithrombin III deficient subjects. Thromb Haemost 1992; 67: 542-4
• 18 Sorensen JV, Lassen MR, Borris LC, Rahr HB, Jensen HP, Hoppensteadt D, Walenga JA, Fareed J. Reduction of plasma levels of prothrombin fragments 1 and 2 during thromboprophylaxis with a low-molecular-weight heparin. Blood Coag Fibrinol 1992; 3: 55-9
• 19 Asakura H, Hifumi S, Jokaji H, Saito M, Kumabashiri I, Uotani C, Morishita E, Yamazaki M, Shibata K, Mizuhashi K, Mifune J, Matsuda T. Prothrombin fragment F 1 + 2 and thrombin-antithrombin complex are useful markers of hypercoagulable state in atrial fibrillation. Blood Coag Fibrinol 1992; 3: 469-73
• 20 Tripodi A, Mannucci PM, Chantarangkul V, Bottasso B, Arbini A, Della Bella S, Scorza R. Markers of procoagulant imbalance in patients with localized melanomas and autoimmune disorders. Brit J Haematol 1993; 84: 670-4
• 21 Fleiss JL. Statistical methods for rates and proportion. Wiley Interseience. New York, NY: 1981: 211-234
• 22 Bauer KA, Barzegar S, Rosenberg RD. Influence of anticoagulants used for blood collection on plasma prothrombin fragment F 1 + 2 measurements. Thrombos Res 1991; 63: 617-28