Thrombin-Based Antithrombin Assays Show Overestimation of Antithrombin III Activity in Patients on Heparin Therapy due to Heparin Cofactor II Influence^[*]

Summary

An extensive comparison has been performed on the clinical chemistry automate Hitachi 717 between thrombin-and Factor X_a-based methods for determination of antithrombin III activity. In 460 patients who did not receive any heparin therapy the agreement between assays was in general close although the thrombin-based methods resulted in slightly higher assignments of 0.3—2.6% antithrombin III activity. The discrepancy was, however, substantial in plasmas from patients receiving heparin of >20000 IU/day, resulting in plasma levels of heparin of 0.8-1.2 IU/ml. Thus, analysis of 102 patients showed that the thrombin-based methods resulted in, on average, 7-16% higher assignment of antithrombin III activity as compared to the Factor X_a-based method used.

Addition of antibodies to antithrombin III and heparin cofactor II revealed that the discrepancy was primarily due to contribution of heparin cofactor II activity in the thrombin-based methods. The results thus suggest that the Factor X_a-based antithrombin III activity method provides more valid results in patients on heparin therapy.

^* In memory of Professor Dr. Manfred Eggstein.

• References

• 1 Abildgaard U. Antithrombin and related inhibitors of coagulation. In: “Recent Advances in Blood Coagulation”. Poller L. Ed. Churchill Livingstone; London, UK: 1981: 151-73
• 2 Kauffman RH, Veltkamp JJ, van Tilburg NH, Vananes LA. Acquired antithrombin III deficiency and thrombosis in nephrotic syndrome. Am J Med 1978; 65: 607-13
• 3 Marciniak E, Farley CH, DeSimone PA. Familial thrombosis due to antithrombin III deficiency. Blood 1974; 43: 219-31
• 4 Thaler E, Lechner K. Antithrombin III deficiency and thromboembolism. Clin Haematol 1981; 10: 369-90
• 5 Jespersen J. Monitoring plasma anti thrombin III levels during infusion of anti thrombin III concentrate. Thromb Haemostas 1984; 52: 214
• 6 Hellgren M, Tengborn L, Abildgaard CF. Pregnancy in women with congenital antithrombin III deficiency: experience of treatment with heparin and antithrombin. Gynecol Obstet Invest 1982; 14: 127-41
• 7 Biiller HR, ten Cate JW. Acquired antithrombin III deficiency: laboratory diagnosis, incidence, clinical implications, and treatment with antithrombin III concentrate. Am J Med 1989; 87 (suppl 3B): 3B-44S 3B-48S.
• 8 Schwartz R, Bauer KA, Rosenberg RD, Kavanaugh EJ, Davies DC, Bogdanoff DA. Clinical experience with antithrombin III concentrate in treatment of congenital and acquired deficiency of antithrombin. Am J Med 1987. (suppl 3B): 3B-53S-3B-60S.
• 9 Odegârd OR, Lie M, Abildgaard U. Heparin cofactor activity measured with an amidolvtic method. Thromb Res 1975; 6: 287-94
• 10 Abildgaard U, Lie M, Odegârd OR. Antithrombin (heparin cofactor) assay with ”new“ chromogenic substrates (S-2238 and Chromozym TH). Thromb Res 1977; 11: 549-53
• 11 Tollefsen DM, Majerus DW, Blank MK. Heparin cofactor II: purification and properties of a heparin-dependent inhibitor of thrombin in human plasma. J Biol Chem 1982; 257: 2162-9
• 12 Wunderwald P, Schrenk WJ, Port H. Antithrombin BM from human plasma: An antithrombin binding moderately to thrombin. Thromb Res 1982; 25: 177-91
• 13 Friberger P, Egberg N, Holmer E, Hellgren M, Blombäck M. Antithrombin assay-the use of human or bovine thrombin and the observation of a “second“ heparin cofactor. Thromb Res 1982; 25: 433-6
• 14 Tran TH, Duckert F. influence of heparin cofactor II (HC II) on the determination of antithrombin III (AT). Thromb Res 1985; 40: 571-6
• 15 Conard J, Bara L, Horellou M-H, Samama MM. Bovine or human thrombin in amidolytic AT III assays. Influence of heparin cofactor II. Thromb Res 1986; 41: 873-8
• 16 Bohner J, von Pape KW, Blaurock M. Chromogener Substrattest zur Bestimmung von Antithrombin III unter Verwendung des Faktors Xa. Lab med 1991; 15: 296-8
• 17 Friberger P, Bohner J, von Pape KW, Blaurock M. Two routine methods for antithrombin III determination. Thromb Res 1992; 67: 473-8
• 18 Demers C, Henderson P, Blajchman MA, Wells MJ, Mitchell L, Johnston M, Ofosu FA, Fernandez-Rachubinski F, Andrew M, Hirsh J, Ginsberg JS. An Antithrombin III assay based on factor Xa inhibition provides a more reliable test to identify congenital antithrombin III deficiency than an assay based on thrombin inhibition. Thromb Haemost 1993; 69: 231-5
• 19 Kristensen HI, Østergaard PB, Nordfang O, Abildgaard U, Lindahl AK. Effect of tissue factor pathway inhibitor (TFPI) in the HEPTEST® assay and in an amidolytic anti factor X_a assay for LMW heparin. Thromb Haemost 1992; 68: 310-4
• 20 Gram J, Jespersen J. Increased concentrations of heparin cofactor II in diabetic patients, and possible effects on thrombin inhibition assay of antithrombin III. Clin Chem 1989; 35: 52-5