A New Enzyme Immunoassay for Soluble Fibrin in Plasma, with a High Discriminating Power for Thrombotic Disorders

Summary

Fibrin formation is a multistep process initiated by thrombin. At first thrombin converts fibrinogen to fibrin molecules which in vivo form soluble complexes with fibrinogen. Soluble fibrin is considered to be an early biochemical marker for intravascular fibrin formation and impending thrombotic events, such as deep venous thrombosis (DVT), pulmonary embolism (PE) and disseminated intravascular coagulopathy (DIC).

A new enzyme immunoassay (EIA) was developed on the basis of a monoclonal antibody directed against a fibrin specific neo-epitope located on the gamma-chain of fibrinogen; γ-(312-324). In addition, it was possible to prepare a lyophilized reference material of thrombin-generated soluble fibrin, that allowed for full antigen recovery after reconstitution with buffer. Assay conditions, e.g. solid phase-Ig concentration and buffer composition, sample and conjugate dilution, and incubation times were optimised.

The present assay was found to be specific (no interference of homologous antigens) and reproducible (intra-assay CV 4-8%, inter-assay CV 4-9%), and therefore highly suited for measuring soluble fibrin levels in a plasma milieu. The median normal value for soluble fibrin was determined in plasma samples obtained from apparently healthy volunteers (n = 81) and found to be 0.040 μg/ml, with a range (10-90 percentiles) of 0.026-0.059 μg/ml.

A retrospective study showed that soluble fibrin levels were highly significantly increased in patients with a confirmed diagnosis of DIC (median 1.042 μg FEU/ml, range 0.160-2.319 μg/ml, n = 21, P <0.0001 vs normal), PE (median 0.527 μg FEU/ml, range 0.084-1.234 μg/ml, n = 29, P <0.0001 vs normal) and DVT (median 0.126 μg FEU/ml, range 0.059-0.878 μg/ml, n = 36, P <0.0001 vs normal), as determined by the Mann-Whitney U-Test.

 

• References

• 1 Doolittle RF. Fibrinogen and fibrin. Sci Am 1981; 245: 92-101.
• 2 Sasaki T, Page IH, Shainoff JR. Soluble complexes of fibrinogen and fibrin. Science 1966; 152: 1069-71.
• 3 Alkajersig N, Fletcher AP. Formation of soluble fibrin oligomers in purifed system and in plasma. Biochem J 1983; 213: 75-83.
• 4 Wieding JU, Hosius C. Determination of soluble fibrin: a comparison of four methods. Thromb Res 1992; 65: 745-56.
• 5 Müller-Berghaus G, Hasegawa H. Pathophysiology of generalized coagulation. Sem Thromb Hemost 1977; 3: 209-46.
• 6 Bowie EJ, Owen CA. The clinical pathology of intravascular coagulation. Bibl Haematol 1983; 49: 217-44.
• 7 Kisker CT, Rush R. Detection of intravascular coagulation. J Clin Invest 1971; 50: 2235-41.
• 8 Vogel G, Spanuth E. Predictive value of fibrin monomers in postoperative deep vein thrombosis. Klin Wochenschr 1990; 68: 1020-6.
• 9 Francis CW, Conaghan DG, Scott WL, Marder VJ. Increased plasma concentrations of cro-linked fibrin polymers in acute myocardial infarction. Circulation 1987; 75: 1170-6.
• 10 Okajima K, Koga S, Okabe H, Inoue M, Takatsuki K. Characterisation of the fibrinolytic state by measuring stable cro-linked fibrin degradation products in disseminated intravascular coagulation associated with acute promyelocytic leukemia. Acta Haematol 1989; 81: 15-8.
• 11 Nieuwenhuizen W. Soluble fibrin as a marker for a pre-thrombotic state: a mini-review. Blood Coag Fibrinol 1993; 4: 93-6.
• 12 Ginsberg JS, Siragusa S, Douketis J, Johnston M, Moffat K, Stevens P, Brill-Edwards P, Panju A, Patel M. Evaluation of a soluble fibrin assay in patients with suspected deep venous thrombosis. Thromb Haemost 1995; 74: 833-6.
• 13 Ginsberg JS, Siragusa S, Douketis J, Johnston M, Moffat K, Donovan D, McGinnis J, Brill-Edwards P, Panju A, Patel M, Weitz JI. Evaluation of a soluble fibrin assay in patients with suspected pulmonary embolism. Thromb Haemost 1996; 75: 551-4.
• 14 Wieding JU, Eisinger G, Köstering H. Determination of soluble fibrin by turbidimetry of its protamin sulphate-induced paracoagulation. J Clin Chem Clin Biochem 1989; 27: 57-63.
• 15 Wiman B, Rånby M. Determination of soluble fibrin in plasma by a rapid and quantitative spectrophotometric assay. Thromb Haemost 1986; 55: 189-93.
• 16 Dempfle C-E, Pfitzner SA, Dollman M, Huck K, Stehle G, Heene DL. Comparison of immunological and functional assays for measurement of soluble fibrin. Thromb Haemost 1995; 74: 673-9.
• 17 Lill H, Spannagl M, Trauner A, Schramm W, Shuller D, Ofenloch-Haehnle B, Draeger B, Naser W, Dessauer A. A new immunoassay for soluble fibrin enables a more sensitive detection of the activation state of blood coagulation in vivo. Blood Coag Fibrinol 1993; 4: 97-102.
• 18 Vogel G, Dempfle C-E, Spannagl M, Leskopf W. The value of quantitative fibrin monomer determination in the early diagnosis of post operative deep vein thrombosis. Thromb Res 1996; 81: 241-51.
• 19 Carville DGM, Dimitreievic N, Walsh M, Digirolamo T, Brill EM, Drew N, Gargan PE. Thrombus precursor protein (TpP): marker of thrombosis early in the pathogenesis of myocardial infarction. Clin Chem 1996; 42: 1537-41.
• 20 Nieuwenhuizen W, Hoegee-de Nobel E, Laterveer R. A rapid monoclonal antibody-based enzyme immunoassay (EIA) for the quantitative determination of soluble fibrin in plasma. Thromb Haemost 1992; 68: 273-7.
• 21 Lee LV, Ewald GA, McKenzy CR, Eisenberg PR. The relationship of soluble fibrin and cro-linked fibrin degradation products to the clinical course of myocardial infarction. Arterioscl Thromb Vasc Biol 1997; 17: 628-33.
• 22 Schielen WJG., Adams HPHM, Van Leuven K, Voskuilen M, Tesser GI, Nieuwenhuizen W. The sequence γ-(312-324) is a fibrin specific epitope. Blood 1991; 77: 2169-73.
• 23 Hoegee-de Nobel E, Voskuilen M, Briët E, Brommer EJP, Nieuwenhuizen W. A monoclonal antibody-based quantitative enzyme immunoassay for the determination of plasma fibrinogen concentrations. Thromb Haemost 1988; 60: 415-8.
• 24 Bos ES, Van der Doelen AA, Van Rooij N, Schuurs AHWM. 3,3’,5,5’-tetramethyl benzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme immunoassay. J Immunoassay 1981; 60: 187-204.
• 25 Koppert PW, Kuipers W, Hoegee-de Nobel E, Brommer EJP, Koopman J, Nieuwenhuizen W. A quantitative enzyme immunoassay for primary fibrinogenolysis products in plasma. Thromb Haemost 1987; 57: 25-8.
• 26 Koopman J, Haverkate F, Koppert PW, Nieuwenhuizen W, Brommer EJP, Van der Werf WGC. New enzyme immunoassay of fibrin-fibrinogen degradation products in plasma using a monoclonal antibody. J Lab Clin Med 1987; 109: 75-84.
• 27 Bos R, Van Leuven CJM, Stolk J, Hiemstra PS, Ronday HK, Nieuwenhuizen W. An enzyme immunoassay for polymorphonuclear leucocyte-mediated fibrinogenolysis. Eur J Clin Inv 1997; 27: 148-56.
• 28 Pfitzner SA, Dempfle C-E, Matsuda M, Heene DL. Fibrin detected in plasma of patients with disseminated intravascular coagulation by fibrin-specific antibodies constists primarily of high molecular weight FXIIIa-crosslinked and plasmin-modified complexes partially containing fibrinopeptide A. Thromb Haemost 1997; 78: 1069-78.
• 29 Emeis JJ, Verheijen JH, Ronday HK, De Maat MPM, Brakman P. Progress in clinical fibrinolysis. Fibrinolysis & Proteolysis 1997; 11: 67-84.
• 30 Nieuwenhuizen W. A reference material for harmonisation of D-Dimer assays. Thromb Haemost 1997; 77: 1031-3.