Hamostaseologie 2010; 30(04): 207-211
DOI: 10.1055/s-0037-1619052
Review
Schattauer GmbH

Discrepancy between one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A phenotype

Diskrepanzen zwischen Einstufen- und chromogenen FVIII-Aktivitätsmessungen können zur Fehldiagnose des Hämophilie-A-Phänotyps führen
J. Oldenburg
1   Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Germany
,
A. Pavlova
1   Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Germany
› Author Affiliations
Further Information

Publication History

Publication Date:
28 December 2017 (online)

Summary

Severity of bleeding phenotype in hemophilia A (HA) depends on the underlying mutation in the F8 gene and, ultimately, on the concentration and functional integrity of the factor VIII (FVIII) protein in circulating plasma. Initial diagnosis for HA and monitoring of treatment is typically performed by measuring of FVIII activity by either one-stage assay or chromogenic assay.

We review evidence for why both types of assay do not give comparable results in a significant proportion of patients with non-severe haemophilia A and why the discrepancy in results between both methods segregates with distinct subclasses of known missense mutations causing haemophilia A. The current understanding of the mechanistic basis for how FVIII:C assay discrepancies arise are discussed.

Conclusion

We propose that both methods should be used in initial patient diagnosis along with follow-up genetic analysis to avoid potential misdiagnosis and to optimize treatment monitoring of patients with HA phenotypes.

Zusammenfassung

Bei Patienten mit Hämophilie A (HA) ist der Schweregrad der Blutungsneigung abhängig von der Mutation im Faktor-8-Gen sowie der Konzentration und der funktionellen Integrität des Faktor-VIII-Proteins im Blutplasma. Die anfängliche Diagnose der HA und die Therapiekontrolle erfolgen normalerweise durch Messung der FVIII-Aktivität, entweder mit Hilfe des Einstufen-Tests (one-stage assay) oder des chromogenen Zwei-Stufen Tests (two-stage assay).

Wir fassen Beobachtungen zusammen, warum die beiden Tests bei einem signifikanten Anteil der Patienten mit leichter oder mittelschwerer HA unterschiedliche Werte liefern. Die Diskrepanz zwischen beiden Testmethoden ist mit bestimmten HA-verursachenden Missense-Mutationen assoziiert. Das gegenwärtige Verständnis der Mechanismen, die zu einer solchen Assay-Diskrepanz führen, wird in diesem Review diskutiert.

Schlussfolgerung

Wir schlagen vor, dass bei der initialen Diagnosestellung beide Methoden sowie eine molekulargenetische Analyse durchgeführt werden sollten, um eine potenzielle Fehldiagnose zu verhindern und die Überwachung der Behandlung von HA-Patienten zu optimieren.

 
  • References

  • 1 Fay PJ. Factor VIII structure and function. Thromb Haemost 1993; 70: 63-67.
  • 2 Lenting PJ, van Mourik JA, Mertens K. The life cycle of coagulation factor VIII in view of its structure and function. Blood 1998; 92: 3983-3996.
  • 3 Brandt JT, Triplett DA. Laboratory assays for factor VIII. In: Zimmerman TS, Ruggeri ZM. (eds). Coagulation and bleeding disorders. The role of factor VIII and vWF. New York: Marcel Dekker; 1989: 343-357.
  • 4 Langdell RD, Wagner RH, Brinkhous KM. Effect of antihemophilic factor on one-stage clotting tests; a presumptive test for hemophilia and a simple one-stage antihemophilic factor assy procedure. J Lab Clin Med 1953; 41: 637-647.
  • 5 Over J. Methodology of the one-stage assay of factor VIII (VIII:C). Scand J Haematol Suppl 1984; 41: 13-24.
  • 6 Biggs R, Eveling J, Richards G. The assay of antihaemophilic-globulin activity. Br J Haematol 1955; 01: 20-34.
  • 7 Barrowcliffe TW. Methodology of the two-stage assay of factor VIII (VIII:C). Scand J Haematol Suppl 1984; 41: 25-38.
  • 8 Rosen S. Assay of factor VIII:C with a chromogenic substrate. Scand J Haematol Suppl 1984; 40: 139-145.
  • 9 Van Dieijen G, van Dieijen-Visser MP, Franssen J, Hemker HC. Spectrophotometric method for the assay of human blood coagulation factor VIII. Haemostasis 1987; 17: 14-24.
  • 10 Hubbard AR, Weller LJ, Bevan SA. Activation profiles of factor VIII in concentrates reflect one-stage/chromogenic potency discrepancies. Br J Haematol 2002; 117: 957-960.
  • 11 Santoro C, Iorio A, Ferrante F. et al. Performance of recalibrated ReFacto laboratory standard in the measurement of FVIII plasma concentration via the chromogenic and one-stage assays after infusion of recalibrated ReFacto (B-domain deleted recombinant factor VIII). Haemophilia 2009; 15: 779-787.
  • 12 Oldenburg J, Ananyeva NM, Saenko EL. Molecular basis of haemophilia A. Haemophilia 2004; 10 Suppl 4: 133-139.
  • 13 Oldenburg J, El-Maarri O. New insight into the molecular basis of hemophilia A. Int J Hematol 2006; 83: 96-102.
  • 14 Oldenburg J, Pavlova A. Genetic risk factors for inhibitors to factors VIII and IX. Haemophilia 2006; 12 Suppl 6: 15-22.
  • 15 Rost S, Löffler S, Pavlova A. et al. Detection of large duplications within the factor VIII gene by MLPA. J Thromb Haemost 2008; 06: 1996-1999.
  • 16 Haemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS). Dr. Geoffrey Kemball-Cook (cited 7 July 2010). http://hadb.org.uk/
  • 17 Poulsen AL, Pedersen LH, Hvas AM. et al. Assay discrepancy in mild haemophilia A: entire population study in a National Haemophilia Centre. Haemophilia 2009; 15: 285-289.
  • 18 Vehar GA, Keyt B, Eaton D. et al. Structure of human factor VIII. Nature 1984; 312: 337-342.
  • 19 Toole JJ, Knopf JL, Wozney JM. et al. Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 1984; 312: 342-347.
  • 20 Kaufman RJ, Wasley LC, Dorner AJ. Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. J Biol Chem 1988; 263: 6352-6362.
  • 21 Tagliavacca L, Moon N, Dunham WR, Kaufman RJ. Identification and functional requirement of Cu(I) and its ligands within coagulation factor VIII. J Biol Chem 1997; 272: 27428-27434.
  • 22 Eaton D, Rodriguez H, Vehar GA. Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry 1986; 25: 505-512.
  • 23 Fay PJ, Anderson MT, Chavin SI, Marder VJ. The size of human factor VIII heterodimers and the effects produced by thrombin. Biochim Biophys Acta 1986; 871: 268-278.
  • 24 Fay PJ, Haidaris PJ, Smudzin TM. Human factor VIIIa subunit structure. Reconstruction of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit. J Biol Chem 1991; 266: 8957-8962.
  • 25 Pittman DD, Kaufman RJ. Proteolytic requirements for thrombin activation of antihemophilic factor (factor VIII). Proc Natl Acad Sci USA 1988; 85: 2429-2433.
  • 26 Fay PJ, Haidaris PJ, Huggins CF. Role of the COOH-terminal acidic region of A1 subunit in A2 subunit retention in human factor VIIIa. J Biol Chem 1993; 268: 17861-17866.
  • 27 O’Brien LM, Huggins CF, Fay PJ. Interacting regions in the A1 and A2 subunits of factor VIIIa identified by zero-length cross-linking. Blood 1997; 90: 3943-3950.
  • 28 Lollar P, Parker CG. PH-dependent denaturation of thrombin-activated porcine factor VIII. J Biol Chem 1990; 265: 1688-1692.
  • 29 Lollar P, Parker ET. Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog. J Biol Chem 1991; 266: 12481-12486.
  • 30 Fay PJ, Smudzin TM. Characterization of the interaction between the A2 subunit and A1/A3-C1-C2 dimer in human factor VIIIa. J Biol Chem 1992; 267: 13246-13250.
  • 31 Van Dieijen G, Tans G, Rosing J, Hemker HC. The role of phospholipid and factor VIIIa in the activation of bovine factor X. J Biol Chem 1981; 256: 3433-3442.
  • 32 Mertens K, van Wijngaarden A, Bertina RM. The role of factor VIII in the activation of human blood coagulation factor X by activated factor IX. Thromb Haemost 1985; 54: 654-660.
  • 33 Fay PJ, Beattie T, Huggins CF, Regan LM. Factor VIIIa A2 subunit residues 558–565 represent a factor IXa interactive site. J Biol Chem 1994; 269: 20522-20527.
  • 34 Lapan KA, Fay PJ. Localization of a factor X interactive site in the A1 subunit of factor VIIIa. J Biol Chem 1997; 272: 2082-2088.
  • 35 Duncan EM, Duncan BM, Tunbridge LJ, Lloyd JV. Familial discrepancy between the one-stage and two-stage factor VIII methods in a subgroup of patients with haemophilia A. Br J Haematol 1994; 87: 846-848.
  • 36 Rudzki Z, Duncan EM, Casey GJ. et al. Mutations in a subgroup of patients with mild haemophilia A and a familial discrepancy between the one-stage and two-stage factor VIII:C methods. Br J Haematol 1996; 94: 400-406.
  • 37 Parquet-Gernez A, Mazurier C, Goudemand M. Functional and immunological assays of FVIII in 133 haemophiliacs – characterization of a subgroup of patients with mild haemophilia A and discrepancy in 1- and 2-stage assays. Thromb Haemost 1988; 59: 202-206.
  • 38 Gaucher C, Jorieux S, Parquet-Gernez A, Mazurier C. Two mutations of the factor VIII (FVIII) gene, R527W and R531H, are identified in 4 unrelated haemophilia A patients with discrepancy in 1- and 2-stage assays of FVIII coagulant activity. Haemophilia 1996; 02 (Suppl. 01) 223a.
  • 39 Montgomery RR, Hathaway WE, Johnson J. et al. A variant of von Willebrand’s disease with abnormal expression of factor VIII procoagulant activity. Blood 1982; 60: 201-207.
  • 40 Schwaab R, Oldenburg J, Kemball-Cook G. et al. Assay discrepancy in mild haemophilia A due to a factor VIII missense mutation (Asn694Ile) in a large Danish family. Br J Haematol 2000; 109: 523-528.
  • 41 Pipe SW, Eickhorst AN, McKinley SH. et al. Mild hemophilia A caused by increased rate of factor VIII A2 subunit dissociation: evidence for nonproteolytic inactivation of factor VIIIa in vivo. Blood 1999; 93: 176-183.
  • 42 Pipe SW, Saenko EL, Eickhorst AN. et al. Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa. Blood 2001; 97: 685-691.
  • 43 Celie PH, Van Stempvoort G, Jorieux S. et al. Substitution of Arg527 and Arg531 in factor VIII associated with mild haemophilia A: characterization in terms of subunit interaction and cofactor function. Br J Haematol 1999; 106: 792-800.
  • 44 Mazurier C, Gaucher C, Jorieux S, Parquet-Gernez A. Mutations in the FVIII gene in seven families with mild haemophilia A. Br J Haematol 1997; 96: 426-427.
  • 45 Keeling DM, Sukhu K, Kemball-Cook G. et al. Diagnostic importance of the two-stage factor VIII:C assay demonstrated by a case of mild haemophilia associated with His1954→Leu substitution in the factor VIII A3 domain. Br J Haematol 1999; 105: 1123-1126.
  • 46 Nesbitt IM, Hinks JL, Sampson B. et al. (eds). Discrepant one and two stage FVIII assays are very common in mild haemophilia A: Correlation with FVIII gene mutations. Proceedings of the XVIIIth ISTH Congress 2001. Thromb Haemost 2001; OV195.
  • 47 Roelse JC, de Laaf RTM, Mertens K. et al. Altered sensitivity towards factor IXa expolains assay discrepancies in plasma of hamophilia A patients with a GLU720 LYS substitution in factor VIII. Thromb Haemost 1999; 82: 5a.
  • 48 Goodeve AC, Hinks JL, Nesbitt IM. et al. (eds). Unusual descrepant factor VIII:C assays in haemophilia A patients with TYR346CYS and GLU321LYS FVIII gene mutations. Proceedings of the XVIIIth ISTH Congress 2001. Thromb Haemost 2001; P1370.
  • 49 Mumford AD, Laffan M, O’Donnell J. et al. A Tyr346→Cys substitution in the interdomain acidic region a1 of factor VIII in an individual with factor VIII:C assay discrepancy. Br J Haematol 2002; 118: 589-594.
  • 50 Trossaert M, Regnault V, Sigaud M. et al. Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype. J Thromb Haemost 2008; 06: 486-493.
  • 51 Cid AR, Calabuig M, Cortina V. et al. One-stage and chromogenic FVIII:C assay discrepancy in mild haemophilia A and the relationship with the mutation and bleeding phenotype. Haemophilia 2008; 14: 1049-1054.
  • 52 Lyall H, Hill M, Westby J. et al. Tyr346→Cys mutation results in factor VIII:C assay discrepancy and a normal bleeding phenotype – is this mild haemophilia A?. Haemophilia 2008; 14: 78-80.