Improvements in Factor VIII Inhibitor Detection: From Bethesda to Nijmegen

ABSTRACT

All methods for the detection of factor VIII (FVIII) inhibitors are based on the measurement of inactivation of FVIII in a mixture of the test plasma containing the putative inhibitor and an exogenous source of FVIII. Various types of assays have been developed since the first inhibitor was described in 1941. Nowadays, two methods are preferably used, the Bethesda assay and the Nijmegen assay. Although the Nijmegen assay shows a better specificity and intra- and interlaboratory variation, it is still hampered by several limitations related to assay characteristics (pH, temperature, and time of incubation), type of control sample, and the von Willebrand factor content of the assay mixture. Epitope specificity plays an important role in the reliability of functional assays because inhibitors against the C2 domain are more difficult to quantify compared with inhibitors against the A2 domain. Finally, lupus anticoagulants can interfere with inhibitor assays, resulting in aberrant results. This report describes in detail the various problems encountered with the assays used in the quantification of functional FVIII inhibitors.

Factor VIII - inhibitors - hemophilia - Bethesda assay - Nijmegen assay

REFERENCES

• 1 Margolius Jr A, Jackson D P, Ratnoff O D. Circulating anticoagulants: a study of 40 cases and a review of the literature.  Medicine (Baltimore). 1961;  40 145-202
• 2 Algiman M, Dietrich G, Nydegger U E, Boieldieu D, Sultan Y, Kazatchkine M D. Natural antibodies to factor VIII (anti-hemophilic factor) in healthy individuals.  Proc Natl Acad Sci U S A. 1992;  89(9) 3795-3799
• 3 Lutz H U, Binder C J, Kaveri S. Naturally occurring auto-antibodies in homeostasis and disease.  Trends Immunol. 2009;  30(1) 43-51
• 4 Lawrence J S, Johnson J B. The presence of a circulating anticoagulant in a male member of a hemophiliac family.  Trans Am Clin Climatol Assoc. 1941;  57 223-231
• 5 Biggs R, Macfarlane R G. Human blood coagulation and its disorders. 2nd ed. Oxford, United Kingdom; Blackwell Scientific Publications 1962
• 6 Biggs R, Bidwell E. A method for the study of antihaemophilic globulin inhibitors with reference to six cases.  Br J Haematol. 1959;  5 379-395
• 7 Biggs R, Austen D E, Denson K W, Rizza C R, Borrett R. The mode of action of antibodies which destroy factor VIII. I. Antibodies which have second-order concentration graphs.  Br J Haematol. 1972;  23(2) 125-135
• 8 Kasper C, Aledort L, Counts R et al.. Letter: A more uniform measurement of factor VIII inhibitors.  Thromb Diath Haemorrh. 1975;  34(3) 869-872
• 9 Rosendaal F R, Nieuwenhuis H K, van den Berg H M, Heijboer H, Mauser-Bunschoten E P, van der Meer J, Smit C, Strengers P F, Briët E. A sudden increase in factor VIII inhibitor development in multitransfused hemophilia A patients in The Netherlands. Dutch Hemophilia Study Group.  Blood. 1993;  81(8) 2180-2186
• 10 Verbruggen B, Novakova I, Wessels H, Boezeman J, van den Berg M, Mauser-Bunschoten E. The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability.  Thromb Haemost. 1995;  73(2) 247-251
• 11 Giles A R, Verbruggen B, Rivard G E, Teitel J, Walker I. Association of Hemophilia Centre Directors of Canada. Factor VIII/IX Subcommittee of Scientific and Standardization Committee of International Society on Thrombosis and Haemostasis . A detailed comparison of the performance of the standard versus the Nijmegen modification of the Bethesda assay in detecting factor VIII:C inhibitors in the haemophilia A population of Canada.  Thromb Haemost. 1998;  79(4) 872-875
• 12 Charles R M, Brown S, Collins P W, Keeling D M, Liesner R. The diagnosis and management of factor VIII and IX inhibitors: a guideline from the United Kingdom Haemophilia Centre Doctors Organization.  Br J Haematol. 2006;  133 591-605
• 13 Brandt J T, Barna L K, Triplett D A. Laboratory identification of lupus anticoagulants: results of the Second International Workshop for Identification of Lupus Anticoagulants. On behalf of the Subcommittee on Lupus Anticoagulants/Antiphospholipid Antibodies of the ISTH.  Thromb Haemost. 1995;  74(6) 1597-1603
• 14 Gawryl M S, Hoyer L W. Inactivation of factor VIII coagulant activity by two different types of human antibodies.  Blood. 1982;  60(5) 1103-1109
• 15 Berntorp E, Ekman M, Gunnarsson M, Nilsson I M. Variation in factor VIII inhibitor reactivity with different commercial factor VIII preparations.  Haemophilia. 1996;  2 95-99
• 16 Astermark J, Voorberg J, Lenk H et al.. Impact of inhibitor epitope profile on the neutralizing effect against plasma-derived and recombinant factor VIII concentrates in vitro.  Haemophilia. 2003;  9(5) 567-572
• 17 Martin P G, Sukhu K, Chambers E, Giangrande P L. Evaluation of a novel ELISA screening test for detection of factor VIII inhibitory antibodies in haemophiliacs.  Clin Lab Haematol. 1999;  21(2) 125-128
• 18 Regnault V, Stoltz J F. Quantitation of factor VIII antibodies by an enzyme-linked immunoassay method.  Blood. 1994;  83(4) 1155-1156
• 19 Mondorf W, Klinge J, Luban N L, Bray G, Saenko E, Scandella D. Recombinate PUP Study Group . Low factor VIII recovery in haemophilia A patients without inhibitor titre is not due to the presence of anti-factor VIII antibodies undetectable by the Bethesda assay.  Haemophilia. 2001;  7(1) 13-19
• 20 Dazzi F, Tison T, Vianello F et al.. High incidence of anti-FVIII antibodies against non-coagulant epitopes in haemophilia A patients: a possible role for the half-life of transfused FVIII.  Br J Haematol. 1996;  93(3) 688-693
• 21 Sahud M A, Pratt K P, Zhukov O, Qu K, Thompson A R. ELISA system for detection of immune responses to FVIII: a study of 246 samples and correlation with the Bethesda assay.  Haemophilia. 2007;  13(3) 317-322
• 22 Verbruggen B, Giles A, Samis J, Verbeek K, Mensink E, Novákovà I. The type of factor VIII deficient plasma used influences the performance of the Nijmegen modification of the Bethesda assay for factor VIII inhibitors.  Thromb Haemost. 2001;  86(6) 1435-1439
• 23 Verbruggen B, van Heerde W, Novákovà I, Lillicrap D, Giles A. A 4% solution of bovine serum albumin may be used in place of factor VIII:C deficient plasma in the control sample in the Nijmegen Modification of the Bethesda factor VIII:C inhibitor assay.  Thromb Haemost. 2002;  88(2) 362-364
• 24 van Den Brink E N, Turenhout E A, Davies J et al.. Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes.  Blood. 2000;  95(2) 558-563
• 25 Triplett D A. Simultaneous occurrence of lupus anticoagulant and factor VIII inhibitors.  Am J Hematol. 1997;  56(3) 195-196
• 26 Blanco A N, Cardozo M A, Candela M, Santarelli M T, Pérez Bianco R, Lazzari M A. Anti-factor VIII inhibitors and lupus anticoagulants in haemophilia A patients.  Thromb Haemost. 1997;  77(4) 656-659
• 27 Arnout J. Antiphospholipid syndrome: diagnostic aspects of lupus anticoagulants.  Thromb Haemost. 2001;  86(1) 83-91
• 28 Tripodi A, Mancuso M E, Chantarangkul V et al.. Lupus anticoagulants and their relationship with the inhibitors against coagulation factor VIII: considerations on the differentiation between the 2 circulating anticoagulants.  Clin Chem. 2005;  51(10) 1883-1885
• 29 Cohen H, Mackie I J, Anagnostopoulos N, Savage G F, Machin S J. Lupus anticoagulant, anticardiolipin antibodies, and human immunodeficiency virus in haemophilia.  J Clin Pathol. 1989;  42(6) 629-633
• 30 al-Saeed A, Makris M, Malia R G, Preston F E, Greaves M. The development of antiphospholipid antibodies in haemophilia is linked to infection with hepatitis C.  Br J Haematol. 1994;  88(4) 845-848
• 31 Kazmi M A, Pickering W, Smith M P, Holland L J, Savidge G F. Acquired haemophilia A: errors in the diagnosis.  Blood Coagul Fibrinolysis. 1998;  9(7) 623-628
• 32 Verbruggen B. Diagnosis and quantification of factor VIII inhibitors.  Haemophilia. 2009;  , February 18 (Epub ahead of print)
• 33 Chantarangkul V, Tripodi A, Arbini A, Mannucci P M. Silica clotting time (SCT) as a screening and confirmatory test for detection of the lupus anticoagulants.  Thromb Res. 1992;  67(4) 355-365
• 34 Triplett D A, Barna L K, Unger G A. A hexagonal (II) phase phospholipid neutralization assay for lupus anticoagulant identification.  Thromb Haemost. 1993;  70(5) 787-793
• 35 Exner T. Diagnostic methodologies for circulating anticoagulants.  Thromb Haemost. 1995;  74(1) 338-344
• 36 Blanco A N, Alcira Peirano A, Grosso S H, Gennari L C, Pérez Bianco R, Lazzari M A. A chromogenic substrate method for detecting and titrating anti-factor VIII antibodies in the presence of lupus anticoagulant.  Haematologica. 2002;  87(3) 271-278
• 37 Chandler W L, Ferrell C, Lee J, Tun T, Kha H. Comparison of three methods for measuring factor VIII levels in plasma.  Am J Clin Pathol. 2003;  120(1) 34-39
• 38 Ling M, Duncan E M, Rodgers S E et al.. Classification of the kinetics of factor VIII inhibitors in haemophilia A: plasma dilution studies are more discriminatory than time-course studies.  Br J Haematol. 2001;  114(4) 861-867
• 39 Prescott R, Nakai H, Saenko E L Recombinate and Kogenate Study Groups et al. The inhibitor antibody response is more complex in hemophilia A patients than in most nonhemophiliacs with factor VIII autoantibodies.  Blood. 1997;  89(10) 3663-3671
• 40 Salvagno G L, Astermark J, Ekman M et al.. Impact of different inhibitor reactivities with commercial factor VIII concentrates on thrombin generation.  Haemophilia. 2007;  13(1) 51-56
• 41 Meijer P, Verbruggen B. The between-laboratory variation of factor VIII inhibitor testing: the experience of the external quality assessment programme of the ECAT Foundation.  Semin Thromb Hemost. 2009;  35(8) xxx
• 42 Kitchen S, Jennings I, Preston F E, Kitchen D P, Woods T AL, Walker I D. Interlaboratory variation in factor VIII:C inhibitor assay results is sufficient to influence patient management: data from the UK NEQAS for blood coagulation.  Semin Thromb Hemost. 2009;  35(8) xxx
• 43 Favaloro E J, Bonar R, Kershaw G, Duncan E, Sioufi J, Marsden K. Investigations from external quality assurance programs reveal a high degree of variation in the laboratory identification of coagulation factor inhibitors.  Semin Thromb Hemost. 2009;  35(8) xxx