PDF herunterladen
Thromb Haemost 2021; 121(01): 027-035
DOI: 10.1055/s-0040-1715443
Coagulation and Fibrinolysis

Genetics and Hemostatic Potential in Persons with Mild to Moderate Hemophilia A with a Discrepancy between One-Stage and Chromogenic FVIII Assays

Annelie Strålfors
1   Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
2   Clinical Chemistry, Karolinska University Laboratory, Karolinska University Hospital, Stockholm, Sweden
,
Danijela Mikovic
3   Hemostasis Department and Hemophilia Center, Blood Transfusion Institute of Serbia, Belgrade, Serbia
,
David Schmidt
4   Coagulation Unit, Department of Hematology, Karolinska University Hospital, Stockholm, Sweden
5   Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden
,
Liselotte Onelöv
2   Clinical Chemistry, Karolinska University Laboratory, Karolinska University Hospital, Stockholm, Sweden
,
Nida Mahmoud Hourani Soutari
1   Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
2   Clinical Chemistry, Karolinska University Laboratory, Karolinska University Hospital, Stockholm, Sweden
,
Maria Berndtson
1   Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
2   Clinical Chemistry, Karolinska University Laboratory, Karolinska University Hospital, Stockholm, Sweden
,
Roza Chaireti
1   Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
4   Coagulation Unit, Department of Hematology, Karolinska University Hospital, Stockholm, Sweden
,
Margareta Holmström
4   Coagulation Unit, Department of Hematology, Karolinska University Hospital, Stockholm, Sweden
5   Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden
,
Jovan P. Antovic
1   Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
2   Clinical Chemistry, Karolinska University Laboratory, Karolinska University Hospital, Stockholm, Sweden
,
Maria Bruzelius
1   Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
4   Coagulation Unit, Department of Hematology, Karolinska University Hospital, Stockholm, Sweden
5   Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden
› Institutsangaben Funding M.B. was supported by funds from Stockholm County Council.
› Weitere Informationen
Auch verfügbar auf
    Lizenzen und Reprints

Abstract

Background Factor VIII (FVIII) activity (FVIII:C) can be measured by different methods including one-stage clotting assays (OSAs) and chromogenic assays (CSAs). Discrepancy between FVIII:C assays is known and associated with genetic variations causing mild and moderate hemophilia A (HA). We aimed to study the discrepancy phenomenon and to identify associated genetic alterations. Further, we investigated if hemostatic global assays could discriminate the group with discrepant FVIII:C from them.

Methods The study contained plasma samples from 45 patients with HA (PwHA) from Hemophilia Centers in Stockholm, Sweden, and Belgrade, Serbia. We measured FVIII:C with OSA and CSA, sequenced the F8 gene, and performed two global hemostatic assays; endogenous thrombin potential and overall hemostatic potential.

Results Nineteen of 45 PwHA had a more than twofold higher FVIII:C using OSA compared to CSA and were considered discrepant. Thirty-four causal mutations were detected, where of five had not previously been associated with assay discrepancy. These novel mutations were p.Tyr25Cys, p.Phe698Leu, p.Met699Leu, p.Ile1698Thr, and Ala2070Val. We found no difference between discrepant and nondiscrepant cases with either of the global assays.

Conclusion There was a discrepancy between FVIII:C assays in almost half of the PwHA, which for some could lead to missed HA diagnoses or misclassification of severity. Genotyping confirmed that mutations associated with FVIII:C discrepancy cluster in the A domains of F8, and five mutations not previously associated with FVIII:C discrepancy was identified. Global hemostatic assays did not contribute to distinguish assay discrepancy in PwHA.

Keywords

clinical laboratory techniques - coagulation factor VIII - DNA mutational analysis - hemophilia A - diagnostic errors

Authors' Contributions

A.S. analyzed and interpreted the data and wrote the manuscript. M.B. was involved in the recruitment of patients, interpretation of the data, and contributed to the writing of the manuscript. D.M. was involved in the recruitment of patients. L.O. and M.B. took part in designing the study. N.M.H.S. performed laboratory measurements and critically revised the manuscript. R.C. and M.H. were involved in the recruitment of patients and critically revised the manuscript. D.E.S. interpreted data and critically revised the manuscript. J.A. took part in designing the study, interpreted data, and critically revised the manuscript.


Supplementary Material



Publikationsverlauf

Eingereicht: 12. Februar 2020

Angenommen: 02. Juli 2020

Artikel online veröffentlicht:
13. August 2020

© 2020. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 Mannucci PM, Tuddenham EG. The hemophilias–from royal genes to gene therapy. N Engl J Med 2001; 344 (23) 1773-1779
    CrossrefPubMedSuche in Google Scholar
  • 2 White II GC, Rosendaal F, Aledort LM, Lusher JM, Rothschild C, Ingerslev J. Factor VIII and Factor IX Subcommittee. Definitions in hemophilia. Recommendation of the scientific subcommittee on factor VIII and factor IX of the scientific and standardization committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 2001; 85 (03) 560-560
    Thieme ConnectPubMedSuche in Google Scholar
  • 3 Bowyer AE, Duncan EM, Antovic JP. Role of chromogenic assays in haemophilia A and B diagnosis. Haemophilia 2018; 24 (04) 578-583
    CrossrefPubMedSuche in Google Scholar
  • 4 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 (04) 846-848
    CrossrefPubMedSuche in Google Scholar
  • 5 Parquet A, Boneu B, Bosser C. et al. Clinical and biological survey of haemophilia A and B patients infused with French heat-treated concentrates. Nouv Rev Fr Hematol 1988; 30 (04) 205-207
    PubMedSuche in Google Scholar
  • 6 Keeling DM, Sukhu K, Kemball-Cook G, Waseem N, Bagnall R, Lloyd JV. 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 (04) 1123-1126
    CrossrefPubMedSuche in Google Scholar
  • 7 Hathaway WE, Christian MJ, Jacobson LJ. Variant mild hemophilia - discrepancy in one stage and 2-stage factor-VIII assays. Thromb Haemost 1983; 50 (01) 357-357
    PubMedSuche in Google Scholar
  • 8 Rudzki Z, Duncan EM, Casey GJ, Neumann M, Favaloro EJ, Lloyd JV. 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 (02) 400-406
    CrossrefPubMedSuche in Google Scholar
  • 9 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 (02) 589-594
    CrossrefPubMedSuche in Google Scholar
  • 10 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 (05) 1049-1054
    CrossrefPubMedSuche in Google Scholar
  • 11 Trossaërt M, Regnault V, Sigaud M, Boisseau P, Fressinaud E, Lecompte T. Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype. J Thromb Haemost 2008; 6 (03) 486-493
    CrossrefPubMedSuche in Google Scholar
  • 12 Bowyer AE, Van Veen JJ, Goodeve AC, Kitchen S, Makris M. Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A. Haematologica 2013; 98 (12) 1980-1987
    CrossrefPubMedSuche in Google Scholar
  • 13 Pavlova A, Delev D, Pezeshkpoor B, Müller J, Oldenburg J. Haemophilia A mutations in patients with non-severe phenotype associated with a discrepancy between one-stage and chromogenic factor VIII activity assays. Thromb Haemost 2014; 111 (05) 851-861
    Thieme ConnectPubMedSuche in Google Scholar
  • 14 Duncan EM, Rodgers SE, McRae SJ. Diagnostic testing for mild hemophilia a in patients with discrepant one-stage, two-stage, and chromogenic factor VIII:C assays. Semin Thromb Hemost 2013; 39 (03) 272-282
    Thieme ConnectPubMedSuche in Google Scholar
  • 15 Castaldo G, D'Argenio V, Nardiello P. et al. Haemophilia A: molecular insights. Clin Chem Lab Med 2007; 45 (04) 450-461
    CrossrefPubMedSuche in Google Scholar
  • 16 Potgieter JJ, Damgaard M, Hillarp A. One-stage vs. chromogenic assays in haemophilia A. Eur J Haematol 2015; 94 (Suppl. 77) 38-44
    CrossrefPubMedSuche in Google Scholar
  • 17 Pipe SW, Eickhorst AN, McKinley SH, Saenko EL, Kaufman RJ. 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 (01) 176-183
    CrossrefPubMedSuche in Google Scholar
  • 18 Pipe SW, Saenko EL, Eickhorst AN, Kemball-Cook G, Kaufman RJ. 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 (03) 685-691
    CrossrefPubMedSuche in Google Scholar
  • 19 Rodgers SE, Duncan EM, Barbulescu DM, Quinn DM, Lloyd JV. In vitro kinetics of factor VIII activity in patients with mild haemophilia A and a discrepancy between one-stage and two-stage factor VIII assay results. Br J Haematol 2007; 136 (01) 138-145
    CrossrefPubMedSuche in Google Scholar
  • 20 Antovic JP, Mikovic D, Elezovic I. et al. Two global haemostatic assays as additional tools to monitor treatment in cases of haemophilia A. Thromb Haemost 2012; 108 (01) 21-31
    Thieme ConnectPubMedSuche in Google Scholar
  • 21 Gilmore R, Harmon S, Gannon C, Byrne M, O'Donnell JS, Jenkins PV. Thrombin generation in haemophilia A patients with mutations causing factor VIII assay discrepancy. Haemophilia 2010; 16 (04) 671-674
    PubMedSuche in Google Scholar
  • 22 Trossaert M, Lienhart A, Nougier C. et al. Diagnosis and management challenges in patients with mild haemophilia A and discrepant FVIII measurements. Haemophilia 2014; 20 (04) 550-558
    CrossrefPubMedSuche in Google Scholar
  • 23 Pezeshkpoor B, Gazorpak M, Berkemeier AC. et al. In silico and in vitro evaluation of the impact of mutations in non-severe haemophilia A patients on assay discrepancies. Ann Hematol 2019; 98 (08) 1855-1865
    CrossrefPubMedSuche in Google Scholar
  • 24 Ljungkvist M, Berndtsson M, Holmström M. et al. Correlation to FVIII:C in two thrombin generation tests: TGA-CAT and INNOVANCE ETP. Mediterr J Hematol Infect Dis 2017; 9 (01) e2017064
    CrossrefPubMedSuche in Google Scholar
  • 25 Antovic A. Screening haemostasis–looking for global assays: the overall haemostasis potential (OHP) method–a possible tool for laboratory investigation of global haemostasis in both hypo- and hypercoagulable conditions. Curr Vasc Pharmacol 2008; 6 (03) 173-185
    CrossrefPubMedSuche in Google Scholar
  • 26 Trossaërt M, Boisseau P, Quemener A. et al. Prevalence, biological phenotype and genotype in moderate/mild hemophilia A with discrepancy between one-stage and chromogenic factor VIII activity. J Thromb Haemost 2011; 9 (03) 524-530
    CrossrefPubMedSuche in Google Scholar
  • 27 Provaznikova D, Houskova K, Radovska A, Salaj P, Hrachovinova I. Novel mutations associated with a discrepancy between one-stage and chromogenic FVIII activity assays. Haemophilia 2015; 21 (04) e330-e332
    CrossrefPubMedSuche in Google Scholar
  • 28 Poulsen AL, Pedersen LH, Hvas AM, Poulsen LH, Thykjaer H, Ingerslev J. Assay discrepancy in mild haemophilia A: entire population study in a National Haemophilia Centre. Haemophilia 2009; 15 (01) 285-289
    CrossrefPubMedSuche in Google Scholar
  • 29 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 (03) 523-528
    CrossrefPubMedSuche in Google Scholar
  • 30 Cutler JA, Mitchell MJ, Smith MP, Savidge GF. The identification and classification of 41 novel mutations in the factor VIII gene (F8C). Hum Mutat 2002; 19 (03) 274-278
    CrossrefPubMedSuche in Google Scholar
  • 31 Lannoy N, Abinet I, Bosmans A, Lambert C, Vermylen C, Hermans C. Computational and molecular approaches for predicting unreported causal missense mutations in Belgian patients with haemophilia A. Haemophilia 2012; 18 (03) e331-e339
    CrossrefPubMedSuche in Google Scholar
  • 32 Casaña P, Martínez F, Cabrera N, Cid AR, Aznar JA. Founder haplotype associated with the factor VIII Asp1241Glu polymorphism in a cohort of mild hemophilia A patients. J Thromb Haemost 2008; 6 (08) 1428-1430
    CrossrefPubMedSuche in Google Scholar
  • 33 van Moort I, Meijer P, Priem-Visser D. et al. Analytical variation in factor VIII one-stage and chromogenic assays: Experiences from the ECAT external quality assessment programme. Haemophilia 2019; 25 (01) 162-169
    CrossrefPubMedSuche in Google Scholar