Identification of factor inhibitors by diagnostic haemostasis laboratories

Summary

We have assessed the proficiency of diagnostic haemostasis facilities to correctly identify coagulation factor abnormalities and inhibitors. Forty-two laboratories participating in the external Quality Assurance Program (QAP) conducted by the RCPA agreed to participate and were each sent a set of eight sample (each 3x1ml) for evaluation. They were asked to blind test these samples for the presence or absence of inhibitors, and wher identified, to perform further analysis (including specific inhibitor analysis). In order to make the exercise more challenging, in addition to true factor inhibitors, samples were provided that reflected potential pre-analytical variables that might arise and complicate inhibitor detection or lead to false inhibitor identification. In brief, the sample set comprised a true high level factor (F)V inhibitor, a true moderate level FVIII inhibitor (but sample was defibrinogenated),a true lupus anticoagulant (LA),a normal (but slightly aged) plasma sample, a normal serum sample, a normal EDTA sample, an oral anticoagulant/vitamin K deficiency sample, and a gross heparin (∼10U/ml) contaminated sample. Sixty-three percent of participants correctly identified the true FV inhibitor as such, although the reported range varied greatly [10 to >250 Bethesda units (BU/ml)] and 46% correctly identi fied the true FVIII inhibitor, despite the complication of the sample presentation, although the reported range also varied (7 to 64 BU/ml). Some laboratories either failed to identify the inhibitor present, or misidentified the inhibitor type. The LA, the oral anticoagulant/vitamin K deficiency, the normal serum sample, and the normal (aged) sample were also correctly identified by most laboratories, as was the absence of specific factor inhibitors in these samples. However, a small subset of laboratories incorrectly identified the presence of specific factor inhibitors in some of these samples. The heparin sample was also correctly identified by most (68%) laboratories. In contrast, the normal EDTA sample was misidentified as a FV and/or FVIII inhibitor by most (68%) laboratories, and only one laboratory correctly identified this as an EDTA sample. Thus, we conclude that although laboratories are able, in most cases, to identify the presence of true factor inhibitors, there is a large variation in identified inhibitor levels and there are also some significant errors in identification (i.e. false negatives and misidentifications). In addition, there is a significant false positive error rate where some laboratories will identify the presence of specific factor inhibitors where no such inhibitor exists (i.e. false positives).

Keywords

Lupus anticoagulant - laboratory assessment - haemostasis testing - diagnostic practice - quality control - Bethesda assa

• References

• 1 Macik BG, Crow P. Acquired autoantibodies to coagulation factors. Cur Opin Hematol 1999; 06: 323-8.
• 2 Key N. Inhibitors in congenital coagulation dis orders. Br J Haematol 2004; 127: 379-91.
• 3 Green D. Spontaneous inhibitors to coagulation factors. Clin Lab Haematol 2000; 22 (Suppl. 01) 21-5.
• 4 Wright J, Paisley S. The epidemiology of inhibitors in haemophilia A: a systematic review. Haemophilia 2003; 09: 418-35.
• 5 Morado M, Villar A, Jimenez VYuste. et al. Prophylactic treatment effects on inhibitor risk: experience in one centre. Haemophilia 2005; 11: 79-83.
• 6 Penner JA. Haemophilic patients with inhibitors to factor VIII or IX: variables affecting treatment re sponse. Haemophilia 2001; 07: 103-8.
• 7 Ljung R, Petrini P, Tengborn L. et al. Haemophilia B mutations in Sweden: a population-based study of mutational heterogeneity. Br J Haematol 2001; 113: 81-6.
• 8 Kasper CK. Laboratory diagnosis of factor VIII in hibitors. In: Acquired Haemophilia. 2nd edition.. Princeton: Excerpta Medica Inc; 1995: 9-24.
• 9 Giles AR, Verbruggen B, Rivard GE. et al. 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. Association of He mophilia Centre Directors of Canada. Factor VIII/IX SSC Subcommittee of ISTH. Thromb Haemost 1998; 79: 872-5.
• 10 Verbruggen B, Novakova I, Wessels H. et al. The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability. Thromb Haemost 1995; 73: 247-51.
• 11 Hay CR, Colvin BT, Ludlam CA. et al. Recommendations for the treatment of factor VIII inhibitors: from the UK Haemophilia Centre Directors’ Organisation Inhibitor Working Party. Blood Coag Fibrinolysis 1996; 07: 134-8.
• 12 Govindaswamy S, Chandler J, Latimer R. et al. Management of the patient with coagulation disorders. Cur Opin Anaesthesiol 2002; 15: 19-25.
• 13 Gringeri A. Mannucci PM (on behalf Italian Association of Haemophilia Centres). Italian guidelines for the diagnosis and treatment of patients with haemophilia and inhibitors. Haemophilia 2005; 11: 611-9.
• 14 Cobas M. Preoperative assessment of coagulation disorders. Int Anesthesiol Clin 2001; 39: 1-15.
• 15 Ortel TL. Clinical and laboratory manifestations of V anti-factor V antibodies. J Lab Clin Med 1999; 133: 326-34.
• 16 Favaloro EJ, Posen J, Ramakrishna R. et al. Factor inhibitors: rare or not so uncommon? A multilaboratory investigation. Blood Coag Fibrinolysis 2004; 15: 637-47.
• 17 Shobeiri SA, West EC, Kahn MJ. et al. Postpartum acquired hemophilia (factor VIII inhibitors): A case report and review of the literature. Obs Gyn Survey 2000; 55: 729-37.
• 18 RCPA Haematology QAP contact and program details. available at: http://www.rcpaqap.com.au/ Last accessed on November 15, 2005.
• 19 Gatti G, Mannucci PM. Use of porcine factor VIII in the management of seventeen patients with factor VIII antibodies. Thromb Haemostas 1984; 51: 379-84.
• 20 Ling M, Duncan EM, Rodgers SE. et al. Low detection rate of antibodies to non-functional epitopes on factor VIII in patients with haemophilia A and negative for inhibitors by Bethesda assay. J Thromb Haemost 2003; 01: 2548-53.
• 21 Sahud M, Ruden S, Soriano C. et al. Characterization of factor VIII autoantibodies with complex reaction kinetics is improved by heat inactivation of the tes plasma to remove residual factor activity. J Thromb Haemost 2005; 03 (Suppl. 01) P0651.
• 22 Austen DEG, Lechner K, Rizza CR. et al. A comparison of the Bethesda and New Oxford Methods of factor VIII antibody assay. Thromb Haemost 1982; 47: 72-5.
• 23 Kitchen S, Preston FB, Raut S. et al. Collaborative study on determination of FVIII inhibitors: SSC working group study on behalf of the SSC FVIII/IX sub comittee. Report to participants. Study summary reported at the 2001 ISTH SSC meeting. Minutes available at http://www.med.unc.edu/isth/ssc Last accessed December 2, 2005.
• 24 Reber G, Aurousseau MH, Dreyfus M. et al. Interlaboratory variability of the measurement of low titer factor VIII:C inhibitor in haemophiliacs: improvement by the Nijmegen modification of the Bethesda assay and the use of common lyophilized plasmas. Haemophilia 1999; 05: 292-3.