Haemophilia A is a bleeding disorder caused by inadequate levels of FVIII.
Watch the following videos to learn about the coagulation tests to use in haemophilia A, what to look for and how to interpret the results.
The videos and accompanying information are from a 2019 question and answer session with Dr Annette Bowyer, Lead Biomedical Scientist at Royal Hallamshire Hospital.
Which coagulation assays are used in patients with haemophilia A, and why?
Dr Bowyer explains:
- The basic coagulation assay for patients with haemophilia A is the activated partial thromboplastin time, or aPTT. This screening test measures the intrinsic pathway of coagulation, including FVIII. In patients with haemophilia A, the aPTT may be prolonged, although some patients with mild haemophilia A may have a normal aPTT
- FVIII activity assays are used both to diagnose patients with haemophilia A, and to monitor treatment with FVIII. The trough level can be measured to ensure that there is adequate treatment on board in between dosing, and peak levels are measured to check that the patient has enough FVIII on board to prevent bleeding
- FVIII prophylaxis regimens can be tailored according to FVIII activity, and the half-life of FVIII in each individual. The half-life is determined by injecting a standard dose of FVIII concentrate into the patient; the level is monitored for the time it takes for the FVIII activity to fall by half
How are coagulation assays in haemophilia A performed, and how are the results interpreted?
Dr Bowyer explains:
- FVIII activity assays are usually monitored using a one-stage aPTT-based clotting test. This is a modified aPTT by inclusion of a FVIII deficient plasma, which makes the test specific for FVIII activity, and by dilution of the patient plasma. FVIII is quantified by comparing the patient clotting time with that of a reference plasma
- FVIII activity can also be monitored using a chromogenic substrate activity assay. This measures the activation of FX by its cofactors, FIXa and FVIIIa, in the Xa complex. Generated FXa cleaves a chromogenic substrate and a colour occurs, which can be measured by optical density. FVIII activity is quantified by comparing optical density of the patient sample with that of a reference plasma. The lower limit of FVIII activity is around 50 u/dl and levels below this may indicate the presence of haemophilia A
- Assays are repeated to check for an expected response to FVIII treatment. Trough levels are measured in between treatment doses to check that levels are above zero to ensure patients are not exposed to the risk of having a bleed. Peak levels are also checked. If FVIII activity is not as expected, this could indicate the development of an inhibitor to FVIII
How are coagulation assays used to indicate the presence of FVIII inhibitors?
Dr Bowyer explains:
- FVIII inhibitor titre is measured by a Bethesda assay. This involves mixing equal volumes of patient plasma and a plasma with a known concentration of FVIII. This is incubated for two hours at 37ºC, and the residual FVIII activity is measured with a one-stage aPTT-based clotting assay. A control tube is set up containing FVIII-deficient plasma and the same plasma with known FVIII concentration
- No loss of residual FVIII in the patient mix indicates the absence of FVIII inhibitor, while a low response may indicate the presence of a FVIII inhibitor. A 50% loss of residual FVIII activity indicates the presence of one Bethesda unit of inhibitor. Inhibitors of <5 Bethesda units/ml are considered low titre; inhibitors of ≥5 Bethesda units are high titre
Biography, Dr Annette Bowyer, Lead Biomedical Scientist, Royal Hallamshire Hospital
Dr Bowyer is the lead biomedical scientist for the haemophilia assays section, in the department of coagulation at the Sheffield Haemophilia and Thrombosis Centre, a designated World Federation of Hemophilia (WFH) International Hemophilia Training Centre. She was awarded her PhD in cardiovascular science from the University of Sheffield with a thesis focusing on the laboratory diagnosis of mild haemophilia A.
With more than 15 years of experience in the diagnosis and monitoring of bleeding disorders, Dr Bowyer has a particular interest in laboratory issues surrounding extended half-life treatment products for haemophilia. Dr Bowyer has also published a review paper, entitled 'Laboratory issues in gene therapy and emicizumab' (Haemophilia, 2020).
Dr Bowyer is a fellow of the Institute of Biomedical Science and a member of the International Society on Thrombosis and Haemostasis (ISTH), the WFH and the UKHCDO laboratory working party.
aPTT, activated partial thromboplastin time; FVIII, Factor VIII; FIX, Factor IX; FIXa, activated Factor IX; FX, Factor X; ISTH, International Society on Thrombosis and Haemostasis; WFH, World Federation of Hemophilia.
- HEMLIBRA Summary of Product Characteristics.
Date of preparation: July 2020