Factor VIII Activity and Inhibitor Assays in the Diagnosis and Treatment of Hemophilia A

Abstract

The treatment of a patient with a factor VIII (FVIII) deficiency can be complicated. The mainstay of therapy is factor replacement. Replacement therapy can be given prophylactically, with the goal of decreasing hemarthroses and spontaneous hemorrhage, or on-demand for the bleeding patient. Intra- and interindividual variability in a patient's response to treatment has been well documented by the differences in observed half-lives of infused product. Although weight-based dosing nomograms are most often used, personalized therapies are coming into use to ease the burden of therapy and cost. The most significant complication of treatment is the formation of inhibitors to FVIII. The role of the laboratory is to provide results for FVIII activity that accurately reflects a patient's baseline level and response to treatment. However, factor activity assays have many components that can contribute to result variability. These include the methodology and reagent components used to measure the FVIII activity, the reference standard employed, algorithm used to interpret the dilutions, and the replacement factor being measured. An understanding of assay variables and their impact will assist in providing accurate factor activity results and appropriate patient care.

Clinical utility and impact of the use of the chromogenic vs one‐stage factor activity assays in haemophilia A and B

“…The aPTT is quantified as the time (seconds) taken for the clot to form from the time point at which calcium is added and is dependent on all of the intrinsic pathway factors, including factor (F) VIII, present in the test plasma (with the exception of FII). A burst of thrombin formation occurs after sufficient levels of activated FVIII (FVIIIa) have been generated through feedback activation by thrombin, leading to the formation of a clot . Adapted with permission from Adcock et al…”

“…Laboratory‐manufactured standards are usually traceable to a secondary standard that has been calibrated against the primary standard. It is recommended that factor activity assays are calibrated at a minimum of once every 6 months, although calibrations are required for new lots of reagents (both deficient plasma and APTT reagents), and some laboratories calibrate more often . The most common commercial factor‐deficient plasmas used in the performance of factor activity assays are immunodepleted for FVIII, FIX or FXI.…”

Postanalytical considerations that may improve the diagnosis or exclusion of haemophilia and von Willebrand disease

“…This may be of additional importance when patients are transferred from primary care facilities, where the former assays are usually the only option, to specialized centres for managing VWD or haemophilia, where chromogenic assays are usually available. A second consideration that will become increasingly important in the future is the recent and ongoing development of extended life replacement products, as some one‐stage tests may under‐ or over‐estimate factor levels depending on the type of replacement therapy . Expert comments on which are the most appropriate tests in patients undergoing different treatments may therefore be valuable to guide prescription (eg chromogenic assays may be more suited for monitoring replacement therapy with β‐domain deletion (BDD) recombinant FVIII) …”

“…An “inhibitor investigation” is always suggested for low FVIII levels detected in a previously undiagnosed patient after preanalytical events have been discounted. Inhibitor investigation may take the form of an initial simple FVIII mixing study to assess the possibility of an inhibitor, or else, a full Bethesda assay may be required in the presence of clinical bleeding, either in a patient with haemophilia or potentially acquired haemophilia . Attributed to the complex kinetics of FVIII inhibitors, incubated assays are generally required …”

“…Failure to achieve the correct diagnosis or exclusion of VWD or haemophilia might be due to analytical issues . Sometimes assays generate inaccurate results (ie reflecting analytical errors) or have limitations in their measurement range of and/or low analytical sensitivity.…”