CONTINUING EDUCATION
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LEARNING OBJECTIVES
1. Describe the pathogenicity of hemophilia.
2. Discuss different assays currently used to diagnose and monitor hemophilia.
3. Discuss the factors that contribute to inaccuracies in different factor activity test methodologies.
4. Describe procedures and ideas to overcome the problematic reporting of inaccurate results in patients with hemophilia.
Hemophilia A and B are X-linked disorders characterized by decreased functional factor VIII (FVIII) and factor IX (FIX) levels, respectively. Disease severity is classified as severe (<1% factor activity), moderate (1%–5%), or mild (6%–<40%).1 Bleeding risk correlates with level of factor activity, and when levels fall below one percent, spontaneous bleeding occurs. The goal of coagulation factor replacement therapy is to elevate and sustain levels in order to achieve a functional factor concentration of one percent or greater.2
Given the importance of accurate measurement of factor activity levels in the diagnosis, classification, and therapeutic monitoring of coagulation factor replacement in hemophilia A and B, accurate assessment of FVIII and FIX activity in the clinical laboratory is critical.3 The coagulation factor activity assay used by the majority of clinical laboratories, the one-stage assay (OSA), may underestimate or overestimate the true FVIII activity in up to 30 percent of patients with mild or moderate (non-severe) hemophilia A, and this has more recently been described as a potential issue in hemophilia B.3-5 Furthermore, certain modified recombinant FVIII and FIX replacement products demonstrate variable and clinically significant differences in post-infusion recovery (that is, the amount of factor measured vs. the actual concentration present), based on the activated partial thromboplastin time (APTT) reagent used in the OSA or assay methodology.6 This article will review when and why discrepancies may occur, as well as what is needed to overcome the potential for inaccurate results in FVIII and FIX measurements, in order to provide optimal patient care.
Approximately 16 percent of patients with mild hemophilia A have a normal FVIII OSA, and the correct diagnosis relies on another less commonly performed activity assay using chromogenic methodology.3,4 Variations in one-stage activity results using different APTT reagents, as well as differences in activity results when measured in the OSA versus chromogenic FIX activity assay, have recently been described in non-severe hemophilia B patients.5 Differences in factor activity measurements between the OSA and chromogenic assay (CSA) in non-severe hemophilia is called discrepant hemophilia. This occurs in 30 percent of patients with non-severe hemophilia A and an as-yet unknown percentage of those with hemophilia B.3,4,7 Such discrepancies may also occur in hemophilia carriers. Although there is no universally accepted definition of what constitutes discrepant hemophilia, the generally accepted criterion is a twofold difference in results between the OSA and CSA. Either OSA results can be greater than CSA or vice versa, depending on the underlying FVIII or FIX gene mutation.3,4,8 Both possibilities can misclassify hemophilia severity, but the former may result in a missed diagnosis.
Hemophilia A
Discrepant hemophilia A has a genetic basis, generally due to missense mutations that affect the stability of the activated form of FVIII (FVIIIa) or the ability of FVIII to successfully bind the activated form of FIX (FIXa), von Willebrand factor, or thrombin.3,8 Missense mutations clustered in the A1-A2-A3 domain interfaces of the FVIII protein cause reduced stability of FVIIIa, which is more apparent in FVIII activity assays where the FVIIIa is generated during a relatively long (for example, two-to-ten-minute) incubation such as the CSA or the infrequently performed two-stage assay.4,8 In the OSA, FVIII is in the activated form for only a brief period. Missense mutations clustered around thrombin cleavage sites or FIXa binding sites are more readily identified in OSA since the factors are present at physiologic concentrations, unlike the CSA, where factor concentrations are optimized. Also, long incubation times in the CSA may help to overcome mutations that interfere with binding. The underlying mutations and discrepancies between OSA and CSA are consistent within and between discrepant hemophilia A families.
In the one-stage FVIII activity assay, patient sample is mixed with FVIII-deficient plasma and APTT reagent, and this mixture is allowed to incubate at 37°C for three to five minutes.7 This allows contact activation but does not activate the FVIII that is present in the mixture. The mixture is recalcified, and the time to clot formation is measured in seconds and compared to a standard curve (Figure 1). In the CSA, patient sample is mixed with purified FIXa and FX, with or without added prothrombin or thrombin. The concentrations of these factors are optimized and are not necessarily physiologic. This mixture is incubated for five to seven minutes in order to generate FXa, and the amount of FXa produced is dependent on the amount of FVIIIa generated during this incubation.7,9 In the second stage of the assay, the amount of FXa generated is determined using a chromogenic substrate, and this is compared to a standard curve in order to determine the FVIII activity present in the sample (Figure 2).