Chromogenic Assay
- Measures hydrolysis of a p-nitroanilide (pNA) substrate
- The rate of release of pNA is proportional to the factor VIII activity in the sample
- Can quantitate factor activity as low as 1% of normal
Chromogenic factor VIII activity is used for diagnosis of mild hemophilia A (in conjunction with one-stage clot-based factor VIII activity) and measurement of factor VIII activity in the presence of interfering drugs or lupus anticoagulants that result in underestimation by clot-based methods. It is also used for measuring factor VIII activity in patients treated with certain modified extended half-life factor VIII replacement products and emicizumab.
Disease Overview
Incidence
Hemophilia A in 1/4,000-5,000 live male births worldwide; rare in females
Inheritance
X-linked recessive, factor VIII deficiency can also be acquired due to autoantibodies
Visit the Hemophilia - Factor VIII or IX Deficiency Consult topic for additional information about factor VIII deficiency
Diagnostic Issues
Hemophilia A may be classified as mild, moderate or severe, based on factor activity.
Disease Classification | Expected Factor Activity |
---|---|
Mild |
6-35% |
Moderate |
1-5% |
Severe |
<1% |
Mild hemophilia A may require both one-stage clot-based factor VIII activity and chromogenic factor VIII activity for diagnosis, due to differences in how the underlying mutations affect factor VIII activity in the tests.
Monitoring Issues
Modified extended half-life factor VIII replacement products may lead to underestimation of factor VIII activity in clot-based factor VIII assays using certain aPTT reagents.
Factor VIII activity cannot be accurately measured using a one-stage clot-based factor VIII activity assay in the presence of emicizumab.
- Emicizumab is a bispecific antibody that bridges factor IX and factor X to produce activated factor X (FXa). Emicizumab effectively replaces the function of factor VIII in secondary hemostasis
- Emicizumab will substitute for factor VIII function in one stage clot-based factor VIII activity assays and will result in overestimation of factor VIII activity (either native factor VIII or factor VIII concentrate administered in an acute care setting)
- Interference may last for up to to 6 months following end of therapy
- In patients receiving emicizumab, patient factor VIII activity (endogenous or factor VIII concentrate) can be accurately measured using chromogenic factor VIII activity with bovine reagents
- Emicizumab can bind to factor IX and X in chromogenic assays using human factor-derived reagents and will still overestimate factor VIII activity
- The chromogenic factor VIII activity assay at ARUP Laboratories uses bovine reagents and is not affected by emicizumab
- Measuring factor VIII inhibitors in patients receiving emicizumab requires use of a Bethesda assay based on a chromogenic factor VIII assay (bovine reagents)
Test Interpretation
Results
- Age-specific reference intervals are provided for each result on the patient chart
- Decreased factor VIII activity is expected in patients with hemophilia A (see table above for disease classification) and is associated with increased risk of bleeding
Limitations
- Decreased chromogenic factor VIII activity results may also be caused by:
- von Willebrand disease
- Specimen collection and storage issues:
- Uncontrolled freeze-thaw cycles
- Prolonged ambient storage
- Activated or clotted specimens
- Anticoagulant medications (assay interference)
- Heparin (>2 U/mL)
- Direct thrombin inhibitors
- Direct Xa inhibitors
- Factor VIII activity may be elevated above usual baseline (normal or high result could mask underlying deficiency) in patients with acute phase responses
- Normal factor VIII activity does not exclude female hemophilia carrier status
References
-
22551712
Fijnvandraat K, Cnossen MH, Leebeek FW , et al. Diagnosis and management of haemophilia. BMJ. 2012;344:e2707.
-
18510526
Verbruggen B, Meijer P, Novákova I , et al. Diagnosis of factor VIII deficiency. Haemophilia. 2008;14 Suppl 3:76-82.
-
29765290
Graf L. Extended Half-Life Factor VIII and Factor IX Preparations. Transfus Med Hemother. 2018;45(2):86-91.
-
28264199
Kitchen S, Tiefenbacher S, Gosselin R. Factor Activity Assays for Monitoring Extended Half-Life FVIII and Factor IX Replacement Therapies. Semin Thromb Hemost. 2017;43(3):331-337.
-
29274194
St Ledger K, Feussner A, Kalina U, et al. International comparative field study evaluating the assay performance of AFSTYLA in plasma samples at clinical hemostasis laboratories. J Thromb Haemost. 2018;16(3):555-564.
-
31203578
Müller J, Pekrul I, Pötzsch B, et al. Laboratory monitoring in emicizumab-treated persons with hemophilia A. Thromb Haemost. 2019;119(9):1384-1393.