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Quest Diagnostics

Factor VIII Activity and Inhibitor Testing

Test codes:

  • 347: Factor VIII Activity, Clotting
  • 15586: Factor VIII Inhibitor, Chromogenic
  • 16049: Factor VIII Activity, Chromogenic
  • 40083: Factor VIII Inhibitor Panel

Factor VIII clotting activity is determined using an aPTT (activated partial thromboplastin time)-based 1-stage clotting assay. The diluted patient sample (a minimum of 3 dilutions) is mixed with factor VIII-deficient plasma, and the clotting time of the mix is then compared to the clotting time of normal pooled plasma.

Hemophilia A and B are X-linked inherited bleeding disorders characterized by decreased factor VIII and IX, respectively. Hemophilia A is the more common disorder, affecting approximately 82% of all patients with hemophilia.1

The severity of hemophilia A is defined by the level of factor VIII activity. Severe, moderate, and mild hemophilia are associated with factor VIII activity levels of <1%, 1% to 5%, and >5% to <40%, respectively.2  Patients with severe or moderate hemophilia A often need specific substitution therapy with plasma-derived or recombinant factor VIII concentrates.  A major complication of replacement therapy is the development of alloantibodies (inhibitors) that inactivate factor VIII.

Autoantibodies may also develop in patients without a congenital disorder (referred to as acquired hemophilia), resulting in life-threatening bleeding symptoms.3 Acquired hemophilia can develop in patients with immunologic disorders such as rheumatoid arthritis, in post-partum women, and in older individuals with no underlying disease.

Factor VIII Inhibitor Panel (test code 40083) utilizes a reflexive approach beginning with the measurement of factor VIII activity (which should be low in the presence of an inhibitor), followed by a chromogenic screen for the detection of IgG factor VIII antibodies. If the chromogenic screen is positive, the antibody titer is determined using the Nijmegen-Bethesda assay. In this method, serial dilutions of the patient specimen are mixed with normal pool plasma, which contains factor VIII, and then incubated.

The aPTT-based 1-stage factor VIII clotting assay is then performed to measure the residual activity in the mixtures. One Bethesda Unit (BU) is defined as the amount of inhibitor that results in 50% residual clotting activity.

Whereas the Bethesda assay detects the presence of a neutralizing antibody (eg, functional activity is decreased), the ELISA assay will detect both neutralizing and non-neutralizing antibodies.

The effect of extended half-life replacement therapies on aPTT-based factor VIII activity levels may vary, depending on the aPTT reagent used by the laboratory. These differences have been attributed to the type of activator in the reagent.6 As an example, the table below illustrates the monitoring requirements for various extended half-life factor VIII therapies, comparing the 1-stage clotting assays (OSA) to another methodology for factor VIII measurement, the chromogenic assay (CSA).

The aPTT reagent in use at Quest Diagnostics utilizes an ellagic acid activator. Therefore, the factor VIII activity and/or inhibitor titer assays performed at Quest are suitable for the replacement therapies listed in this table.

click the table to open up in a new window (enlarged)

No. Emicizumab is a bispecific factor IXa- and factor X-directed antibody. While not a direct factor VIII replacement, it restores the missing cofactor activity to propagate the clotting cascade. Therefore, coagulation laboratory tests based on the aPTT may be grossly elevated (eg, factors VIII, IX and XI, XII) and there may be false-negative inhibitor studies by the Nijmegen-Bethesda assay.

Factor VIII chromogenic-based assays using bovine coagulation factors (test code 16049) are insensitive to emicizumab and can be used to monitor endogenous or infused factor VIII activity. As stated in the Hemlibra package insert, due to the long half-life of the drug, the effect on aPTT-based assays may persist for up to 6 months after the last dose.10

In addition, Quest Diagnostics offers a chromogenic bovine factor VIII inhibitor assay (test name: Factor VIII Inhibitor, Chromogenic; test code 15586) that is not affected by emicizumab. Patients can get accurate factor VIII inhibitor levels while on this medication.

Factor VIII is a positive acute-phase reactant. Persistent elevation of factor VIII has been associated with an increased risk for venous thrombosis, but the exact mechanism is unclear. Levels will increase in response to stress, inflammatory conditions, surgery, and strenuous exercise. Increased levels are also physiologic with pregnancy.

It is appropriate to use a chromogenic factor VIII assay, particularly one that uses bovine coagulation factors, to avoid inference to drugs such as emicizumab, in order to monitor endogenous or infused factor VIII activity. Chromogenic factor VIII assays are also useful in the case of acquired inhibitors such as lupus anticoagulant that can interfere in clot based assays.

In addition, in patients with a suspected bleeding disorder, it is essential that both a 1-stage factor VIII clot-based assay and a chromogenic assay be performed to avoid missing the diagnosis by either assay.

References

  1. Soucie JM, Miller CH, Kelly FM, et al. A study on prospective surveillance for inhibitors among persons with haemophilia in the United States. Haemophilia. 2014;20(2):230-237. doi:10.1111/hae.12302
  2. Blanchette VS, Key NS, Ljung LR, et al.  Definitions in hemophilia: communication from the SSC of the ISTH. J Thromb Haemost. 2014;12(11):1935-1939. doi:10.1111/jth.12672
  3. Franchini M, Lippi G. Acquired factor VIII inhibitors. Blood. 2008;112(2):250-255. doi:10.1182/blood-2008-03-143586
  4. Adynovate [package insert]. 2017.
  5. Altuviiio [package insert]. 2025.
  6.  Pipe S, Sadeghi-Khomami A, Konkle BA, et al. A global comparative field study to evaluate the factor VIII activity of efanesoctocog alfa by one-stage clotting and chromogenic substrate assays at clinical haemostasis laboratories. Haemophilia. 2024;30(1):214-223. doi:10.1111/hae.14831
  7. Eloctate [package insert]. 2017.
  8. Church N, Leong L, Katterle Y, et al. Factor VIII activity of BAY 94-9027 is accurately measured with most commonly used assays: results from an international laboratory study. Haemophilia. 2018;24(5):823-832. doi:10.1111/hae.13564
  9.  Jivi [package insert]. 2018.
  10. Hemlibra [package insert]. 2018.


This FAQ is provided for informational purposes only and is not intended as medical advice. Test selection and interpretation, diagnosis, and patient management decisions should be based on the clinician’s education, clinical expertise, and assessment of the patient.


Document FAQS.210 Version: 1

Version 1: Effective 12/15/2025 to present

Version 0: Effective 01/28/2019 to 12/15/2025

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