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Factor VIII Activity, Clotting

Test code(s) 347(X)

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, post-partum women, and in older individuals with no underlying disease.

Test code 40083 (Factor VIII Inhibitor Panel) utilizes a reflexive approach beginning with the measurement of factor VIII activity (which should be low in the presence of an inhibitor), followed by an ELISA-based assay for the detection of IgG factor VIII antibodies. If 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 (containing 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.

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 will yield overly shortened clotting times, and therefore grossly elevated factor VIII activity and false-negative inhibitor studies by the Nijmegen-Bethesda assay.

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.8

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


  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;20:230-237.
  2. Blanchette VS, Key NS, Ljung LR, et al. Definitions in hemophilia: communication from the SSC of the ISTH. J Thromb Haemost. 2014;12:1935-1939.
  3. Franchini M, Lippi G. Acquired factor VIII inhibitors. Blood. 2008;112:250-255.
  4. Adynovate [package insert]. Westlake Village, CA: Baxalta US; March 2017.
  5. Eloctate [package insert]. Waltham, MA: Bioverativ Therapeutics; December 2017.
  6. 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;24:823-832.
  7. Jivi [package insert]. Whippany, NJ: Bayer HealthCare; August 2018.
  8. Hemlibra [package insert]. South San Francisco, CA: Genentech; October 2018.


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

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Effective 01/28/2019 to present