What is the physiologic importance of factor VIII?

The activated form of coagulation factor VIII (FVIII) is an essential cofactor that together with its partner, the protease FIXa, cleaves factor X to form the active Xa. This is an essential and critical step in the propagation phase of coagulation. A hereditary deficiency of FVIII is the cause of the X-linked disorder hemophilia A, a bleeding disorder that affects approximately 1 in 5,000 male infants. Hemophilia is classified as mild, moderate, or severe depending on the measured activity of FVIII, with 6%-50% activity considered mild, 1%-5% moderate, and < 1% severe.

What role do clinical laboratory tests play in hemophilia treatment?

Hemophilia is commonly treated with an infusion of recombinant FVIII, but one problem with this form of therapy is that patients might form alloantibodies to the infused factor. FVIII antibodies (also known as inhibitors) are diagnosed most commonly in male patients with severe hemophilia A who are taking factor replacement therapy; the prevalence in this patient group is about 15%-20%. These inhibitors result from exposure to human FVIII in a setting where endogenous FVIII is absent or near-absent. In normal individuals who have had no prior bleeding problems, factor VIII inhibitors can also appear in the form of spontaneous autoantibodies in an autoimmune condition known as acquired hemophilia.

Whatever the cause, it is important to measure the titer of these inhibitors. Labs do this by incubating the diluted patient plasma with normal plasma for 2 hours at 37°C. A titer < 5 Bethesda units (BU) indicates that the inhibitor can be overcome by administering additional FVIII. For higher titers (≥ 5 BU), such therapy is no longer effective, and treatment moving forward must include a bypassing agent. This might be recombinant FVIIa or a prothrombin complex concentrate, the latter of which is a preparation of mainly non-activated factors II, IX, and X mixed with mostly activated factor VII (VIIa).

What do labs need to know about assays for measuring FVIII?

The most common method for measuring FVIII activity is the one-stage activity assay. An essential component of this assay is a factor-VIII deficient substrate plasma, which is mixed 1-to-1 with the diluted patient plasma specimen. An activated partial thromboplastin time is then performed on the mix. One-stage assays are calibrated using a standard reference plasma.

For patients taking new hemophilia A replacement factor therapies, however, the one-stage assay may underestimate FVIII activity. New therapies partially overcome the relatively short (8-12 hour) and problematic half-life of recombinant FVIII using several mechanisms: PEGylated molecules (FVIII linked to polyethylene glycol), FVIII fused to immunoglobulin Fc domains or albumin, or single-chain FVIII molecules. With some of these new FVIII preparations—particularly the single-chain form—another method known as a chromogenic activity assay is more likely than a one-stage assay to give the correct activity.

At this time, the reasons for this discrepancy are still unclear. Suggested mechanisms include more rapid proteolytic inactivation of the single-chain FVIII or other forms of clearance, as well as differential responses to the phospholipid used in the one-stage assay reaction. Conversely, there are some mild forms of hemophilia A in which the opposite is true: The one-stage assay reads higher than the chromogenic assay. At this time, there are no Food and Drug Administration-approved chromogenic assays available in the U.S., but the assay is available for ordering at specialized reference laboratories.

Dr. Harris will delve more into “Coagulation Factor VIII—Evolution, Biosynthesis, Biology, and Monitoring in the Clinical Laboratory,” during two brown bag sessions at the 70th AACC Annual Scientific Meeting on Wednesday, August 1, from 7:30-8:30 a.m. and 12:30-1:30 p.m. at McCormick Place, Chicago.

Neil Harris, MBChB, MD, DABCC, is a clinical associate professor in the department of pathology, immunology, and laboratory medicine and core laboratory medical director at the University of Florida College of Medicine in Gainesville.+Email: harris[at]pathology.ufl.edu