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Coagulation Factor Testing
Coagulation factors may be measured by methods assessing both their presence, as antigens, and their ability to function, as activity. Inherited coagulation factor deficiencies may be of two types. Type I defects (quantitative) have decreased absolute amounts of the factor, resulting in both decreased activity and antigen levels. They result from lack of production or increased clearance of the gene product. Type II defects (qualitative) have a defective gene product, resulting in decreased activity but normal or slightly decreased levels of antigen. Type II defects result from a mutation changing the gene product or altered posttranslational modification. Acquired defects may mimic either type by decreased or faulty production, increased clearance or inactivation, or the presence of a circulating inhibitor, usually an antibody, which reacts with the factor to mask its activity or remove it from the circulation. Fig. 131.1 illustrates the relationship among functional and immunologic tests and how they reveal underlying structure and function. This chapter reviews both the coagulation factor activity (functional) and antigen (immunologic) assays for factors II, V, VII, VIII, IX, X, XI, XII, and XIII, which are indicated in the diagnosis of congenital and acquired coagulation factor deficiencies, characterization of coagulation factor defects, and monitoring of coagulation factor therapy.
Functional and immunologic tests of coagulation factors.
Methods
Activity assays may be clot-based or chromogenic (amidolytic). Clot-based factor assays are modifications of the screening tests to allow a single factor to be rate-limiting in clot formation. Each compares the relative abilities of a test plasma and a control plasma to correct the defect of plasma deficient in a given factor. Correction may be measured in an activated partial thromboplastin time (PTT)–based or prothrombin time (PT)–based assay. Chromogenic assays also provide all components needed except for the factor to be measured; the endpoint is the cleavage of a synthetic substrate through an enzymatic reaction, detected colorimetrically. Immunologic tests for measuring coagulation factors include enzyme-linked immunosorbent assay, latex immunoassay, and other standard methods. The measurement of most clinical relevance is activity; immunologic methods are used primarily to characterize the type of defect present as quantitative or qualitative.
FVIII Activity
FVIII activity (VIII:C) can be measured by a one-stage assay based on the PTT, a two-stage assay (rarely used now), or a chromogenic method. Results are comparable when testing patient plasma, except for a small number of mild hemophilia A patients who may give higher or lower values with the two-stage and chromogenic assays than with one-stage assays and those treated with certain hemophilia treatment products. Recombinant FVIII lacking the B domain, whether or not it is modified to last longer, may be more accurately measured with a chromogenic assay. For accurate quantitation of low levels of VIII:C, a second “low” curve may be generated using VIII:C reference plasma at concentrations of 0.5–20 U/dL. Measurement of VIII:C concentrates requires use of a concentrate standard.
FVIII Antigen
FVIII antigen (VIII:Ag) can be measured using only human or monoclonal antibodies and does not precipitate. It is not routinely measured in clinical practice. The old term “factor VIII–related antigen” or VIII:RAg seen in the literature actually refers to von Willebrand factor antigen (VWF:Ag).
FIX Activity
FIX activity (IX:C) may be measured by one-stage assay based on the PTT or by chromogenic assay. A low curve may also be used to quantitate more accurately the levels of IX:C seen in hemophilia patients.
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