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Factor VIII Chromogenic Activity

Factor VIII is a large glycoprotein cofactor with a molecular weight of 320 kilodaltons that is synthesized mainly in hepatocytes, but also in hepatic macrophages, megakaryocytes, and endothelial cells. Factor VIII circulates in plasma bound to von Willebrand factor (vWF) at a concentration of approximately 0.1 mg/mL. Plasma half-life of factor VIII is 8 to 12 hours. Factor VIII deficiency should be suspected when a patient with excessive bleeding has a normal protime (PT) and an extended activated partial thromboplastin time (aPTT).

Congenital deficiency of factor VIII causes hemophilia A. Hemophilia A is the second most common inherited bleeding abnormality after von Willebrand disease. Prevalence is approximately 1 of every 5000 live male births and is the same in all ethnic groups. It is transmitted as an X chromosome-linked hereditary disorder. Most cases occur in men whose mothers are carriers of the genetic defect. About 30% of factor VIII deficiencies arise in men as spontaneous mutations. Female carriers of hemophilia A may rarely experience excessive bleeding.

Severity of hemophilia A is defined by the level of factor VIII activity. Severe hemophilia, which represents approximately half the cases, is associated with a factor VIII level <1%. Moderate hemophilia occurs in about 10% of cases and has factor VIII levels between 1% and 5%. The remaining 30% to 40% of hemophiliacs have mild disease with factor VIII levels above >5%.

Factor VIII levels can be decreased by the development of antibodies, which are also called inhibitors, because they inhibit Factor VIII activity. Inhibitors may develop after replacement therapy with factor VIII concentrates or spontaneously after the formation of autoantibody. Factor VIII may be decreased in some types of von Willebrand disease.

Factor VIII levels are elevated at birth and increase during pregnancy. Factor VIII is an acute phase reactant, meaning that levels rise during stress, surgery, and inflammation. Several drugs including epinephrine, DDAVP, and estrogen increase factor VIII levels. Persistent elevation of factor VIII above 150% is associated with a fivefold increased risk for venous thromboembolism. The higher the factor VIII activity, the higher is the risk. Factor VIII levels >150% are observed in 20% of individuals with venous thrombosis or thromboembolism.

The coagulation factor activity assay used by the majority of clinical laboratories is the one-stage clotting assay. Factor VIII activity can also be measured by a two stage chromogenic substrate assay. The first step involves the addition of patient plasma to a reaction mixture containing thrombin or prothrombin, Factor IXa, FX, calcium and phospholipid. Factor VIII is converted to FVIIIa, which works with FIXa to convert Factor X to Factor Xa. The amount of Factor Xa generated is proportional to the amount of functional FVIII present in the patient's sample. The amount of activated factor X generated is determined with a chromogenic substrate and the activity is determined from a standard curve.

The one stage clotting assay may underestimate or overestimate the true FVIII activity in up to 30% of patients with mild or moderate hemophilia A when compared to a chromogenic substrate assay. This difference in factor activity measurements between the clotting and chromogenic substrate assays is called discrepant hemophilia and is caused by missense mutations that reduce stability of activated FVIII (FVIIIa). This reduced stability is more apparent in clot based FVIII activity assays where FVIIIa is generated during a longer incubation period compared to the chromogenic assay.

Some of the new modified recombinant FVIII concentrates are not accurately measured by the activated partial thromboplastin time (APTT) reagent used in the clot based assay. Overestimation of post-infusion plasma factor activity can lead to underdosing of the replacement factor and an increased risk of bleeding. Conversely, underestimation of factor activity in the post-infusion sample may lead to overdosing of replacement factor, which increases cost and risk for thrombosis. Most of these recombinant FVIII products are accurately measured using a chromogenic assay.

Factor VIII activity may be spuriously decreased by lupus anticoagulant in the one stage clotting assay, but not in the chromogenic substrate assay. The chromogenic substrate assay uses a high dilution of plasma and is not phospholipid dependent.

Reference range of factor VIII is 50 to 170% using a chromogenic substrate assay.

Factor VIII is a labile protein. Improper handling of a specimen may give a falsely low result. Factor VIII activity in plasma specimens that have been frozen and thawed may be 10% to 20% lower than fresh specimens.

Specimen requirement is a light-blue top tube of blood for citrated plasma.

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