Factor VIII Chromogenic Activity

Factor VIII is a large glycoprotein cofactor (320 kilodaltons) that is produced mainly in hepatocytes, but also to some extent by liver macrophages, megakaryocytes, and endothelial cells.^8,9 Factor VIII circulates in the plasma bound to von Willebrand factor (vWF) at a concentration of approximately 0.1 mg/mL.⁹ The plasma half-life of factor VIII is short at about 8 to 10 hours.⁹ 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).

Hemophilia A, or classic hemophilia, occurs as the result of congenital deficiency of factor VIII.^8,10 Clinical features of hemophilia A are the same as for hemophilia B, which is caused by factor IX deficiency (see Factor IX Activity [086298]). Hemophilia A is the second most common inherited bleeding abnormality (second only to von Willebrand disease), occurring in approximately 1 of every 5000 live male births.^8,10 Hemophilia A accounts for approximately 85% of all hemophilia cases.¹⁰ This condition is transmitted as an X chromosome-linked hereditary disorder.¹⁰ The majority of cases occur in men whose mothers are carriers of the genetic defect. About 30% of factor VIII deficiencies arise in men as spontaneous mutations.^8,10 The prevalence of hemophilia A is equal in all ethnic groups.^8,10 Female carriers of hemophilia A may rarely present with excessive bleeding.⁸ Hemophilia symptoms can also occur in female carriers who have a high degree of lyonization of the factor VIII alleles.¹⁰ Females with Turner syndrome karyotype XO, can also be symptomatic.¹⁰

The severity of hemophilia A can be defined by the level of factor VIII activity.^10,11 Severe hemophilia, which represents approximately half the cases, is associated with a factor VIII level <1%. About 10% of cases are moderate with factor VIII levels of 1% to 5% and the remaining 30% to 40% of hemophiliacs have the mild condition with factor VIII levels above >5%.

The coagulation factor activity assay used by the majority of clinical laboratories, the one-stage assay (OSA), may underestimate or overestimate the true FVIII activity in up to 30% of patients with mild or moderate (nonsevere) hemophilia A.^6,7 Approximately 16% of patients with mild hemophilia A have a normal FVIII OSA, and the correct diagnosis relies on the chromogenic factor VIII assay.^6,7 Differences in factor activity measurements between the OSA and chromogenic substrate assay (CSA) in nonsevere hemophilia is called discrepant hemophilia. This occurs in 30% of patients with nonsevere hemophilia A.^6,7,12 Such discrepancies may also occur in hemophilia carriers. Although there is no universally accepted definition for what constitutes discrepant hemophilia, the generally accepted criterion is a twofold difference in results between the OSA and CSA. Either OSA results can be greater than CSA or vice versa, depending on the underlying FVIII gene mutation.^6,7,12,13 Both possibilities can misclassify hemophilia severity, but the former may result in a missed diagnosis.

Discrepant hemophilia A has a genetic basis, generally due to missense mutations that affect the stability of the activated form of FVIII (FVIIIa) or the ability of FVIII to successfully bind the activated form of FIX (FIXa), von Willebrand factor, or thrombin.^6,13 Missense mutations clustered in the A1-A2-A3 domain interfaces of the FVIII protein cause reduced stability of FVIIIa, which is more apparent in FVIII activity assays where the FVIIIa is generated during a relatively long (for example, 2-to-10-minute) incubation such as the CSA or the infrequently performed two-stage assay.^7,13 In the OSA, FVIII is in the activated form for only a brief period. Missense mutations clustered around thrombin cleavage sites or FIXa binding sites are more readily identified in OSA since the factors are present at physiologic concentrations, unlike the CSA, where factor concentrations are optimized. Also, long long incubation times in the CSA may help to overcome mutations that interfere with binding. The underlying mutations and discrepancies between OSA and CSA are consistent within and between discrepant hemophilia A families.

Certain modified recombinant FVIII replacement products demonstrate variable and clinically significant differences in post-infusion recovery (that is, the amount of factor measured vs. the actual concentration present), based on the activated partial thromboplastin time (APTT) reagent used in the OSA or assay methodology.¹⁴ 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 the replacement factor, which not only has cost implications but also may place the patient at risk for thrombosis. Most recombinant FVIII products may be accurately measured using a chromogenic assay, even when this is performed with a plasma calibrator rather than a product-specific calibrator.^15,16

Factor VIII activity may be spuriously decreased on the one stage activity assay in the presence of a lupus anticoagulant. A chromogenic factor activity assay does not demonstrate lupus anticoagulant interference due to the high initial dilution used in the assay and the decreased phospholipid dependence of the assay. Factor VIII levels are elevated at birth and increase during pregnancy.⁸ Factor VIII is an acute phase reactant with levels that rise during periods of acute stress, following surgery, and in inflammatory conditions.⁸ Levels can also increase as the result of strenuous exercise or the administration of several drugs including epinephrine, DDAVP, or estrogen (for birth control or hormone replacement therapy). Factor VIII levels can be elevated in a number of clinical conditions including carcinoma, leukemia, liver disease, renal disease, hemolytic anemia, diabetes mellitus, deep vein thrombosis, and myocardial infarction.⁸ Persistent elevation of factor VIII above 150% is associated with an increased risk for venous thrombosis of more than fivefold.^9,17 Elevated factor VIII is also associated with an increased risk for recurrence of venous thromboembolism. Risk is graded such that the higher the factor VIII activity, the higher the risk.¹⁸ The basis for this increased risk is not well understood as genetic studies of the factor VIII and von Willebrand factor genes failed to identify a genetic basis for this increased risk.⁹ Values >150% are observed in 20% to 25% of individuals with venous thrombosis or thromboembolism in the absence of other known causes of factor VIII elevation.¹⁷