Factor VIII Assay 

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Factor VIII (antihemophilic factor) is a key factor of the intrinsic clotting cascade. Normal hemostasis requires at least a quarter (25%) of factor VIII activity.

Symptomatic hemophiliacs usually have levels 5% of normal level. Disease severity are categorized as severe if the level is less than 1%, moderate if the level is between1 to 5%, and mild if it is more than 5%.

The reference range for factor VIII is 50-150% of normal (varies with age). ^[1]

Factor VIII is increased in the following:

Inflammatory states as it is an acute phase reactant

Use of oral contraceptives

Last trimester of pregnancy

Factor VIII is decreased in the following:

Hemophilia A

Disseminated intravascular coagulation (DIC)

Acquired factor VIII inhibitor

von Willebrand disease

In some female carriers of hemophilia gene (mild) ^[2]

See the list below:

Specimen: Plasma

Container: Blue top

Collection method: Routine venipuncture

Deliver the sample (on ice) immediately to the laboratory. Keep stable for 2 hours; freeze if the assay is delayed more than 2 hours. ^[1]

All samples must be sent in a sealed, leak-proof container marked with a biohazard sticker to comply with Occupational Safety and Health Administration (OSHA) safety standards.

See the list below:

Quantitative functional assays of coagulation factors

Factor VIII (antihemophilic factor) is a key factor of the intrinsic clotting cascade. Normal hemostasis requires at least a quarter (25%) of factor VIII activity.

Symptomatic hemophiliacs usually have levels 5% of normal level. Disease severity are categorized as severe if the level is less than 1%, moderate if the level is between1 to 5%, and mild if it is more than 5%.

Factor VIII assays are used to help replacement therapy in patients with hemophilia.

Factor deficiency can be distinguished from factor inhibitor by an inhibitor screen. ^[3]

Factor VIII is produced in the liver, ^[4] perhaps not in hepatocytes. ^[5] Endothelial cells of liver are the major site of biosynthesis. ^[6] The notion of cure of hemophilia A by liver transplantation in human and canine subjects supports this statement. ^[7]

Factor VIII circulates with von Willebrand factor (VWF) in a noncovalent complex. VWF is a complex glycoprotein that works as a carrier for factor VIII. When associated with VWF, the half-life of factor VIII is 8-12 hours. The half-life is shortened without VWF. In many patients with a deficiency of VWF, low level of factor VIII has been found. ^[8] In a mouse model, isolated hepatocytes transplantation did not correct hemophilia A, but transplantation of a cellular fraction enriched in liver endothelial cells did. ^{[5, 9]}

The factor VIII gene is located on the X chromosome. Hemophilia A is transmitted as a sex-linked recessive manner due to deficiency of factor VIII. Activated factor X or thrombin activates factor VIII. This activation also results in the release of factor VIIIa from VWF. Factor VIIIa is inactivated by thrombin or activated protein C (APC). ^[5]

Bleeding episodes in patients with hemophilia A can be managed by replacing factor VIII. Several products are available for use in raising factor VIII. Both of cryoprecipitate and fresh-frozen plasma (FFP) contain factor VIII and were the only products available for treatment in the past. A large volume of plasma must be infused to achieve and maintain even lowest factor VIII levels. The highest factor VIII level achieved with plasma is about 20% of normal, which may not be adequate for hemostasis. ^[5] Several commercial lyophilized factor VIII concentrates that use cryoprecipitate of pooled normal human plasmas are available. Because of the risk of transmission of viruses, factor VIII concentrates have been sterilized by heating in solution, by superheating to 80° C, and by exposure to organic solvent-detergents that inactivate lipid-enveloped viruses, including HIV, hepatitis B, and hepatitis C viruses.

However, these procedures do not inactivate parvovirus or hepatitis A. ^{[10, 11, 12]} As parvovirus is transmitted by cellular elements of the blood, such infection does not occur frequently in patients with hemophilia A. Nonetheless, seroconversion to parvovirus B19 has been found in patients receiving plasma-derived concentrates treated with pasteurization or solvent-detergent extraction. ^[12]

Currently a dozen or so factor VIII products are available and are regarded as safe from the perspective of transmission of viruses. ^[12] Factor VIII produced by available recombinant DNA techniques is safe and effective. The severity and site of bleeding determine the frequency and dose of factor VIII infusion. In mildly or moderately affected hemophilia A patients, 1-desamino-8-D-arginine vasopressin (DDAVP; desmopressin) increases factor VIII levels 2-fold to 3-fold above baseline. However, patients with severe hemophilia A do not respond to DDAVP. ^[13] Mechanism of action of DDAVP in raising factor VIII level remains unknown. As DDAVP is a potent antidiuretic, hyponatremia may result by its use. Tachyphylaxis does occur with repeated administration.

Factor VIII testing is indicated when classic hemophilia is suspected.

Although factor assays are usually PT-based or PTT-based (automated clotting tests), immunogenic factor or chromogenic assays are also available for factor VIII.

Argatroban, heparin, and hirudin may interfere with specific factor assays. Quantitative functional assays of coagulation factors and of physiological inhibitor proteins are based on parallel-line or slope ratio bioassays. With the modern analyzers the test procedure follows the typical example of clinical chemistry: a single test plasma dilution read from an actual calibration curve, regular internal and external quality control.

If the results are unexpected or if hemophilia is suspected, the usual recommendation is to repeat the assay with 3 different predilutions of the test plasma. The resulting potency estimates should not differ by more than 10-15% from their average. If it deviates more than this range, the assay is not valid, and further investigation searching for inhibitors should be performed. ^[14]

Limitations of the test include partially clotted specimens due to poor mixture of anticoagulant (3:2 sodium citrate as per manufacturer’s blue topped tube); overfilled or underfilled test tubes altering the ratio of blood to anticoagulant (9:1); improperly stored plasma; contamination with heparin or dilution of collected sample if indwelling catheters are used; or analytical errors such as lipemic, icteric, or hemolyzed plasma that may interfere with photoelectric measuring instruments. ^[2] Markedly increased level of factor VIII may predispose to thromboembolism. ^[2]

Nicoll Diana MSJ, Pignone Michael, Lu Chuanyi Mark. Pocket Guide to Diagnostic Tests,. 5e ed: http://www.accessmedicine.com/pocketDiagnostic.aspx.

Williamson MA, Snyder LM, Wallach JB. Wallach’s interpretation of diagnostic tests. 9th ed. Wolters Kluwer/Lippincott Williams & Wilkins Health: Philadelphia; 2011.

Lindgren A, Wadenvik H, Tengborn L. Characterization of inhibitors to FVIII with an ELISA in congenital and acquired haemophilia A. Haemophilia. 2002 Sep. 8(5):644-8. [Medline].

Shaw E, Giddings JC, Peake IR, Bloom AL. Synthesis of procoagulant factor VIII, factor VIII related antigen and other coagulation factors by the isolated perfused rat liver. Br J Haematol. 1979 Apr. 41(4):585-96. [Medline].

Monroe DM HM, Roberts HR. Molecular Biology and Biochemistry of the Coagulation Factors and Pathways of Hemostasis. Prchal JT KK, Lichtman MA, Kipps TJ, Seligsohn U, ed. Williams Hematology. 8th ed. New York: McGraw-Hill; 2010.

Hellman L, Smedsrod B, Sandberg H, Pettersson U. Secretion of coagulant factor VIII activity and antigen by in vitro cultivated rat liver sinusoidal endothelial cells. Br J Haematol. 1989 Nov. 73(3):348-55. [Medline].

Bontempo FA, Lewis JH, Gorenc TJ, Spero JA, Ragni MV, Scott JP. Liver transplantation in hemophilia A. Blood. 1987 Jun. 69(6):1721-4. [Medline].

Marchioro TL, Hougie C, Ragde H, Epstein RB, Thomas ED. Hemophilia: role of organ homografts. Science. 1969 Jan 10. 163(3863):188-90. [Medline].

Kumaran V, Benten D, Follenzi A, Joseph B, Sarkar R, Gupta S. Transplantation of endothelial cells corrects the phenotype in hemophilia A mice. J Thromb Haemost. 2005 Sep. 3(9):2022-31. [Medline].

Santagostino E, Mannucci PM, Gringeri A, Azzi A, Morfini M, Musso R. Transmission of parvovirus B19 by coagulation factor concentrates exposed to 100 degrees C heat after lyophilization. Transfusion. 1997 May. 37(5):517-22. [Medline].

Robertson BH, Alter MJ, Bell BP, Evatt B, McCaustland KA, Shapiro CN. Hepatitis A virus sequence detected in clotting factor concentrates associated with disease transmission. Biologicals. 1998 Jun. 26(2):95-9. [Medline].

Roberts HR KN, Escobar MA. Hemophilia A and Hemophilia B. Prchal JT KK, Lichtman MA, Kipps TJ, Seligsohn U, ed. Williams Hematology. New York: McGraw-Hill; 2010.

Rodeghiero F, Castaman G, Di Bona E, Ruggeri M. Consistency of responses to repeated DDAVP infusions in patients with von Willebrand’s disease and hemophilia A. Blood. Nov 1 1989. 74(6):1997-2000.

Marbet GA. Quantification of coagulation factors and inhibitors. Still a special task. Hamostaseologie. 2006 Jan. 26(1):38-41. [Medline].

Bishnu Prasad Devkota, MD, MHI, FRCS(Edin), FRCS(Glasg), FACP Professor of Medicine, St Louis University School of Medicine

Bishnu Prasad Devkota, MD, MHI, FRCS(Edin), FRCS(Glasg), FACP is a member of the following medical societies: American College of Physicians, American Medical Informatics Association, Royal College of Physicians and Surgeons of Glasgow, Healthcare Information and Management Systems Society, Royal College of Surgeons of Edinburgh

Disclosure: Nothing to disclose.

Eric B Staros, MD Associate Professor of Pathology, St Louis University School of Medicine; Director of Clinical Laboratories, Director of Cytopathology, Department of Pathology, St Louis University Hospital

Eric B Staros, MD is a member of the following medical societies: American Medical Association, American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology

Disclosure: Nothing to disclose.

Factor VIII Assay 

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