Factor VIII Chromogenic Activity

CPT: 85240
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Test Details


  • Antihemophilic Factor (AHF)


Used in the diagnosis of nonsevere hemophilia A.6, 7, 13 Useful in the accurate determination of factor VIII (FVIII) activity in the presence of a lupus anticoagulant or when certain modified recombinant FVIII replacement products are present. This assay can also be used in place of the one-stage (standard) FVIII activity assay for any indication.


Factor VIII is an acute phase reactant and can be elevated in a number of clinical conditions. This can affect the accuracy of the test in diagnosing hemophilia. Factor VIII levels should not be used to determine the carrier status of females. Genetic testing should be used for this purpose. Factor VIII inhibitors (both autoantibodies that develop after replacement therapy and autoantibodies that develop spontaneously) can result in low factor VIII levels. A lupus anticoagulant may cause factor VIII activity to appear spuriously low and a chromogenic factor VIII activity is recommended in this circumstance. Direct Xa inhibitor therapy may cause factitiously low results.


Factor VIII activity is determined by a two-stage chromogenic substrate assay where the amount of activated factor X generated is proportional to the amount of functional FVIII present in the test plasma in the presence of excess activated FIX. The amount of activated factor X generated is read with a chromogenic substrate and the activity is determined from a standard curve.

Additional Information

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

Specimen Requirements


Plasma, frozen


1 mL

Minimum Volume

0.5 mL


Blue-top (sodium citrate) tube

Patient Preparation

Ideally the patient should not be on anticoagulant therapy. Avoid heparin and direct Xa inhibitor therapies for about three days prior to testing. Do not draw from an arm with a heparin lock or heparinized catheter.


Citrated plasma samples should be collected by double centrifugation. Blood should be collected in a blue-top tube containing 3.2% buffered sodium citrate.1 Evacuated collection tubes must be filled to completion to ensure a proper blood to anticoagulant ratio.2,3 The sample should be mixed immediately by gentle inversion at least six times to ensure adequate mixing of the anti- coagulant with the blood. A discard tube is not required prior to collection of coagulation samples except when using a winged blood collection device (i.e. 'butterfly'), in which case a discard tube should be used.4,5 When noncitrate tubes are collected for other tests, collect sterile and nonadditive (red-top) tubes prior to citrate (blue-top) tubes. Any tube containing an alternate anticoagulant should be collected after the blue-top tube. Gel-barrier tubes and serum tubes with clot initiators should also be collected after the citrate tubes. Centrifuge for 10 minutes and carefully remove 2/3 of the plasma using a plastic transfer pipette, being careful not to disturb the cells. Deliver to a plastic transport tube, cap, and recentifuge for 10 minutes. Use a second plastic pipette to remove plasma, staying clear of the platelets at the bottom of the tube. Transfer the plasma into a LabCorp PP transpak frozen purple tube with screw cap (LabCorp N° 49482). Freeze immediately and maintain frozen until tested. To avoid delays in turnaround time when requesting multiple tests on frozen samples, please submit separate frozen specimens for each test requested. Please print and use the Volume Guide for Coagulation Testing to ensure proper draw volume.

Storage Instructions


Stability Requirements



Room temperature

4 hours


4 hours


2 weeks

Freeze/thaw cycles

Stable x2

Causes for Rejection

Severe hemolysis; improper labeling; clotted specimen; specimen diluted with IV fluids; samples thawed in transit; improper sample type; sample out of stability.

Clinical Information

Special Instructions

If the patient's hematocrit exceeds 55%, the volume of citrate in the collection tube must be adjusted. Refer to Coagulation Collection Procedures for directions.


1. Adcock DM, Kressin DC, Marlar RA. Effect of 3.2% vs 3.8% sodium citrate concentration on routine coagulation testing. Am J Clin Pathol. 1997 Jan;107(1):105-110.8980376
2. Reneke J, Etzell J, Leslie S, Ng VL, Gottfried EL. Prolonged prothrombin time and activated partial thromboplastin time due to underfilled specimen tubes with 109 mmol/L (3.2%) citrate anticoagulant. Am J Clin Pathol. 1998 Jun;109(6):754-757.9620035
3. National Committee for Clinical Laboratory Standardization. Collection, Transport, and Processing of Blood Specimens for Coagulation Testing and General Performance of Coagulation Assays; Approved Guideline. 5th ed. Villanova, Pa: NCCLS; 2008. Document H21-A5:28(5).
4. Gottfried EL, Adachi MM. Prothrombin time and activated partial thromboplastin time can be performed on the first tube. Am J Clin Pathol. 1997;107(6):681-683.9169665
5. McGlasson DL, More L, Best HA, Norris WL, Doe RH, Ray H. Drawing specimens for coagulation testing: Is a second tube necessary? Clin Lab Sci. 1999 May-Jun;12(3):137-139.10539100
6. Oldenburg J, Pavlova A. Discrepancy between one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A phenotype. Hemostaseologie. 2010 Nov;30(4):207-211.21057709
7. Kamphuisen PW, Eikenboom JC, Rosendaal FR, et al. High factor VIII antigen levels increase the risk of venous thrombosis but are not associated with polymorphisms in the von Willebrand factor and factor VIII gene. Br J Haematol. 2001 Oct;115(1):156-158.11722428
8. Adcock DM, Bethel MA, Macy PA. Coagulation Handbook. Aurora, Colo: Esoterix-Colorado Coagulation; 2006.
9. Chandler WL, Rodgers GM, Sprouse JT, Thompson AR. Elevated hemostatic factor levels as potential risk factors for thrombosis. Arch Pathol Lab Med. 2002 Nov;126(11):1405-1414.12421150
10. Cohen AJ, Kessler CM. Hemophilia A and B. In: Kitchens CS, Alving BM, Kessler CM, eds. Consultative Hemostasis and Thrombosis. Philadelphia, Pa: WB Saunders Co;2002:43-56.
11. Triplett DA. Coagulation abnormalities. In: McClatchey KD, ed. Clinical Laboratory Medicine. 2nd ed. Philadelphia, Pa: Lippincott Williams and Wilkins; 2002:1033-1049.
12. Peyvandi F, Oldenburg J, Friedman KD. A critical appraisal of one-stage and chromogenic assays of factor VIII activity. J Thromb Haemost. 2016 Feb;14(2):248-261.26663865
13. Pavlova A, Delev D, Pezeshkpoor B, Muller J, Oldenburg J. Haemophilia A mutations in patients with nonsevere phenotype associated with a discrepancy between one-stage and chromogenic factor VIII activity assays. Thromb Haemost. 2014 May 5;111(5):851-861.24452774
14. Dodt J, Hubbard AR, Wicks SJ, et al. Potency determination of factor VIII and factor IX for new product labeling and postinfusion testing: challenges for caregivers and regulators. Haemophilia. 2015 Jul;21(4):543-549.25623631
15. Tiefenbacher S, Robinson MM, Ross EL, et al. Comparison of FVIII activity of select novel recombinant FVIII replacement products in commonly used FDA approved one-stage clot assay systems. J Thromb Haemost. 2015;13(Suppl 2):566 (abstract).
16. Kitchen S, Kershaw G, Tiefenbacher S. Recombinant to modified factor VIII and factor IX - chromogenic and one-stage assays issues. Haemophilia. 2016 Jul;22(Suppl 5):72-77.27405680
17. Kamphuisen PW, Eikenboom JC, Rosendaal FR, et al. High factor VIII antigen levels increase the risk of venous thrombosis but are not associated with polymorphisms in the von Willebrand factor and factor VIII gene. Br J Haematol. 2001 Oct;115(1):156-158.11722428
18. Cristina L, Benilde C, Michela C, Mirella F, Giuliana G, Gualtiero P. High plasma levels of factor VIII and risk of recurrence of venous thromboembolism. Br J Haematol. 2004 Feb;124(4):504-510.14984502


Adcock DM, Gosselin R. Direct oral anticoagulants (DOACs) in the laboratory: 2015 Review. Thromb Res. 2015 Jul;136(1):7- 12.25981138


Order Code Order Code Name Order Loinc Result Code Result Code Name UofM Result LOINC
500192 FVIII Chromogenic 500193 FVIII Chromogenic % 49865-9

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