From: Elizabeth M. Van Cott, M.D., and Michael Laposata, M.D., Ph.D., “Coagulation.” In: Jacobs DS et al, ed. The Laboratory Test Handbook, 5th Edition. Lexi-Comp, Cleveland, 2001; 327-358.

Related Information

Activated Partial Thromboplastin Time
Antiphospholipid Antibody (Lupus Anticoagulant and/or Anticardiolipin Antibody)
Factor Inhibitors
Factor XIII
Fibrinogen
High-Molecular Weight Kininogen
Mixing Studies
Prekallikrein
Prothrombin Time
von Willebrand Factor

Synonyms Ac-Globulin (Factor V); Antihemophilic Factor (Factor VIII); Autoprothrombin I (Factor VII); Autoprothrombin II (Factor IX); Christmas Disease Factor (Factor IX); Hageman Factor (Factor XII); Labile Factor (Factor V); Plasma Thromboplastin Antecedent (Factor XI); Plasma Thromboplastin Component (Factor IX); Proaccelerin (Factor V); Proconvertin (Factor VII); Prothrombin (Factor II); Stable Factor (Factor VII); Stuart Factor (Factor X); Stuart-Prower Factor (Factor X)

Applies to DDAVP; Desmopressin; Factor(s) II, V, VII, VIII, IX, X, XI, XII; Factor VIII:von WIllebrand Factor Ratio; Hemophilia A (Factor VIII Deficiency); Hemophilia B (Factor IX Deficiency); INR; International Normalized Ratio

Abstract Isolated factor deficiencies can be hereditary, but this is much more rare than multiple, acquired factor deficiencies produced by liver disease, disseminated intravascular coagulation, warfarin, or the inhibitory effects in factor assays from lupus anticoagulants, heparin, or other anticoagulants.

Specimen Plasma

Container Three blue top (sodium citrate) tubes if all factor assays are requested

Collection Routine venipuncture. If multiple tests are being drawn, draw blue top tubes after any red top tubes but before any lavender top (EDTA), green top (heparin), or gray top (oxalate/fluoride) tubes. Immediately invert tube gently at least 4 times to mix. Tubes must be appropriately filled. Deliver tubes immediately to the laboratory.

Avoid heparin contamination during specimen collection. Heparin, hirudin, or argatroban anticoagulation can interfere with factor assays by acting as an “inhibitor”, resulting in falsely decreased factor levels. Heparin, if present, must be removed from specimens by the laboratory. Warfarin decreases factors II, VII, IX, and X.

Storage Instructions Separate plasma from cells as soon as possible. Store plasma at room temperature for up to 2 hours, at 2degrees C to 8degrees C for up to 4 hours, or store frozen. Factor VIII, and to a lesser extent factor V, degrade if specimens are kept unfrozen for prolonged periods.

Causes for Rejection Specimen received more than 4 hours after collection, tubes not filled, clotted specimen

Turnaround Time Less than 1 day, unless test has to be sent out to a reference laboratory

Reference Interval Factor levels are expressed as percent of normal plasma concentrations. By definition, normal plasma contains 100% (1 unit/mL) of each factor. The reference range is approximately 60% to 140%. Factor VIII levels are not decreased at birth or throughout childhood. The other factor levels are below adult reference range at birth, ranging approximately from 10% to 100%. The levels increase toward the adult reference range by age 6 months, although they may remain mildly below adult normal range throughout childhood.1,2 However, newborns and children do not normally experience bleeding, because a balance between coagulation factors and natural coagulation inhibitors is maintained throughout development. Factor XI can decrease during pregnancy, whereas fibrinogen and factor VIII increase.

Use To determine the etiology of a prolonged PT or PTT. Usually performed after a mixing study has been completed (see Mixing Studies), to identify specific factor deficiencies or inhibitors. Assays for factors VIII, IX, XI, and XII are performed to evaluate a prolonged PTT (with normal PT). Assays for fibrinogen, factors II, V, VII, and X are performed to evaluate a prolonged PT (with normal PTT). If PT and PTT are both prolonged, all eight factors and fibrinogen may be performed to establish the cause for the prolongations. Because acquired causes of factor deficiencies are generally more common than hereditary causes, a patient found to have a factor deficiency should be evaluated for possible acquired etiologies, especially if multiple factor deficiencies are present (see Tables 1 and 2). If a hereditary etiology for the decrease appears likely, the diagnosis can be confirmed by measuring the factor in relatives.

Chromogenic factor X assays are useful for monitoring warfarin in the presence of a lupus anticoagulant, hirudin, or argatroban. Lupus anticoagulants, hirudin or argatroban can prolong the PT and therefore the international normalized ratio (INR).3 In these situations, the INR can overestimate the amount of warfarin anticoagulation and lead to inappropriate reductions in warfarin dose. Warfarin decreases factor X (as well as factors II, VII, and IX), and a chromogenic assay is available for factor X which has no interference from lupus anticoagulants, hirudin or argatroban. When the INR is 2-3, the chromogenic factor X level is approximately 20% to 40%. Each laboratory should determine its own chromogenic factor X therapeutic range.

Relatives of patients with a known hereditary factor deficiency may choose to have PT, PTT, and/or factor assay(s) performed to determine if they also have the deficiency or if they are a carrier.

Factor VIII assays are part of a von Willebrand disease evaluation. Factor VIII assays, together with von Willebrand factor assays, can also assess for hemophilia A carrier status in females. In hemophilia A carriers, the factor VIII:von Willebrand factor ratio is approximately 0.5.

An increased risk for thrombosis has been reported with elevated levels of certain coagulation factors (eg, fibrinogen, factor VII, factor VIII, factor XI).4,5,6,7 In addition, factor VII levels may be affected by dietary lipids, and factor VII levels correlate with triglyceride and cholesterol levels.8 However, factor assays have not yet been added to most hypercoagulation panels.

Table 1. Effects of Hereditary or Acquired Factor Deficiencies on PT and PTT

PTT Prolonged, PT Normal

Deficiencies of factor(s) VIII, IX, XI, and/or XII (intrinsic pathway)

PT Prolonged, PTT Normal

Deficiency of factor VII (extrinsic pathway)

Occasionally, mild-to-moderate deficiencies of factor(s) II, V, X, and/or fibrinogen (common pathway)

Both PT and PTT Prolonged

Deficiencies of factor(s) II, V, X, and/or fibrinogen (common pathway)

Multiple factor deficiencies

Table 2. Acquired Causes of Factor Deficiencies

Acquired Conditions Affecting PT Sooner and More Significantly Than PTT
Warfarin or vitamin K deficiency	Decreased function of factors II, VII, IX, and X
Liver dysfunction	Decreases hepatic synthesis of coagulation factors. All factors may be decreased except for factor VIII. Factor VII has the shortest half-life and therefore is often the earliest and most severely decreased factor. Factors XI and XII have the longest half-lives and therefore are often the last to be affected.
Disseminated intravascular coagulation (DIC)	All factors can be variably decreased, including factor VIII, due to factor activation and consumption.
Amyloidosis	Factor X, and occasionally other factors, can be decreased, due to binding of factor(s) to amyloid.
Acquired Conditions Affecting PTT Sooner and More Significantly Than PT
Prolonged specimen transit to laboratory	Degradation of factors V and VIII
Proteinuria	Occasionally, decreased factors XI and XII. PT usually normal.
Acquired Conditions That Can Interfere With Factor Assays
Heparin	Inhibits activated factors II, X, IX, XI, and XII, prolonging the PTT earlier and more than PT, and interfering in PTT-based factor assays more than PT-based factor assays, without causing a true decrease in factor levels.
Lupus anticoagulants	Inhibits the phospholipid cofactor function in coagulation, often prolonging the PTT. PT is usually normal. Can interfere in PTT-based factor assays, without causing a true decrease in factor levels. Rarely, a lupus anticoagulant can bind to factor II and cause a true decrease in factor II, prolonging the PT.
Hirudin and argatroban	Inhibit activated factor II (thrombin), prolonging both the PT and PTT, and interfering in PT- or PTT-based factor assays without causing a true decrease in factor levels.
Factor inhibitors

Methodology Factor assays are PT- or PTT-based reactions. These assays are performed by mixing patient plasma with plasma that is deficient in the factor that is being measured. Based on the resulting PT or PTT of this mixture, the amount of factor can be determined by comparing the PT or PTT clotting time to a standard curve that plots known factor levels against clotting times. Factor VIII, IX, XI, and XII assays are PTT-based. Factor II, VII, and X assays are PT-based. PT- and PTT-based assays are both available for factor V.

The presence of an inhibitor, such as a lupus anticoagulant, can cause artifactual decreases in the in vitro factor level. Therefore, laboratories should perform factor assays at multiple dilutions. At higher dilutions, the inhibitor interference will decrease due to dilution of the inhibitor.

Chromogenic factor assays and immunoassays (antigen assays) are commercially available for some of the coagulation factors. For example, chromogenic assays are commercially available for factors II, VII, VIII, and X; antigen assays are commercially available for factors VII, VIII, IX, and X. Therapeutic anticoagulants, lupus anticoagulants, and other inhibitors do not interfere with these assays (except in some instances when the inhibitor or anticoagulant is directed specifically against the factor being assayed or against another factor that participates in the assay).

Additional Information Factor deficiencies can be quantitative or qualitative. In quantitative disorders, the factor level determined by routine PT- or PTT-based methods (functional activity assays) is similar to the result obtained by immunological (antigen) assays. In qualitative disorders, the PT- or PTT-based (functional) assay result is decreased, but the antigen level is normal or significantly higher than the functional level, indicating the presence of a dysfunctional protein. Antigen assays are not available for some factors.

Hereditary Deficiencies of Factor VIII (Hemophilia A) or Factor IX (Hemophilia B)

Hemophilia A (factor VIII deficiency) is the most common severe hereditary bleeding disorder, affecting 1 in 5000-10,000 males.9,10 Hemophilia B (factor IX deficiency) is also a severe hereditary bleeding disorder, affecting 1 in 25,000-30,000 males. Hemophilia A and B are X-linked recessive disorders, because the factor VIII and factor IX genes are located on the X chromosome. Therefore, typically only males are affected. Females carrying the hemophilia mutation on one of their two X chromosomes are carriers. Female carriers with factor VIII levels <50% and bleeding symptoms have been reported.

The clinical severity of hemophilia A or B depends on the factor level. In hemophilia, <1% factor VIII or IX produces severe hemophilia with spontaneous bleeding, 1% to 5% produces moderate bleeding, and >5% is considered mild hemophilia in which bleeding occurs primarily with trauma or surgery rather than spontaneously. The baseline factor level remains relatively constant within an individual and within a kindred. Bleeding manifestations include hemarthrosis, soft tissue hematomas including bleeding into muscles, easy bruising, excessive bleeding with surgery, trauma, dental extractions, and circumcision, bleeding in the gastrointestinal or genitourinary tract, epistaxis, poor wound healing, and uncommonly, umbilical stump bleeding. Intracranial hemorrhages can occur, particularly following trauma.

Hemophilia testing is suggested for male patients with unexplained bleeding, especially if the PTT is prolonged with a normal PT and platelet count. Decreases in factor VIII or IX to <20% to 30% can cause PTT prolongations, depending on the PTT reagent and instrument. Since up to 30% of hemophilia A or B cases arise from new mutations, a positive family history will not always be present. If a family history is present, the inheritance pattern is X-linked recessive. The initial tests for hemophilia are the factor VIII and factor IX assays. A von Willebrand test panel is usually also performed. In von Willebrand disease, factor VIII levels are decreased secondary to a decrease in von Willebrand factor. Thus, the tests for von Willebrand factor assess whether a decrease in factor VIII represents von Willebrand disease or hemophilia A. The von Willebrand panel is also useful in predicting hemophilia A carrier status in females. In hemophilia A carrier females, the ratio of factor VIII to von Willebrand factor is approximately 0.5:1. In normal persons, the ratio is approximately 1:1, since factor VIII circulates in the plasma with von Willebrand factor. Confirmation of carrier status often requires family or genetic studies (see below). Both factor VIII and von Willebrand factor can be elevated during acute phase reactions, including pregnancy. Therefore, if a patient is determined to have elevated acute phase reactants, testing should be repeated at a time when the acute phase reaction has subsided. Lastly, factor VIII is labile at room temperature. Consequently, a mild to moderate decrease in factor VIII may be seen in specimens that have not been processed and stored appropriately.

The factor VIII gene is quite large, and numerous mutations causing hemophilia A have been identified. Therefore, genetic testing can be difficult. An inversion mutation of intron 22 has been shown to cause up to 40% of severe hemophilia A in Caucasians, which simplifies genetic testing in these families.11 Restriction fragment length polymorphism (RFLP) studies or methods that directly identify the mutation may be useful in families without the intron 22 inversion.

Numerous mutations causing hemophilia B have also been identified. Like hemophilia A, genetic testing for female carrier status or prenatal detection can often be achieved with restriction fragment length polymorphism (RFLP) analysis or methods that directly demonstrate the mutation.

Many patients with hemophilia became infected with human immunodeficiency virus (HIV) as a result of treatment with factor concentrates prior to the availability of HIV testing of blood donors. Currently, factor VIII and IX concentrates are treated to destroy HIV and other viruses, and blood donors are screened for HIV. Desmopressin (DDAVP) elevates factor VIII (and von Willebrand factor) levels approximately two- to threefold over baseline for 6-12 hours. Therefore, it is often used to treat bleeding episodes in patients with mild hemophilia.

Hereditary Deficiencies of Other Coagulation Factors (Factors II, V, VII, X, XI, XII)

Unlike factor VIII and IX deficiencies, which have X-linked recessive inheritance, hereditary deficiencies of the other coagulation factors have autosomal inheritance. Hereditary deficiencies of factors II, V, VII, and X are rare. Factor XI deficiency is common among individuals of Ashkenazi Jewish descent. Factor XII deficiency is relatively common, but it is not associated with any bleeding risk. With the other factor deficiencies, bleeding symptoms may include easy bruising, epistaxis, menorrhagia, bleeding with surgery, trauma, dental extractions, postpartum, or circumcision, umbilical stump bleeding (especially with factor XIII deficiency or afibrinogenemia, described separately) and bleeding in the gastrointestinal or genitourinary tract. Intracranial hemorrhage has been reported with severe deficiencies of factor II, V, VII, or X. Hemarthrosis and bleeding into muscles, characteristic of factor VIII and IX deficiencies, are less common but can occur in other factor deficiencies.12,13,14,15,16 Factor deficiencies may prolong the PT and/or PTT, depending on the factor and the severity of the decrease in factor level (see Table 1).

In general, with hereditary factor deficiencies, heterozygous deficient individuals have approximately 50% (most commonly within 30% to 60%) of the normal value for the affected factor. Homozygous deficient individuals have a more severe decrease in the affected factor. As previously mentioned, heterozygous or homozygous deficiencies of factor XII do not cause bleeding.17 Heterozygous deficiencies of the other factors are usually either asymptomatic or have a milder bleeding tendency than homozygous deficiencies (see Table 3). Factor XI deficient heterozygotes may have bleeding symptoms.16 Factor II or factor X deficient heterozygotes sometimes have mild bleeding symptoms. With rare exceptions, heterozygous factor V or VII deficiencies are asymptomatic. In contrast, homozygous deficiencies of these factors (II, V, VII, X, XI) do have an increased incidence of bleeding symptoms. However, factor V, VII, or XI levels do not always correlate with severity of symptoms. In general, factor VIII and IX deficiencies tend to be the most severe, while deficiencies of factors II, V, or XI tend to be milder than factor VIII or IX deficiencies. Severe deficiencies of factor VII or X can have a clinical presentation as severe as hemophilia A or B.

Table 3. Coagulation Factor Deficiencies andFactorHalf-Lives

Factor Deficiency	Level Required for Surgical Hemostasis	Bleeding Risk in Homozygous Deficiency?	Bleeding Risk in Heterozygous Deficiency?	Biologic Half-life of Factor
Fibrinogen (factor I)	100 mg/dL	Yes	Sometimes	72-120 h
Prothrombin (factor II)	10%-40%	Yes	Sometimes	72 h (48-120 h)
Factor V	10%-30%	Yes*	No (rare exceptions)	12-36 h
Factor VII	10%-25%	Yes*	No (rare exceptions)	4-7 h
Factor VIII	Major surgery or major bleeding: 80%-100%	Yes (X-linked recessive)	No (rare exceptions; heterozygotes are carrier females)	8-12 h
	Postoperative: 30%-50%
	Minor bleeding: 30%-50%
Factor IX	Major surgery or major bleeding: 50%-80%	Yes (X-linked recessive)	No (rare exceptions; heterozygotes are carrier females)	18-24 h
	Postoperative: 40%
	Minor bleeding: 30%-50%
Factor X	10%-40%	Yes	Sometimes	24-48 h
Factor XI	15%-50%	Sometimes*	Sometimes	40-84 h
Factor XII	0%	No	No	48-52 h
Factor XIII	>5%-50%**	Yes	Sometimes	9-12 d
*Factor V, VII, or XI levels do not always correlate well with severity of bleeding.
**Factor XIII levels >1%-5% have traditionally been considered asymptomatic; however, recent evidence suggests heterozygous deficiencies with levels up to 50% can be associated with excess bleeding.
Adapted from Van Cott EM and Laposata M, “Coagulation, Fibrinolysis, and Hypercoagulation,” Clinical Diagnosis and Management by Laboratory Methods, 20th ed, Henry JB, ed, New York, NY: WB Saunders Co, 2001, 642-59.
References:
Menitove 1995, Edmunds 1994, Laposata 1989, Roberts 1995, Roberts 1994, and see text for references regarding bleeding risk with heterozygous and homozygous deficiencies.

Hereditary Combined Coagulation Factor Deficiencies

Combined factor deficiencies are very rare. A combined deficiency of factors V and VIII is an autosomal recessive disorder arising, in most families studied so far, from a mutation in an endoplasmic reticulum-Golgi intermediate compartment gene on chromosome 18 which appears to decrease intracellular transportation of factors V and VIII.18 A combined deficiency of the vitamin K-dependent factors II, VII, IX, and X has been described which, in at least some families, is due to a mutation in the gamma-glutamyl carboxylase gene.19 The gene codes for an enzyme that carboxylates glutamate residues in the vitamin K dependent coagulation factors, a reaction that is necessary for normal function of these vitamin K-dependent factors.

Footnotes

1. Andrew M, Vegh P, Johnston M, et al, “Maturation of the Hemostatic System During Childhood,”Blood, 1992, 80(8):1998-2005.

2. Andrew M, Paes B, and Johnston M, “Development of the Hemostatic System in the Neonate and Young Infant,”Am J Pediatr Hematol Oncol, 1990, 12(1):95-104.

3. Moll S and Ortel TL, “Monitoring Warfarin Therapy in Patients With Lupus Anticoagulants,”Ann Intern Med, 1997, 127(3):177-85.

4. Iacoviello L, Di Castelnuovo A, de Knijff P, et al, “Polymorphisms in the Coagulation Factor VII Gene and the Risk of Myocardial Infarction,”N Engl J Med, 1998, 338(2):79-85.

5. Ma J, Hennekens CH, Ridker PM, et al, “A Prospective Study of Fibrinogen and Risk of Myocardial Infarction in the Physician’s Health Survey,”J Am Coll Cardiol, 1999, 33(5):1347-52.

6. van der Meer FJ, Koster T, Vandenbroucke JP, et al, “The Leiden Thrombophilia Study (LETS),”Thromb Haemost, 1997, 78(1):631-5.

7. Meijers JC, Tekelenburg WLH, Bouma BN, et al, “High Levels of Coagulation Factor XI as a Risk Factor for Venous Thrombosis,”N Engl J Med, 2000, 342(10):696-701.

8. Mennen LI, Schouten EG, Grobbee DE, et al, “Coagulation Factor VII, Dietary Fat and Blood Lipids: A Review,”Thromb Haemost, 1996, 76(4):492-9.

9. Hoyer LW, “Hemophilia A,”N Engl J Med, 1994, 330(1):38-47.

10. Ljung R, Petrini P, and Nilsson M, “Diagnostic Symptoms of Severe and Moderate Haemophilia A and B. A Survey of 140 Cases,”Acta Paediatr Scand, 1990, 79(2):196-200.

11. Naylor JA, Green PM, Rizza CR, et al, “Factor VIII Gene Explains All Cases of Haemophilia A,”Lancet, 1992, 340(8827):1066-7.

12. Giangrande PLF, “Other Inherited Disorders of Blood Coagulation,” Rizza C and Lowe G, eds, Haemophilia and Other Inherited Bleeding Disorders, London, WB Saunders Co, 1997, 291-307.

13. Kane WH and Davie EW, “Blood Coagulation Factors V and VIII: Structural and Functional Similarities and Their Relationship to Hemorrhagic and Thrombotic Disorders,”Blood, 1988, 71(3):539-55.

14. Cooper DN, Millar DS, Wacey A, et al, “Inherited Factor VII Deficiency: Molecular Genetics and Pathophysiology,”Thromb Haemost, 1997, 78(1):151-60.

15. Cooper DN, Millar DS, Wacey A, et al, “Inherited Factor X Deficiency: Molecular Genetics and Pathophysiology,”Thromb Haemost, 1997, 78(1):161-72.

16. Modi GJ and Musclow CE, “Factor XI: A Piece of the Coagulation Puzzle,”Lab Med, 1993, 24:353-6.

17. Halbmayer WM, Haushofer A, Schon R, et al, “The Prevalence of Moderate and Severe FXII (Hageman Factor) Deficiency Among the Normal Population: Evaluation of the Incidence of FXII Deficiency Among 300 Healthy Blood Donors,”Thromb Haemost, 1994, 71(1):68-72.

18. Nichols WC, Seligsohn U, Zivelin A, et al, “Mutations in the ER-Golgi Intermediate Compartment Protein ERGIC-53 Cause Combined Deficiency of Coagulation Factors V and VIII,”Cell, 1998, 93(1):61-70.

19. Brenner B, Sanchez-Vega B, Wu SM, et al, “A Missense Mutation in Gamma-Glutamyl Carboxylase Gene causes Combined Deficiency of all Vitamin K-Dependent Blood Coagulation Factors,”Blood, 1998, 92(12):4554-9.

References

Edmunds LH and Salzman EW, “Hemostatic Problems, Transfusion Therapy, and Cardiopulmonary Bypass in Surgical Patients,”Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd ed, Colman RW, Hirsh J, Marder VJ, et al, eds, Philadelphia, PA: Churchill Livingstone, 1994, 958.

Laposata M, Connor AM, Hicks DG, et al, The Clinical Hemostasis Handbook, Chicago, IL: Yearbook Medical Publishers, Inc, 1989.

Menitove JE, Gill JC, and Montgomery RR, “Preparation and Clinical Use of Plasma and Plasma Fractions,”William’s Hematology, 5th ed, Beutler E, Lichtman MA, Coller BS, et al, eds. New York, NY: McGraw-Hill, 1995, 1657.

Roberts HR and Hoffman M, “Hemophilia and Related Conditions – Inherited Deficiencies of Prothrombin (Factor II), Factor V, and Factors VII to XII,”William’s Hematology, 5th ed, Beutler E, Lichtman MA, Coller BS, et al, eds, New York, NY: McGraw-Hill, 1995, 1413-39.

Roberts HR and Lefkowitz JB, “Inherited Disorders of Prothrombin Conversion,”Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd ed, Colman RW, Hirsh J, Marder VJ, et al, eds, Philadelphia, PA: Churchill Livingstone, 1994, 200-18.