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 Protein C Resistance and the Factor V Leiden Mutation
Heparin Antifactor Xa Assay
Hypercoagulation Panel
Protein C
Protein S
Applies to Heparin; Heparin Cofactor II; Heparin Resistance
Replaces Antithrombin III Assay
Abstract A deficiency of antithrombin, a natural anticoagulant protein, leads to a hypercoagulable state with an increased risk for venous thrombosis. Acquired antithrombin deficiencies are more common than hereditary deficiencies.
Specimen Plasma
Container One blue top (sodium citrate) tube
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.
Storage Instructions Separate plasma from cells as soon as possible. Plasma may be stored on ice for up to 4 hours, otherwise, store frozen.
Causes for Rejection Specimen received more than 4 hours after collection, tubes not filled, clotted specimens
Turnaround Time 2-4 hours; longer if testing is batched
Reference Interval Results are often reported as a percent of the amount expected in normal plasma. By definition, the mean value in normal plasma is 100%. The reference range is approximately 80% to 130%.
Results may also be reported in mg/dL; reference range is approximately 17-39 mg/dL (SI: 170-390 mg/L).
At birth, antithrombin levels average 63% (range 39% to 87%) of adult levels. Antithrombin increases to adult values within 6 months.1 Spontaneous thromboses do not develop in normal infants because a balance between procoagulants and inhibitors is maintained.
Use A functional assay should be performed first, because both type I and type II hereditary antithrombin deficiencies will be detected. If the result of the functional assay is decreased, the antigenic assay is needed to differentiate between type I and type II deficiencies. If the antigen assay is performed without the functional assay, type II deficiencies will not be detected (see Additional Information).
Antithrombin Levels
| Increased With | Decreased With |
| Coumadin® (possibly) | Heparin |
| Liver disease | |
| Thrombosis (eg, pulmonary embolism, acute myocardial infarction, thrombophlebitis) | |
| Disseminated intravascular coagulation (DIC) | |
| Surgery | |
| Nephrotic syndrome | |
| Oral contraceptives, pregnancy (possibly) |
Limitations
Chromogenic (functional) assays: Heparin cofactor II, another natural thrombin inhibitor, produces falsely elevated levels of antithrombin in some thrombin-based assays.2 One commercially available kit uses heparin that has been treated with a bacterial enzyme (chondroitinase) such that the heparin no longer enhances heparin cofactor II activity, and heparin cofactor II interference is thus essentially eliminated.3 In factor Xa-based assays, high levels of heparin cofactor II will not lead to an overestimate of antithrombin, because heparin cofactor II does not inhibit factor Xa.
Hirudin or argatroban anticoagulation can interfere with thrombin-based assays.
Antigenic assays: If used without the functional assay, type II antithrombin deficiencies will not be detected. An antigenic test is usually performed only if the functional test result is decreased, to determine if the patient has type I or type II deficiency (see Additional Information).
Methodology Functional (activity) and antigenic (immunologic) tests are available. Functional assays are usually chromogenic. To perform the chromogenic test, heparin and thrombin are added to the patient’s plasma. Antithrombin in the patient’s plasma will bind and inhibit thrombin. A chromogenic substrate that resembles thrombin’s natural substrate is then added. Any unbound thrombin will cleave the substrate, liberating a chromogenic substance that can be measured spectrophotometrically. The amount detected is inversely proportional to the amount of antithrombin in the patient.4 Factor Xa-based methods are also available; these are similar in principle to the thrombin-based assays described above, except that factor Xa is used instead of thrombin.2 Antigenic (immunologic) assay: A commonly used automated method involves latex particles coated with antibodies directed against antithrombin. In the presence of antithrombin, the latex particles form aggregates that absorb light passing through the specimen. The amount of light absorbance is directly related to the amount of antithrombin in the specimen.5
Additional Information Antithrombin is a natural inhibitor of thrombin as well as factors Xa, IXa, XIa, XIIa, and kallikrein. The activity of antithrombin is greatly accelerated by interaction with the glycosaminoglycans, heparan sulfate or heparin. Heparan sulfate is naturally located in vivo on the endothelial cell surface. Antithrombin deficiency is present in 0.17% of the general population.6 It accounts for 1.1% of unselected patients with venous thrombosis and up to 5% of patients younger than age 70 years with thrombosis.7,8 Over 127 mutations in the antithrombin gene are known to cause hereditary antithrombin deficiency.9 Individuals heterozygous for antithrombin deficiency have a fivefold increased risk for venous thrombosis.10 Homozygous deficiencies are incompatible with life, except for patients with type II deficiency due to heparin-binding mutations. Heterozygotes generally have antithrombin levels between 45% to 75%,11 although levels as high as 78% have been observed. The risk for thrombosis is further increased in the presence of a second risk factor.12 The age at onset of thrombosis is usually between 10-50 years (peak 15-35 years) in heterozygous individuals. The risk of arterial thrombosis remains uncertain, but 2% of individuals developed arterial thrombosis in one study.13
Decreased antithrombin also arises from acquired conditions, such as:
* decreased hepatic synthesis from liver disease or L-asparaginase treatment
* consumption from thrombosis, DIC (disseminated intravascular coagulation) or surgery
* increased clearance from full-dose heparin use14
* proteinuria
Mild decreases occasionally result from elevated estrogen levels (eg, pregnancy or oral contraceptive use). Colitis has been associated with low antithrombin levels. If a patient with low antithrombin has any of the conditions listed above, the test should be repeated once the condition is no longer present, if possible. Confirmation of a hereditary antithrombin deficiency may require documenting antithrombin deficiency in a relative. In contrast to protein C and protein S, which are decreased by Coumadin®, antithrombin levels may increase while on Coumadin®. Antithrombin levels in premenopausal women may be somewhat lower than in men, but postmenopausal women have higher levels than men.15
Antithrombin deficiencies are quantitative (type I) or qualitative (type II).11 In type I deficiencies, normal antithrombin molecules are made, but in reduced quantity. In type II deficiencies, normal amounts of antithrombin are made, but the antithrombin is defective. Accordingly, type I deficiencies have decreased antithrombin in both functional and antigenic assays. Type II deficiencies have normal (or near normal) antigenic antithrombin levels, with decreased functional antithrombin. Thus, if only antigenic assays are performed, type II deficiencies will not be detected. Therefore, a functional assay should be used as the initial screening assay. If the result is decreased, an antigenic assay should be performed to determine if the deficiency is type I or type II. According to one study, 0.02% of the general population have type I antithrombin deficiency and 0.15% have type II.6 The heparin-binding variant, which is one of the mutations that causes type II deficiency, has a low risk of thrombosis in comparison to the other mutations, and it is present in at least 0.01% of the general population.6 For patients with test results suggesting type II deficiency, a method has been described that tests for the heparin-binding mutation.16
Patients with marked decreases in antithrombin may demonstrate heparin resistance, in which very high doses of heparin are required to obtain a therapeutic PTT prolongation. Antithrombin concentrates are available for the treatment of hereditary antithrombin deficiency. The use of antithrombin concentrates for certain acquired antithrombin deficiencies, such as DIC, is under investigation.17
See Hypercoagulation Panel.
Footnotes
1. Andrew M, Paes B, Milner R, et al, “Development of the Human Coagulation System in the Full-Term Infant,”Blood, 1987, 70(1):165-72.
2. Demers C, Henderson P, Blajchman MA, et al, “An Antithrombin III Assay Based on Factor Xa Inhibition Provides a More Reliable Test to Identify Congenital Antithrombin III Deficiency Than an Assay Based on Thrombin Inhibition,”Thromb Haemost, 1993, 69:231-5.
3. Triscott MX and Eggerding VC, “Improved Differentiation Between Normal and Abnormal Antithrombin Levels Using a Thrombin Based Chromogenic Assay,”Thromb Haemost, 1999, (Suppl):379.
4. Odegard O, Lie M, and Ablidgaard U, “Heparin Cofactor II Activity Measured With an Amidolytic Method,”Thromb Res, 1975, 6:287-94.
5. Laroche P, Plassart V, and Amiral J, “Rapid Quantitative Latex Immunoassays for Diagnosis of Thrombotic Disorders,”Thromb Haemost, 1989, 62:379.
6. Tait RC, Walker ID, Perry DJ, et al, “Prevalence of Antithrombin Deficiency in the Healthy Population,”Br J Haematol, 1994, 87(1):106-12.
7. Rodeghiero F and Tosetto A, “The Epidemiology of Inherited Thrombophilia: The VITA Project,”Thromb Haemost, 1997, 78(1):636-40.
8. Melissari E, Monte G, Lindo VS, et al, “Congenital Thrombophilia Among Patients With Venous Thromboembolism,”Blood Coagul Fibrinolysis, 1992, 3(6):749-58.
9. Bayston TA and Lane DA, “Antithrombin: Molecular Basis of Deficiency,”Thromb Haemost, 1997, 78(1):339-43.
10. van der Meer FJM, Koster T, Vandenbroucke JP, et al, “The Leiden Thrombophilia Study (LETS),”Thromb Haemost, 1997, 78(5):631-5.
11. Lane DA, Bayston T, Olds RJ, et al, “Antithrombin Mutation Database: 2nd (1997) Update,”Thromb Haemost, 1997, 77(1):197-211.
12. van Boven HH, Vandenbroucke JP, Briet E, et al, “Gene-Gene and Gene-Environment Interactions Determine Risk of Thrombosis in Families With Inherited Antithrombin Deficiency,”Blood, 1999, 94:2590-4.
13. Demers C, Ginsberg JS, Hirsh J, et al, “Thrombosis in Antithrombin III-Deficient Persons: Report of a Large Kindred and Literature Review,”Ann Intern Med, 1992, 116(9):754-61.
14. Rao AK, Niewiarowski S, Guzzo J, et al, “Antithrombin III Levels During Heparin Therapy,”Thromb Res, 1981, 24:181-6.
15. Meade TW, Dyer S, Howarth DJ, et al, “Antithrombin III and Procoagulant Activity: Sex Differences and Effects of the Menopause,”Br J Haematol, 1990, 74(1):77-81.
16. Sas G, Pepper DS, and Cash JD, “Further Investigations on Antithrombin III in the Plasmas of Patients With the Abnormality of Antithrombin III Budapest,”Thromb Diath Haemorrh, 1975, 33:564-72.
17. Eisele B and Lamy M. “Clinical Experience With Antithrombin III Concentrates in Critically Ill Patients With Sepsis and Multiple Organ Failure,”Semin Thromb Hemost, 1998, 24:(1)71-80.
References
Blajchman MA, Austin RC, Fernandez-Rachubinski F, et al, “Molecular Basis of Inherited Human Antithrombin Deficiency,”Blood, 1992, 80(9):2159-71.
De Stefano V, Finazzi G, and Mannucci PM, “Inherited Thrombophilia: Pathogenesis, Clinical Syndromes, and Management,”Blood, 1996, 87(9):3531-44.
