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.
Index of Tests
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.
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,
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).
Thrombosis (eg, pulmonary embolism, acute myocardial infarction,
Disseminated intravascular coagulation (DIC)
Oral contraceptives, pregnancy (possibly)
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
Hirudin or argatroban anticoagulation can interfere with thrombin-based
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
Decreased antithrombin also arises from acquired conditions, such
* decreased hepatic synthesis from liver disease or L-asparaginase
* consumption from thrombosis, DIC (disseminated intravascular
coagulation) or surgery
* increased clearance from full-dose heparin use14
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.
1. Andrew M, Paes B, Milner R, et al, "Development of the Human
Coagulation System in the Full-Term Infant,"Blood, 1987,
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,
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,
6. Tait RC, Walker ID, Perry DJ, et al, "Prevalence of Antithrombin
Deficiency in the Healthy Population,"Br J Haematol, 1994,
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,
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,
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.
Blajchman MA, Austin RC, Fernandez-Rachubinski F, et al, "Molecular
Basis of Inherited Human Antithrombin Deficiency,"Blood,
De Stefano V, Finazzi G, and Mannucci PM, "Inherited Thrombophilia:
Pathogenesis, Clinical Syndromes, and Management,"Blood,