Thrombosis Risk Tests
Thrombosis risk tests check for defects in the anti-coagulant system (hypercoagubility) that can cause a predisposition to thrombosis. The D-dimer test is used to screen for the presence of fibrin associated with deep vein and other forms of thrombosis.
The purpose of thrombosis risk tests is to establish whether someone has a predisposition for developing thrombosis or has suffered a thrombotic episode, so that appropriate interventions can be instituted. The most common thrombosis risk tests are the D-dimer test, protein C test, protein S test, factor V Leiden test, prothrombin 1+2 (prothrombin 1.2) test, and the antithrombin test. These tests can be ordered individually but are usually ordered as a part of a panel.
Common indications for testing include:
- venous thrombosis
- pulmonary embolism
- cerebral brain thrombosis
- transient ischemic attack or premature stroke
- peripheral vascular disease
- prior to pregnancy, oral contraceptive prescription, estrogen therapy or major surgery if there is a family history of thrombosis
- relative with known genetic predisposition to thrombosis
- history of thrombosis and presence of a known genetic predisposition to thrombosis
- previous laboratory finding of activated protein C resistance (indication for factor V Leiden DNA test)
- premature myocardial infarction in a female patient (indication for prothrombin DNA test)
- history of multiple unexplained miscarriages
Treatment with Coumadin, an anticoagulant, can interfere with the protein C and protein S tests. Ideally, the patient should discontinue treatment with Coumadin two weeks prior to undergoing these tests. If this is not possible then, an alternate panel of risk tests should be used. Alternatively heparin therapy can replace Coumadin therapy for two weeks prior to the tests, although heparin anticoagulant therapy can sometimes result in false positive antithrombin III test results. Protein S assays are not reliable during pregnancy. Heterophilic antibodies and rheumatoid factor are known to cause false positive reactions for D-dimer.
The D-dimer test is the only laboratory test that is used to screen for the presence of deep vein thrombosis. The test is positive only when fibrin has formed. The D fragment of fibrinogen is produced by the action of plasmin on fibrinogen. Thrombin activates factor XIII, which stabilizes the fibrin clot by dimerizing the D fragments. In disseminated intravascular coagulation, pulmonary embolism, deep vein thrombosis, sickle cells disease and other conditions such as post surgical thrombus formation, the D-dimer level will be elevated in serum or plasma. D-dimer is measured by immunoassay, either latex agglutination or enzyme immunoassay (EIA). Latex agglutination is a qualitative assay that is not sufficiently sensitive to screen for deep vein thrombosis. Levels measured by EIA below 200 ng/ml indicate that thrombosis is unlikely in patients with no apparent signs of deep vein thrombosis.
Prothrombin fragment 1.2 (1+2), like D-dimer, is a marker for thrombotic disease. Prothrombin fragment 1+2 (1.2) can be measured by enzyme immunoassay. This fragment is produced when factor Xa activates prothrombin. The prothrombin fragment is increased in persons at risk for thrombotic episodes.
Other thrombosis risk tests check for mutations in the genes or proteins that are involved in the anticoagulant system. The anticoagulant system is designed to regulate coagulation and prevent excess blood clotting. Each anticoagulant protein is produced by a different gene. Each person possesses two copies of each anticoagulant gene. Mutation in an anticoagulant gene can cause it to produce abnormal protein, an increased or decreased amount of normal protein or can cause it to stop producing protein altogether. The common anticoagulant abnormalities (protein S, protein C, antithrombin III, prothrombin and factor V Leiden) are autosomal dominant, since only one gene of a pair needs to be
In some cases a thrombosis risk test checks for a change in the anticoagulant gene. In other cases, it is not feasible to check for a gene change and the activity of the protein is assayed.
Proteins C and S
Mutation in the genes that produce protein C and protein S can cause an increased risk of thrombosis. The frequency of protein C deficiency in the general population is 0.5% or less and the frequency of protein S deficiency is approximately 0.7%. Activated protein C (APC) is involved in inactivating blood coagulation factors V and VIII. Inactivation of these factors decreases blood coagulation. Activated protein S is a cofactor that enhances the activity of protein C. A deficiency in activated factors C or S can result in increased levels of factor Va and VIIIa, which increases the risk of thrombosis.
As of 2001, DNA testing for proteins C and S deficiencies is not available on a clinical basis. Proteins C and S can be measured by immunoassay which determines the mass of protein present, or by one of two functional tests. Protein C is a serine protease that inactivates factors Va and VIIIa. In the chromogenic substrate assay, plasma is mixed with Agkistrodon snake venom, an activator of protein C. The activated protein C splits a synthetic anilide substrate producing a yellow product. The amount of color is proportional to the concentration of functional protein C. However, this test does not detect all abnormal forms of protein C and will be normal in those cases where the defect occurs in the binding of protein C to protein S. All forms of protein C deficiency can be detected using a coagulation test in which protein C deficient plasma is mixed with Agkistrodon snake venom and the patient's plasma. Calcium chloride and activated thromboplastin are added and the time required for clot formation is measured. The clotting time is proportional to the concentration of functional protein C in the sample.
Protein S is a cofactor required for enzymatic activity of protein C. Protein S can be measured by immunoassay or by a coagulation test using protein S deficient plasma, activated protein C, activated factor V, and calcium. The time required for a clot to form is proportional to protein S activity.
Factor V Leiden
A mutation in the gene that produces factor V protein, called a factor V Leiden mutation, causes this factor to become resistant to inactivation by protein C (APC resistance). APC resistance increases the risk of thrombosis. If another type of factor V mutation, called an R2 mutation, is found in one copy of the factor V gene, and a Leiden mutation is found in the other copy, the risk of thrombosis is further increased. An R2 mutation alone does not cause an increased risk of thrombosis. R2 mutation testing is, therefore, only performed if a Leiden mutation is found in one copy of the factor V gene. Factor V Leiden has normal coagulation activity when activated, and therefore, does not affect clotting tests such as the prothrobin time. It is detected by the polymerase chain reaction (PRC) using a probe that recognizes the point mutation in the factor V gene. Factor V Leiden is the most common inherited risk factor for hypercoagulability. Its prevalence is 2–7% in the general population.
Prothrombin (factor II)
A mutation in the gene that produces prothrombin can also result in an increased risk of thrombosis. Prothrombin is the precurser to thrombin. Thrombin when activated converts fibrinogen to fibrin which forms the clot. A mutation, called G20210A, in the gene that produces prothrombin results in increased prothrombin plasma levels and an increased risk of thrombosis. Prothrombin mutation is the second most common inherited risk factor for hypercoagulability; the point mutation occurs in approximately 2% of the general population. The changed gene is detected by PCR analysis of DNA.
Mutation in the gene that produces Antithrombin III can result in increased thrombosis. Antithrombin III (AT), when activated by heparin, neutralizes thrombin and other activated coagulation factors. A deficiency in this protein results in increased levels of coagulation factors
DNA tests require 5 mL of whole blood in an EDTA (lavender top) tube and protein activity tests require 3 mL of fresh or frozen citrated plasma. Thrombosis risk panels require 5 mL of whole blood in an EDTA (lavender top)tube and 3 mL of fresh or frozen citrated plasma in 1 mL aliquots. The turn around time for thrombosis risk tests range from one to five days.
Thrombosis risk panels
Two thrombosis risk panels are used, one for patients not receiving Coumadin therapy and one for those who are.
Panel for patients not on Coumadin therapy:
- factor V Leiden DNA test
- prothrombin (Factor II) DNA test
- antithrombin activity
- protein C activity
- protein S activity
(This panel is less accurate and should only be used if discontinuation of therapy is not possible.) Panel for patients on Coumadin therapy:
- factor V Leiden DNA test
- prothrombin (Factor II) DNA test
- antithrombin activity
- protein C/factor IX antigen ratio
- protein S/factor IX antigen ratio
If possible, Coumadin anticoagulant therapy should be discontinued at least two weeks prior to undergoing the thrombosis risk tests.
There are no post-test procedures required.
Excessive bleeding, bruising, and soreness around the puncture site, as well as fainting and feeling light-headed are possible complications of the blood draw. Infection is also an occasional complication.
The type of results, interpretation, and management recommendations vary by type of thrombosis risk. Factor V and prothrombin DNA testing is fairly definitive. Test results for protein S, C, and antithrombin deficiencies are more difficult to interpret since environmental factors can influence the results. The clinical history and family history should be used to aid in the interpretation. It is important to rule out acquired protein S, C, and antithrombin deficiency prior to establishing a diagnosis. Acquired protein S deficiency is quite common and can be caused by factors such as: the lupus anticoagulant, pregnancy, liver disease, inflammatory conditions, nephritic syndrome, and thomboembolism. Liver disease can decrease protein C levels and oral contraceptives can increase protein C levels. Acquired antithrombin deficiency can result from mild liver disease, acute thrombosis, and heparin anticoagulant therapy. When the results are borderline, repeat testing and comparative studies of other family members may be appropriate. Protein activity testing cannot definitively differentiate those with one abnormal copy from those with two abnormal copies of an anticoagulant gene.
These may be defined in mass units for immunoassay methods or as the percentage of normal for functional assays. Values presented below are representative of immunoassay and functional assays but will vary depending upon the method employed.
- antithrombin III: 20-30 mg/dL or 80-120% of normal
- D-dimer: less than 200 ng/mL
- protein C: 3-4 μg/mL or greater than 65% of normal
- protein S: 0.7-1.4 μg/mL or greater than 65% of normal
Anticoagulant—A medication that prevents blood clotting.
Blood clot—The solid clump of accumulated blood factors that results when blood coagulates.
Cerebral brain thrombosis—Thrombus that forms within a blood vessel in the brain.
DNA testing—Testing for a change or changes in a gene or genes.
Embolism—A blood clot that has traveled from a different location.
Gene—A building block of inheritance, made up of a compound called DNA (deoxyribonucleic acid) and containing the instructions for the production of a particular protein.
Heterozygous—Changes in one copy of a gene.
Homozygous—Changes in both copies of a gene.
Mutation—Change in a gene.
Peripheral vascular disease—Narrowing of the blood vessels that carry blood to the extremities such as the arms and legs.
Neonatal purpura fulminans—A life-threatening condition in the neonate that results in small hemorrhages in the skin.
Placental infarction—An area of dead tissue in the placenta that is due to an obstruction of circulation in the area.
Protein—A substance produced by a gene that is involved in creating the traits of the human body or is involved in controlling the basic functions of the human body such as blood coagulation.
Thrombosis—The development of a thrombus.
Thrombus—An accumulation of blood factors that often causes a vascular obstruction. Often used synonymously with the term blood clot.
Transient ischemic attack—A temporary blockage of an artery which supplies blood to the brain and lasts less than 24 hours. Often called a "mini-stroke."
Health care team roles
The main role for the nurse is patient education. Patients with positive results need to be informed of the increased risk of thrombosis. Patients need to be reassured, however, that many people with a genetic predisposition to thrombosis remain free of symptoms for their entire life. Women should be informed that they have an increased risk of second-or third-term pregnancy loss and obstetrical complications such as preeclampsia, fetal growth retardation, and placental infarction. Patients also need to be counseled about the common environmental risk factors for thrombosis. Thrombosis risk tests are performed by a clinical laboratory scientist, CLS(NCA)/medical technologist, MT(ASCP) or clinical laboratory technician, CLT(NCA) or medical laboratory technician, MLT(ASCP). Results of a thrombosis risk panel or test is interpreted by a physician. The physician also determines if further tests (e.g. Doppler ultrasound) are needed and directs any anticoagulant therapy.
Common environmental risk factors for thrombosis include:
- oral contraceptive use
- estrogen therapy
- medications that are estrogen receptor modulators such as Tamoxifan and Raloxifene
- diabetes mellitus
- presence of lupus anticoagulant
- prolonged bed rest
Smoking should be discouraged in all patients with positive test results. Oral contraceptive use should be strongly discouraged in patients who are homozygous for the prothrombin or factor V Leiden mutations or who have a severe C, S, or antithrombin deficiency. Patients who are heterozygous for factor V Leiden or prothrombin G20210A or who have a mild deficiency in protein C, S, or antithrombin should be informed of the risks associated with oral contraceptive use.
It is important that the patient be informed of the hereditary nature of the disorder. Heterozygotes have a 50% chance of passing on the changed gene to their offspring and homozyotes have a 100% chance of passing on a changed gene. Homozygotes have inherited a changed gene from each parent. Heterozygotes have usually inherited the changed gene from either their father or mother. In some cases the gene change will occur spontaneously
Patients with positive test results should be encouraged to inform first degree relatives of their risks. It can sometimes be helpful to provide the patient with an informational letter about their test results that they can give to other family members.
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Rodgers, George. "Thrombosis and Antithrombotic Therapy." In Wintrobe's Clinical Hematology, 10th ed. Edited by Lee, Richard, John Foerster, John Lukens, Frixos Paraskevas, John Greer, and George Rodgers. Baltimore, MD: Williams and Wilkins, 1999. pp. 1781-1818.
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Barger, A. P. and R. Hurley. "Evaluation of the hypercoagulable state. Whom to screen, how to test and treat." Postgraduate Medicine 108 (4), (September 15, 2000): 59-66.
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Lisa Maria Andres, M.S., GCG