Hypercoagubility tests measure three proteins produced by the liver that when deficient increase the risk of thrombosis. These proteins are antithrombin III, protein C, and protein S.
The blood coagulation process prevents people from bleeding excessively. Normally, this process occurs whenever the blood vessel wall incurs damage, and the resulting clot keeps the blood from escaping from the damaged vessel. The coagulation factors responsible for clot formation are mainly enzymes, and their activity is down regulated by other proteins that inactivate them. Abnormal clot formation will occur when one of these proteins is defective or diminished in concentration. The purpose of hypercoagubility tests is to determine whether a person is at increased risk for abnormal clot formation. A person with a deficiency of antithrombin III, protein C, or protein S is at increased risk for thrombus formation. This can result in occlusion of a vessel and diminished blood flow to dependent tissues.
When functional activity is being measured for protein S or C, anticoagulant therapy with warfarin (Coumadin) will interfere with the results. Heparin therapy will interfere with functional assays for antithrombinIII. Blood for these tests must be collected in sodium citrate. The nurse or phlebotomist collecting blood for this test should observe universal precautions for the prevention of transmission of bloodborne pathogens. Shaking the tube vigorously, collecting an insufficient sample, or using the wrong tube required for hypercoagubility tests are not acceptable and will require a second venipuncture.
Hypercoagulable conditions that allow the blood to clot inappropriately pose a life-threatening risk to patients. A thrombus that forms in the absence of bleeding can obstruct the normal flow of blood to tissues beyond the clot site. If a portion of the mass breaks off and travels to another body site or organ, it is called an embolus. A thrombus or embolus can be fatal if it lodges in a vital area like the heart or lungs. When antithrombin III, protein C, or protein S is deficient or defective, there will be an increased risk of thrombus or embolus formation.
Deficiencies of antithrombin III, protein C, and protein S may be either inherited or acquired. All three deficiencies are inherited as an autosomal dominant condition. The heterozygote (one defective gene) generally has 40-50% of the normal level of functional protein. Such persons usually have a history of thrombotic events such as deep or superficial vein thrombosis, pulmonary or cerebral embolism, ischemic heart disease or coronary thrombosis. Since the liver is the site of synthesis, a deficiency of each of these proteins can result from liver disease. Antithrombin III deficiency may also be caused by oral contraceptives, nephrotic syndrome, and disseminated intravascular coagulation. Acquired protein S deficiency is also associated with the lupus anticoagulant (an antibody to phospholipid), pregnancy, oral contraceptives, and vitamin K deficiency.
Hypercoagubility testing is performed when a patient has risk factors that increase the chance of thrombosis. These factors include:
Antithrombin III binds to factor Xa and thrombin (serine proteases involved in coagulation) and prevents them from acting on their natural substrates. Antithrombin III requires heparin for its activity. The heparin is released from the vessel wall cells and complexes the antithrombin III. This causes changes in the conformation of antithrombin III that allow it to bind to the active sites of Xa and thrombin. Two types of assays are available for measuring antithrombin III. Immunoassays such as nephelometry determine the mass of antithrombin III in the plasma based upon its reaction with specific antibody. Functional assays are usually based upon its activity against a chromogenic substrate. The plasma is incubated with excess factor Xa and heparin. The factor Xa that remains active is measured by
Immunoassay—A laboratory analysis that uses an antibody-antigen reaction. A specific antibody is used that will react with the protein of interest, for example antithrombin III. In this reaction, the antithrombin III functions as the antigen.
Nephelometry—A laboratory technique used to measure light scattering by small immune complexes suspended in the reaction solution. Light scattering is proportional to the antigen concentration with the antibody is present in excess.
its ability to split a synthetic analide substrate. This produces aniline, which is yellow in color and can be measured at 405 nm. The amount of aniline produced is inversely proportional to antithrombin III activity. Results are expressed as the percentage of a normal control plasma. It is important to note that immunoassays and functional assays may give different results. When the quantity of antithrombin III is decreased, both tests will be abnormal. However, when the protein is defective, the immunoassay result will be normal, but the functional assay will be abnormal. Some functional deficiencies are heparin dependent. When the assay is performed without heparin, the activity is equal to that of the normal control. These persons have an antithrombin III with a defective heparin binding site, and their condition is usually milder.
Protein C is a serine protease that enzymatically inactivates both factor Va and factor VIIIa. Protein C is activated when a receptor on the blood vessel cell surface called thrombomodulin binds to and neutralizes the coagulating activity of thrombin. The activity of protein C requires phospholipids from platelets adhering to the vessel wall and also protein S. As with antithrombin III, protein C may be measured by immunoassay or by a functional assay. When immunoassay is used, only a quantitative deficiency of protein C will be detected. Two types of functional assays are used, the chromogenic substrate assay and the clot formation (activated partial thromboplastin) test. In the clot formation test, plasma is mixed with an activator of protein C, usually Agkistrodon snake venom. After incubation, calcium chloride and activated thromboplastin are added and the time required for a clot to form is measured. The time required for the clot to form is proportional to protein C activity. Results are expressed as the percentage activity of the normal control. All forms of protein C deficiency are detected by this method. In the chromogenic substrate assay, plasma is mixed with Agkistrodon snake venom and incubated. A synthetic analide substrate is added. The activated protein C splits the substrate liberating analine. The concentration of aniline is measured at 405 nm and is proportional to protein C activity. This assay detects quantitative deficiencies of protein C and some functional defects, but it does not detect defective binding of protein C by protein S.
Protein S is a cofactor necessary for protein C activity. Approximately 60% of the circulating protein S is bound to complement and the remainder is free. Only the free protein S is physiologically active. Protein S can be measured by immunoassay or by a clot formation test. The immunoassay measures total protein S unless the protein-bound fraction has been removed prior to assay. Some persons with protein S deficiency have low levels of free protein S but normal levels of the protein-bound form. Others have a decreased concentration of both free and bound protein S. The functional assay will be abnormal in both types. The assay is performed by mixing the patient's plasma with protein S deficient plasma, activated protein C, and activated factor V. After incubation, calcium chloride is added. The time required for a clot to form is proportional to the protein S activity of the sample. The result for the patient is compared to normal plasma to determine the percentage of protein S activity.
The reason for testing should be explained by the health care provider. If the patient is taking an anticoagulant medication, the physician may require that the medication be discontinued for a specific time period prior to hypercoagubility testing.
Discomfort or bruising may occur at the puncture site, or the person may feel dizzy or faint. Pressure to the puncture site until the bleeding stops reduces bruising. Applying warm packs to the puncture site relieves discomfort.
Minor temporary discomfort may occur with any blood test, but there are no complications specific to hypercoagubility testing. In normal circumstances, a blood draw for these tests takes only a few minutes, and the patient experiences only minor discomfort.
- Antithrombin III activity assessed by immunoassay: 20– 30 mg/dL or 0.85-1.22 U/mL.
- Antithrombin III antigen assessed by chromogenic substrate assay: 80–120% of normal activity.
- Protein C activity assessed by photo-optical clot detection: 70–140% of normal activity.
- Protein C antigen assessed by immunoassay: 0.8-2.3 U/mL.
- Protein S activity assessed by photo-optical clot detection: (Male) 70–150% of normal activity; (Female) 58–130% of normal activity.
- Protein S antigen assessed by immunoassay: 0.6-1.8 U/mL.
If the level of any of these regulatory proteins is below normal, the patient has an increased risk of developing serious blood clots.
Health care team roles
A physician orders the tests and interprets the results. Usually a nurse or phlebotomist performs the venipuncture procedure. Clinical laboratory scientists/medical technologists perform the assays for antithrombin III, protein C, and protein S. Persons with family history of thrombosis and abnormal test results may be referred for genetic testing. A genetic counselor will explain the genetic findings and the chance of passing the abnormal gene to an offspring.
The health care team must provide clear and accurate information for patients who require hypercoagubility testing. The medical implications of having a blood clotting abnormality are serious and the patient must understand the importance of treatment. If an abnormal gene is responsible he or she will require anticoagulant medication for life. Since these deficiencies are inherited as autosomal dominant traits an affected person has a 50% chance of passing the condition to their offspring. Additional members of the patient's family will also require testing.
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The American Society for Clinical Laboratory Science. 7910 Woodmont Ave., Suite 523, Bethesda, MD 20814. (301) 657-2768. <www.ascls.org>.
The American Thrombosis Association. PO Box 6494, Denver, CO 80206. (303) 384-9239. <www.bloodclot.org>.
"Normal Laboratory Values, Table 296-2." In The Merck Manual of Diagnosis and Therapy. 17th ed., internet edition, 1999. <www.merck.com/pubs/mmanual>.
Linda D. Jones, B.A., PBT (ASCP)