THROMBOTIC-DISORDERS-LAB-ASSESSMENT.pptx

Medical Technology

1. Thrombotic Disorders and Laboratory Assessment

2. DEVELOPMENTS IN THROMBOSIS RISK TESTING Before 1992, medical laboratory professionals performed assays to detect only three inherited venous thrombosis risk factors: deficiencies of the coagulation control factors antithrombin, protein C, and protein S. Taken together, these three deficiencies accounted for no more than 7% of cases of recurrent venous thromboembolic disease and bore no relationship to arterial thrombosis. The list of current assays includes antithrombin, protein C, protein S, APC resistance, FVL mutation, prothrombin G20210A mutation, lupus anticoagulant (LA), and several additional markers of both venous and arterial thrombotic disease described in this chapter.

3. Thrombosis Thrombosis is a multifaceted disorder resulting from abnormalities in blood flow, such as stasis, and abnormalities in the coagulation system, platelet function, leukocyte activation molecules, and the blood vessel wall. Thrombosis is the inappropriate formation of platelet or fibrin clots that obstruct blood vessels. These obstructions cause ischemia (loss of blood supply) and necrosis (tissue death) Thrombophilia (once called hypercoagulability ) is defined as the predisposition to thrombosis secondary to a congenital or acquired disorder. The theoretical causes of thrombophilia are the following: p hysical, chemical, or biologic events such as chronic or acute inflammation that release prothrombotic mediators from damaged blood vessels or suppress blood vessel production of normal antithrombotic substances i nappropriate and uncontrolled platelet activation u ncontrolled blood coagulation system activation u ncontrolled fibrinolysis suppression

4. THROMBOSIS RISK FACTORS Acquired Thrombosis Risk Factors Thrombosis Risk Factors Associated with Systemic Diseases Congenital Thrombosis Risk Factors Double Hit

5. Acquired Thrombosis Risk Factors In life, we acquire a legion of habits and circumstances that either help maintain or damage our hemostasis systems. Their variety and interplay make it difficult to pinpoint the factors that contribute to thrombosis or to determine which have the greatest influence. These factors seem to contribute to venous and arterial thrombosis in varying degrees.

6. Thrombosis Risk Factors Associated with Systemic Diseases Chronic APL antibodies confer a risk of venous or arterial thrombosis—a condition called the antiphospholipid syndrome (APS). Chronic APL antibodies often accompany autoimmune connective tissue disorders, such as lupus erythematosus, some appear in patients without any apparent underlying disease. Malignancies often are implicated in venous thrombosis. One mechanism is tumor production of tissue factor ana logues that trigger chronic low-grade disseminated intravascu lar coagulation (DIC).

7. Myeloproliferative neoplasms such as essential thrombocythemia and polycythemia vera may trigger thrombosis, probably through platelet hyperactivity. Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a stem cell mutation that modifies membrane-anchored platelet activation suppressors. Chronic inflammatory diseases cause thrombosis through a variety of mechanisms, such as elevation of fibrinogen and fac tor VIII, suppressed fibrinolysis, promotion of atherosclerotic plaque formation, and reduced free protein S activity secondary to raised C4b-binding protein (C4bBP) levels.

8. Congenital Thrombosis Risk Factors Clinicians suspect congenital thrombophilia when a thrombotic event occurs in young adults; occurs in unusual sites such as the mesenteric, renal, or axillary veins; is recurrent; or occurs in a patient who has a family history of thrombosis. Because thrombosis is multifactorial, even patients with congenital thrombophilia are most likely to experience thrombotic events because of a combination of constitutional and acquired conditions Double Hit Thrombosis often is associated with a combination of genetic defect, disease, and lifestyle influences. Just because someone possesses protein C, protein S, or antithrombin deficiency does not mean that thrombosis is inevitable.

9. LABORATORY EVALUATION OF THROMBOPHILIA When thrombophilia is suspected, it is important to assess all known risk factors because it is the combination of positive results that determines the patient’s cumulative risk of thrombosis. Antiphospholipid Antibodies Activated Protein C Resistance and Factor V Leiden Mutation Prothrombin G20210A Antithrombin Protein C Control Pathway

10. Antiphospholipid Antibodies APL antibodies comprise a family of immunoglobulins that bind protein-phospholipid complexes. APL antibodies in clude LAs, detected by clot-based profiles, and ACL and anti– b 2 -GPI antibodies, detected by immunoassay. Chronic autoim mune APL antibodies are associated with APS, which is characterized by transient ischemic attacks, strokes, coronary and peripheral artery disease, venous thromboembolism, and recurrent pregnancy complications, including spontaneous abortions. Lupus Anticoagulant Test Profile Anticardiolipin Antibody Immunoassay Anti– b 2-Glycoprotein I Immunoassay Antiphosphatidylserine Immunoassay

11. Lupus Anticoagulant Test Profile Clot-based assays with reduced reagent phospholipid concentrations are sensitive to LA. The two most recommended test systems are the dilute Russell viper venom time (DRVVT) and the silica-based partial thromboplastin time (PTT), both formulated with low- phospholipid concentrations designed to be LA sensitive. A confirmed positive result in one system is conclusive despite a negative result in the other. Anticardiolipin Antibody Immunoassay ACL test is an immunoassay that may be normalized among laboratories and is not affected by heparin therapy, oral anticoagulant therapy, current thrombosis, or factor deficiencies.

12. Anti– b 2-Glycoprotein I Immunoassay The practitioner performs IgM and IgG anti– b 2 -GPI immuno assays as a part of the profile that includes ACL assays. An anti– b 2 -GPI result of greater than 20 IgG or IgM anti– b 2 -GPI units correlates with thrombosis more closely than the presence of ACL antibodies. Any ACL or anti– b 2 -GPI assay yielding positive results is repeated on a new specimen collected after 12 weeks to distinguish a transient alloantibody from a chronic autoantibody. Antiphosphatidylserine Immunoassay For cases in which an APL antibody is suspected but the routine LA, ACL, and b 2 -GPI assay results are negative, the clinician may wish to order the antiphosphatidylserine immunoassay to detect APL antibodies specific for phosphatidylserine. A result greater than or equal to 16 IgG or 22 IgM antiphosphatidylserine units is considered positive.

13. Activated Protein C Resistance and Factor V Leiden Mutation The activated protein C (APC)– protein S complex normally hy drolyzes activated factors V and VIII (factors Va and VIIIa ). A mutation in the factor V gene substitutes glutamine for argi nine at position 506 of the factor V molecule (FV R506Q). The arginine molecule is a normal cleavage site for APC, so the substitution slows or resists APC hydrolysis. Activated Protein C Resistance Clot-Based Assay Factor V Leiden Mutation Assay

14. Activated Protein C Resistance Clot-Based Assay Factor V Leiden Mutation Assay Most laboratories confirm the APC resistance diagnosis using the molecular FVL mutation test. The determination of zygosity is important to predict the risk for thrombosis and establish a treatment regimen.

15. Prothrombin G20210A A guanine-to-adenine mutation at base 20210 of the 3 9 un- translated region of the prothrombin (factor II) gene has been associated with mildly elevated plasma prothrombin levels, averaging 130%. The increased risk of thrombosis in those with the mutation seems to be related to the elevated prothrombin activity. The risk of venous thrombosis in heterozygotes is only two to three times the baseline risk. Although the mutation may cause a slight prothrombin elevation, a phenotypic prothrombin activity assay is of little diagnostic value because there is considerable overlap between normal prothrombin levels and prothrombin levels in people with the mutation.

16. Antithrombin Antithrombin (previously named antithrombin III or ATIII) is a serine protease inhibitor (SERPIN) that neutralizes factors IIa (thrombin), IXa , Xa , XIa , and XIIa , all the serine proteases except factor VIIa . First of the plasma coagulation control proteins to be identified and the first to be assayed routinely in the clinical hemostasis laboratory. Acquired antithrombin deficiency occurs in liver disease, in nephrotic syndrome, with prolonged heparin therapy, with asparaginase therapy, with the use of oral contraceptives, and in DIC, where antithrombin is rapidly consumed. Laboratory practitioners screen for antithrombin deficiency using a clot-based or chromogenic functional assay , but most laboratory directors choose the chromogenic assay because of its stability and reproducibility. Antithrombin may become decreased during prolonged or intense heparin therapy and may be largely consumed if the patient has a congenital antithrombin deficiency. In this in stance, heparin may be administered in therapeutic or higher dosages, but it neither exerts an anticoagulant effect nor is detected by the PTT, known as heparin resistance.

17. Protein C Control Pathway Thrombin is an important coagulation factor because it cleaves fibrinogen, activates platelets, and activates factors V, VIII, XI, and XIII. In the intact vessel where clotting would be pathological, thrombin binds endothelial cell membrane thrombomodulin and becomes an anticoagulant. Protein C Assays Functional assays detect both quantitative and qualitative protein C deficiencies, either chromogenic or clot-based protein C activity assays are available. Agkistrodon contortrix a southern copperhead serpent which activates protein C.

18. ARTERIAL THROMBOSIS PREDICTORS Arterial thrombotic disease, including peripheral vascular occlusion, myocardial infarction (heart attack), cerebrovascular ischemia (stroke), arises from atherosclerotic plaque. The traditional predictors of arterial thrombosis risk are elevated total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), or an elevated ratio of total cholesterol to high-density lipoprotein cholesterol (TC:HDL-C) secondary to HDL-C defi ciency. Researchers have sought additional arterial thrombosis pre dictors by performing prospective randomized studies of lipo- protein subtypes, fibrinolytic pathway components, and markers of inflammation. The results of these studies have led to the identification of potential markers of arterial thrombosis risk.

19. C-Reactive Protein is a calcium-dependent pentameric ligand-binding member of the pentraxin family produced in the liver that circulates in plasma at a concentration below 0.55 mg/L. l aboratory professionals continue to use this simple and inexpensive assay to confirm inflammation and monitor the effectiveness of anti-inflammatory therapy. Plasma Homocysteine Homocysteine is a naturally occurring sulfur-containing amino acid formed in the metabolism of dietary methionine. Fasting homocysteinemia is an independent risk factor for arterial thrombosis, with relative risk ratios of 1.7 for coronary artery disease, 2.5 for cerebrovascular disease, and 6.8 for peripheral artery disease. Several theories link homocysteinemia with coronary artery disease, most citing damage to the endothelial cells.

20. Fibrinogen Activity laboratory professionals measure fibrinogen using immunoassay, nephelometry, or the Clauss clot-based method to detect dysfibrinogenemia, hypofibrinogenemia, or afibrinogenemia f ibrinogen concentration correlates with relative risk of myocardial infarction in asymptomatic persons or patients with angina pectoris Lipoprotein (a) is low-density lipoprotein that may be used to predict arterial thrombosis. l ipoprotein (a) may contribute to thrombosis by its antifibrinolytic property. i t also may contribute to the overall concentration of LDL-C. Levels can be lowered with statin drugs, as can LDL-C levels.

21. DISSEMINATED INTRAVASCULAR COAGULATION DIC, also named defibrination syndrome or consumption coagulopathy , is the generalized activation of hemostasis secondary to a systemic disease. It is an acquired condition in which normal physiology of coagulation is disturbed leading to widespread intravascular coagulation process associated with injury to microvasculature which results in organ dysfunction, capillary leak and shock. Occurs due to simultaneous action of the following 4 mechanisms: Increased thrombin generation Suppressed physiological anticoagulant pathways Activation & subsequent impairment of fibrinolysis Activation of inflammatory pathways

22. DIC CAUSES: Infection/sepsis Trauma Obstetric, e.g. amniotic fluid embolism, placental abruption, preeclampsia S evere liver failure M alignancy, e.g. solid tumors and leukemias T issue destruction, e.g. pancreatitis, burns V ascular abnormalities, e.g. vascular aneurysms, liver hemangiomas T oxic/immunological, e.g. ABO incompatibility, snake bite, recreational drugs

23. DIC CLINICAL MANIFESTATIONS Petechiae Purpura Arterial line oozing Venipuncture site bleeding TREATMENT Elimination of the precipitating factor if possible Replacement of coagulation factors and platelets Inhibition of the clotting process with heparin or other agents

24. HEPARIN-INDUCED THROMBOCYTOPENIA Heparin-induced thrombocytopenia (HIT), also called heparin- induced thrombocytopenia with thrombosis, is an adverse effect of heparin treatment. Patients receiving heparin must have platelet counts performed every other day, a platelet count decrease during heparin ad ministration indicates HIT. LABORATORY TEST: Most acute care laboratories provide a heparin-induced antibody immunoassay but seldom as a stat assay. Microbial contamination, lipemia, and hemolysis may also invalidate immunoassay results. Many laboratories provide aggregometry or lumiaggregome try methods to confirm HIT The reference method is the washed platelet. TREATMENT: A dministra tion of UFH or LMWH is immediately discontinued, and the physician chooses an alternate form of anticoagulation.