Coagulation Testing

1. Coagulation Testing Diane Jette BioMedica Diagnostics Inc.

2. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0

3. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Hemostasis is the arrest of bleeding from an injured blood vessel Vasoconstriction and compression of injured vessels Platelets adhere to the site of injury and form a platelet plug Platelets release factors to augment vasoconstriction and initial vessel wall repair Platelets provide surface membrane sites and components for the formation of enzyme/cofactor complexes in blood coagulation reactions

4. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Coagulation Reactions Lead to the Formation of a Blood Clot Two pathways: Intrinsic and Extrinsic - Coagulation Cascade Formation of a prothrombin activator - complex of Factor Xa, Factor Va and procoagulant phospholipid on surface of platelets. Prothrombin activator cleaves prothrombin into two fragments to give Thrombin. Thrombin cleaves small peptides from fibrinogen to form fibrin monomers that polymerize. Thrombin activates Factor XIII to cross-link the fibrin to form an insoluble clot.

5. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Coagulation Cascade Intrinsic Pathway: (APTT) Factors VIII, IX, XI, and XII. Activated on surface of exposed endothelium. Complexes form on platelet phospholipids. Extrinsic Pathway: (PT) Factors IV, V, VII, X Activated by Tissue phospholipids (Tissue Factor or Tissue thromboplastin) released into blood as a result of tissue damage.

6. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Coagulation Cascade Common Pathway (Thrombin Time) Factors I and II Leads to the formation of Fibrin Clot Thrombin time does not measure deficiencies in Intrinsic or Extrinsic pathway

7. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 The Role of Calcium Ca ions are needed for most of the reactions in the Coagulation Cascade Ca-chelating agents are used in vitro as anticoagulants (Citrate, EDTA, Oxalate) When Coagulation Factors are synthesized without Vitamin K they cannot bind Ca and lose enzymatic function

8. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Regulatory Mechanisms Inhibition of Factor Activity Plasma protease inhibitors: anti-thrombin III (ATIII), ?2-macroglobulin, ?1 - antiprotease Heparin converts ATIII from a slow acting inhibitor to an instantaneous inhibitor of Thrombin, Factor Xa and Factor IXa Protein C and Protein S are serine proteases that cleave Factors VIII and Factor Va rendering them inactive

9. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Fibrolysis Fibrin clot is degraded by protolytic enzymes and fragments dissolved in blood Process is catalyzed by Plasmin Plasminogen is converted to Plasmin Activation by tissue plasminogen activator (tPA) and urokinase Fibron degrades into large fragments X and Y then smaller fragments D and E

10. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Regulation of Fibrolysis Plasminogen activator inhibitors (PAIs) and plasmin inhibitors slow the fibrolysis process tPA and urokinase have short half-lives and are rapidly cleared through the liver Unbound plasmin is instantaneously neutralized by ?2-antiplasmin

11. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Hereditary Coagulation Disorders Hemophilia A Factor VIII deficiency 80% of all Hemophilia cases Hemophilia B Factor IX deficiency Prolonged ATPP Recovered by dilution 1:1 with normal plasma Normal PT and Normal Bleeding Time Factor XI Deficiency 5 to 9% of European Jews ?2-antiplasmin Deficiency

12. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Acquired Coagulation Disorders Liver Disease Impaired clotting Factor synthesis Increased fibronolysis Thrombocytopenia Desseminated Intravascular Coagulation (DIC) Something enters the blood that activates factors Complication of obstetrics, infection, malignancy, shock, severe brain trauma Elevated PT, APTT, D-Dimer and other fibron degradation products

13. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Circulating Anticoagulants Antibodies that neutralize clotting factor activity Factor VIII Anticoagulants Antibody Same profile as Hemophilia A Clotting time not restored by mixing with normal plasma Life-threatening condition

14. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Lupus Anticoagulants Antibodies to phospholipid binding sites on clotting factors Prevent factors from accumulating on phospholipid surfaces Elevated APTT clotting times not corrected with mixing with normal plasma PT normal or slightly elevated. Non-specific depression of clotting factor activities (Factors VIII, IX, XI, XII) Test sensitivity increased by using diluted reagent Dilute ATPP reagent, Russell�s viper venom time, Kaolin time Clotting times corrected with the addition of phospholipids

15. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Oral Anticoagulant Therapy Coumadin or Warfarin Inhibitor of Vitamin K dependant Factor synthesis Oral anticoagulant Dose regulated by therapeutic effect PT assay to measure INR INR range established for optimum therapeutic effect (typically 2.0 to 3.0)

16. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Prothrombin Time: PT PT reagent contains Calcium ions and Thromboplastin from brain tissue (Rabbit). Thromboplastin (Tissue Factor) protein-lipid complex found in tissues outside blood vessels. Measures the function of the Extrinsic Pathway. Sensitive to Factors IV, V, VII, X. Provided as a lyophilized reagent. Used to monitor oral anticoagulant therapy (Warfarin / Coumadin).

17. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 PT Reagent Calibration Reagents are calibrated against standard PT reagent established by the WHO. ISI = International Sensitivity Index. ISI is assigned by the manufacturer for each lot of reagent using reference material traceable to WHO. The lower the ISI the more sensitive the Reagent ISI of 1.8 to 2.4 = Low sensitivity (North American Standard PT) ISI of 1.4 to 1.8 = Average sensitivity ISI 1.0 to 1.4 = High Sensitivity

18. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 PT: INR Values INR = International Normalised Ratio. MNP = Mean Normal Plasma. INR = (PT / MNP)ISI An INR of 1.0 means that the patient PT is normal. An INR greater then 1.0 means the clotting time is elevated.

19. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 INR Calculation Example 1 MNP = 12.0 s ISI = 1.25 Patient Plasma = 20 s INR = (20.0 / 12.0)1.25 = 1.9 Example 2 MNP = 12.0 s ISI = 1.85 Patient Plasma = 17 s INR = (17.0 / 12.0)1.85 = 1.9 Example 3 MNP = 12.0 s ISI = 1.4 Patient Plasma = 20 s INR = (20.0 / 12.0)1.4 = 2.0 Example 4 MNP = 12.0 s ISI = 2.0 Patient Plasma = 20 s INR = (20.0 / 12.0)2.0 = 2.8

20. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Expected PT Values Mean Normal Plasma = 10 to 14 seconds. Mean Normal Plasma value varies with PT sensitivity. A high sensitivity (Low ISI) PT will give a high normal PT value (13 to 15 seconds). Oral anticoagulant monitoring = Target INR of 2.0 to 3.0. INR of greater than 5 or 5.5 = unacceptable high risk of bleeding. %CV between duplicates less than 5%

21. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Performing a PT test Pre-warm PT reagent and sample to 37 oC Add 100 ?L sample to cuvette Add 200 ?L of PT reagent to cuvette Start timer Record time to clot in seconds Calculate INR see product insert for PT

22. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Activated Partial Thromboplastin Time APTT or PTT Reagent contains phospholipids and a �surface activator�; (Ellagic Acid, Micronized Silica) Calcium Chloride reagent added to start the reaction. APTT reagent mimics the surface of a platelet. Measures activity of clotting factors in the Intrinsic Pathway, factors VIII, IX, XI and XII No WHO calibration standard

23. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Uses of APTT Sensitive to 30 to 40% deficiencies of all factors except VII and XIII. Heparin inhibits the activity of clotting factors in the Intrinsic Pathway. A standard curve (APTT time vs Heparin concentration) is prepared using a heparin standard. APTT is also sensitive to other non-specific Factor inhibitors such as Lupis Anticoagulant. Can be influenced by Vitamin K deficiency and Coumadin therapy. Negative APTT result usually rules out Hemophilia.

24. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Expected APTT Values Normal Range: 26 to 40 seconds Slightly Elevated: 45 to 65 seconds Extremely Elevated = > 70 seconds %CV less than 7%

25. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Performing an APTT Test Pre-warm Calcium Chloride reagent to 37 oC. Add 100 ?L of sample to cuvette. Add 100 ?L of APTT to cuvette and incubate for 3 minutes. Add 100 ?L of Calcium Chloride reagent and start timer. Record the time to clot in seconds. See APTT product Insert

26. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Heparin Monitoring Prepare Heparin Standards Prepare stock heparin (10 USP units/mL) Prepare working heparin dilutions (0.1 to 0.8 U/mL) by diluting stock in normal plasma Run heparin dilutions as samples in APTT assay Plot the results as U/mL vs Log Clotting times Run patient samples in APTT assay and determine heparin concentration from the plot.

27. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Heparin Calibration Curve

28. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Factor Substitution Tests: PT and APTT Dilute patient sample one to one with adsorbed plasma and serum to determine if normal clotting time is restored. Serum: Source of Factors IX, X, XI & XII Adsorbed Plasma: Source of Factors VIII, V, XI, & XII

29. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Factor Plots: PT and APTT Dilute a normal plasma sample or control 1 to 10 in saline Dilute in saline: 100%, 50%, 25%, 12.5%. 6.25%, 3.12%, Mix one to one with a factor deficient plasma. Measure PT or APTT and plot Log % Factor vs Log clotting time. Dilute patient plasma 1 to 10 in saline. Mix one to one with a factor deficient plasma. All factors will be restored except the deficient factor. If the factor is present in test sample no reduction in clotting will be seen. (Same clotting time as the 100% standard) Determine percent factor from standard curve.

30. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Factor Calibration Curve

31. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Plasma Controls Used to monitor assay performance (QC) Made from pooled normal human plasma with some factors selectively adsorbed. Three levels available: Level 1 control represents a normal plasma Level 2 represents a slightly elevated plasma (INR ~ 1.5) Level 3 represents a severely elevated plasma (INR ~ 2.5) Each laboratory should establish expected ranges for PT and APTT.

32. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Thrombin Time (TT or TAT) Measures common pathway. Fibrinogen --> Fibron Clot Not sensitive to deficiencies in Intrinsic or Extrinsic pathways. Reagent consists of animal thrombin. Normal Clotting time is 15 seconds. If elevated sample is mixed one to one with normal and re-tested. If normal clotting is not restored then an anti-coagulant is present.

33. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Bleed Time Test An incision is made Time to stop bleeding is measured Normal clotting time is 7.5 minutes

34. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 D-Dimer Test D-Dimer is a fibrin monomer Product of fibrolysis Latex agglutination assay is used Anti-D-Dimer antibody coated on micro-latex beads Cardiac Infarction Marker

35. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Activated Clotting Time (ACT) Clotting time of whole blood in the presence of silica based activator. Normal clotting times = 90 to 170 sec. Used to monitor heparin doses from 1 to 10 U/mL (APTT is sensitive to heparin at 0.2 to 1 U/mL). Used with invasive procedures that require on-site adjustment of heparin and protamine dosage. (ex. Cardiopulmonary bypass surgery). Not amenable for use with an optical instrument, too cloudy. Also called HMT, Heparin Management Test

36. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Fibrin-1 Clot Detection

37. BioMedica Diagnostics Inc. / March 2002 / Rev. 1.0 Clot Detection