Introduction to Coagulation Testing

1. Introduction to Coagulation Testing Laura Worfolk, Ph.D. Scientific Director, Hematology Quest Diagnostics Nichols Institute, Chantilly, VA

2. Normal Hemostasis Absence of overt bleeding/thrombosis Bleeding Thrombosis Hemostasis • Intricate system maintaining blood in fluid state • Reacts to vascular injury to stop blood loss and seal vessel wall • Involves platelets, clotting factors, endothelium, and inhibitory/control mechanisms • Highly developed system of checks and balances

3. Interested Specialties Anesthesiology Anticoagulant Management HIT Vascular Surgery Graft Occlusion PAD Cardiology Premature CAD Hematology Hemophilia Thrombophilia Primary Care Practice patterns vary Bleeding & Thrombosis OB/GYN Fetal loss, Infertility, Menorrhagia Nephrology AV Graft Occlusion Neurology Stroke

4. Hemostasis Statistics • #1 cause of death is CVD (includes heart attack & stroke)* • ~1-2% of population w/ von Willebrand’s disease† • ~18,000 Americans w/ hemophilia† • ~600,000/year w/ venous thromboembolism‡ • ~½ with long-term health consequences; ~60,000 fatalities† • ~5-8% of population w/ thrombophilia† *WHO. †CDC ‡www.dvt.org..

5. Primary Hemostasis • Platelet role: • Adhesion (via vWF), post injury to vessel wall • Activation: shape changed, contents released • Aggregation, ie, “plug formation” • Formation of surface for coagulation reactions - “fibrin glue” vWF, von Willebrand factor.

6. IX IXa Prothrombin Thrombin Coagulation Cascade XII XIIa Injury XI XIa HMWK/Prekallikrein VII TF VIII VIIIa X Xa TF/VIIa V Va XIII XIIIa Fibrinogen Fibrin (soluble) Fibrin (insoluble)

7. Cascade Simplified Activation/Injury Intrinsic Pathway (XIIa, XIa, IXa, VIIIa) Extrinsic Pathway (TF, VIIa) Common Pathway (Xa, Va, IIa, Fibrinogen)

8. Thrombin Regulation • Activity and formation tightly controlled • Antithrombin III • Inactivation of IIa and other enzymes involved in its formation • Protein C and protein S pathway • Inactivation of cofactors Va and VIIIa • Tissue factor pathway inhibitor • Turns off extrinsic pathway (TF, VIIa) Defects in regulatory mechanisms: thrombosis

9. PAI-1 Alpha-2 AP Fibrinolytic Pathway Clot lysis vital in prevention of vessel occlusion uPA, tPA Plasmin Plasminogen Fibrin Clot Fibrin(ogen) Degradation Products Defects: bleeding or thrombosis

10. Hemostasis Balance Thrombin Generation (ie, Factors II – XII, cells) Plasmin Generation (ie, tPA, uPA, cells) Healing Coagulation Fibrinolysis Thrombin Regulation (ie, PC/PS, AT, TFPI, cells) Plasmin Regulation (ie, PAI-1, cells) Cellular contribution: platelets, endothelium, monocytes

11. Bleeding Thrombosis Alteration of Balance Laboratory testing indicated if • Factor deficiencies • Acquired inhibitors • Anticoagulant therapy • Consumption (DIC) • Dysfibrinogenemia • Platelet defects • von Willebrand’s disease DIC, Disseminated intravascular coagulation

12. Bleeding Thrombosis Alteration of Balance Laboratory testing indicated if • Inhibitor deficiencies • Acquired inhibitors (eg, lupus anticoagulant) • DIC • Heparin induced thrombocytopenia

13. Case Study #1 • 21 y/o female with vague family history of bleeding disorder; evaluated prior to taking scuba diving lessons • Has nose bleeds following aspirin ingestion • Differential diagnosis? • Role of laboratory testing???

14. Case #1: Lab Testing • Screening assays • aPTT: assesses intrinsic & common pathways • PT: assesses extrinsic & common pathways • Fibrinogen: hypo- or dysfibrinogenemia? • CBC: platelet count • von Willebrand’s disease (vWD) evaluation • Multiple tests required to classify vWD type • Antigenic and functional assays

15. Case #1: Test Results *Acute phase proteins.

16. Case #1: Test Results

17. Normal Type 1 Type 2A Case #1: Multimer Analysis Shown is representative gel of normal and type 1 and 2A vWF deficiencies Patient results demonstrated absence of high and intermediate molecular weight multimers consistent with type 2A vWD

18. Case #1: Summary Probable diagnosis von Willebrand’s disease type 2A (bleeding disorder)

19. Case Study #2 • 38 y/o Caucasian man admitted for evaluation of portal hypertension; history of recurrent thrombosis (>10 years) PT, aPTT, fibrinogen: normal • Positive family history; father and sister with venous thrombotic episodes, but no laboratory investigation • Differential diagnosis??

20. Inherited Thrombophilia Risk Factors VTE, venous thromboembolism; RR, relative risk; APC, activated protein C; AT, antithrombin.

21. Case #2: Lab Testing

22. Case #2: Summary Probable diagnosis Thrombosis caused by APC resistance/factor V Leiden mutation

23. Value of Thrombophilia Testing • Testing does not affect management of acute events • Test results may influence decisions • How long & how intensively to treat • Prevention of recurrence • Prophylaxis during high-risk procedures • Need to evaluate family members • Estimate future risk (ie, risk associated with HRT) HRT, hormone replacement therapy.

24. Case Study #3 • 40 y/o woman with iron deficiency anemia due to menorrhagia; hysterectomy delayed due to prolonged screening test aPTT: 47.8 sec (elevated) PT: 13.0 sec (normal) Fibrinogen: 300 mg/dL (normal) • No history of bleeding or bruising; no family history • Differential diagnosis??

25. Lupus Anticoagulants • Antiphospholipid antibodies (APA) are directed against proteins bound to phospholipid membrane surfaces • Lupus anticoagulants (LA) are a type of APA • Associated with thrombosis & recurrent fetal demise • Characterized by prolongation of phospholipid dependent clotting assays (ie, aPTT)

26. ISTH Criteria for Lupus Anticoagulants • Prolongation of a phospholipid dependent clotting assay (ie, aPTT) • Evidence of inhibition in mixing studies • Evidence that inhibition is phospholipid dependent • Lack of specific inhibition by any one coagulation factor or other circulating inhibitor (ie, FVIII inhibitors, heparin)

27. Case #3: Lab Testing

28. Case #3: Summary Probable diagnosis: Lupus anticoagulant LA may be asymptomatic or associated with thrombotic events or recurrent abortion. A bleeding history requires other coagulopathies be excluded. Since LA may be transient, international consensus guidelines suggest waiting at least 12 weeks before retesting to confirm antibody persistence. J Thromb Haemost. 2006;4:295.

29. Role of Laboratory Testing • Assist in diagnosis of bleeding and thrombotic disorders; for example: • Screen for von Willebrand’s disease in patients with menorrhagia (ACOG recommendation) • Test for thrombophilia risk factors in patients with recurrent spontaneous abortion or thrombotic events • Monitor anticoagulant therapy • Oral anticoagulants, heparin, thrombin inhibitors

30. Role of Laboratory Testing • Monitor replacement therapy • Factor levels (ie, FVIII, vWF) • Pre-op screening • Risk assessment

31. Pre-analytical Considerations • Proper specimen handling, processing, and storage is critical for accurate and precise results • General specimen requirements available • www.questdiagnostics.com(click on Test Menu) • www.nicholsinstitute.com(click on lab information specimen requirements) • Quest Diagnostics Nichols Institute Directory of Services (contact your local representative)

32. Resources • Laboratories performing routine, specialty, and esoteric hemostasis testing • Consultative services available @ Quest Diagnostics Nichols Institute • Quest Diagnostics Interpretive Guide: http://www.questdiagnostics.com/hcp/intguide/hcp_ig_main.html Jeffrey Dlott, MD Chantilly, VA 703-802-6900, x7259 Mervyn Sahud, MD San Juan Capistrano, CA 949-728-4794

33. Case-Oriented Symposium on Bleeding & Thrombosis • October 11-12, 2007, Renaissance Hotel, Washington DC; topics: • Pediatric hemostasis issues • Thrombophilia • Platelet disorders • Thrombotic thrombocytopenia purpura • FVIII Inhibitors • Point of Care testing • New technologies & more For more information on this CME approved symposium, go to: http://www.nicholsinstitute.com/Coagulation/Default.htm.

34. References • ACOG committee opinion. von Willebrand’s disease in gynecologic practice. Int J Gynaecol Obstet. 2002;76:336. • Brandt JT, et al. Laboratory identification of lupus anticoagulants: Results of the Second International Workshop for Identification of Lupus Anticoagulants. On behalf of the Subcommittee on Lupus Anticoagulants/ Antiphospholipid Antibodies of the ISTH. Thromb Haemost. 1995;74:1597. • Miyakis et al. International consensus statement on an update of the classification criteria for definite APS. J Thrombo Haemost. 2006;4:295.

35. References • Press et al. Clinical utility of FV Leiden testing for the diagnosis and management of thromboembolic disorders. Arch Pathol Lab Med. 2002;126:1304. • Sadler et al. Update on the pathophysiology & classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. J Thrombo Haemost. 2006;10:2103. • Thrombophilia: Laboratory support of risk assessment and diagnosis. Available at: http://www.questdiagnostics.com/hcp/intguide/jsp/showintguidepage.jsp?fn=CF_Thrombophilia/CF_Thrombophilia.htm. Accessed March 21, 2007.

36. Thank you.