1. Tests of Hemostasis Path 430/826 David Lillicrap Department of Pathology and Molecular Medicine Queen’s University, Kingston, Canada

2. Inherited Bleeding Disorders Hemophilia A and B von Willebrand disease “Rare Bleeding Disorders” Factor deficiencies:ie. FXI, FVII, FX Platelet disorders:ie. Glanzmann’s Disease, Bernard-Soulier

3. Acquired Bleeding Disorders Liver dysfunction Vitamin K deficiency DIC: sepsis, cancer, obstetric pathologies Drugs: anticoagulants/anti-platelet agents

4. Clinical Evaluation of Bleeding

5. Excessive Mucocutaneous Bleeding Bruising Epistaxis Oral cavity bleeding GI/GU bleeding Menorrhagia

6. Musculoskeletal Bleeding Hemarthroses Soft Tissue/Muscle Bleeds

7. Prior Challenges to the Hemostatic System Surgery Tonsillectomy Dental Procedures Wisdom teeth extraction

8. 1.Anecdotal bleeding histories vs 2.Validated bleeding scores (bleeding assessment tools)

9. 2005 Vicenza 0 to +3 40 min 2006 MCMDM-1VWD -1 to +4 40 min 2008 Condensed MCMDM-1VWD -1 to +4 10 min 2009 PBQ -1 to +4 20 min 2010 ISTH BAT 0 to +4 20 min Rydz and James Nov 2012 JTH Recent Evolution of Bleeding Assessment Tools

11. p<0.001 p=0.173p<0.005 Previously Diagnosed with VWD (n=42) ANOVA p<0.001

12. Utility of Bleeding Assessment Tools 1. Facilitate caregiver communication concerning severity of bleeding phenotype. 2. Justification for intensity of laboratory investigation.

13. Laboratory Tests of Hemostasis Test Analyte Platelet poor plasma

14. Routine Hemostasis Testing Platelet poor plasma Activator + Phospholipid Thromboplastin Ca 2+ ++ APTTPT

16. Initiation Phase

17. TFPI Extrinsic Pathway Inhibition TFPI Inhibits TF/FVIIa/FXa complex

18. Amplification Phase Initiated by positive thrombin feedback * * *

19. Extrinsic Pathway (prothrombin time - PT)

20. Intrinsic Pathway (aPTT)

21. Final Reaction Thrombin Time Addition of Exogenous Thrombin

22. Limitations to Current Hemostasis Tests Insensitive to many bleeding pathologies No detection of hypercoagulability Standardization challenging Mild hemophilia, VWD Antithrombin, Protein C and S deficiency TFPI, Thrombomodulin

23. Assessment of Platelet Contribution to Hemostasis 1. Platelet number 2. Platelet morphology 3. Platelet function Platelet aggregation studies with panel of agonists

24. Light Transmission Aggregometry

25. Light Transmission Platelet Aggregation Testing Result in Bernard-Soulier Syndrome – Absent GPIb receptor

26. Development of New “Global” Hemostasis TestsDevelopment of New “Global” Hemostasis Tests Enhanced sensitivity Reflection of complete hemostatic system More physiological But equally (if not more) difficult to standardize

27. Global Tests of Hemostasis a) Thrombin generation assays(TGA) a) Thromboelastography

28. IIa Thrombin Pro-coagulant effects Fibrinogen Fibrin FVIII FVIIIa FV FVa FXIII FXIIIaTAFI TAFIa PAR1 PAR4 FXI FXIa

29. TAT = thrombin – antithrombin complexes Absent in Hemophilia

30. 0 100 200 300 400 500 600 700 800 12345678910111213 Subjects Total thrombin (nM) Thrombin at 20 Minutes Over 6 Months Brummel et al 2008

31. After – Confirmation of a clinical bleeding phenotype Extensive hemostasis laboratory investigation 30-40% of bleeding conditions are without a definitive diagnosis Potential role for genome-wide searches Whole exome/Whole genome analysis