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

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

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

Clinical Evaluation of Bleeding

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

Musculoskeletal Bleeding Hemarthroses Soft Tissue/Muscle Bleeds

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

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

2005 Vicenza 0 to min 2006 MCMDM-1VWD -1 to min 2008 Condensed MCMDM-1VWD -1 to min 2009 PBQ -1 to min 2010 ISTH BAT 0 to min Rydz and James Nov 2012 JTH Recent Evolution of Bleeding Assessment Tools

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

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

Laboratory Tests of Hemostasis Test Analyte Platelet poor plasma

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

Initiation Phase

TFPI Extrinsic Pathway Inhibition TFPI

Amplification Phase * * *

Extrinsic Pathway (prothrombin time - PT)

Intrinsic Pathway (aPTT)

Final Reaction Thrombin Time

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

Assessment of Platelet Contribution to Hemostasis 1. Platelet number 2. Platelet morphology 3. Platelet function Aggregation studies with panel of agonsists

Light Transmission Platelet Aggregation Testing

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

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

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

TAT = thrombin – antithrombin complexes Absent in Hemophilia

Subjects Total thrombin (nM) Thrombin at 20 Minutes Over 6 Months Brummel et al

Flourogenic Thrombin Generation Assay Current detection limit

After – Confirmation of a clinical bleeding phenotype Extensive hemostasis laboratory investigation 30-40% of bleeding conditions are without a definitive diagnosis