Tests of Hemostasis

Path 430/826

David LillicrapDepartment 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

• SurgeryTonsillectomy

• Dental ProceduresWisdom teeth extraction

1.Anecdotal bleeding histories

vs

2.Validated bleeding scores (bleeding assessment tools)

2005Vicenza0 to +340 min 2006

MCMDM-1VWD-1 to +440 min 2008

CondensedMCMDM-1VWD

-1 to +410 min 2009

PBQ-1 to +420 min

2010ISTH BAT

0 to +420 minRydz and James Nov 2012 JTH

Recent Evolution of Bleeding Assessment Tools

Type 1 Type 2 Type 30

10

20

30

Ble

edin

g S

core

(-1

to

+

4)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

Ca2+ Ca2+

+ +

APTT PT

Initiation Phase

TFPI

Extrinsic Pathway Inhibition

TFPI

AmplificationPhase

**

*

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 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

ThrombinPro-coagulant

effects

Fibrinogen Fibrin

FVIII

FVIIIa

FV

FVa

FXIII FXIIIa TAFI TAFIa PAR1PAR4

FXI FXIa

TAT = thrombin – antithrombin complexes

Absent in Hemophilia

0

100

200

300

400

500

600

700

800

1 2 3 4 5 6 7 8 9 10 11 12 13

Subjects

Tot

al th

rom

bin

(nM

)Thrombin at 20 Minutes Over 6 Months

Brummel et al

Factor VIII FTGT on Normal Plasma: Dose Response

-500

0

500

1000

1500

2000

2500

3000

2 7 12 17 22 27 32 37 42 47 52 57 62

Time (min)

Fre

e T

hro

mb

in (

RF

U)

Normal Plasma (NP)

10% NP

1% NP

0.1% NP

0.01% NP

FVIII Deficient Plasma

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