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Tests of Hemostasis
Path 430/826
David LillicrapDepartment of Pathology and Molecular Medicine
Queen’s University, Kingston, Canada
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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
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Acquired Bleeding Disorders
• Liver dysfunction
• Vitamin K deficiency
• DIC: sepsis, cancer, obstetric pathologies
• Drugs: anticoagulants/anti-platelet agents
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Clinical Evaluation of Bleeding
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Excessive Mucocutaneous Bleeding
• Bruising
• Epistaxis
• Oral cavity bleeding
• GI/GU bleeding
• Menorrhagia
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Musculoskeletal Bleeding
• Hemarthroses
• Soft Tissue/Muscle Bleeds
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Prior Challenges to the Hemostatic System
• SurgeryTonsillectomy
• Dental ProceduresWisdom teeth extraction
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1.Anecdotal bleeding histories
vs
2.Validated bleeding scores (bleeding assessment tools)
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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
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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
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Utility of Bleeding Assessment Tools
1. Facilitate caregiver communication concerning severity of bleeding phenotype.
2. Justification for intensity of laboratory investigation.
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Laboratory Tests of Hemostasis
Test Analyte
Platelet poor plasma
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Routine Hemostasis Testing
Platelet poor plasma
Activator +Phospholipid
Thromboplastin
Ca2+ Ca2+
+ +
APTT PT
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Initiation Phase
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TFPI
Extrinsic Pathway Inhibition
TFPI
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AmplificationPhase
**
*
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Extrinsic Pathway
(prothrombin time - PT)
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Intrinsic Pathway(aPTT)
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Final Reaction
Thrombin Time
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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
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Assessment of Platelet Contribution to Hemostasis
1. Platelet number
2. Platelet morphology
3. Platelet function
Aggregation studies with panel of agonsists
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Light Transmission Platelet Aggregation Testing
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Development of New “Global” Hemostasis Tests
• Enhanced sensitivity
• Reflection of complete hemostatic system
• More physiological
• But equally (if not more) difficult to standardize
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Global Tests of Hemostasis
a) Thrombin generation assays (TGA)
a) Thromboelastography
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IIa
ThrombinPro-coagulant
effects
Fibrinogen Fibrin
FVIII
FVIIIa
FV
FVa
FXIII FXIIIa TAFI TAFIa PAR1PAR4
FXI FXIa
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TAT = thrombin – antithrombin complexes
Absent in Hemophilia
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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
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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
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After –
•Confirmation of a clinical bleeding phenotype
•Extensive hemostasis laboratory investigation
30-40% of bleeding conditions are without a definitive diagnosis