International Hemostasis VIP Meeting
China, 2006
Armando Tripodi
<armando.tripodi@unimi.it>
Angelo Bianchi Bonomi Hemophilia and
Thrombosis Center
University of Milan
Italy
TRIPODI
TRIPODI
Settings where the Laboratory can help
Clinicians
• Diagnosis of congenital hemorrhagic
coagulopathies (pre-surgical screening)
• Thrombophilia testing and aPL/LA Syndrome
• Diagnosis of acute venous thromboembolism
• Heparin monitoring
• Oral anticoagulant monitoring
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Settings where the laboratory
can help clinicians
Diagnosis of Congenital
Hemorrhagic Coagulopathies
(pre-surgical screening)
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Congenital Hemorrhagic
Coagulopathies
Aims of Laboratory Investigation
• To establish the causes of bleeding in
patients who have shown evidence of
abnormal bleeding
• To detect mild defects in asymptomatic
patients
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Congenital Hemorrhagic
Coagulopathies
Most Important Screening Test
Good Clinical History
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Congenital Hemorrhagic
Coagulopathies
Why should clinical history be collected
• Poor sensitivity of screening tests to
detect mild defects
• The type of bleeding may provide
valuable clue to its etiology
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Aims of the Clinical History
To establish
•
•
•
•
Type of bleeding
Location, frequency, duration, severity
Whether it is spontaneous or post-traumatic
Whether other family members have the
same symptoms
• The age of appearence of the first symptoms
• Whether other diseases are present
• Whether the patients is taking drugs
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Main Bleeding Symptoms
• Bleeding from mucous membranes is a typical
feature of platelet disorders
• Soft-tissue bleeding is a typical feature of
coagulation disorders
• Umbilical cord and delayed bleeding are typical
feature of factor XIII deficiency
• Simultaneous bleeding from multiple sites
suggests an acute, acquired systemic
coagulation or fibrinolytic disorders
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Laboratory Tests
They should be
•
•
•
•
Sensitive
Limited in number
Easy to do
Their results clinically-relevant
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Diagnosis of Congenital Hemorrhagic
Coagulopathies
• First Step (Simple Screening Tests)
- To detect most frequent and well established
causes of bleeding
• Second Step (Specific Tests)
- To detect less common causes of bleeding
due to abnormalities to which the screening
tests are insensitive
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Diagnosis of Congenital Hemorrhagic
Coagulopathies
First Step
• Primary Hemostasis
- Platelet count
- Bleeding Time (or alternative tests)
• Coagulation
- Prothrombin time (PT)
- Activated partial thromboplastin time (APTT)
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Further Evaluation of Primary
Hemostasis
• Low Platelet Count
- Investigation of thrombocytopenia
• Prolonged Bleeding Time
- Measurement of plasma Willebrand factor
- Platelet aggregation studies
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Further Evaluation of Coagulation
PT/APTT Prolongation
Mixing
Correction
Factor assay
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No correction
Screening for LA Inhibitor assay
Diagnosis of Congenital Hemorrhagic
Coagulopathies
• First Step (Simple Screening Tests)
- To detect most frequent and well established
causes of bleeding
• Second Step (Specific Tests)
- To detect less common causes of bleeding
due to abnormalities to which the screening
tests are insensitive
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Diagnosis of Congenital Hemorrhagic
Coagulopathies
Second Step
• Factor XIII deficiency
• Fibrinolysis defects
- tPA, PAI, α2PI
• Von Willebrand factor deficiency
• Dysfibrinogenemia
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Settings where the laboratory
can help clinicians
Thrombophilia Testing
and aPL/LA Syndrome
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Thrombophilia
• It may be defined as a condition
characterized by an increased risk of
thromboembolism at relatively young age
• It may secondary to congenital, or acquired
causes and some of them may be detected
by laboratory investigation
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Laboratory Diagnosis of Thrombophilia
Conditions to be investigated
• Congenital
- Antithrombin, protein C, protein S deficiencies
- APC-resistance (factor V Leiden)
- Hyperprothrombinemia (prothrombin mutation)
- Dysfibrinogenemia
• Acquired
- Moderate hyperhomocysteinemia
- Antiphospholipid antibody syndrome
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Naturally Occurring Anticoagulants
HMKW
PK
XIIa
XI
XIa
Antithrombin-Heparin
IX
IX VIIa-TF
IXa
VIIIa
X
X
Xa
Va
II
Fibrinolysis
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Fibrin
IIa
Fibrinogen
Main Characteristics of Congenital
Antithrombin Deficiency
Inheritance
Values in affected members
Thrombotic symptoms
Possible predisposing factors
Autosomal dominant
~ 50% (functional assay)
Deep vein thrombosis
Pregnancy, surgery,
oral contraceptives, etc.
Prevalence in patients
with venous thrombosis
2-4%
Prevalence in the general population
Rare
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Naturally Occurring Anticoagulants
HMKW
PK
XIIa
XI
Antithrombin-Heparin
XIa
IX
Protein C
Protein S
IXa
VIIIa
X
IX VIIa-TF
X
Xa
Va
II
Fibrinolysis
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Fibrin
IIa
Fibrinogen
Main Characteristics of Congenital
Protein C/Protein S Deficiency
Inheritance
Values in affected members
Thrombotic symptoms
Possible predisposing factors
Prevalence in patients
with venous thrombosis
Prevalence in the general population
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Autosomal dominant
Heterozygous:
Homozygous:
(functional assay)
~ 50%
< 10%
Deep vein thrombosis,
superficial thrombophlebitis
Pregnancy, surgery,
oral contraceptives, etc.
4-8%
Rare
APC Resistance
Control
Patient
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Dahlbäck et al, 1993
Main Causes of APC Resistance
• Congenital (85% of all cases)
- Factor V Leiden mutation (the vast majority)
- Factor V Cambridge mutation (very rare)
• Acquired
- Elevated coagulation factor levels
- Pregnancy
- Oral contraceptives intake
- Lupus anticoagulants
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APC Resistance
Types of measurement
• Plasma analysis (APTT-based method)
- Simple
- Cheap
- Sensitive to acquired APC resistance, not only to FV Leiden
• Plasma analysis (APTT-based method with FV-def. plasma)
- 100% specific for FV Leiden
• DNA analysis
- Does not detect acquired APC resistance
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APC Resistance
APTT-based Method
• It consists of two APTT
- With APC
- Without APC
• Results Expression
- APC ratio =
APTT with APC
APTT without APC
• Interpretation
- Lower than normal APC ratio suggests
“APC resistance”
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Main Characteristics of Congenital
APC Resistance (FV Leiden)
Inheritance
Values in affected members
Thrombotic symptoms
Possible predisposing factors
Prevalence in patients
with venous thrombosis
Prevalence in the general population
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Autosomal dominant
Heterozygous: low APC-ratio
Homozygous: very low
APC-ratio
Deep vein thrombosis
Pregnancy, surgery,
oral contraceptives, etc.
20-60%
3-15% in Caucasians
Prothrombin mutation
• Genetic transition
- G-to-A at position 20210 in the prothrombin
gene (untranslated region)
• Phenotypic expression
- High levels of plasmatic prothrombin
• Clinical expression
- Increased risk of venous thromboembolism
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Main Characteristics of Congenital
Hyperprothrombinemia
(Prothrombin mutation 20210)
Inheritance
Values in affected members
Thrombotic symptoms
Possible predisposing factors
Prevalence in patients
with venous thrombosis
Prevalence in the general population
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Autosomal dominant
Heterozygous: 110-130%
Homozygous: > 130%
Deep vein thrombosis
Pregnancy, surgery,
oral contraceptives, etc.
6-18%
2-3% in Caucasians
Main Characteristics of Congenital
Dysfibrinogenemia
Inheritance
Main laboratory features
Symptoms
Prevalence in patients
with venous thrombosis
Prevalence in the general population
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Autosomal recessive
Discrepancy between
immunologic and functional
fibrinogen, prolonged thrombin
clotting time
None, hemorrhage, venous
and arterial thrombosis
Rare
Very rare
Main Characteristics of Hyperhomocysteinemia
Congenital
Deficiency of CBS, MS, abnormal
(absent or thermolabile variant)
MTHFR.
Acquired
Vitamin deficiency (folate, B12),
age, gender, chronic renal failure.
Values in affected
subjects
Symptoms
Prevalence in patients
with venous thrombosis
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Moderate:
Medium:
Severe:
15-30 µM
30-100 µM
> 100 µM
Moderate: arterial, venous thrombosis
Severe: homocystinuria syndrome
10-20%
Main Characteristics of
Antiphospholipid Antibody syndrome
Clinical features
Laboratory features
Prevalence in patients with
thrombosis
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Arterial and/or venous thrombosis,
pregnancy loss
repeated positive solid-phase
antiphospholipid-(protein) antibodies
(anticardiolipin, anti-b2GPI) and/or
lupus anticoagulant tests
Unknown
Antiphospholipid Antibody Syndrome
Laboratory diagnosis
• LA and solid-phase antiphospholipid
antibodies coexist in about 2/3 of the
patients with the syndrome
• Diagnosis must be based on both LA and
solid-phase antiphospholipid antibodies
detection
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Phospholipid-dependent tests for
lupus anticoagulants
•
•
-
APTT and dilute PT
Relatively insensitive
KCT (or SCT) and dRVVT
Sensitive
Patients with higher dRVVT-ratio than KCTratio are more likely to develop thrombosis??
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Antiphospholipid Antibody Syndrome
Solid-phase antibodies
•
•
•
•
•
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Anti-cardiolipin
Anti-b2-Glycoprotein I
Anti-phosphatidylserine
Anti-prothrombin
Anti-PS, anti-PC, anti-Annexin V
Laboratory Diagnosis of
Antiphospholipid Syndrome
Recommendations
• Search for LA
- At least two phospholipid-dependent tests
(screen and confirm)
• Search for solid-phase antiphospholipid
antibodies
- Anti-cardiolipin
- Anti-β2-GPI
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Laboratory Diagnosis of Thrombophilia
Who should be tested
• Patients with history of thrombosis
• Family members
• No general screening of the population for
APC resistance
• Is prophylactic APC resistance testing
beneficial in association with risk situation?
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Laboratory Diagnosis of Thrombophilia
When is it appropriate to test
• After (and far from) a thrombotic episode
• After discontinuation of oral anticoagulation
• After delivery and puerperium
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Settings where the laboratory
can help clinicians
Diagnosis of Acute Venous
Thromboembolism
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Diagnosis of Deep Vein Thrombosis
• Clinical
- Unrealiable
• Plebography
- Gold Standard
• Compression ultrasonography (CUS)
- Reliable (if thrombosis is proximal)
• D-Dimer measurement
- High negative predictive value when used in
combination with clinical probability
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D-Dimer
• D-dimer results from the plasmin-mediated
degradation of cross-linked fibrin
- It is an index of fibrin deposition
- It is not specific for venous thromboembolism
- It has a high negative predictive value for the
diagnosis of venous thromboembolism, especially
if used in combination with the clinical probability
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Diagnosis of Venous Thromboembolism (VTE)
Combination of Clinical Probability and D-dimer
Symptomatic VTE
D- Dimer
Clinical probability
Negative D-Dimer and
Low clinical probability
Negative D-dimer and
High clinical probability
Positive D-Dimer
Exclude VTE
Further Investigation
Further investigation
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D-Dimer to Establish the Optimal Duration of
Oral Anticoagulant Treatment (OAT)
G. Palareti et al, NEJM 2006
• Patients with a first episode of unprovoked VTE were
on OAT for a minimum of 3 months
• D-Dimer was measured after 1 month after cessation
of OAT
• Patients with normal D-Dimer did not continue OAT
• Patients with elevated D-Dimer were randomized either
to stop or resume OAT
• All patients were followed up for an average of 1.15
years to assess for recurrent VTE
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D-Dimer to Establish the Optimal Duration of OAT
Cumulative incidence of outcomes
Palareti et al. NEJM 2006
0.20
Elevated D-Dimer No OAT
11.7% patient-years
0.15
0.10
Normal D-Dimer
4.2% patient-years
0.05
2.0% patient-years
Elevated D-Dimer + OAT
0.00
0
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100
200
300
Days
400
500
600
Settings where the Laboratory
can help Clinicians
Heparin monitoring
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Type of Heparins
•
•
-
Unfractionated Heparin
Treatment of acute venous thromboembolism
Prophylaxis
Low Molecular Weight Heparin
Treatment of acute venous thromboembolism
Prophylaxis
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Monitoring Unfractionated Heparin
• Treatment of acute venous thromboembolism
- APTT (therapeutic interval: from 1.5 to 2.5
the basal value)
• Prophylaxis
- In general no monitoring is required
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Monitoring Low Molecular Weight
Heparin
• Treatment of acute venous thromboembolism
- In general no monitoring is required
- When monitoring is required, the test of
choice is the anti-factor Xa activity
• Prophylaxis
- No monitoring
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Acute Venous Thromboembolism
Epidemiology
•
•
-
Incidence
1.6:1000 inhabitants per year
Causes
Acquired (surgery, cancer, pregnancy, oral
contraceptives, etc.)
- Congenital (Deficiency of Anticoagulant
mechanisms)
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Acute Venous Thromboembolism
Incidence Following:
• Surgery
- General
- Orthopedic
- Cancer
• Medical diseases
• Stroke (affected limb)
• Trauma
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25%
50-70%
50%
16%
60%
>60%
Deep Vein Thrombosis (DVT) Rates after
Major Orthopedic Surgery in Asia (1)
Piovella F et al, JTH 2005
• Aim
- To assess the incidence of DVT in patients
undergoing major orthopedic surgery of the
lower limbs without prophylaxis
• Design
- Epidemiological study based on postoperative
screening with centrally adjudicated bilateral
venography
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Deep Vein Thrombosis (DVT) Rates after
Major Orthopedic Surgery in Asia
Piovella F et al, JTH 2005
• Participating Centers
- 19 across Asia (China, Indonesia, South Korea,
Malaysia, Philippines, Taiwan and Thailand)
• Results
- DVT was diagnosed in 41% (95% CI 35-47%) of the
patients
• Conclusions
- The rate of DVT in the absence of prophylaxis in Asia is
similar to that reported in Western countries
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Settings where the Laboratory
can help Clinicians
Oral anticoagulant
monitoring
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Oral Anticoagulant Treatment
• Optimal level of anticoagulation
- To prevent thrombotic recurrences
- Still adequate to ensure hemostasis
• Laboratory control
- To adjust dosage in individual patients
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Prothrombin Time (PT)
Defined as
- Clotting time of citrated plasma (or whole blood)
upon addition of tissue extract (thromboplastin)
and calcium ions
Advantages
- Simple and cheap
- Sensitive to most of the clotting factors
depressed by oral anticoagulation (VII, X, II)
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Prothrombin Time (PT)
Main drawback in monitoring oral anticoagulants
• Different responsiveness of commercial PT
systems to the defect induced by oral
anticoagulants
• Different ways of expressing results
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Prothrombin Time (PT)
Expression of Results
• Coagulation time
- Seconds
• Percentage activity
- Interpolated from a calibration curve
• Ratio
- Patient-to-normal coagulation time
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Prothrombin time (PT)
Consequences of the Different
Responsiveness of PT Systems
• Different degree of anticoagulation in
different hospital
• Difficult establishment of “universal”
therapeutic intervals
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Prothrombin Time (PT)
Possible solutions to the different
responsiveness of PT systems
• To use the same PT system in all laboratories
• To calibrate commercial PT systems against
an International Standard
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Log-PT with IS
manual technique
Calibration of PT Systems
ISIWorking system = Slope x ISIIS
Log-PT with Working System
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Hierarchy of WHO Standards
67/40
BCT/253
OBT/79
RBT/90
rTF/95
RBT/05
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RBT/79
How to Convert PT into INR
PTRatio =
PT patients
Mean Normal PT
ISI
INR = (PTRatio)
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Degree of Anticoagulation and INR
Inadequate
Adequate
Excessive
1
2
3
4
INR
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5
6
Implementation of the INR System
Responsibility (1)
• Manufacturers of thromboplastins
- They should provide the ISI for their systems
• National control Laboratories
- They should check the reliability of the ISI by
occasional calibrations and organization of
regular external quality control schemes
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Implementation of the INR System
Responsibility (2)
• Laboratory workers
- They should favor implementation by expressing
results for patients on oral anticoagulants as INR
and informing clinicians on the INR system
• Clinicians
- They should use the INR for patients on oral
anticoagulants
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Numbers of Patients on Oral
Anticoagulant Treatment
• About 1% of the general population in
Western countries is on oral anticoagulant
treatment
- Prevention of venous thromboembolism
- Prosthetic cardiac valves
- Atrial fibrillation
• The current rate of increase is about 10%
per year
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Organization of Oral Anticoagulant
Treatment
• Established
- Anticoagulation clinic
- Specialists (cardiologists, hematologists, etc.)
• Being Developed (fast growing)
- Self-testing
- Self-management
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Anticoagulation Clinics
UK, Netherlands and Italy
•
-
Main Task
Patient education
Laboratory monitoring
Clinical monitoring
Assistance on the occasion of adverse
events
- Assistance on the occasion of surgery or
other potentially hemorrhagic events
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Anticoagulation Clinics
UK, Netherlands and Italy
•
•
-
Personnel
Physicians
Nurses
Medical technologists
Equipment
Coagulometers
Computer software for automated drug prescription
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Anticoagulation Clinic
registration
prescription
Computer-asssisted
dosage
Blood
sampling
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Protrombin time (PT)