International Hemostasis VIP Meeting China, 2006

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International Hemostasis VIP MeetingChina, 2006

• Armando Tripodi
• ltarmando.tripodi_at_unimi.itgt
• Angelo Bianchi Bonomi Hemophilia and Thrombosis
  Center
• University of Milan
• Italy

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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
CoagulopathiesFirst 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
No correction
Correction
Factor assay
Screening for LA
Inhibitor assay
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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
CoagulopathiesSecond Step

• Factor XIII deficiency
• Fibrinolysis defects
• - tPA, PAI, a2PI
• 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 ThrombophiliaConditions
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
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Naturally Occurring Anticoagulants
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Main Characteristics of Congenital Protein
C/Protein S Deficiency
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APC Resistance
Control
Patient
Dahlbäck et al, 1993
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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 ResistanceTypes 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 ResistanceAPTT-based Method
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Main Characteristics of Congenital APC Resistance
(FV Leiden)
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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 Hyperprothrombi
nemia (Prothrombin mutation 20210)
Inheritance Values in affected
members Thrombotic symptoms Possible
predisposing factors Prevalence in
patients with venous thrombosis Prevalence in
the general population
Autosomal dominant Heterozygous 110-130 Homozyg
ous gt 130 Deep vein thrombosis Pregnancy,
surgery, oral contraceptives, etc. 6-18 2-3
in Caucasians
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Main Characteristics of Congenital Dysfibrinogenem
ia
Inheritance Main laboratory features Symptoms
Prevalence in patients with venous
thrombosis Prevalence in the general population
Autosomal recessive Discrepancy between
immunologic and functional fibrinogen,
prolonged thrombin clotting time None,
hemorrhage, venous and arterial
thrombosis Rare Very rare
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Main Characteristics of Antiphospholipid Antibody
syndrome
Clinical features Laboratory features Preval
ence in patients with thrombosis
Arterial and/or venous thrombosis, pregnancy
loss repeated positive solid-phase antiphospholip
id-(protein) antibodies (anticardiolipin,
anti-b2GPI) and/or lupus anticoagulant
tests Unknown
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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 KCT-ratio
  are more likely to develop thrombosis??

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Antiphospholipid Antibody Syndrome
Solid-phase antibodies

• Anti-cardiolipin
• Anti-?2-Glycoprotein I
• Anti-phosphatidylserine
• Anti-prothrombin
• Anti-PS, anti-PC, anti-Annexin V

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Laboratory Diagnosis of Antiphospholipid
SyndromeRecommendations

• Search for LA
• At least two phospholipid-dependent tests (screen
  and confirm)
• Search for solid-phase antiphospholipid
  antibodies
• Anti-cardiolipin
• Anti-ß2-GPI

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Laboratory Diagnosis of ThrombophiliaWho 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 ThrombophiliaWhen 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
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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 Palareti et al. NEJM 2006
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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 ThromboembolismEpidemiology

• Incidence
• 1.61000 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 25
• - Orthopedic 50-70
• - Cancer 50
• Medical diseases 16
• Stroke (affected limb) 60
• Trauma gt60

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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 AsiaPiovella 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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Hierarchy of WHO Standards
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How to Convert PT into INR
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Degree of Anticoagulation and INR
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Implementation of the INR SystemResponsibility
(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 SystemResponsibility
(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 ClinicsUK, 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 ClinicsUK, Netherlands and Italy

• Personnel
• Physicians
• Nurses
• Medical technologists
• Equipment
• Coagulometers
• Computer software for automated drug prescription

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