tripodi international hemostasis vip meeting china, 2006 armando tripodi angelo bianchi bonomi...

TRIPODI

International Hemostasis VIP MeetingChina, 2006

Armando Tripodi<[email protected]>

Angelo Bianchi Bonomi Hemophilia and Thrombosis CenterUniversity 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

• To establish the causes of bleeding in patients who have shown evidence of abnormal bleeding

• To detect mild defects in asymptomatic patients

Aims of Laboratory Investigation

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Most Important Screening Test

Good Clinical History

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• Poor sensitivity of screening tests to detect mild defects

• The type of bleeding may provide valuable clue to its etiology

Why should clinical history be collected

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Aims of the Clinical History

• 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

To establish

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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

• Sensitive

• Limited in number

• Easy to do

• Their results clinically-relevant

They should be

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• 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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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

No correction

Screening for LA Inhibitor assay

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• 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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Second Step

• Factor XIII deficiency

• Fibrinolysis defects

- tPA, PAI, α2PI

• Von Willebrand factor deficiency

• Dysfibrinogenemia

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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

HMKW

XIa

IXa

Xa

IIa

IX

Antithrombin-Heparin

Fibrinolysis Fibrin

PK

XI

XIIa

IX

X

VIIIa

Va

II

VIIa-TF

X

Fibrinogen

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Main Characteristics of CongenitalAntithrombin Deficiency

Inheritance

Values in affected members

Thrombotic symptoms

Possible predisposing factors

Prevalence in patientswith venous thrombosis

Prevalence in the general population

Autosomal dominant

~ 50% (functional assay)

Deep vein thrombosis

Pregnancy, surgery,oral contraceptives, etc.

2-4%

Rare

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Naturally Occurring Anticoagulants

HMKW

XIa

IXa

Xa

IIa

IX

Antithrombin-Heparin

Fibrinolysis

Protein CProtein S

Fibrin

PK

XI

XIIa

IX

X

VIIIa

Va

II

VIIa-TF

X

Fibrinogen

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Main Characteristics of CongenitalProtein C/Protein S Deficiency

Inheritance


Thrombotic symptoms




Autosomal dominant

Heterozygous: ~ 50%Homozygous: < 10%(functional assay)

Deep vein thrombosis,superficial thrombophlebitis


4-8%

Rare

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APC Resistance

Dahlbäck et al, 1993

Patient

Control

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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

• It consists of two APTT - With APC - Without APC

• Results Expression

APTT with APC

APTT without APC- APC ratio =

• Interpretation - Lower than normal APC ratio suggests “APC resistance”

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Main Characteristics of CongenitalAPC Resistance (FV Leiden)

Inheritance


Thrombotic symptoms




Autosomal dominant

Heterozygous: low APC-ratioHomozygous: very low APC-ratio



20-60%

3-15% in Caucasians

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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 CongenitalHyperprothrombinemia

(Prothrombin mutation 20210)

Inheritance


Thrombotic symptoms




Autosomal dominant

Heterozygous: 110-130%Homozygous: > 130%



6-18%

2-3% in Caucasians

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Main Characteristics of CongenitalDysfibrinogenemia

Inheritance

Main laboratory features

Symptoms



Autosomal recessive

Discrepancy between immunologic and functional fibrinogen, prolonged thrombin clotting time

None, hemorrhage, venousand arterial thrombosis

Rare

Very rare

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Main Characteristics of Hyperhomocysteinemia

Congenital

Acquired

Values in affected subjects

Symptoms


Deficiency of CBS, MS, abnormal(absent or thermolabile variant) MTHFR.

Vitamin deficiency (folate, B12),age, gender, chronic renal failure.

Moderate:Medium:Severe:

Moderate: arterial, venous thrombosisSevere: homocystinuria syndrome

10-20%

15-30 µM30-100 µM

> 100 µM

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Main Characteristics ofAntiphospholipid Antibody syndrome

Clinical features

Laboratory features

Prevalence in patients withthrombosis

Arterial and/or venous thrombosis,pregnancy loss

repeated positive solid-phaseantiphospholipid-(protein) antibodies(anticardiolipin, anti-2GPI) and/orlupus anticoagulant tests

Unknown

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Antiphospholipid Antibody Syndrome

• 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

Laboratory diagnosis

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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

• Anti-cardiolipin

• Anti-2-Glycoprotein I

• Anti-phosphatidylserine

• Anti-prothrombin

• Anti-PS, anti-PC, anti-Annexin V

Solid-phase antibodies

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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 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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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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Symptomatic VTE

D- Dimer Clinical probability

Negative D-Dimer andLow clinical probability

Negative D-dimer and High clinical probability

Positive D-Dimer

Exclude VTE Further Investigation Further investigation

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 OATPalareti et al. NEJM 2006

0 100 200 300 400 500 6000.00

0.05

0.10

0.15

0.20

Elevated D-Dimer No OAT

Elevated D-Dimer + OAT

Normal D-Dimer

Days

Cu

mu

lati

ve in

cid

enc

e o

f o

utc

om

es

4.2% patient-years

11.7% patient-years

2.0% patient-years

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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

- 1.6:1000 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 25%

- Orthopedic 50-70%

- Cancer 50%

• Medical diseases 16%

• Stroke (affected limb) 60%

• Trauma >60%

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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 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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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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Calibration of PT Systems

Log-PT with Working System

Lo

g-P

T w

ith

IS

man

ual

tec

hn

iqu

e

ISIWorking system = Slope x ISIIS

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Hierarchy of WHO Standards

67/40

BCT/253 OBT/79 RBT/79RBT/90

rTF/95

RBT/05

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INR = (PTRatio)ISI

PTRatioPT patients

Mean Normal PT=

How to Convert PT into INR

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Degree of Anticoagulation and INR

Inadequate

INR

AdequateExcessive

1 2 3 4 5 6

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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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Anticoagulation Clinic

registration

Blood sampling

Protrombin time (PT)

Computer-asssisted dosage

prescription

tripodi international hemostasis vip meeting china, 2006 armando tripodi angelo bianchi bonomi...

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