1. International Hemostasis VIP Meeting China, 2006
Armando Tripodi
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center
University of Milan
Italy
TRIPODI

2. TRIPODI

3. 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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4. Settings where the laboratory can help clinicians
Diagnosis of Congenital Hemorrhagic Coagulopathies (pre-surgical screening)
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5. 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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6. Congenital Hemorrhagic Coagulopathies
Most Important Screening Test
Good Clinical History
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7. 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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8. 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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9. 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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10. Laboratory Tests Sensitive Limited in number Easy to do
They should be
Sensitive
Limited in number
Easy to do
Their results clinically-relevant
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11. 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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12. 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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13. 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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14. Further Evaluation of Coagulation
PT/APTT Prolongation
Mixing
Correction
No correction
Factor assay
Screening for LA
Inhibitor assay
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15. 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
TRIPODI

16. Diagnosis of Congenital Hemorrhagic Coagulopathies Second Step
Factor XIII deficiency
Fibrinolysis defects
- tPA, PAI, α2PI
Von Willebrand factor deficiency
Dysfibrinogenemia
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17. Settings where the laboratory can help clinicians
Thrombophilia Testing and aPL/LA Syndrome
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18. 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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19. 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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20. Antithrombin-Heparin
Naturally Occurring Anticoagulants
HMKW
XIa
IXa Xa
IIa IX
Antithrombin-Heparin
Fibrinolysis
Fibrin PK XI
XIIa X
VIIIa Va II
VIIa-TF
Fibrinogen
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21. Main Characteristics of Congenital Antithrombin Deficiency
Inheritance
Values in affected members
Thrombotic symptoms
Possible predisposing factors
Prevalence in patients
with 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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22. Antithrombin-Heparin
Naturally Occurring Anticoagulants
HMKW
XIa
IXa Xa
IIa IX
Antithrombin-Heparin
Fibrinolysis
Protein C
Protein S
Fibrin PK XI
XIIa X
VIIIa Va II
VIIa-TF
Fibrinogen
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23. 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
Autosomal dominant
Heterozygous: ~ 50%
Homozygous: < 10%
(functional assay)
Deep vein thrombosis,
superficial thrombophlebitis
Pregnancy, surgery,
oral contraceptives, etc.
4-8%
Rare
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24. APC Resistance
Control
Patient
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Dahlbäck et al, 1993

25. 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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26. 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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27. APC Resistance APTT-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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28. 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
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
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29. Prothrombin mutation Genetic transition
- G-to-A at position in the prothrombin gene (untranslated region)
Phenotypic expression
High levels of plasmatic prothrombin
Clinical expression
- Increased risk of venous thromboembolism
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30. 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
Autosomal dominant
Heterozygous: %
Homozygous: > 130%
Deep vein thrombosis
Pregnancy, surgery,
oral contraceptives, etc.
6-18%
2-3% in Caucasians
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31. Main Characteristics of Congenital Dysfibrinogenemia
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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32. Prevalence in patients with venous thrombosis
Main Characteristics of Hyperhomocysteinemia
Congenital
Acquired
Values in affected
subjects
Symptoms
Prevalence in patients
with venous thrombosis
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 thrombosis
Severe: homocystinuria syndrome
10-20% µM µM
> 100 µM
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33. Prevalence in patients with
Main Characteristics of
Antiphospholipid Antibody syndrome
Clinical features
Laboratory features
Prevalence in patients with
thrombosis
Arterial and/or venous thrombosis,
pregnancy loss
repeated positive solid-phase
antiphospholipid-(protein) antibodies
(anticardiolipin, anti-b2GPI) and/or
lupus anticoagulant tests
Unknown
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34. 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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35. 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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36. 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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37. 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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38. 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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39. 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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40. Settings where the laboratory can help clinicians
Diagnosis of Acute Venous Thromboembolism
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41. 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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42. 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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43. 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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44. 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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45. D-Dimer to Establish the Optimal Duration of OAT
Palareti et al. NEJM 2006
100
200
300
400
500
600
0.00
0.05
0.10
0.15
0.20
Elevated D-Dimer No OAT
Elevated D-Dimer + OAT
Normal D-Dimer
Days
Cumulative incidence of outcomes
4.2% patient-years
11.7% patient-years
2.0% patient-years
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46. Settings where the Laboratory can help Clinicians
Heparin monitoring
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47. Type of Heparins Unfractionated Heparin
Treatment of acute venous thromboembolism
Prophylaxis
Low Molecular Weight Heparin
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48. 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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49. 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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50. 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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51. Acute Venous Thromboembolism Incidence Following:
Surgery
- General %
- Orthopedic %
- Cancer 50%
Medical diseases 16%
Stroke (affected limb) %
Trauma >60%
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52. 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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53. 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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54. Settings where the Laboratory can help Clinicians
Oral anticoagulant monitoring
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55. 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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56. 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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57. 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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58. 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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59. 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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60. 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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61. Log-PT with Working System
Calibration of PT Systems
Log-PT with Working System
manual technique
Log-PT with IS
ISIWorking system = Slope x ISIIS
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62. Hierarchy of WHO Standards
67/40
BCT/253
OBT/79
RBT/79
RBT/90
rTF/95
RBT/05
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63. PTRatio = INR = (PTRatio)ISI How to Convert PT into INR PT patients
Mean Normal PT =
INR =
(PTRatio)ISI
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64. Degree of Anticoagulation and INR
Inadequate
INR
Adequate
Excessive 1 2 3 4 5 6
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65. 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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66. 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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67. 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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68. Organization of Oral Anticoagulant Treatment
Established
- Anticoagulation clinic
- Specialists (cardiologists, hematologists, etc.)
Being Developed (fast growing)
- Self-testing
- Self-management
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69. 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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70. Anticoagulation Clinics UK, Netherlands and Italy
Personnel
Physicians
Nurses
Medical technologists
Equipment
Coagulometers
Computer software for automated drug prescription
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71. Anticoagulation Clinic
registration
Blood sampling
Protrombin time (PT)
Computer-asssisted dosage
prescription
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