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 TRIPODI

Settings where the laboratory can help clinicians Diagnosis of Congenital Hemorrhagic Coagulopathies (pre-surgical screening) TRIPODI

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 TRIPODI

Congenital Hemorrhagic Coagulopathies Most Important Screening Test Good Clinical History TRIPODI

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 TRIPODI

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 TRIPODI

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 TRIPODI

Laboratory Tests Sensitive Limited in number Easy to do They should be Sensitive Limited in number Easy to do Their results clinically-relevant TRIPODI

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

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) TRIPODI

Further Evaluation of Primary Hemostasis Low Platelet Count Investigation of thrombocytopenia Prolonged Bleeding Time Measurement of plasma Willebrand factor Platelet aggregation studies TRIPODI

Further Evaluation of Coagulation PT/APTT Prolongation Mixing Correction No correction Factor assay Screening for LA Inhibitor assay TRIPODI

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

Diagnosis of Congenital Hemorrhagic Coagulopathies Second Step Factor XIII deficiency Fibrinolysis defects - tPA, PAI, α2PI Von Willebrand factor deficiency Dysfibrinogenemia TRIPODI

Settings where the laboratory can help clinicians Thrombophilia Testing and aPL/LA Syndrome TRIPODI

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 TRIPODI

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 TRIPODI

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 TRIPODI

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 TRIPODI

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 TRIPODI

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 TRIPODI

APC Resistance Control Patient TRIPODI 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 TRIPODI

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 TRIPODI

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” TRIPODI

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 TRIPODI

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 TRIPODI

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: 110-130% Homozygous: > 130% Deep vein thrombosis Pregnancy, surgery, oral contraceptives, etc. 6-18% 2-3% in Caucasians TRIPODI

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 TRIPODI

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% 15-30 µM 30-100 µM > 100 µM TRIPODI

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 TRIPODI

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 TRIPODI

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?? TRIPODI

Antiphospholipid Antibody Syndrome Solid-phase antibodies Anti-cardiolipin Anti-2-Glycoprotein I Anti-phosphatidylserine Anti-prothrombin Anti-PS, anti-PC, anti-Annexin V TRIPODI

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 TRIPODI

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? TRIPODI

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 TRIPODI

Settings where the laboratory can help clinicians Diagnosis of Acute Venous Thromboembolism TRIPODI

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 TRIPODI

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 TRIPODI

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 TRIPODI

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 TRIPODI

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 TRIPODI

Settings where the Laboratory can help Clinicians Heparin monitoring TRIPODI

Type of Heparins Unfractionated Heparin Treatment of acute venous thromboembolism Prophylaxis Low Molecular Weight Heparin TRIPODI

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 TRIPODI

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 TRIPODI

Acute Venous Thromboembolism Epidemiology Incidence 1.6:1000 inhabitants per year Causes Acquired (surgery, cancer, pregnancy, oral contraceptives, etc.) - Congenital (Deficiency of Anticoagulant mechanisms) TRIPODI

Acute Venous Thromboembolism Incidence Following: Surgery - General 25% - Orthopedic 50-70% - Cancer 50% Medical diseases 16% Stroke (affected limb) 60% Trauma >60% TRIPODI

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 TRIPODI

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 TRIPODI

Settings where the Laboratory can help Clinicians Oral anticoagulant monitoring TRIPODI

Oral Anticoagulant Treatment Optimal level of anticoagulation - To prevent thrombotic recurrences - Still adequate to ensure hemostasis Laboratory control - To adjust dosage in individual patients TRIPODI

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) TRIPODI

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 TRIPODI

Prothrombin Time (PT) Expression of Results Coagulation time Seconds Percentage activity - Interpolated from a calibration curve Ratio - Patient-to-normal coagulation time TRIPODI

Prothrombin time (PT) Consequences of the Different Responsiveness of PT Systems Different degree of anticoagulation in different hospital Difficult establishment of “universal” therapeutic intervals TRIPODI

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 TRIPODI

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 TRIPODI

Hierarchy of WHO Standards 67/40 BCT/253 OBT/79 RBT/79 RBT/90 rTF/95 RBT/05 TRIPODI

PTRatio = INR = (PTRatio)ISI How to Convert PT into INR PT patients Mean Normal PT = INR = (PTRatio)ISI TRIPODI

Degree of Anticoagulation and INR Inadequate INR Adequate Excessive 1 2 3 4 5 6 TRIPODI

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 TRIPODI

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 TRIPODI

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 TRIPODI

Organization of Oral Anticoagulant Treatment Established - Anticoagulation clinic - Specialists (cardiologists, hematologists, etc.) Being Developed (fast growing) - Self-testing - Self-management TRIPODI

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 TRIPODI

Anticoagulation Clinics UK, Netherlands and Italy Personnel Physicians Nurses Medical technologists Equipment Coagulometers Computer software for automated drug prescription TRIPODI

Anticoagulation Clinic registration Blood sampling Protrombin time (PT) Computer-asssisted dosage prescription TRIPODI