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Almazov Federal Medical Research Centre St.Petеrsburg, Russia Laboratory tests in blood coagulation disorders Vavilova Tatiana September 18, 2014 Moscow.

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Presentation on theme: "Almazov Federal Medical Research Centre St.Petеrsburg, Russia Laboratory tests in blood coagulation disorders Vavilova Tatiana September 18, 2014 Moscow."— Presentation transcript:

1 Almazov Federal Medical Research Centre St.Petеrsburg, Russia Laboratory tests in blood coagulation disorders Vavilova Tatiana September 18, 2014 Moscow

2 Conflict of interests is absent

3 Thrombosis Disorders of haemostasis Bleeding hereditary acquired hereditary acquired DIC

4 Basic principle in diagnostic and laboratory assessment of hemostatic disorders None of the components can be prioritized Clinician + Laboratory staff = correct diagnosis and benefits for patient Clinical since Laboratory testing Individual and family history of thrombotic disorders

5 The investigation of haemostasis disorder requires a stepwise approach Clinical signs Laboratory results Laboratory results Anamnesis Laboratory results Laboratory results Laboratory results Laboratory results Clinical signs

6 Essential aspects of haemostasis diagnosis plasma factors testing

7 THROMBUS Vessel wall Blood cells Plasma factors  Coagulation  Platelets and vessel wall – microcirculation, small arteries  Plasma factors – veins, cardiac chambers  Anticoagulation  Fibrinolysis

8 ADP, serotonin Adgisive proteins Mitogenic Factors Coagulation factors Proteases inhibitors β -Thromboglobulin Platelets factor 4 … CD62P (P-selectin), CD40L – exposure on membrane Jurk K,Kehrel BE // Sem Thromb Hemost (2005) 31,

9 Platelet receptors

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11 Preanalitical phase ➙ The blood sampling procedure for haemostasis test is a critical issue. ➙ Avoiding the prolonged application of a venous cuff can reduce artifacts. ➙ Blood should be taken carefully into the tube without foam formation and the tube should be gently inverted in order to completely mix the citrate and blood. ➙ Samples in whith incorrect ratio of blood to anticoagulant or samples with visible fibrin strands, must not be used for testing because the results will be inaccurate. ➙ The main screening tests need to be performed within 4 hours

12 Platelet Function Assays

13 Traditional Evaluation of Platelets

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17 Functional assays for platelet activation 1.Measuring of spontaneous and induced aggregation of platelets in PRP and whole blood (light transmission, impedance measuring) 2.Laser light dispersion for aggregate size measuring in PRP 3.Flow cytometry (microparticles, platelet/leucocytes aggregates, platelet receptors …) 4.…

18 Platelet Aggregometry

19 Platelet aggregation testing assess: platelet adherence (Glycoprotein Ibα [GPIbα]), secretion (secondary wave or increase in luminescence), aggregation (αIIb/β3). Agonists are used to activate platelets via various receptors: 1. arachidonic acid (thromboxane pathway via cyclooxygenase), 2. collagen (integrin α2/β1 & GPVI receptors), 3. ADP (receptors P2Y1 & P2Y12), 4. epinephrine (α2-adrenergic receptor), 5. ristocetin (GPIbα), 6. thrombin receptor activating peptide (Protease Activated Receptor [PAR] 1 and 4).

20 Standard aggregometry (light transmittion) Born GVR. Nature 1962; 194: Born GVR, Cross MJ. J Physiol 1963; 168: Manufacturers Bio/Data, (PAP-4) Chrono-Log, Labitec, Germany (APACT) Solar (Belorussia)

21 Understanding Platelet Aggregation Tracings

22 Standard aggregation Application: Detection of platelet function defects Detection of platelet function defects Visualization of aggregation Widely available Use >40 years Very flexible Continuous monitoring “Real Time” Poor reproducibility Need of platelet preparation Sample processing time Low sensitivity + -

23 LTR Laser aggregometry (light dispersion) Manufacturers: Biola, Moscow, Russia; (LA220, LA230); Kowa, Japan; (AG-10, PA-20, PA-100, PA-200)

24 Miyamoto S et al. Thromb Haemost 2003; 89(4):681 Microaggregates and coronary risk factors

25 Plasma markers of the platelet activity Requirements: Must be specific marker for platelets Must be resistant to preanalitical artifacts Must be measured by cheap, reproducible, simple laboratory technique, such as ELISA, immunoturbidimetry or latex aggregation Possible candidate molecules: Substances that are released from the platelet granules  Platelet factor 4 (PF 4)  β-thromboglobulin (β-TG) Molecules that are exposed on, and then shed from, platelet surface  P-selectin Secreted metabolic molecules  Thromboxane B 2

26 Laboratory tests for plasma coagulation, anticoagulation and fibrinolisis

27 Haemostasis starts with the interaction between TF and FVIIa on the surface of subendothelial cells. The small amount of thrombin generated during the amplification phase activates platelets locally on whose surface the subsequent reactions take place. The resulting thrombin burst results in the formation of a stable clot.

28 TF : VIIa IX IX XIXa Xa : Va IIa (thrombin) Activated platelet vWF : VIII → VIIIa V → Va X XXXаXаXXXаXа VIIIa : IXa Va : Xa II IIa (thrombin) FIBRINOGEN FIBRIN I phase II phase III phase VII XI XIa initiation amplificationpropagation thrombin and fibrin generation fibrin generation

29 Laboratory coagulation tests and technological principals 1.Clotting tests 2.Tests with chromogenic substrates 3.Immunochemical methods 4.Methods of molecular genetics

30 The most common coagulometers principles – clotting assays mechanical (steel ball) turbidimetry nephelometry/light scatter

31 Immunochemical methods The principle of latex enhanced immunoassays (antibody coated particles agglutinate in the presence of antigen) The principle of the enzyme linked immunosorbent assay (ELISA)

32 Screening assays for either the extrinsic or intrinsic pathways are performed in order to get an overview of the enzymes, cofactors and inhibitors, involved in the respective pathway, and also of the influence of drugs or autoantibodies. The most important tests are prothrombin time and aPTT.

33 TF : VIIa IX IX XIXa Xa : Va IIa (thrombin) Activated platelet vWF : VIII → VIIIa V → Va X XXXаXаXXXаXа VIIIa : IXa Va : Xa II IIa (thrombin) FIBRINOGEN FIBRIN I phase II phase III phase VII XI XIa initiation amplificationpropagation thrombin and fibrin generation fibrin generation

34 TF : VIIa IX IX XIXa Xa : Va IIa (thrombin) Activated Platelet vWF : VIII → VIIIa V → Va X XXXаXаXXXаXа VIIIa : IXa Va : Xa II IIa (thrombin) FIBRINOGEN FIBRIN VII XI XIa aPTT PT

35 The principle of the aPTT red arrows = positive feedback reactions A prolonged APTT is found in: Contact activator, phospholipids Ca Cl 2

36 Heparin sensitivity of different aPTT reagents

37 Prolongation of aPTT Number of platelets, PT and BT are normal Prolongation of aPTT Number of platelets, PT and BT are normal 1:1 mixt test aPTT aPTT Correction of aPTT Correction No correction of aPTT of aPTT No correction of aPTT of aPTT Factors deficiency Presence of inhibitors FVIII, FIX activity activity FXII, HMWK, PK activity activity HemophiliaHemophilia FXII, HHMWK, PK Insuff. (clinical negative) FXII, HHMWK, PK Insuff. (clinical negative) Detection of inhibitors to FVIII Detection of inhibitors to FVIII LА/аPL Inhibitor-dependentHemophiliaInhibitor-dependentHemophilia APS (if clinical and lab. symtoms are combined) APS (if clinical and lab. symtoms are combined)

38 The prothrombin time assay An abnormal PT is found in: red arrows = feedback reactions Tissue factor, Ca 2+

39 The calibration of thromboplastins in the ISI/INR system The INR is calculated according to: INR = PR ISI WHO reference thromboplastin (human, rabbit or bovine) House standard thromboplastin ISI determination (instrument specific) Thromboplastin lot With assigned ISI value Determination of Normal PT Clotting time patient Calculation of INR Industry Laboratory

40 “International Normalised Ratios (INR) – the First 20 Years” “International Normalised Ratios (INR) – the First 20 Years” ( Poller L.// JTH 2004; 2: ) Benefits Decreasing of oral anticoagulants dosage Decreasing the number of bleeding complications Improvement anticoagulant therapy - became more safety Possibility to computerize the management of anticoagulation Problems Heterogeneous of referent materials Complexity of calibration process ISI determination must be instrument specific Differences in INR level because of reagent and equipment distinction

41 Vitamin K reductase (VKORC) Vit K-OHVit K=O CarboxylaseCarboxylase Warfarin PIVKA PIVKA – proteins induced in vitamin K absence (Factors II, VII, IX, X; protein С, protein S) Proenzymes (Factors II, VII, IX, X; PС, PS) Active Factors (II, VII, IX, X; PС, PS) CYP2C9

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43 Example of a patient with high anticoagulation variability

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45 Markers of coagulation activity (hyprcoagulability, prethrombotic stage) Prothrombin Fragment 1+2 Thrombin-antithrombin complex Fibrinopeptide A Fibrin-monomer Fibrinogen/fibrin degradation products D-dimer ResearchClinical practice  Fibrinogen  vWF  FVIII activity  D=dimer

46 Step 1 Thrombin converts fibrinogen to soluble fibrin by cleaving the fibrinopeptides A and B. The fibrin monomers polymerize spontaneously Step 2 Active factor XIII links tow D- domains and generates a solid fibrin clot. A new plasmin- resistant antigenic determinant (D-Dimer) is produced Step 3 Thus, fragments containing D- Dimer are formed during the degradation of the fibrin clot by plasmin An Introduction to D-Dimer A summary of the D-Dimer origin

47 47 Sensitivity, % (frequency of true positive results) 1 – specificity, % (frequency of false positive results) Specificity, % 1 - sensitivity, % Optimal division point cut-off

48 VTE suspition Unlikely Likely or non indicated DD DD DD < 500 mg/l DD > 500 mg/l STOP CT Scan PE?DVT?US STOP « Neg » Prox TVP Clinical probability ccording Well’s score

49 1 st Trim: 139 – 602 mg/l 2 nd Trim: 291 – th Trim: Delivery, D1-3: > 500 Utility first 4W PP? DD: pregnancy? Chabloz, 2001 – Epinay 2005

50 Source: Lawrie A, Béguin S, Hemker H C, Henckel T, Samama M, Woodhams B, Gray E. (2005). The Thrombin Generation Test (TGT); On behalf of the International Society on Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SSC) Working Group on Thrombin Generation Tests www.blood.com Thrombin Generation Test

51 Calibrated Automated Thrombogram - CAT Invented by the “father” of TGT - Prof C Hemker (Maastricht) The “Gold Standard Method” A Fluorogenic Assay Uses PPP or PRP

52 Thank you for your attention!


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