Transcript Document
Almazov Federal Medical Research Centre St.Petеrsburg, Russia
Vavilova Tatiana
Laboratory tests in blood coagulation disorders
September 18, 2014 Moscow
Conflict of interests is absent
Disorders of haemostasis Bleeding
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hereditary acquired DIC Thrombosis
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hereditary
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acquired
Basic principle in diagnostic and laboratory assessment of hemostatic disorders Laboratory testing Clinical since Individual and family history of thrombotic disorders None of the components can be prioritized Clinician + Laboratory staff = correct diagnosis and benefits for patient
The investigation of haemostasis disorder requires a stepwise approach Clinical signs Laboratory results Anamnesis Clinical signs Laboratory results Anamnesis Anamnesis Clinical signs Laboratory results
2 1
Essential aspects of haemostasis diagnosis
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plasma factors testing
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Vessel wall THROMBUS Blood cells Plasma factors
Coagulation Platelets and vessel wall – microcirculation, small arteries Plasma factors – veins, cardiac chambers Anticoagulation Fibrinolysis
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, 381-92
Platelet receptors
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
Platelet Function Assays
Traditional Evaluation of Platelets
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Functional assays for platelet activation
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.
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Flow cytometry (microparticles, platelet/leucocytes aggregates, platelet receptors …) …
Platelet Aggregometry
www.clot-ed
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.
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collagen (integrin α2/β1 & GPVI receptors), ADP (receptors P2Y1 & P2Y12), epinephrine (α2-adrenergic receptor), 5.
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ristocetin (GPIbα), thrombin receptor activating peptide (Protease Activated Receptor [PAR] 1 and 4).
Standard aggregometry (light transmittion) Born GVR. Nature 1962; 194: 927-929 Born GVR, Cross MJ. J Physiol 1963; 168:178-195
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Manufacturers Bio/Data, www.biodatacorp.com (PAP-4) Chrono-Log, www.chronolog.com Labitec, Germany www.labitec.de (APACT) Solar (Belorussia)
Understanding Platelet Aggregation Tracings
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Standard aggregation
• Visualization of aggregation • Widely available • Use >40 years • Very flexible • Continuous monitoring “Real Time”
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• Poor reproducibility • Need of platelet preparation • Sample processing time • Low sensitivity •
Application: Detection of platelet function defects
LT
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Laser aggregometry (light dispersion)
Manufacturers: Biola, Moscow, Russia; www.biola.ru (LA220, LA230); Kowa, Japan; www.kowa.com (AG-10, PA-20, PA-100, PA-200)
5.0
2.5
1.25
R 5.0
2.5
0.63
1.25
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Microaggregates and coronary risk factors
Miyamoto S et al. Thromb Haemost 2003; 89(4):681
Plasma markers of the platelet activity
Requirements: Possible candidate molecules: 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
Substances that are released from the platelet granules
Molecules that are exposed on, and then shed from, platelet surface
Platelet factor 4 (PF 4) β-thromboglobulin ( β-TG) P-selectin
Secreted metabolic molecules
Thromboxane B 2
Laboratory tests for plasma coagulation, anticoagulation and fibrinolisis
• 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.
XI XIa TF : VIIa IX X IXa Xa : Va IIa (thrombin) VII I phase initiation vWF : VIII → VIIIa VIIIa : IXa FIBRINOGEN V → Va II phase Activated platelet X Va : Xa X а II IIa (thrombin) amplification propagation III phase thrombin and fibrin generation FIBRIN
Laboratory coagulation tests and technological principals
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Clotting tests Tests with chromogenic substrates Immunochemical methods Methods of molecular genetics
The most common coagulometers principles – clotting assays
mechanical (steel ball) turbidimetry nephelometry/light scatter
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)
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.
XI XIa TF : VIIa IX X IXa Xa : Va IIa (thrombin)
aPTT
vWF : VIII → VIIIa V → Va VIIIa : IXa
Activated Platelet
X X а Va : Xa II IIa (thrombin) VII FIBRINOGEN FIBRIN
PT
The principle of the aPTT
Contact activator, phospholipids
A prolonged APTT is found in:
Ca Cl 2
red arrows = positive feedback reactions
Heparin sensitivity of different aPTT reagents
FVIII, FIX activity Correction of aPTT Prolongation of aPTT Number of platelets, PT and BT are normal 1:1 mixt test aPTT Factors deficiency No correction of aPTT Presence of inhibitors Hemophilia FXII, HMWK, PK activity Detection of inhibitors to FVIII L А/аPL FXII, HHMWK, PK Insuff. (clinical negative) Inhibitor-dependent Hemophilia APS (if clinical and lab. symtoms are combined)
The prothrombin time assay Tissue factor, Ca 2+
An abnormal PT is found in: red arrows = feedback reactions
The calibration of thromboplastins in the ISI/INR system
WHO reference thromboplastin (human, rabbit or bovine) House standard thromboplastin
The INR is calculated according to:
ISI determination (instrument specific) Thromboplastin lot With assigned ISI value Determination of
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Normal PT
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Clotting time patient Calculation of INR
INR = PR
ISI
“International Normalised Ratios (INR) – the First 20 Years” ( Poller L.// JTH 2004; 2: 849-60) Problems Benefits
Decreasing of oral anticoagulants dosage
Heterogeneous of referent materials
Decreasing the number of bleeding complications
Complexity of calibration process
Improvement anticoagulant therapy - became more safety
ISI determination must be instrument specific
Possibility to computerize the management of anticoagulation
Differences in INR level because of reagent and equipment distinction
Proenzymes (Factors II, VII, IX, X; P С, PS) Active Factors (II, VII, IX, X; P С, PS) Carboxylase CYP2C9
Warfarin
Vit K=O Vit K-OH Vitamin K reductase (VKORC) PIVKA – proteins induced in vitamin K absence (Factors II, VII, IX, X; protein С, protein S)
Example of a patient with high anticoagulation variability
Markers of coagulation activity (hyprcoagulability, prethrombotic stage)
Research Clinical practice
Prothrombin Fragment 1+2 Thrombin-antithrombin complex
Fibrinogen vWF FVIII activity D=dimer Fibrinopeptide A Fibrin-monomer
Fibrinogen/fibrin degradation products D-dimer
An Introduction to D-Dimer
A summary of the D-Dimer origin
Thrombin converts fibrinogen to soluble fibrin by cleaving the fibrinopeptides A and B. The fibrin monomers polymerize spontaneously Active factor XIII links tow D domains and generates a solid fibrin clot. A new plasmin resistant antigenic determinant (D-Dimer) is produced Thus, fragments containing D Dimer are formed during the degradation of the fibrin clot by plasmin
Optimal division point
cut-off Specificity, % 1 – specificity, % (frequency of false positive results) 47
DD < 500 mg/l Unlikely VTE suspition
Clinical probability ccording Well’s score
Likely or non indicated DD DD PE?
DVT?
DD > 500 mg/l US STOP « Neg » Prox TVP STOP CT Scan
DD: pregnancy?
1 st 2 nd Trim: 139 – 602 mg/l Trim: 291 – 1231 3 th Trim: 489 - 2217 Delivery, D1-3: > 500 Utility first 4W PP?
Chabloz, 2001 – Epinay 2005
Thrombin Generation Test
120 Time to peak 100 Peak Hight 80 60 Area Under the Curve 'ETP' 40 20 Lag-time 0 0 5 10 Time (min) 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
2005.
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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
Thank you for your attention!