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Coag testing for hema fellows mskcc 10 15 2015 dr peerschke
- 1. Introduction to Coagulation Testing Ellinor I. Peerschke, Ph.D., F.A.H.A. Vice Chair, Laboratory Medicine Chief, Hematology & Coagulation Laboratory Services MSKCC
- 2. Hemostasis Balance between bleeding and clotting Cellular Components • Vascular endothelial cells • Platelets – Primary hemostasis Fluid phase components • Coagulation proteins and enzymes • Secondary Hemostasis
- 3. Learning Objectives List major Clinical Laboratory Coagulation Screening Tests Discuss use of screening tests and their principles in the evaluation of patients with bleeding disorders or for monitoring patients receiving anticoagulation therapy Interpret test results
- 4. Coagulation Screening Tests PT APTT Fibrinogen Thrombin Time ( D-dimer)
- 5. Coagulation Cascade Prothrombin Time Activated Partial Thromboplastin Time Thrombin Time
- 6. Preanalytical Considerations Specimen Collection 3.2% sodium citrate 9:1 volume of blood to anticoagulant Hct <25% or >50% may affect results
- 7. Preanalytical Considerations Specimen Stability PT (stable up to 72 h, closed tube at RT) APTT (stable up to 4 h, closed tube at RT) Special tests – plasma must be frozen at –80C, if not assayed within 4 h of collection Specimen Processing Preparation of Platelet Poor Plasma • Plt < 10,000/ µl • Centrifugation (10 – 20 min, 1000g) • Assays performed on Plasma
- 8. Analytical Considerations Types of Assays Functional • clot based – Optical clot detection – Mechanical clot detection • chromogenic Immunologic • ELISA • Latex immuno agglutination assay (LIA)
- 9. Types of Assays • Functional Assays Clot-based assays Good screening assays Based on a functioning coagulation cascade Subject to exogenous and intrinsic interferences Chromogenic assays Discreet measure of the activity of a specific enzyme Not affected by most preanalytical variables Enzyme of interest Peptide pNA Peptide pNA Color develops Quantify spectrophotometrically Absorbance correlates with activity (Substrate) +
- 10. Types of Assays Immunologic assays LIA- or ELISA-based technologies Measure the amount of protein present rather than functional activity • Sandwich ELISA • Immuno capture • Immuno detection with enzyme conjugated secondary antibody • Substrate cleaved by conjugated enzyme • Color development • Spectrophotometric quantification • Latex Agglutination • Antibody coated latex beads • Agglutination in presence of antigen • Agglutination is measured optically
- 11. Screening Tests of Hemostasis PT APTT Fibrinogen Thrombin Time D-Dimer Identify underlying coagulation defect Monitor/assess anticoagulation therapy Evaluate for DIC Rule out DVT/PE Clot based Optical LIA Test
- 12. Major Uses: Hemostasis Screening Monitoring Warfarin Anticoagulation From: Jesty & Morrison, Stony Brook University, Stony Brook, NY
- 13. INR INR= International Normalized Ratio (patient PT/mean normal PT)ISI ISI= International Sensitivity Index
- 14. Major Uses: Hemostasis Screening Monitoring UFH heparin therapy From: Jesty & Morrison, Stony Brook University, Stony Brook, NY
- 15. APTT reagents are variably sensitive to UFH Laboratories establish reagent specific therapeutic range Reagent standardization has not been successful APTT: Monitoring UFH Therapy
- 16. APTT response to heparin may be exaggerated by Conditions that elevate the APTT: • Concomitant warfarin therapy • Lupus anticoagulant • Liver disease APTT response to heparin may be blunted by Conditions that shorten the APTT: • Elevated Factor VIII • Antithrombin deficiency Under-estimates level of anticoagulation • Cause of in vitro drug “resistance” APTT: Monitoring UFH Therapy
- 17. Thrombin Time Highly sensitive to UFH Contamination Highly sensitive to Direct Thrombin Inhibitors Evaluates 3rd Stage of Coagulation Dysfibrinogenemia (follow with Reptilase Time) Thrombin: FPA & FPB Reptilase: FPA Need fibrinogen result for interpretation Hypo/Dysfibrinogenemia Compare functional fibrinogen with fibrinogen antigen
- 18. Fibrinogen & Thrombin Time Fibrinogen Assay Patient plasma + excess thrombin Thrombin Time Patient plasma+ limited thrombin Reaction Fibrinogen + thrombin • Cleavage of FPA, FPB Fibrin monomers Fibrin monomer polymerization CLOT Factor XIII + thrombin F XIIIa F XIIIa crosslinking of fibrin into stable clot
- 19. Fibrinogen Assay and Thrombin Time • Neither assay measures crosslinked fibrin or F XIII activity • Reptilase Time • Reptilase cleaves FPA • Insensensitive to heparin
- 20. Limitations of Coagulation Screening Tests Do not detect defects in F XIII Do not detect defects in fibrinolysis Fibrinolysis inhibitors • Tissue plasminogen activator • Plasmin inhibitor
- 21. Sensitivity of Screening Tests PT/APTT : prolonged by single factor deficiency <30% (variable) PT: highly sensitive to Vit K dependent factor deficiencies Variably sensitive to DOAC APTT: sensitive to unfractionated heparin – Variably sensitive to LMWH – Variably sensitive to DOAC
- 22. Monitoring LMWH: Anti-Xa Heparin Assay Specifically determines anticoagulant activity of LMWH (and UFH) by measuring ability of heparin-bound antithrombin to inhibit F Xa More specific than aPTT since it measures inhibition of a single enzyme Major advantage is lack of biologic interference Limitations of Heparin Assay Clinical data examining outcomes is limited Eikelboom JW. Thromb Haemost 2006;96:547-52. Francis JL. Pharmacotherapy 2004;24:108S-19S. Color development is Inversely proportional to the anticoagulant concentration in the plasma sample Excess FXa Plasma [heparin] + (Antithrombin) [AT-Heparin-Xa] + Residual FXa Chromogenic substrate pNA
- 23. Specific Assays for DOAC Rivaroxaban Level Chromogenic Xa Dabigatran Level Dilute thrombin time • Diluted Patient plasma NYS approved at MSKCC
- 24. Short PT/APTT In vitro sample activation traumatic venopuncture under anticoagulation/low Hct High F VIII (APTT) PCCs, rFVIIa
- 25. D-dimer D-dimer = crosslinked fibrin degradation product Presence indicates activation of both coagulation and fibrinolysis (plasmin)
- 26. Quantitative D-dimer Assay MoAb to D-dimers linked to microbeads Agglutination of beads occurs in the presence of D-dimers Agglutination is measured optically Cut off: <230 ng/ml Rule out thrombotic event: • NPV 100% • Specificity 49%
- 27. Elevated D-dimers Recent thrombosis DIC Cancer Inflammatory conditions
- 28. Interpretation of Prolonged PT and/or APTT Results Factor Deficiency Single vs multiple deficiencies Specific Anticoagulants Specific factor inhibitor F VIII, F V Global Anticoagulant Lupus anticoagulant Paraproteins Therapeutic Anticoagulants: UFH, LMWH, Direct Oral
- 29. Prolonged PT/APTT Work-Up: Mixing Studies Compare Clotting Time Results Patient Plasma Pooled Normal Plasma (PNP) Patient : PNP mix (1:1 mix) Interpretation: Correction • Factor deficiency Lack of Correction • Circulating anticoagulant
- 30. Mixing Studies Immediate Mix Incubated Mix (60 min, 37o C) To detect time dependent inhibitors • F VIII Inhibitors – Less correction of Incubated Mix than Immediate Mix
- 31. Mixing Study at MSKCC APTT Actin FS Lupus anticoagulant insensitive reagent Normal result rules out a significant factor deficiency
- 32. Clot Based Specific Factor Assays PT or APTT based Constituents Patient plasma Factor deficient plasma Reference Plasma Assay Principle Patient plasma reconstitutes factor deficient plasma Assayed reference plasma is used for quantitation
- 33. Factor Assays Determines factor level as % activity relative to reference plasma Procedure Patient plasma or reference plasma (PNP) Dilute plasma 1/10, 1/20/ 1/40 with deficient plasma (deficient in a single factor) Perform PT or aPTT and compare clotting time (seconds) of patient plasma to reference plasma (standard curve) Patient plasma is run in multiple dilutions (usually 3) to check for the presence of an inhibitory substance Reference Plasma Patient 1 Patient 2 Patient 1: Factor Deficiency Patient 2: Inhibitor Effect
- 34. Lupus Anticoagulant Heterogeneous antibodies against phospholipids and phospholipid binding proteins Not usually associated with bleeding • Arterial/venous thrombosis • Rarely patients may also have antibodies against F II – Check PT for prolongation Prolongs screening APTT • Reagent dependent – APTT Actin FS » LA insensitive • Most clinical APTT reagents are moderately sensitive to LA – Normal APTT does not rule out a LA
- 35. ISTH Guidelines for Lupus Anticoagulant Testing • Two tests based on different principles • dRVVT • sensitive aPTT (low phospholipids and silica as activator) • LA should be considered positive if one of the two tests gives a positive result (Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, de Groot PG. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost 2009; 7: 1737–40) • False negative rate • ~20% for low and intermediate titer antibodies • False positive rate ~ 10% • Repeat testing in 12 weeks for confirmation (Dembitzer et al, Am J Clin Pathol 2010; 134:764-773)
- 36. Lupus Anticoagulant Testing: dRVVT Screen Xa X Va Xa Prothrombin dRVVT Thrombin Ca2+ Fibrinogen Fibrin Low Phospholipid Content Prolonged Clotting Time
- 37. dRVVT Confirm (LA) Xa X Va Xa Prothrombin dRVVT Thrombin Ca2+ Fibrinogen Fibrin High Phospholipid Content DRVVT TEST RESULT: Ratio SCREEN/CONFIRM Positive: ratio >1.2 Shortened Clotting Time as Compared to Screen
- 38. Questions?
Notas del editor
- What types of assays are there for identifying patients at risk and how good are they? Functional assays for type I deficiencies Antigenic assays for type II deficiencies DNA-based assays for confirmation The general principle of a chromogenic assay, in its simplest form: A protein is converted to its active formThe cleaved peptide releases the chromophore, in this example pNA, which gives off a color. In this example, PNA is used. PNA = para-nitroaniline, which appears as a yellow color.
- What types of assays are there for identifying patients at risk and how good are they? Functional assays for type I deficiencies Antigenic assays for type II deficiencies DNA-based assays for confirmation The general principle of a chromogenic assay, in its simplest form: A protein is converted to its active form. the. The cleaved peptide releases the chromophore, in this example pNA, which gives off a color. In this example, PNA is used. PNA = para-nitroaniline, which appears as a yellow color.