![200
150
100
50
0
0 0,2 0,4 0,6 0,8 1
aPTT [sec]
UFH: Correlation of APTT and
Anti-FXa poor;
LMWH: very low sensitivity
Fondaparinux: insensitive
UFH
LMWH
Monitoring Heparin
Therapy
APTT
Responsiveness varies with
reagent, instrument, drug
Standardization attempts
(INR like) have failed](https://image.slidesharecdn.com/introdtocoagtestingforfellowsmskcc2014-140922155653-phpapp01/75/Introd-to-coag-testing-for-fellows-mskcc-2014-16-2048.jpg)
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Introd to coag testing for fellows mskcc 2014
- 1. Introduction to CoagulationTesting Ellinor I. Peerschke, Ph.D., F.A.H.A. Vice Chair, Laboratory Medicine Head, Laboratory Hematology & Coagulation Service MSKCC
- 2. Coagulation Screening Tests PT APTT Fibrinogen Thrombin Time HIT screen PFA-100 Platelet Function Von Willebrand’s Disease
- 3.
- 4. Preanalytical Variables Specimen Collection 3.2% sodium citrate 9:1 volume of blood to anticoagulant Hct 25% or 50% may affect results
- 5. Preanalytical Variables Stability PT (good up to 72 h, closed tube at RT) APTT (good 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/ ml • Centrifugation (10 – 20 min, 1000g) • Assays performed on Plasma
- 6. Plasma vs Serum Plasma Anticoagulated • Only citrate is acceptable Common Abbreviations: PPP – platelet poor plasma NPP – normal pool plasma PNP – pooled normal plasma Serum Not anticoagulated Consumption of coagulation factors, particularly fibrinogen, F V, F VIII, F II
- 7. Analytical Variables Types of Assays clot based chromogenic antigen assays
- 8. Screening Tests of2o Hemostasis PT APTT Fibrinogen Thrombin Time
- 10. INR correlation betweenanalytical systems
- 12. Monitoring Heparin Clot based assays: aPTT (sec) aPTT reagent sensitivity varies aPTT Ratio (1.5 – 2.5 x) • use median value of normal range • use patient’s baseline aPTT
- 13. Monitoring Heparin Therapy Using the APTT APTT response to heparin therapy may be exaggerated Numerous factors may elevate the APTT: • Concomitant warfarin therapy • Lupus anticoagulant • Factor deficiency • Liver disease
- 14. Monitoring Heparin Therapy Using the APTT APTT response to anticoagulants may be blunted Factor VIII and fibrinogen elevate • Can shorten the APTT in a clinically significant manner • Increase in factor VIII from 100% to 250% can shorten APTT by 10% Under-estimates level of anticoagulation Cause of in vitro drug “resistance”
- 15. Monitoring Heparin (LMWH) Chromogenic assays Anti Xa assay= Heparin Assay • Specify anticoagulant • Need separate assay for Fondaparinux Role of AT III
- 16. 200 150 100 50 0 0 0,2 0,4 0,6 0,8 1 aPTT [sec] UFH: Correlation of APTT and Anti-FXa poor; LMWH: very low sensitivity Fondaparinux: insensitive UFH LMWH Monitoring Heparin Therapy APTT Responsiveness varies with reagent, instrument, drug Standardization attempts (INR like) have failed
- 17. Direct Oral Anticoagulants aI Ia aXa
- 18. DOACs Dabigatran etexilate Rivaroxaban Apixaban Target Thrombin F Xa F Xa Prodrug Converted via esterase catalyzed hydrolysis to dabigatran No No Bioavailability 6.5% 80% ~ 66% Peak 2h 2.5 – 4 h 3 h ½ life 12-14h 7-13h 8-13h Routine monitoring No No No Dosing Fixed, BID Fixed, BID Fixed, BID Elimination 80% renal 67% renal, 33% fecal 25% renal, 75% fecal
- 19. PT (Recombiplastin): MSKCC Dabigatran Rivaroxaban 25 20 15 10 5 0 0 100 200 300 400 500 600 PT (sec) (Recombiplastin) Dabigatran (ng/ml) 45 40 35 30 25 20 15 10 5 0 0 100 200 300 400 500 PT (sec) Recombiplastin Rivaroxaban (ng/ml)
- 20. APTT (SynthasIL): MSKCC Dabigatran Rivaroxaban 100 90 80 70 60 50 40 30 20 10 0 0 100 200 300 400 500 600 APT (sec) SynthasIL Dabigatran (ng/ml) 70 60 50 40 30 20 10 0 0 100 200 300 400 500 APT (sec) SynthasIL Rivaraxaban (ng/ml)
- 21. APTT (Actin FS):MSKCC Dabigatran Rivaroxaban 80 70 60 50 40 30 20 10 0 0 100 200 300 400 500 APT Sec) (Actin FS) Rivaroxaban (ng/ml) 90 80 70 60 50 40 30 20 10 0 0 100 200 300 400 500 600 APT (sec) Actin FS Dabigatran (ng/ml)
- 22. Thrombin Time: MSKCC Dabigatran ng/ml Sec 0 24.9 55 300 182 300 268 300 303 300 355 300 480 300 550 300 Rivaroxaban ng/ml Sec 0 30.5 140 28.5 190 30.6 230 32.3 260 29.2 370 30.7 460 33.0
- 23. Thrombin Time Evaluates 3rd Stage of Coagulation Dysfibrinogenemia (follow with Reptilase Time) Thrombin: FPA FPB Reptilase: FPA Need fibrinogen result for interpretation (Hypofibrinogenemia/DIC) better alternatives: fibrinogen, D-dimers Heparin Contamination
- 24. Fibrinogen Assay and Thrombin Time 3rd Stage of Coagulation Neither assay measures crosslinked fibrin To measure F XIII activity- F XIII assay or quantitative F XIII antigen
- 25. Sensitivity of ScreeningTests PT/APTT : prolonged by single factor deficiency 30% (variable) PT: highly sensitive to multiple Vit K dependent factor deficiencies APTT: more sensitive to heparin – Variably sensitive to LMWH
- 26. Short PT/APTT In vitro sample activation problematic venopuncture under anticoagulation/low Hct High F VIII (APTT) FEIBA, r F VIIa
- 27. D-dimers vs FDPs D-dimer = crosslinked degradation product FPD = fibrinogen or fibrin degradation product primary fibrinolysis tPA uPA SK Fibrinolysis
- 28. Quantitative D-dimer Assays MoAb to D-dimers on microbeads Agglutination of beads in the presence of D-dimers Reference Range: 230 ng/ml Rule out thrombotic event: NPV 100% Specificity 49%
- 29. Elevated D-dimers Recent thrombosis DIC Inflammatory conditions Cancer
- 30. Interpretation of ProlongedPT and/or APTT Results Factor Deficiency Single vs multiple deficiencies Circulating Anticoagulant Lupus-like anticoagulant Specific factor inhibitor Paraproteins Anticoagulants: UFH, LMWH, Direct Oral Anticoagulants
- 31. Mixing Studies Patient Results PNP Patient : PNP Interpretation: What is correction? Factor deficiency vs. circulating anticoagulants APTT Actin FS Lupus anticoagulant insensitive reagent Normal result rules out a significant factor deficiency
- 32. Case Study 23 Y Female, newly diagnosed Hodgkins Lymphoma with widespread metastatic disease to bones, liver, spleen, and lymphadenopathy Scheduled for possible right pleural biopsy and mediastinal lymph node biopsy PT: 22.1 sec (9.4 – 12.8 sec) APTT: 79.6 sec (23.8-36.3 sec)
- 33. Mixing Studies PT MIX Immediate PT patient: 22.1 sec NPP: 10.7 sec MIX: 12.2 sec Incubated PT patient: 22.5 sec NPP: 10.8 sec Mix 12.4 sec APTT MIX Immediate APTT patient: 79.6 sec NPP: 33.4 sec MIX 58.2 sec Incubated APTT patient: 82.9 sec NPP: 33.4 sec Mix: 57.9 sec
- 34. Additional Studies APTT Actin FS: 39.7 sec (29.1 sec) Factor sensitive Lupus anticoagulant insensitive Thrombin Time: 22.9 sec (17-24 sec) Conclusion Suspect common pathway factor deficiency Suspect lupus anticoagulant
- 35. Confirmatory Assays Factor Assays Factor VII: 31% Factor X: 49% Factor V: 97% Factor VIII: 166% Factor IX: 39% Factor XI: 67% Lupus Anticoagulant Studies DRVVT Ratio: 2.0 (1.2) Positive
- 36. Clot Based SpecificFactor Assays PT or APTT based Constituents Patient plasma Factor deficient plasma Assay Principle Patient plasma reconstitutes factor deficient plasma Pooled normal plasma/assayed reference plasma is used for quantitation
- 37. Effect of anticoagulantson factor assays Heparin Direct thrombin or Xa inhibitors – all clot based assays are invalid falsely DECREASED Factors I - XII falsely INCREASED anticoagulation factors
- 38. Detection of Lupus Anticoagulant No single screening test Requires a panel of screening and confirmatory assays demonstrating neutralization of lupus anticoagulant with excess phospholipid Dilute Russel Viper Venom Time APTT based tests • Silica Clotting Time (IL) • StaClot LA (Stago) Results affected by anticoagulation therapy
- 39. Sensitivity of ScreeningAPTT to Lupus Anticoagulant A normal APTT does not rule out the presence of a lupus anticoagulant Most commercial APTT reagents are moderately sensitive to LA Proficiency Testing Survey data ~20% False Negative on samples with low titer LAC
- 40. HIT Testing Screening ELISA Antibodies to heparin-PF4 complexes • IgG titers – IgG titer (OD) more specific – Involved in platelet activation • Specificity: heparin spike • High Negative Predictive Value • Poor specificity (false positives) – Improves with consideration of pre-test probability
- 41. 4T Scoring Systemfor Pretest Probability 2 1 0 Thrombocytopenia 50% fall in plt or plt nadir of 20K-100K 30-50% fall in plt or plt nadir 10K-19K 30% fall in plt or plt nadir of 10K Timing 5-10 d post heparin 1 day if previous heparin within 100 days unclear or plt fall after 10 days Plt fall 5 days and without recent heparin Thrombosis New thrombosis, skin necrosis Progressive or recurrent thrombosis, some skin lesions e.g. erythema None Other causes of Thrombocytopenia Score 3: 5% chance of HIT Score 4-5: Intermediate risk Score 6: Very high risk of HIT None Possible Other causes clearly identified (Lo GK, Juhl D, Warkentin TE, Sigouin CS, Eichler P, Greinacher A. Evaluation of pretest clinical score (4 T’s) for the diagnosis of heparin-induced thrombocytopenia in two clinical settings. J Thromb Haemost 2006; 4: 759–65.)
- 42. ConfirmatoryHIT/T Testing Serotonin Release Assay Gold standard • Send-out test • Use to clarify ELISA test result – intermediate positives (O.D. 0.8 and 1.0), or – high positives (O.D. 1.0) not responsive to heparin spike
- 43.