Laboratory tests of hemostasis and coagulation system (dr ellinor peerschke 9 5 13)

4,789 views

Published on

Published in: Business, Technology
0 Comments
17 Likes
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
4,789
On SlideShare
0
From Embeds
0
Number of Embeds
152
Actions
Shares
0
Downloads
0
Comments
0
Likes
17
Embeds 0
No embeds

No notes for slide

Laboratory tests of hemostasis and coagulation system (dr ellinor peerschke 9 5 13)

  1. 1. Introduction to Coagulation Testing Ellinor I. Peerschke, Ph.D., F.A.H.A. Vice Chair, Laboratory Medicine Head, Hematology & Coagulation Laboratories MSKCC 1
  2. 2. Preanalytical Variables Specimen Collection 3.2% sodium citrate 9:1 volume of blood to anticoagulant Hct <25% or >50% may affect results 2
  3. 3. 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/ µl • Centrifugation (10 – 20 min, 1000g) • Assays performed on Plasma 3
  4. 4. Plasma vs Serum Plasma Anticoagulated • Only citrate is acceptable Common Abbreviations: Serum Not anticoagulated Consumption of coagulation factors, particularly fibrinogen, F V, F VIII, F II PPP – platelet poor plasma NPP – normal pool plasma PNP – pooled normal plasma 4
  5. 5. Coagulation Cascade 5
  6. 6. Analytical Variables Types of Assays clot based chromogenic antigen assays 6
  7. 7. Coagulation Screening Tests PT APTT Fibrinogen Thrombin Time 7
  8. 8. 8
  9. 9. INR correlation between analytical systems 9
  10. 10. 10
  11. 11. Monitoring Heparin Clot based assays: aPTT Ratio (1.5 – 2.5 x) • use median value of normal range • use patient’s baseline aPTT Chromogenic assays Anti Xa assay= Heparin Assay Role of AT III 11
  12. 12. 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 12
  13. 13. Monitoring HeparinTherapy 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” 13
  14. 14.  Responsiveness varies with reagent, instrument, drug  Standardization attempts (INR like) have failed UFH LMWH 150 aPTT [sec] Monitoring Heparin Therapy APTT 200 100 50 0 0 0,2 0,4 0,6 0,8 UFH: Correlation of APTT and Anti-FXa poor; LMWH: very low sensitivity Fondaparinux: insensitive 14 1
  15. 15. New Anticoagulants aXa aIIa 15
  16. 16. New direct Xa inhibitors Rivaroxaban Mw 436 g/mole Rapid onset Apixaban MW 459.5 g/mole Rapid onset • ~ 3h • 3-4 h Half-life Half-life • ~ 12h • 10-14h Excretion Excretion • Renal • Hepatic metabolism – Substrate for CYP 3A4 • Renal • Hepatic metabolism – Substrate for CYP 3A4 16
  17. 17. Oral Direct Xa inhibitors: Effect on clot based tests Variable effects depending on reagents Prolonged PT • 2x control at ~138 -764nM Prolonged APTT • 2x control at ~897-2050nM Thrombin time • Insensitive ~2500nM All clot based factor assays show a concentration dependent effect 17
  18. 18. Fibrinogen Assay and Thrombin Time Neither assay measures crosslinked fibrin To measure F XIII activity- F XIII assay or quantitative F XIII antigen 18
  19. 19. Thrombin Time Heparin Contamination Dysfibrinogenemia (follow with Reptilase Time) Thrombin: FPA & FPB Reptilase: FPA (Hypofibrinogenemia/DIC) better alternatives: fibrinogen, D-dimers Need fibrinogen result for interpretation! 19
  20. 20. Sensitivity of Screening Tests PT/APTT : prolonged by single factor deficiency <30% (variable) PT: highly sensitive to multiple Vit K dependent factor deficiencies APTT: more sensitive to heparin – sensitive to LMWH 20
  21. 21. Short PT/APTT In vitro sample activation problematic venopuncture under anticoagulation/low Hct High F VIII (APTT) FEIBA, r F VIIa 21
  22. 22. D-dimers vs FDPs Fibrinolysis tPA uPA SK D-dimer = crosslinked degradation product FPD = fibrinogen or fibrin degradation product primary fibrinolysis 22
  23. 23. 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% 23
  24. 24. Elevated D-dimers Recent thrombosis DIC Inflammatory conditions Cancer 24
  25. 25. Interpretation of Prolonged PT and/or APTT Results Factor Deficiency Single vs multiple deficiencies Circulating Anticoagulant Lupus-like anticoagulant Specific factor inhibitor Paraproteins Anticoagulants: UFH, LMWH, Direct Xa Inhibitors, DTI 25
  26. 26. Mixing Studies Patient Results PNP Patient : PNP Interpretation: What is correction? Factor deficiency vs. circulating anticoagulants APTT Actin FS Lupus anticoagulant insensitive reagent 26
  27. 27. 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: 19.1 sec (9.4 – 12.8 sec) APTT: 79.6 sec (23.8-36.3 sec) 27
  28. 28. Mixing Studies PT MIX Immediate PT patient: 19.1 sec NPP: 10.7 sec MIX: 12.2 sec Incubated PT patient: 19.5 sec NPP: 10.8 sec Mix 12.4 sec APTT MIX Immediate PT patient: 79.6 sec NPP: 33.4 sec MIX 58.2 sec Incubated PT patient: 82.9 sec NPP: 33.4 sec Mix: 57.9 sec 28
  29. 29. Additional Studies APTT Actin FS: 39.7 sec (<29.1 sec) Factor sensitive Lupus anticoagulant insensitive Thrombin Time: 23.9 sec (17-24 sec) Conclusion Suspect common pathway factor deficiency Suspect lupus anticoagulant 29
  30. 30. Confirmatory Assays Factor Assays Factor VII: 31% Factor X: 59% Factor V: 97% Factor VIII: 166% Factor IX: 109% Factor XI: 67% Lupus Anticoagulant Studies DRVVT Ratio: 2.0 (<1.2) 30
  31. 31. Clot Based Specific Factor Assays PT or APTT based Patient plasma Deficient plasma Pooled normal plasma/assayed reference plasma Patient plasma assayed at 3 dilutions Anticoagulant effect: rising factor level with increasing dilution 31
  32. 32. Effect of anticoagulants on factor assays Heparin Direct thrombin or Xa inhibitors – all clot based assays are invalid falsely DECREASED Factors I - XII falsely INCREASED anticoagulation factors 32
  33. 33. Detection of Lupus Anticoagulant Dilute Russel Viper Venom Time Silica Clotting Time • (APTT based test) Tests based on neutralization of lupus anticoagulant with excess phospholipid 33
  34. 34. QUESTION DOES A NORMAL APTT RULE OUT A LUPUS ANTICOAGULANT? 34
  35. 35. Sensitivity of Screening APTT to Lupus Anticoagulant A normal APTT does not rule out the presence of a lupus anticoagulant 20% False Negative on samples with low titer LAC depending on APTT reagent 35
  36. 36. Evaluating a Lupus Anticoagulant (ISTH Recommendations) Prolonged Coagulation Screening Test (PT, APTT-LA, dRVVT) Uncorrected Screening Test by Mixing Study (R/O factor deficiency) Shortening of Abnormal Clotting Time with Addition of Excess Phospholipid Exclusion of specific factor inhibitor 36
  37. 37. 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 37
  38. 38. 4T Scoring System for Pretest Probability (T.E. Warkentin, 2006) 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 unclear or plt fall after 10 days Plt fall <5 days and without recent heparin <1 day if previous heparin within 100 days Score <3: < 5% chance of HIT Score 4-5: Intermediate risk Score > 6: Very high risk of HIT Thrombosis New thrombosis, skin necrosis Progressive or recurrent thrombosis, some skin lesions e.g. erythema None Other causes of Thrombocytopenia None Possible Other causes clearly identified 38
  39. 39. ConfirmatoryHIT/T Testing Heparin Induced Platelet Aggregation ~ 50% sensitive Serotonin Release Assay Gold standard • not available locally • 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 39
  40. 40. Questions? 40

×