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Innovations in coagulation testing

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  • 1. Innovations in Coagulation Testing: State of the Art of Coagulation Testing ELLINOR I. PEERSCHKE, PH.D., F.A.H.A. VICE CHAIR, LABORATORY MEDICINE HEAD, HEMATOLOGY & COAGULATION LABORATORY SERVICES MSKCC
  • 2. OverviewDiscuss performance characteristics of screening tests, and utilization of the APTTDiscuss the state-of-the art of current coagulation test performance  F VIII, F IX, XI  Lupus Anticoagulant Detection  Case Studies
  • 3. Hemostasis StudiesTypes of Assays  clot based  chromogenic  immunologic assays
  • 4. Screening TestsPTAPTTFibrinogenThrombin Time  Heparin Contamination  Hypo/Dysfibrinogenemia (follow with Reptilase Time)  Need fibrinogen result for interpretation!
  • 5. APTT1954 (K.M. Brinkaus et al.) developed a recalcification time assay+ Platelet substitute = Partial thromboplastin time  Rabbit brain  Newer assays use soy based synthetic phospholipids+ negatively charged activators = APTT  Better reproducibility  Kaolin, silica, ellagic acid, celite  Colloidal silica
  • 6. Selection of APTT ReagentScreen for Factor DeficiencyMonitor HeparinDetect Lupus AnticoagulantSpecialized Testing: Factor AssaysA single reagent may not be able to fulfill all requirements!
  • 7. APTT MethodsClot based method  Original  Technologist + water bath+ stopwatch  Current Automated  Optical  Mechanical
  • 8. Sensitivity of Screening TestsPT/APTT : prolonged by single factor deficiency <30% (variable) Upper limit reference range : 30 secPT: highly sensitive to multiple Vitamin K dependent factor deficiencies; monitor warfarin anticoagulation with INRAPTT: sensitive to heparin  Heparin therapeutic range (0.3 – 0.7 IU/ml)  sensitive to LMWH
  • 9. Screening APTT Reagent SelectionDo we want to detect a lupus anticoagulant during routine APTT screening?Consequences of using a screening APTT reagent with moderate sensitivity to LAC  Delay in patient care (surgery, invasive procedures) until factor deficiency is excluded  Expense  Hospital  Laboratory
  • 10. Case Study49 y.o. female with cardiomyopathy and stage D systolic heart failure – requires a biventricular pacemakerScreening APTT 132.3 sec (23.6 -35.7 sec) Stago/Roche STA PTT A reagentThrombin Time: 17.6 sec (<21 sec) no heparin contaminationNo Bleeding History
  • 11. Mixing Study1:1 Mix Patient Plasma: PNP  APTT 58 sec  APTT PNP 28 sec  Interpretation: Partial Correction  Circulating anticoagulant; Factor deficiency
  • 12. Additional StudiesReflex APTT with LAC insensitive reagent Dade Behring Actin FS: 31.1 sec (reference range 23.2 -32.0 sec)Interpretation: No significant factor deficiency High likelihood of lupus anticoagulant LAC confirmed by StaClot LA
  • 13. APTT ReagentsReagent Lupus Anticoagulant SensitivitySiemens Actin FSL SensitiveHemosil APTT-SPStago STA-PTT A ModerateHemosil SynthasisSiemens Actin FS InsensitiveStago CK Prest
  • 14. Lupus Anticoagulant Sensitivity of APTT reagents used by laboratories subscribed to CAP proficiency surveys 4500 Number of Laboratories 4000 2 3500 3000 2500 1 2000 1500 1000 3 500 0 Sensitive Moderate Insensitive Reagent Sensitivity to Lupus Anticoagulant 1= Siemens Actin FSL 2= Diag Stago STA-PTT A Hemosil APTT-SP Hemosil Synthasil 3=Siemens Actin FS
  • 15. Survey of Clinical Coagulation LaboratoriesEvaluate Coagulation Laboratory Practices  Survey design:  APTT reagents &  George Fritsma,  The Fritsma Factor utilization  Ankush Randhawa  Sponsored in part by  Precision Biologic Precision Biologic &  Dr. Marissa Marques Fritsma Factor  University of Alabama,Dialog about best Birmingham  Dr. Dorothy Adcock practices  Esoterix, Denver, CO  Meeting clinical  Dr. Elizabeth Van Cott expectations  Mass General Hospital, Boston, MA  Sponsored by Stago
  • 16. RespondentsSupervisorManagerPathologistLab Director Technologist Lead Technologist Technical Specialist N=93
  • 17. Daily APTT Volume for Responding Laboratories APTT volume Laboratories (#) > 300 32 150-300 15 10-80 45
  • 18. APTT UtilizationIndication % of local APTT volumeMonitor Unfractionated 49%heparin therapyScreen for Coagulation 41%Factor DeficiencyScreen for Lupus 14%Anticoagulant
  • 19. What is the lupus anticoagulant sensitivity level of your high- volume routine screening APTT reagent?HIGHModerateLOWDon’t know
  • 20. What is the lupus anticoagulant sensitivity level of your high-volume routine screening APTT reagent? N=93 responses
  • 21. Does a normal APTT rule out a lupus anticoagulant?YESNODon’t Know
  • 22. A normal APTT rules out a lupus anticoagulant.
  • 23. Sensitivity of Screening APTT to Lupus AnticoagulantNASCOLA 2008 surveys  20% False Negative on sample with low titer LAC (n=58)MSH – (Stago Roche PTT Automate) ~25% False Negative (normal APTT with positive LA work-up)  DRVVT  StaClot LA
  • 24. Detection of Lupus AnticoagulantISTH guidelines 2009  Perform at least 2 screening tests which demonstrate prolongation of a phospholipid-dependent clotting time using different testing principles (APTT, DRVVT)  Perform mixing studies to confirm the presence of a circulating anticoagulant and rule out factor deficiency  Perform confirmatory tests that demonstrate phospholipid dependent inhibitory activity
  • 25. Why do laboratories chose reagents with LA sensitivity?Instrument – reagent compatibility  QA programs  Troubleshooting test performance issuesNeed for larger commercial reagent portfolio
  • 26. Approach to abnormal PT or APTTFactor Deficiency Mixing StudiesCirculating anticoagulant 1:1 Immediate Mix  Lupus anticoagulant  Patient Results  acquired inhibitors  PNP  F VIII, F V ( F II)  Patient : PNP  Paraproteins Interpretation: What is  Anticoagulants correction? Factor deficiency(ies) vs. lupus anticoagulant  Repeat APTT with LAC insensitive reagent
  • 27. Abnormal APTT work-upPartial/No Correction of 1:1 Mixing StudyLA studies & Factors VIII, IX, XI  if LA neg, normal factors:  continue with F XII, PK, HMWKIf LA positive with normal Factors  check PT  if abnormal, perform F II assay
  • 28. Factor Assays: Interlab Variability (NASCOLA PT/EQA Results) 2003 Proficiency Testing ProgramSurvey ID Factor Method Mean (%) Range (%) CV (%)01-03 VIII Clot (n= 97) 23 12-36 20% Chrom 22 17-27 20% (n= 4)02-03 Clot 80 50-116 14% (n= 101) Chrom 79 68-94 14% (n= 4) IX Clot 57 38-83 14% (n= 111) 23 12-38 22%02-03 XI Clot 55 32-70 14% (n= 107) 84 58-117 14%
  • 29. Evaluation of Severe DeficienciesF VIII , 1.0 %  CV high  Recommend extended curves  Use reference plasma that is calibrated against a WHO standard
  • 30. High Factor Level: Patient PlasmaFactor VIII# Reporting 51Labs High Factor VIIIExpected 178Result (%)Mean 188% oCV highRange 134- 280% oRecommend extended curveCV 16%Classification oUse reference plasma that is calibrated to WHO standardNormal 57%Borderline 2%NormalBorderlineAbnormalAbnormal 34%
  • 31. Intrinsic Pathway Factor Assay VariablesAssay Type  clot based  One stage assay  chromogenic assaysActivatorDeficient PlasmaCalibrator PlasmaEquipment
  • 32. Intrinsic Pathway Factor Assays: Assay Conditions (NASCOLA) Activator (14- 15different activators used)  24% Stago/Roche PTT Automate  20% Siemes/Dade Behring Actin FSL  14% Siemes/Dade Behring Actin FS Deficient Plasma ( 8-10 different deficient plasmas used)  25% Precision Biologics  20% George King  16% Siemens Dade Behring  16% HRF Inc. Calibrator (9-10 different calibrators used)  27% Precision Biologic Normal Reference Plasma  27% Stago Roche Unicalibrator  20% Siemens Dade Behring Standard Human Plasma  12% I.L. Calibration Plasma Equipment (7-10 different analyzers used)  30% Stago Roche STA, STA Compact, STA-R  22% Siemens Dade Behring BCS  12% Stago/Roche STA-R Evolution  12% MDA BioMerieux  10% Abbott IMx
  • 33. Impact of Assay Variables on Factor Results Variable Data Sets % Data Sets with Findings(from D. D. Castellone, (n)*Analysis of APTT Reagent/ActivatorECAT/NASCOLA Ellagic Acid 96 32% Inter laboratory CV >20%proficiency testing data Micronized Silica 128 31% Mean factor level 20% below targetfor factors VIII, IX, XI,XII from 2003-2007) Cephalin Silica 128 16% Mean factor level 10-15% above target Clot Detection Method* Participant results Electromechanical 96 33% Inter laboratory CV >20%submitted for each factor Optical 96 40% Inter laboratory CV >20%VIII, IX, XI, and XII assaychallenge represented one Reference Plasma Standarddata set Fresh Frozen 96 29% Inter laboratory CV >20% Lyophilized 96 40% Inter laboratory CV >20% Deficient Plasma Source Congenitally Deficient 96 35% Inter laboratory CV>20% Immunoabsorbed 96 53% Inter laboratory CV >20%
  • 34. Performance of Major LAC Screening Tests: NASCOLA ASSAY 2008-1 2008-2 2008-3 2008-4 2009-2 Medium Titer LAC High Titer LAC Plasma Medium Titer LAC Low Titer LAC Plasma Plasma Normal Plasma Pool Pool Plasma Pool (Diluted) False Negative False Negative False Negative False Negative False Positive All 0% 0% 5.9% 9.6% 6.6% APTT (combined) 0% 0% 4.5% 2.4% 5.4% APTT (LAC sensitive) 0% 0% 3.0% 3.2% 7.4% APTT (LAC moderate 0% 0% 7.1% 0% 0% sensitivity)
  • 35. Performance of Confirmatory Assays: NASCOLA ASSAY 2008-1 2008-2 2008-3 2008-4 2009-2 Intermediate Titer High Titer LAC Intermediate Titer Low Titer LAC LAC Plasma Normal Plasma Pool Plasma Pool LAC Plasma Plasma Pool (Diluted) False Negative False Negative False Negative False Negative False Positive Integrated- APTT based 0% 82% 5% 89% 31% dRVVT 3% 29% 26% 62% 5%
  • 36. Overall State of the Art of Hemostasis Testing in North Americao Test peformance characteristics have not changed significantly since 2003o Imprecision of assay results remains higho Will greater STANDARDIZATION improve performance? reagents test systems testing algorithms interpretive reporting
  • 37. 75 y F – ICU - PneumoniaPT 18.5 s (12.2 – 13.5s)APTT 220s (24.8 – 35.5s)Fibrinogen 334 mg/dl (180 – 400 mg/dl)D-dimer: 5.4 µg/ml (< 1.1 µg/ml)Plt: 136K/µl (150 – 450 K/µl)
  • 38. 75y F- ICUConsider:  PT 18.5 sDIC (12.2 – 13.5s)  APTT 220slupus anticoagulant with F II deficiency, (24.8 – 35.5s)  Fibrinogen 334 mg/dl Liver dysfunction, (180 – 400 mg/dl)Vit K deficiency,  D-dimer: 5.4 µg/mlheparin contamination (< 1.1 µg/ml)  Plt: 136K/µl (150 – 450 K/µl)
  • 39. 75y F- ICUMixing Study  Patient APTT 156s  Pooled Normal Plasma 30.7 s  1:1 immediate mix 113.2  Patient PT 18.8s  Pooled Normal Plasma 11.4 s  1:1 immediate mix 14.2 s No Correction: Circulating Anticoagulant Present o APTT Actin FS: 120s (normal 23.2 -32.0 sec)
  • 40. 75y F- ICU Thrombin Time  >300 sec (normal <21 sec) Reptilase Time  22 sec (normal < 21 sec) Factor Assays  F II 83%  F V 121%  F VII 114% Heparin contamination, no evidence of Vit K deficiency, or acute liver dysfunction  ? Prolonged PT
  • 41. 81 y F -ICU - Bleeding after colonoscopyPT 12.6 s (12.2 – 13.5s)Immediate Mix: APTT 48 s (24.8 – 35.5s)  PNP 28.7 s  1:1 mix 32.0 secIncubated Mix: APTT 51 s  PNP 30.2 s  1:1 mix 50 sAPTT Actin FS 39 sec (normal 23.2 -32.0 sec)
  • 42. 81 y F - ICU - Bleeding after colonoscopy  F VIII 12%Suspect acquired F VIII inhibitor  Bethesda Titer 1.8 unitsNB: F VIII inhibitors are often not detected with immediate mixing study