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Lab Screening for Hemorragic Congenital Coagulopathies Strategy and approach

Lab Screening for Hemorragic Congenital Coagulopathies Strategy and approach. VWD Panel: VWF Antigen & Activity Assays FII, FV, F VII, FX Clotting Assays FVIII, FIX, FXI, FXII Clotting Assays ACL TOP and ACL Elite Factor Parallelism FXIII Antigen. VWD Panel: VWF Antigen & Activity Assays.

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Lab Screening for Hemorragic Congenital Coagulopathies Strategy and approach

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  1. Lab Screening for Hemorragic Congenital Coagulopathies Strategy and approach

  2. VWD Panel: VWF Antigen & Activity Assays • FII, FV, F VII, FX Clotting Assays • FVIII, FIX, FXI, FXII Clotting Assays • ACL TOP and ACL Elite Factor Parallelism • FXIII Antigen

  3. VWD Panel: VWF Antigen & Activity Assays

  4. VWD types

  5. VWD Diagnosis VWD typing is a complex and multi-technology process.

  6. HemosIL VWD Panel • HemosIL VWF Antigen • HemosIL VWF Activity • Automated Latex immunoturbidrimetic • Applications on ACL Elite/Elite Pro, ACL Advance, ACL TOP • VWF Antigen and Activity values assigned on the HemosIL Plasma Calibrator and Controls

  7. HemosIL VWF Antigen Latex particles coated with rabbit polyclonal antibody specific for VWF antigen HemosIL VWF Activity Latex particles coated with anti-VWF mouse monoclonal antibody directed against the platelet binding site of VWF (Glycoprotein Ib receptor) HemosIL VWD Panel Plasma VWF triggers the agglutination of the latex particles measured at 405 nm.

  8. HemosIL VWD panel

  9. VWF Activity: definition • The activity of VWF is: • Binding of circulating FVIII with the function of stabilizing it and protecting from degradation • Adhesion of platelets to injured vessels • By interacting with the collagen present in the sub-endothelium • This bound induces VWF to interact with platelet receptors: Glycoprotein Ib of inactivated platelets and glycoprotein IIb-IIIa of activated platelets Multimer size is important for normal biological function: the larger is the most active

  10. VWF: Rco Platelet aggregation VWF causes agglutination of stabilized platelets in the presence of ristocetin Ristocetin, in vitro, mimics the action of collagen in modifying the VWF conformation, thus facilitating the binding with the platelet receptor glycoprotein Ib. The agglutination degree can be measured in Aggregometers (Biodata, Helena, Dade) or Coagulation analyzers (Dade) HemosIL VWF: Ac Latex immunoturbidimetric Anti-VWF antibody coated on Latex particles and directed against the platelet binding site of VWF (glycoprotein Ib receptor) Concept usually applied to ELISA (by Axis-Shield; Corgenix). HemosIL VWF Activity

  11. Correlation with RCo

  12. VWD Diagnosis

  13. VWF Activity: References • New Automated von Willebrand Factor Activity Assay to Distinguish Type 1 and Type 2 von Willebrand Disease.M. Piñol1, M. Costa, M. Sales, M.T. Canciani, A.B. FedericiXIX ISTH, Birmingham, UK July 12 – 18, 2003 • Comparison of a new rapid automated VWF:Activity assay with a visual agglutination VWF:RCo assay in screening for VWD.JM Smith, A Bowyer, J Storr, M Makris, S KitchenHaemophilia 2004 : 10; 19 • Comparison of a von Willebrand Factor Collagen Binding Assay with a Latex-Enhanced Turbidimetric Immunoassay for the Determination of von Willebrand Factor activity in Plasma.Telting d. et al.XVIIth International Symposium on Technologycal Innovation in Laboratory Hemtalogy, May 13-15, 2004 Spain.

  14. VWF Activity: References • Determination of von Willebrand Factor Activity: Evaluation of the HemosIL Assay in Comparison with established Procedures .Sucker C. et al.Clinical and Applied Thrombosis/Hemostasis, 12:3, 305 – 310, 2006 • Comparison of a new Automated von willebrand Factor Activity Assay With an Aggregation von Willebrand Ristocetin Cofactor Activity Assay for the Diagnosis of von Willebrand Disease.Vleeschawer etal.Blood Coagulation & Fibrinolysis 17(5): 353-358, 2006 • Evaluation of a new turbidimetric assay for von Willebrand factor activity useful in the general screening of von Willebrand diseaseM. PIÑOL, M. SALES, M. COSTA, J. SERRA, A. TOSETTO, M. T. CANCIANI, A.B. FEDERICIHaematologica (submitted 2006)

  15. Intrinsic and Extrinsic clotting Assays

  16. HemosIL Factor Def PlasmaPanel

  17. HemosIL Factor Def PlasmaPanel • Main Features • Immuno-depleted Factor Deficient Plasma • Factor level < 1% (all remaining at optimal level) • Format: 5 x 1 mL (lyo) • Stability at 2-8 °C : 24h after reconstitution • Stability on-board : 24h after reconstitution

  18. ACL TOPFactor Parallelism

  19. Factor Parallelism • What is Factor Parallelism? • Measurement of Factor Activity Levels at different sample dilutions • Comparison of Plasma sample results at different dilutions with Calibration results at different dilutions • Determination of Factor Activity levels • Uncover presence of inhibitors

  20. Factor Parallelism • Why is Factor Parallelism used? • Increase the quality of the results for factor assays • Uncover possible interferences that might affect the test results of the “single dilution test” • Lupus Anticoagulants • Heparin • Factor Inhibitors

  21. Factor Parallelism • How to use Factor Parallelism: • 2-3 dilutions of the test plasma • The result of a sample dilution should fall within the working range of the assay • Results of the various test plasma dilutions should demonstrate parallelism with respect to those of the calibration plasma • Ideally test plasma and calibration plasma dilutions should be the same

  22. Factor Parallelism • Criteria to define factor parallelism: • No official guidelines • Based on mathematical or statistical analysis • Examples: • Comparison of slopes (test plasma dilution curve vs calibration plasma dilution curve) • Check of R2 of test plasma dilution curve • Check variance of recalculated results between first dilution (100%) and subsequent dilutions

  23. Factor Parallelism • Effects of Heparin & Lupus Anticoagulant interference: • Prolongation of aPTT clotting times may result on falsely detected low factor concentration results • With test plasma at increasing dilutions may decrease final heparin/Lupus concentration, thus recalculated results may increase with the sample dilution

  24. Factor Parallelism • Factor inhibitors interference: • Factor inhibitors may be weak or strong inhibitors, therefore the effect of sample dilution may or may not uncover their presence

  25. Factor Parallelism: ACL TOP User definable report: may define or select up to 4 Reporting Units

  26. Factor Parallelism: ACL TOP • Factor Parallelism Criteria:

  27. ACL TOP: Normal sample

  28. ACL TOP: Sample with inhibitors

  29. ACL Elite/Elite ProFactor Parallelism

  30. ACL Elite/Elite Pro:Factor Parallelism • 3 Sample Dilutions (100%; 50%; 25%) • Dilutions Automatically done by the instruments • Calculations and Results Evaluation automatically performed by the system and reported • 1 Replicate/Level • Available for FVIII and FIX • APTT -SP Library 1.01 – 1.09 • APTT Synthasil Library in development • Representation of results numerical

  31. Parallelism - Calculation Setup Elite Series performs 3 Fixed Dilutions on Samples(1 replicate/Level) Dilution 1 = 1:5 Dilution 2 = 1:10 Dilution 3 = 1:20

  32. Parallelism - Calculation Setup User can select to display and print 4 of the following: • CR% = Corrected Result % • AveCR% = Average of the Corrected Results in % • CV-CR% = CV of the Corrected Results in % • Slope = Slope of the linear regression(seconds vs. uncorrected % result) • Intercept = Intercept - linear regression line • R2 = Correlation coefficient - linear regression line

  33. Parallelism Screen Report • Results for 3 dilutions are reported in • Seconds • % • Recalculated % • Mean Recalculated % • CV Recalculated % • Slope • Intercept • R2

  34. Parallelism Printed Detail Report This report displays the individual dilution results plus the corrected % activity results.

  35. Parallelism Printed Sample Report This report displays recalculated results

  36. FXIII Antigen Assay

  37. FXIII Biochemistry

  38. FXIII Biochemistry

  39. FXIII in the Coag Cascade

  40. FXIII Deficiency • Inherited • Frequency 1: 5,000,000 • Types: Subunit A or Subunit B • In type A usually undetectable levels of FXIII • No cases of normal Factor XIII antigenic levels with impaired FXIII activity levels have been reported so far • The bleeding tendency typically becomes obvious when FXIII levels are <1-2%. • However severe bleeding episodes may occur with FXIII levels in the range of 30% - 50%.

  41. FXIII Deficiency • Acquired • malignant hematological diseases • severe liver disease • DIC • acute stages of inflammatory gastrointestinal disease • Henoch Schoenlein purpura

  42. Measurement methods COMPETITIVE ASSAYS • Katona (Thromb Haemost 2000: 83: 268-73) • Lim W et al ( J Thromb Haemost 2004; 2: 1017-1018) • the clot solubility tests only detect severe FXIII deficiencies with FXIII activity levels of 0 – 3%, thus making these tests unsuitable for detecting FXIII activity levels >5%. • Concerns regarding the Berichrom FXIII assay analytical sensitivity. Katona reports that the Berichrom assay is relatively insensitive and below FXIII levels of 5% the accuracy and the imprecision are not reliable. • As presented by the survey conducted by the FXIII working party of European Thrombosis Research Organization (ETRO), following a study on 72 patients a FXIII activity >5% can also be severe (Seitz R et al. Sem thromb Hemost 1996; 22: 415-8).

  43. HemosIL FXIII Antigen CONCEPT: Immunoturbidimetric assay which measures specifically the subunit A of the FXIII tetramer • The inherited FXIII deficiencies reported so far have decreased antigenic and activity levels. There have been no known cases in which the deficiency was caused by decreased activity, with normal antigenic levels. • In addition, the very rare deficiencies affecting the subunit B also influence the plasma levels of the subunit A (Katona E. et al. Thromb Haemost 2000: 83: 268-73). In these cases, the tetramer is not detectable, while the subunit A is detectable but with low concentrations.

  44. HemosIL FXIII Antigen CONCEPT: Immunoturbidimetric assay which measures specifically the subunit A of the FXIII tetramer • In acquired deficiencies, the measured antigenic levels of FXIII-A would be proportional to the activity levels even though they may not be identical (Lim W et al J Thromb Haemost 2004; 2: 1017-1018) particularly after replacement therapy.

  45. HemosIL FXIII Antigen

  46. HemosIL FXIII Antigen

  47. HemosIL FXIII Antigen • CONCLUSIONS • HemosIL FXIII Antigen is a liquid ready to use immunoturbidimetric assay • Automated on ACL Futura/Advance and ACL TOP • Detects the Active subunit of FXIII • Simplify and facilitates the laboratory investigation of FXIII deficiency

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