1. Performances of Platelet Microparticle Generation Assay for the diagnosis of HIT F. Mullier, V. Minet, N. Bailly, C. Chatelain, I. Elalamy, J-M. Dogné and B. Chatelain SSC meeting ISTH 2014 June 25 th, 2014

2. Physiopathology of HIT Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion Physiopathology of heparin-induced thrombocytopenia FP4 : platelet factor 4 ; HS : heparan sulfate ; CS : chondroitin sulfate. Gruel et al, 2013

3. Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion Physiopathology of heparin-induced thrombocytopenia FP4 : platelet factor 4 ; HS : heparan sulfate ; CS : chondroitin sulfate. Gruel et al, 2013 Physiopathology of HIT

4. Generation of microparticles (MPs) HIT serum in the absence of heparin Platelets were incubated with HIT serum in the presence of 0.1 U/mL heparin Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion Generation of platelet-derived microparticles occurs when platelets are activated by HIT sera. Hughes et al. Blood 2001

5. Micropaticles Sub-micrometer sized vesicles released from cell membranes Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets. Sinauridze et al. 2007 Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion JM Freyssinet, F.Toti 2010 Flow cytometry

6. Objectives The ultimate goal is to provide a validated routine functional test to diagnose HIT with good performances. 1) To evaluate performances of platelet microparticles assay (PMPGA) compared to clinical outcome 2) To compare PMPGA with performances of 14 C-serotonin release assay (SRA) on the same series of patients. Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion

7. Protocol Citrated WB healthy platelet donor + patient’s serum (53 patients) + UFH at 1 IU/mL and 500 IU/mL Incubation 20’ 37°C 1200rpm on PACKS-4 Antibodies labeling : Anti-CD41-PE and Annexin-V FITC Platelet microparticles quantification (Flow rate, acquisition time) by flow cytometry (FACSAria®) Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion

8. Gating strategy Size defined using a blend of monodisperse fluorescent beads of 3 diameters (Megamix bead subsets). Robert et al., 2009; Lacroix et al., 2010 Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion Threshold set on the forward scatter according to ISTH recommendations Threshold on side scatter set at the lower limit (i.e. 200 AU)

9. Results : example Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion HIT patient Not HIT patient

10. Results : example Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion Expression of results : Rule 1 : Ratio, Q2 1 IU UH/ml/Q2 500 IU UH/ml Rule 2 : Concentration, Q2 1 IU UH/ml

11. Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion Performances Clinical outcome ROC Curves were performed to determine the optimal cut-offs of PMPGA for rule 1 (2,4) and rule 2 ( 4,835 MPs/µL) compared to clinical outcome No statistical difference between rule 1 and rule 2  larger prospective study needed PMPGA presented at least similar performances than SRA

12. Advantages and limitations Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion AdvantagesLimitations Whole blood procedure : mimics the in vivo HIT reaction Non radioactive Good performances Concern of a potential lack of diagnostic sensitivity using whole blood assays versus washed platelet assays Limited number of patients Absence of a positive control Immediate availability of a healthy compatible blood group donor

13. Conclusion and perspectives Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion PMPGA is a reliable assay mimicking in vivo HIT reaction and could be implemented in routine clinical laboratories. In our sample of patients, PMPGA showed good correlation with 14C-SRA performances and with clinical outcome. A prospective study on a large cohort of suspected HIT patients would be valuable to confirm the use of PMPGA as a new promising biological reference assay to diagnose HIT.

14. Thank you for your attention and thank you to all people involved

15. Introduction – Heparin-induced thrombocytopenia – Fondaparinux-associated HIT – Conclusion Positive type-II HIT: Rule 1: PMPs PS+ between 1IU heparin/ml and 500IU heparin/ml >2.4 Rule 2: [PMPs PS+ at 1IU heparin/ml] > 4,834/µl (data set n°2) RATIO: 7.8 Back-up slides

16. Introduction – Objective – Subjects and methods – Results and discussion – Conclusion Back-up slides

17. Introduction – Heparin-induced thrombocytopenia – Fondaparinux-associated HIT – Conclusion Reference: Clinical outcome PMPs PS pos and ratio PMPs PS pos > other MP subpopulations Back-up slides

18. Preanalytical and analytical conditions A wide variety of preanalytic and analytic variables have been reported in the literature, resulting in a wide range of PMP values.  Take care about standardization of preanalytical and analytical conditions ! S. Robert et al., 2009 R.Lacroix et al., 2010 R.Lacroix et al., 2013 F.Mullier et al., 2013 Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion

19. Gating strategy Size defined using a blend of monodisperse fluorescent beads of 3 diameters (Megamix bead subsets). Robert et al., 2009; Lacroix et al., 2010 Introduction – Objectives – Subjects and methods – Results and discussion – Conclusion Threshold set on the forward scatter according to ISTH recommendations Threshold on side scatter set at the lower limit (i.e. 200 AU)