Absolute quantification of platelets and microparticles

1. Using Accuri C6 Absolute quantification of platelets and microparticles Nicolas Bailly – Pr B. Chatelain – Pr F.Mullier Laboratoire d’hématologie CHU Dinant-Godinne UCL Namur- Belgique Accuri Users Meeting Aalst 20 may 2014

2. Introduction: Flow Cytometry and absolutecounting • What are the techniques available for absolute couting in Flow Cytometry?

4. Disadvantages: • Method has a lot of limits • Needscontrols for both instruments • Inter-laboratorystandardizationisdifficult • Results are lessaccurate: errors are multiplied • Advantages • Cheap, user friendly, fast • Couldbeused on every flow cytometer • Stilloftenused and described in papers

5. One plateform: the flow cytometer • Adaptedwith syringe, peristalticpump, … • Could determine with accuracy the volume of analyzed sample. • Is related to the number of events • Advantages: • Low CV on quantifications • Full auto • No interference on/withsample • Disadvantages: • Needs calibration and adaptation • Calibrations are performedwithbeads

6. Principle: • A knownamount of beadsisadded to a known volume of blood • What kind of beads • SureCount • FlowCount • TruCount • ………….. • All thesebeads are fluorescent

7. Advantages: • User friendly • Compatible with all instrument • Adjustable to all kind of application • Disadvantage: • Bead’ssedimentationduring acquisition • Needs fluorescence: spillover • Pipettingdependant • Depends on proteins in plasma • Leads to aggregate • Events to records ≤ #beadsx 10

8. If the flow is steady • For a same T-Time, the acquired volume of the sample will be the same. • If this volume could be determined • We could correlate it with the number of events • So we have events per volume!

9. Poseuille’s Law • Flow Rate (Q) depends on: • Radius of the flow (r) • Distance run by the flow into the flowcell (l) • Difference of pressure in the FlowCell (p) • Sampleviscosity(η) π r 4 Q = X p 8 ηl

10. The flow rate is inversely proportional to the sample’s viscosity • If • viscosity  then flow rate • viscosity  then flow rate π r 4 Q = X p 8 ηl

11. Platelet immunocounting on Accuri C6 How & why?

12. The platelet - Introduction • One of the three cellular elements of the blood. • Size: ± 3µm • Could be activated • Modification in size, shape, functionnality • Why do weneedabsolute quantification of platelet? • Definethrombocytopenia • Hereditary/acquireddisorders • Transfusion threshold • PLT concentrate

13. How to performplatelet count? • Platelet Count • Historically: BurkerChamber • Time consuming • Variability • Usually: Impedanceprinciple • Lack of sensitivity • withmacrothrombocytes • Low MCV (Red Blood Cell Volume) • Most sensitive: FCM • Immunophenotyping (CD41) • Coulddetectgiantplateletfrom RBC

14. Flow cytometryoffers a high sensitivity • The use of monoclonal antibody Backgating

15. Platelet count determination on Accuri C6 • Objectives: • Determination of relevant variables on platelet count on Accuri C6 • Definition and validation of a standardized protocol • Perform comparisons with hematocytometer

16. What are the variables? • Variables thatcould affect plateletcounting • Presence of protein in buffer/sample. • Height of the sample (volume in the tube) • Viscosity of the sample • Kind of tube used • (Non-)Expression of some markers • ………………………..

17. Presence of protein in sample • Why do we need to use BSA? • Like latex beads, platelets leads to agregate together • Inducedby lack of electric charge • The add of 1% of proteins prevents over/underestimation

18. Height of volume • Hypothesis: the height of the sample in the tube affect the absolutecounting Calibratefluidicswith the same volume as used in experiment Use a constant volume • 3mL of sample (Diluted Whole Blood) – 12 consecutive runs on n= 7– ratio Runx/Run1

19. Viscosity of the sample • PlateletRich Plasma and PlateletConcentratedon’t have sameviscosity as wholeblood. Solution : Dilute the sample (PRP or Whole Blood) in PBS.

20. We define the protocol • BSA: Avoid platelet agregates • Dilution factor: 1734  viscosity ≈ PBS • Use of polypropylene tube. • Acquisition limited to time/volume  same volume aspired. 1 minute FAST (66µl/min) Threshold: FSC-H 750µL in a tube Acquisition

21. Evaluation of the protocol • Timing between sampling and analysis • Linearity on whole blood • Check the thrombopenic blood • Precision • Specificity • Accuracy

22. Impact of time Effect of time between sampling and analysis on PLT Count in whole blood Platelets per microliter 0 min 120 min 240 min 360 min 840 min n=8 – sameresults on PRP (data not shown)

23. Linearity Linearity of Platelet Quantification on whole blood dilution

24. Linearity on thrombopenicblood Linearity of Platelet Quantification on whole blood dilution (low platelet count)

25. Conclusions - Platelet • Plateletcountingcouldbeperformedon Accuri C6 with high sensitivity and specificity. • Flow Cytometry should be used for enumeration on Platelet Concentrates • Some variables affect the absolute counting • Knowing them and taking them in consideration is necessary to perform reliable numeration • Immunocounting of plateletson AccuriC6 offer a fast and reliable alternative to plateletcountingwhenhematocytometerreachesitslimits.

26. Microparticules & cytometry

27. What is a microparticle? • Whatisit? • Biological structure derivedfromcells • Which size? • 100nm – 1000nm • If<100nm  exosome

28. Somecharacteristics: • They include a variable amount of phosphatidylserine on their surface • They have multiple origins and express the same clusters of differenciationthat the original cell. • PlateletMicrovesicles (PMP): CD41+ / CD9+ • MonocyticMicrovesicles (MPM): CD14+ • ErythrocyticMicrovesicles (Ery-MP): CD235a+ • May be derived from activation or apoptosis

29. Functions • What are the major functions of a MV • Procoagulantactivity • By linking coagulation factors • By expressing Tissue Factor • Pro-inflammatoryactivity • Involved in cell’ssurvival • Interactions withothercells • transmembranereceptor • RNAmessager • RNAmicro • ………..

30. Flow Cytometry in MP analysis Advantages Disadvantages Limited Sensitivity Detectable differences between biological material and beads, calibrators, ... • AbsoluteCounting • Beads, Constant Flow, pumps • Cellular origin • Monoclonal Antibodies • Lowcost and availability. • User friendly • Possible standardization

31. Flow Cytometry • Sensitivity of the current FCM islimited • Microvesicles: • [100 ;1000nm]. • CurrentsFCM’slimits of detection: • >500nm • >300nm 1000nm 500nm 100nm

32. What are the applications/interests?

33. Interest of MP absolutecounting by FCM • HereditarySpherocytosis • Red Blood Celldisorder • Gold Standard • Membrane Electrophoresis • Ektacytometry • Not user friendly and lowavailability

34. Interest of MP absolutecounting by FCM • HeparinInducedThrombocytopenia (HIT) • Couldinduce high risk of thrombosis • Gold-standard: Serotonin Release Assay • Only performed at select reference laboratories • Require use of radioactivity • Lack of standardization • No quality control • Objective: develop a test by FCM based on MP generation to diagnose HIT • Citrated WB healthy platelet donor + patient’s serum or PPP • Incubation 20’ 37°C 1200rpm with • - 0 IU heparin/ml • 1IU heparin/ml • 500 IU heparin/ml

35. E. De Maistre and coll. Can J Anaesth. 2006

36. PMPs PS+: 3,453/μl PMPs PS+: 1,004/μl PMPs PS+: 7,809/μl Positive Type-II HIT PMPs PS+: 635/μl PMPs PS+: 622/μl PMPs PS+: 744/μl Negative Type-II HIT

37. What are the relevant variables? • Pre-analytical steps are fundamental. • Analytical steps are also important.

38. Preanalytical variables Solution: STANDARDIZATION !!!!

39. Preanalytical variables • Impact of the kind of tube used for the counting • There is a 2:1 ratio betweeneach population of beads • What’s the impact of polypropylène and polystyrene tube on beads? Polystyrene Polypropylene Theorically 900nm 900nm X 2 X 1,43 X 1,98 500nm 500nm X 2 X 2,83 X 1,995 300nm 300nm

40. In microparticles analyses, we • Overload the sample with monoclonal antibodies • We couldn’t use washing procedure • MoAb leads to aggregate together. • Centrifugation step required

41. No centrifugation Centrifugation

42. Standardization of the FCM • Why? • Have the same results on different instrument • How • Use of Megamix Beads to define a MP Area on Side/Forward Scatter • Fix the threshold according rules • Standardization submitted and validated by ISTH workgroup.

43. How to standardize? • Use of MegamixBeads to standardize MP Area on FSC/SSC • Standardization of the position of the threshold. • Cut the peak of the smallestbeadsatitsmedian

44. Standardization • Twocytometers: • FACSAria • Accuri C6 • Twodifferentkinds of opticalbench • Twodifferentkinds of resolution • … … … PMP : 382/µL PMP : 387/µL

45. Quality Control • Laboratory tests require QC • Evaluate a FCM for MP analysis? • What’s the impact of the instrument’sdegradation on the MP count?

46. QC = Separation Index • Mathematical parameter • Based on Megamix Beads (Biocytex)

47. Quality Control • Laboratory tests require QC • Evaluate a FCM for MP analysis? • What’s the impact of the instrument’sdegradation on the MP count?

48. Following of a FACSAria without service April 2010 February 2010 November 2009 S.I. : 11.2 S.I. : 14.6 S.I. : 6.3 May 2010 July 2010 S.I. : 3.4 S.I. : 10.9 S.I. : 1.8 Maintenance

49. QC – Need of specific monitoring Weeks

50. Quality Control • Laboratory tests require QC • Evaluate a FCM for MP analysis? • What’s the impact of the instrument’sdegradation on the MP count?