1. Microparticle analysis by flow cytometry: principles and pitfalls
Nicolas Bailly, Pr François Mullier, Pr Bernard Chatelain CHU Dinant-Godinne UCL Namur, Belgium
Insérer lieu et endroit

2. The size range of microvesicles is from 30nm to 1μm
Figure 2 Size ranges of membrane vesicles (Adapted from B.Gyorgy and colleagues (11)). Exosomes share size distribution with viruses. Microparticles overlap in size with bacteria and protein aggregates (i.e immune complexes).
The MV nomenclature is still a mattter of debate since there is no consensus on size distribution.
That’s why I will use the term microvesicles throughout this presentation if not otherwise specified
Adapted from B.Gyorgy and coll. Cell. Mol. Life Sci. 2011 2

3. Some characteristics
They include a variable amount of phosphatidylserine on their surface
They have multiple origin and often express the same clusters of differenciation that the original cell
Platelets MicroParticles (PMP) are CD41+, CD9+,…
Erythrocyte derived MicroParticles are CD235a+
………………………
Microparticles may be derived from activation or apoptosis
JM. Freyssinet, F.Toti. Thrombosis Research 2010

4. Functions Procoagulant activity Pro Inflammatory activity
Substrate for the binding with the coagulation’s factors.
By expressing tissue factor
Pro Inflammatory activity
Involved in cell’s survival
Interaction and communication with other cells
Transmembrane Receptor
RNAmessenger
MicroRNA
PL.Gross. Blood 2012

5. Applications Cancer: Sepsis Hemostasis: Hematology:
Thrombotic risk, cell survival, invasiveness,..
Sepsis
Hemostasis:
Heparin Induced Thrombocytopenia
Hematology:
Hereditary Spherocytosis, PNH,...

6. What are the available techniques?

7. Techniques available Flow Cytometry Nanoparticles Tracking Analysis
Impedance based FCM
Dynamic Light Scattering
Electronic Microscopy
Atomic Force Microscopy
Functionnal Tests

8. Flow cytometry Requires cell suspension Hydrofocusing of cells
Cells are hit by a laser
Measurement of scatter
Measurement of fluorescence
Light is transformed in electrons
PMT/PD
We measure a « pulse » in volt by time

9. We use submicron beads (i. e
We use submicron beads (i.e. Megamix, Megamix+) to define the area of interest
Once calibrated, we could run the samples

10. Actual sensitivity of FCM
Reminder:
100nm < MP < 1000nm
Current FCM’s limits of detection:
> 500nm
> 300nm
>100nm
1000nm
500nm
100nm

11. Advantages & disadvantages of flow Cytometry
Absolute Counting
Beads, flow rate,…
Cellular origin
Antibodies
Low cost and availability
User friendly
Possible standardization
Limited Sensitivity (not on new generation of instrument)
Detectable differences
Between biological material and beads, calibrators,…
Poor informations regarding activity

12. Comparison of techniques
Flow Cytometry
Impedance FCM
AFM
Dynamic Light Scattering
Nanotrack Analyser
Functionnal tests
Electronic Microscopy
Accuracy and sensitivity
(+)
++(+)
+++ ++
Specificity
(cell. Orig.)
AcMo
+(+) + -
(functions)
Practicability and costs -+
A combination of methods is required for a full study of MVs

13. What are the pitfalls…
…and how to avoid them?

14. Pitfall 1 : Size
In Flow Cytometry, the size is mainly associated to the Forward Scatter
What’s the Forward Scatter?
FL1
FL4
SSC
FL2
FL3
FSC

15. Pitfall 1 : Size Forward Scatter is the RELATIVE size
The perception of the FSC depends on many parameters:
Refractive Index
Kind of beads/standards
Collection angle

16. Pitfall 1 : Size ≠ Polarized light Non Polarized Light MPs
MPs closer to 0.5µm than 0.9
MPs closer to 0.9µm
BD Influx
BD AccuriC6

17. Pitfall 1 : Size Mainly depends on refractive index
How to solve the problem
Use calibration material with the same refractive index as microparticles
Use mathematical/physical tool to extrapolate the relative size
This solution is currently under study by an International team (see ISTH Workshop called exometry)
Standardization efforts allows you to mesure the same population on different cytometers

18. Pitfall 2 : Pre-analytical steps
As with many laboratory tests: importance of pre-analytical steps!
The steps between sample collection and FCM analysis can change everything
Many different points of the preanalytical steps could affect the MP analysis:
Needle & tourniquet
Anticoagulant(EDTA, Citrate, CTAD, Heparine,…)
Tube haulage (vertical, horizontal, pneumatic system,..)
Sample type (PFP, whole blood,…)
Time and delay
Between sampling and centrifugation step(s)
Between centrifugation step and analysis
Centrifugation (speed, time, temperature, brake,…)

19. Pitfall 2 : Pre-analytical steps
Solution: Standardization!!!!

20. Pitfall 2 : Pre-analytical steps
Not only specific for Flow Cytometry
These piftalls are encountered also for several methods
AFM
TEM
… …

21. Pitfall 3 : signal detectors
Photodiode
Most of the instrument
FSC = Photodiode
SSC = PMT
Some instruments allows a double measurement of the FSC by using
Photodiode
PMT
When available, use PMT as detector for FSC
Could be compensate by High Dynamic Range
PMT
BD Fortessa

22. Pitfall 4: signal’s measuring parameter
A signal could be measured on
Height of the peak
Area of the peak
Width of the peak
For small events
Work on primary signal = Height of the peak
Height = Maximum Digitized Value of the Pulse
Area: Sum of all pulse heights
Width: Scaled Area Divided by height

23. Pitfall 4: signal’s measuring parameter
Area
Height
BD FACSCanto
BD FACSAria

24. Pitfall 5: Fluidic Noise
Sheath must be filtered
Use of a 0.22 vs 0.10μ filter?
Some filters generate more noise.
Impact of the noise?
Electronic Abort
Lack of resolution
Possible free fluorescence
How to decrease the noise?
Sedimentation!

25. Pitfall 5: Fluidic Noise
Few instruments are able to perform the acquisition of the sheath
Sheath filtered at 0.22µm
Six hours of sedimentation
Decreasing of general noise: 75%
BD Influx

26. Pitfall 6: Free Fluorescence
Use of procedure without washing steps
Free fluorescence is possible
PE +++
Possibility of antibodies aggregates (Immune complex)
Solution: centrifugation of the antibodies
15,0000g x 4°C (Current Protocols of Cytometry – Titering Antibodies – Unit 4.1)
13,0000g x 2’

27. Pitfall 6: Free Fluorescence
Core Stream
Core Stream
Laser

28. Pitfall 6: Free Fluorescence
Without centrifugation
With centrifugation
BD AccuriC6

29. Pitfall 7: Flow Rate
An increase of the flow rate has an impact on the resolution between two populations of small events
BD FACSAria

30. Quality Control Each lab test needs QC
How to know if my FCM is able to measure microparticles?
How to compare performance from differents instruments
What is the impact of weak performances on the counting of microparticles?

31. Separation Index Adaptation of a mathematical parameter
Use of Megamix beads (Biocytex) as reference beads
What are the interests?

32. Instrument monitoring
November 2009
February 2010
April 2010
S.I. : 14.6
S.I. : 11.2
S.I. : 6.3
Daily monitoring of the instrument
LJ graphs
Define the need of maintenance
Sensitivity required > WBC sensitivity
May 2010
July2010
S.I. : 3.4
S.I. : 1.8
S.I. : 10.9
Maintenance
Lymphocyte QC = OK

33. Day by day following of an Accuri C6
Time (weeks)

34. Perspective and future
Biological Standard:
Bacteria? Lipidic emulsion?
Generic labelling
Antibody? Protein? QC
Separation Index
Technological improvement:
Manufacturer’s duty

35. Perspective - TOF Refractive Index is our best ennemy
Is it possible to be independant of R.I.?
Yes!
How?
Remember:
On (nearly) every instrument: -width parameter is calculated
It could be measured
Height = Maximum Digitized Value of the Pulse
Area: Sum of all pulse heights
Width: Scaled Area Divided by height

36. Perspective - TOF The –Width parameter could be measured
Witdh is… Time Of Flight
Actually, only the Accuri C6 measures the width
Threshold of
detection
Laser
Δ Time
Time In
Time Out

37. Perspective - TOF Time Of Flight:
Time (in µs) it takes for a particles to cross the threshold

38. Perspective - TOF Get the real size, independent of material
Define area of interest by size
Get better separation of small particles

39. Take Home Message Know your instrument, its possibility and limits
Don’t think with a conventional FCM mind: flow cytometry for small events shake up some principles
Standardization

40. Take Home Message Flow cytometry offers: To be followed…
Reliable & low cost techniques for MP counting and characterization.
Multi(colors) characterization (up to 4-5 antibodies).
Rapidity: 2 minutes of analysis could be enough
Very affordable techniques
A lot of laboratory uses it
A lot of improvement are/will be done in the previous/next few years
A lot of scientific societies are working for an harmonization of the technique
To be followed…

41. Support from scientific societies
ISEV + Researchagte

42. Acknowledgments Thanks For Your Attention Mont-Godinne (Belgique)
Pr François Mullier
Pr Bernard Chatelain
Yvan Cornet
Marseille (France)
Pr Romaric Lacroix
Dr Philippe Poncelet
Pr Françoise Dignat-George
Students/Grants
Dr Berangère Devalet
Damien Gheldof
Adeline Wannez
Peter Engel (data on Time Of Flight), Jolita Hendriksen (BD BeNeLux) and all the Applications Engineer