1. Diagnostic des pathologies plaquettaires par cytométrie en flux: aspects préanlytiques et analytiques
François MULLIER, Véronique LATGER-CANNARD, Thomas LECOMPTE, Bernard CHATELAIN
November 19th 2010
The Belgian-Luxembourgish Cytometry Meeting 2010

2. Michelson AD. Nature Reviews 2010;9:154-169
Platelet
Michelson AD. Nature Reviews 2010;9:

3. Preanalytical requirements and standardized analytical procedures
Evolution of flow cytometry
Leucocytes
Platelets
Microparticles
Preanalytical requirements
and standardized analytical procedures

4. Clinical applications
P.Harrisson. Blood Reviews 2005;19:

5. LA Krueger et al. In: Current Protocols in Cytometry (2002) Unit 6.10
Blood collection and anticoagulant
Blood collection: - Careful
- Processing within 30min
- Light tourniquet or not at all
- 21-G (or larger bore) needle
- Smooth draw (good flow)
- Discard the first 2 ml of blood drawn
Anticoagulant
LA Krueger et al. In: Current Protocols in Cytometry (2002) Unit 6.10

6. Sample handling, whole blood
Sample handling: - Minimize time between drawing and preparation to reduce spontaneous platelet activation
- Properly mix antocoagulant
- Avoid unnecessary agitation prior testing
- Container: nonwettable surfaces: siliconized glass or polypropylene  to avoid initiation of clotting resulting from contact activation
- Transport: No pneumatic (platelet activaton)
No extreme temperatures
Whole blood 6

7. LA Krueger et al. In: Current Protocols in Cytometry (2002) Unit 6.10
Platelet activation
LA Krueger et al. In: Current Protocols in Cytometry (2002) Unit 6.10

8. Platelet quantification
Dual platform system
[RBC] Hematimetry (ex: XE-2100®)
FCM
RBC
Doublets
PLT
CD41 - PE
PLT eventsFCM + Doublets eventsFCM X
RBCCBC =
PLT / μL in whole blood
RBC eventsFCM + Doublets eventsFCM

9. Anti-platelet antibody
Reticulated platelets
Enhanced destruction:
ITP
Decreased formation:
CIT and aplastic anemia
TPO
Anti-platelet antibody
Spleen
Young, reticulated
platelet
Why?
- Bone marrow megakaryocyte activity and platelet kinetics
- Differentiate low platelet production from enhanced consumption
- Decreases the invasive BM aspiration need and eliminates superfluous platelet transfusions 9

10. Reticulated platelets
Flow cytometry
Lack of standardisation
Flow cytometer and a cytometrist required
Not widespread as a daily routine test
Hematimetry (ex: XE 2100)
Precise, automated, relatively inexpensive, non-ivasive
Good reproducibility and stability (48 hours)
Reference range: %
Autoimmune thrombocytopenia:
IPF=17-22%
ITP Aplastic Anemia
Healthy subject 10

11. Diagnosis of platelet disorders
I.Salles et al. Blood Reviews 2008;22(3);

12. Diagnosis of platelet disorders
PHB. Bolton-Maggs et al. British Journal of Haematology 2005;135;

13. Diagnosis of platelet disorders: Analytic
Measure the platelet surface receptor(s) from gated events
Platelets size < RBC, lymphocytes  high-voltage used RBC, leukocytes: not always appropriated for platelets
Diluted sample +
Low flow rate:
platelet
events/sec
Why?
To minimize platelet
coincidence
Threshold:
Platelet identifier
(and not the FSC!!!)
Light scatter:
Logarithmic mode
Why?
Heterogeneity in
Size and morphology
Identification:
Light scatter + positive labeling with platelet-specific identifier 13

14. Diagnosis of platelet disorders: Analytic
Antibody titration
Minimum 2 platelet markers coupled with 2 different fluorochromes
Glanzmann Thrombasthenia and Bernard-Soulier syndrome:
1 healthy subject in //

15. Diagnosis of platelet disorders: Analytic
MFI MESF or ABC or AB/C
≈ antigen density
MFI: Mean Fluorescent intensity
MESF: Mean Equivalent Soluble Fluorochrome
ABC: Antigen Binding Capacity
AB/C: Antigen Binding per cell

16. Diagnosis of platelet disorders: Analytic
MFI ABC (saturation) or AB/C (no saturation)
Ag-Ab reaction:
cross-reactions
Unspecific bindings
Affinity and avidity of the receptor
Steric obstruction
Valence
F/P
Fluorochrome size

17. Diagnosis of platelet disorders: Analytic
Platelet GpScreen or Platelet GP Receptors : QIFI
Monoclonal antibodies against glycoproteins:
GpScreen: GpIIIa (CD61), GpIb (CD42b), GpIa(CD49b)
GpReceptors: GpIIb-IIIa, GpIb, GpIIIa,GPM140 before and after TRAP activation
Fluorescence intensity  sites by platelet by a calibration curve
Calibrated beads with an increasing amount of IgG
Revelation by mouse anti-IgG-FITC

18. Mean Fluorescence Intensity Number of GpIb sites/cell
Diagnosis of platelet disorders: Analytic
Healthy
subject
Patient
Subject
Mean Fluorescence Intensity
Number of GpIb sites/cell
Normal range
Healthy subject
20825
33850
Patient
27927
43450 18 18

19. Mean Fluorescence Intensity Number of GpIa sites/cell
Diagnosis of platelet disorders: Analytic
Healthy
subject
Patient
Sujet
Mean Fluorescence Intensity
Number of GpIa sites/cell
Normal range
Ratio GpIb/GpIa
Sujet sain
2576
4805
7,0
Pauline
8873
14937
2,9 19 19

20. Mean Fluorescence Intensity Number of GpIIIa sites GpIIIa/cell
Diagnosis of platelet disorders: Analytic
Healthy subject
Patient
BSS Heterozygous
GpIbβ: Pro105Leu
Subject
Mean Fluorescence Intensity
Number of GpIIIa sites GpIIIa/cell
Normal range
Ratio GpIb/GpIIIa
Healthy subject
27020
43173
0,8
Pauline
77614
112567
0,4
Unpublished data (collaboration with KUL Kortrijk H. Deckmyn and K.Vanhoorelbeke) 20 20

21. Analytic: clone importance
Glycoprotein (CD)
Clone
Epitope
Function
GpIa (CD49b)
Gi9
GpIbα (CD42b)
SZ2
Narrow epitope of GpIbα (Tyr276-Glu282) in the anionic sulfated sequence flanking the LRR region at its C-terminal end
Conformation-sensitive for BSS Bolzano
Lj-Ib1
Epitope located between His1-Arg293 of GpIbα
Platelet binding absent in BSS Bolzano
Variant Padova
Lj-Ib10
Epitope located between residues Ala238 and Ala293 of GpIbα
AK2
His1-Thr81 fragment of the GpIbα: near first LRR where Asn41 lies
Highly sensitive to Glu40 mutation
HIP1
His1-Thr81 fragment of the GpIbα: in vicinity of the second LRR (HIP1)
MB45
BSS Bolzano
4F8:strictement IIIa:épitope aa de la GpIIIa

22. Analytic: clone importance:GpIIb
JL. Miller et al. Seminars in Thrombosis and Haemostasis 2009;35(2):

23. Variant Padova: GpIb: Asn41His (N41H)
Discrepancy between impaired binding of N-term specific anti-CD42b (FC) and increased amount of GpIb α (WB)
S.Vettore et al. Haematologica 2008;93(11):

24. GpVI
B.Dumont et al. Blood 2009;114:

25. Mepacrine testing: storage pool disease

26. Mepacrine testing: storage pool disease
Preanalytic: - anticoagulant, type of buffer, reagents filtration,whole
blood or PRP, platelets fixation,TRAP dilution
Analytic:- Ab, coincidence
- Capture: - [mepacrine], incubation, PMT voltage, stability of
mepacrine labelling, gating strategy, compensation
- Release: - platelet activator: which, []
- incubation
- before or after mepacrine labelling
Results: - biexponential
- difference or quotient
- intern control
- platelet volume

27. Autofluorescence Capture Autofluorescence Relâche
Mepacrine testing: storage pool disease
Compensation
Capture
Relâche
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
Tube 6
Tube 7
Comp 7,6µM
Autofluorescence Capture
Capture à 7,6µM
Capture à 15,2µM
Autofluorescence Relâche
Relâche à 7,6µM
Relâche à 15,2µM
Sang dilué
300µL
TRAP 1mM
---
6µL
Mélanger doucement et incuber exactement 15 minutes à température ambiante
CD41-PE
10µL
Mépacrine à 7,6µM
20µL
Mépacrine à 15,2µM

28. Mepacrine testing: storage pool disease
Release 16μM
Capture 16μM
Unpublished data (collaboration with CHU Nancy V.Latger-Cannard, T.Lecompte

29. Mepacrine testing: storage pool disease
Unpublished data (collaboration with CHU Nancy V.Latger-Cannard, T.Lecompte 29

30. Mepacrine testing: storage pool disease
Unpublished data (collaboration with CHU Nancy V.Latger-Cannard, T.Lecompte 30

31. Mepacrine testing: storage pool disease
Woman 15 years
Hemorragic score: 1-2
Absence of familial context
Thromcytopenia: 126*103/µl (MPV:9,9µm3)
Hypoagregability to ADP, collagen and
ristocetin high dose
Unpublished data (collaboration with CHU Nancy V.Latger-Cannard, T.Lecompte 31

32. Mepacrine testing: storage pool disease
Unpublished data (collaboration with CHU Nancy V.Latger-Cannard, T.Lecompte 32

33. Mepacrine testing: storage pool disease
Unpublished data (collaboration with CHU Nancy V.Latger-Cannard, T.Lecompte 33

34. Type-II Heparin-induced thrombocytopenia
Watch over [PLT] before and during treatment by heparin
True thrombocytopenia must me confirmed
No gold standard  Arguments
Clinical Biological 34
BH Chong. Thromb Haemost 2009
A Greinacher. J Thromb Haemost 2009 34

35. Type-II Heparin-induced thrombocytopenia
The pathogenesis had been
thought to involve circulating PF4/heparin immune complexes
that bind to the FccRIIA receptor on platelets and related Fc
receptorsonmonocytes, endothelial cells andother vascular cells
that promote platelet activation and generation of thrombin,
setting up an explosive feed forward, prothrombotic loop
E. De Maistre et al. Can J Anesth, 2006

36. Modified from F. Mullier et al. Thromb Haemost 2010;103(6)
Type-II HIT: Platelet Microparticle Generation Assay
healthy platelet donor citrated WB + patient’s serum or PPP
Incubation 20’ 37°C 1200rpm with:
1 IU heparin/ml
500 IU heparin/ml
Platelet Microparticle Numeration by
Flow cytometry:
GpIIb (CD41) and PS (Annexin V) positive Microparticles 
Modified from F. Mullier et al. Thromb Haemost 2010;103(6)

37. F. Mullier et al. Thromb Haemost 2010;103(6)
Platelet Microparticle Generation Assay (PMPGA): Positive
RATIO:30,3
F. Mullier et al. Thromb Haemost 2010;103(6)

38. F. Mullier et al. J.Thromb Haemost 2010; in preparation
PMPGA: clinical study (47 patients)
PMPGA Sensitivity: 70% (95% CI: 34,8-93,0%)
PMPGA Specificity: 97,9% (95% CI: 88,7-99,6%)
First patient: 4T:3, serology and LTA negative
Second patient: 4T:6 , serology negative, LTA positive
LMWH since > 3 months
Thrombocytopenia increased after switch to danaparoid and hirudine
(Voluminous hematoma  hypovolemic shock)
F. Mullier et al. J.Thromb Haemost 2010; in preparation

39. PMPGA: conclusions
- Rapid (1h) and reliable test which mimics the in vivo type II HIT reaction
- Detects clinically important HIT antibodies.
- Improves the performances of LTA , applicable in labs without LTA
- Comparable results with 14C-SRA
- Comparison of specificity of PMPGA and SRA requires larger studies.

40. JM. Freyssinet, F.Toti. J Thromb Haemost 2010:125:s46-s48
Microparticles
0.1-1µm
JM. Freyssinet, F.Toti. J Thromb Haemost 2010:125:s46-s48

41. Hemorragic syndromes:
Microparticles
Lowered synthesis
Increased synthesis
Hemorragic syndromes:
 Scott Syndrome
 Castaman Syndrome
 Wiskott-Aldrich Syndrome
 Glanzman Thrombasthenia
Positive:
Recovering of an efficient haemostasis
Auto-immune thrombocytopenia
Haemophilia …
Negative:
Procoagulant overactivity and contribution to vascular injuries :
Sickle cell disease
Sepsis
PTT, HIT, VTE
Aplastic anaemia, PNH
Diabetes
Vascular and cardiovascular diseases
Cancer
Other: platelet activation, bacterial contamination in platelet concentrates

42. Mackman et al. Journal of Clinical Oncology October 2009
Microparticles
Mackman et al. Journal of Clinical Oncology October 2009

43. B. Valeur. Lumière et luminescence. Editions Belin. December 2005
F.Mullier et al. Joint meeting Belgian Society of Thrombosis and Haemostasis and Belgian Society of Clinical Biology. June 2010 43 43

44. Advanced Flow cytometry?
Microparticles: detection of the tip of the iceberg by FCM
Flow cytometry
≤ % ≤1000PMP/µl
1µm
But variations seem to remain significant under pathological circumstances
0.5µm
AFM
DLS EM
MP/µl
100nm
Advanced Flow cytometry?
B.Chatelain et al.International Society of Thrombosis and Hemostasis, Cairo 2010 44

45. M.Bigos. Current Protocols in Cytometry (2007) 1.21.1-1.21.6
Microparticles: detection of the tip of the iceberg by FCM
Resolution or Separation index (SI): 1 2
(Median2 – Median1 )
(SD2 + SD1 )
M.Bigos. Current Protocols in Cytometry (2007)
Figure 2: Method of calculation of Separation index (SI). Median of MB 0,9µm’s peak (green peak) minus median of MB 0,5µm’s peak (red peak) is divided by the sum of standard deviation of MB 0,5µm’s peak and the standard deviation of MB 0,5µm’s peak.
F.Mullier et al. In preparation 45

46. Microparticles: detection of the tip of the iceberg by FCM
Table 1 showed comparison of separation index (SI) on different flow cytometers for FSC-H, FSC-A,SSC-H, SSC-A, FL-H and FL-A. Parameters indicated in red are those providing the best resolution between MB 0,5µm and MB 0,9µm
Instrument
FSC-H
FSC-A
SSC-H
SSC-A
FL-H
FL-A
BD LSRII Fortessa PD ND
21,2
17,1
BC Navios
32,0
6,6 13
BC Gallios W²
28,3
8,8 12
BD Influx FS Perp
20,7
5,8
7,5
Apogee A50
18,2
20,6
9,8
9,2
6,7
BD FACS Aria
17,2
13,5
18,5
20,2
8,5
10,3
BC Gallios W
15,1 8
BD LSRII Fortessa PMT
13,8
11,7
12,8
Accuri C6
9,5
9,4
4,4
5,3
3,6
BC Gallios N
6,9 9
BD Influx FS Par
6,1
Partec Cyflow ML
2,3 6
BD FACS CantoII
2,2
0,9
10,2
5,2
Millipore Easycyte 8HT (prototype)
1,1
1,3
2,8
Miltenyi MACSQuant
0,8 5
3,2
Choice between FSC-H, FSC-A,SSC-H and SSC-A depends on the instrument
However, sensitivity is required to have the whole information
F.Mullier et al. In preparation 46

47. Flow cytometry is a powerfull tool in the field of hemostasis
diagnosis and monitoring of hemorragic and thrombotic disorders
Need of well designed and standardised methods
Based on the expertise in Hemostasis and Flow cytometry
Clinically validated

48. Thank you!! Bernard Chatelain Jean-Michel Dogné
Christian Chatelain Lutfiye Alpan
Jean-Claude Osselaer Philippe Devel
Jacques Jamart Damien Gheldof
Nicolas Lufin
Séverine Robert
Nicolas Bailly
Justine Baudar
Yvan Cornet Claire Pouplard
Françoise Dignat-George
Ismail Elalamy
Yves Gruel
Romaric Lacroix
Véronique Latger-Cannard
Thomas Lecompte
Philippe Poncelet
Thank you!!