Immunophenotyping by flow cytometry...Immunophenotyping by flow cytometry Pr François Mullier Pr...
Transcript of Immunophenotyping by flow cytometry...Immunophenotyping by flow cytometry Pr François Mullier Pr...
Immunophenotyping by flow cytometry
Pr François Mullier
Pr Bernard Chatelain
BHS course, October 12th, 2013
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
INTRODUCTION: Principles
Tutorial: http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html
Multiple parameters of
individual cells within a
heterogenous
preparation
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
INTRODUCTION: Principles
More and more colors….so more
and more complex
SAMPLE PREPARATION:
QUALITY CONTROL CONCLUSION APPLICATIONS INTRODUCTION SAMPLE PREPARATION
• Individualized cells cell suspension
• Whole blood: Little preparation (already in suspension)
RBC lysis (NH4Cl, [RBC])
Enrichment (Ficoll, Percoll)
Anticoagulant: EDTA or heparin
• Washing sometimes required:
- cell concentration
- eliminate cell fragments
- mandatory if presence of molecules able to fix the detection
antibodies (ex:λκ)
:
Hematology
SAMPLE PREPARATION:
QUALITY CONTROL CONCLUSION APPLICATIONS INTRODUCTION SAMPLE PREPARATION
Bone marrow
27g needle
Ganglion
fragment
'
Saline
Glass rod
Ganglion fragment
:
Hematology
Bone marrow Ganglion
SAMPLE PREPARATION:
QUALITY CONTROL CONCLUSION APPLICATIONS INTRODUCTION SAMPLE PREPARATION
• Blood collection: - Careful
- Light tourniquet or not at all
- 21-G (or larger bore) needle
- Smooth draw (good flow)
- Discard the first 2 ml of blood drawn
- Processing within 30min
- Container: nonwettable surfaces: siliconized glass or
polypropylene to avoid initiation of clotting resulting from contact activation
• Anticoagulant
:
Hemostasis, preanalytic
LA Krueger et al. In: Current Protocols in Cytometry (2002) Unit 6.10
SAMPLE PREPARATION:
QUALITY CONTROL CONCLUSION APPLICATIONS INTRODUCTION SAMPLE PREPARATION
• Sample handling: - Minimize time between drawing and preparation to
reduce spontaneous platelet activation
- Properly mix anticoagulant
- Avoid unnecessary agitation prior testing
- Transport: No pneumatic (platelet activaton)
No extreme temperatures
• Whole blood
:
Hemostasis, preanalytic
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
:
Current applications, Hematology
Cell blood Counting
Platelet quantification
Diagnostic, Prognosis and follow-up of hematological
diseases
Red Blood Cells pathologies diagnosis: PNH, HS
Primary and Acquired Immune defects (AIDS,…)
BCR-ABL qualification
Liquids
DNA Analysis
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Current applications, Hemostasis
Platelet disorders diagnosis
Platelet activation
Antiaggregants follow-up (VASP)
Reticulated platelets
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Current applications, Transfusion
Anti-platelet antibodies
Transplants, grafts: CD34 quantification
Transplants follow-up
Kleihauer
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Acute leukemia
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Acute leukemia
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Acute leukemia
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
FCM
Promyelocytes
Nucleus
Cytoplasm
Auer rods
Peroxydase
t(15;17) t(15;17)
variant t(11;17)
ZBTB16
ATRA
Leucocytosis
Sensitive Sensitive Resistant
+++ +++ +++
Reg/Lob
Presents
Bilob Reg/ pelger
HyperG MicroG
Absents
HyperG
Absents
t(15;17) bcr3
transcript
CD34-/DR-
CD33+
CD15-
CD34-/DR-
CD33+
CD15-
CD34+/CD2+
CD33+/DR-
CD15-
CD34+/CD2+
DR-/CD33+
CD15-
Reg/Lob
Presents
HyperG
+++
Sensitive
t(5;17)
NPM1
HyperG++
MicroG+
Absents
Reg/Lob
Sensitive
CD34-/DR-
CD33+
CD15-
+++
CD56+ : bad prognosis
AML M3
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Chronic Lymphoid Leukemia
Infinicyte
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Chronic Lymphoid Leukemia
On CD19+ Ly Matutes 4/5 (CD22+ et CD79b+)
CLL cut-off:
5000 Monoclonal B
lymphocytes/µl
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Mantle Lymphoma
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Mantle Lymphoma
Other applications:
Multiple myeloma
Extrahematological invasions
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
T lymphomas
• Remains a challenge in 2013
• Clinical information required:
- skin rash, extensive lymphadenopathy, hypergammaglobulinemia AILT
- neutropenia, rheumatoid arthritis LGL
• >3000/μl with CD4/CD8 inversion morphology + serology
• CD4+CD8+: reported if >1%, TCR gene rerrangement study indicated
• Aberrant expression is suggestive for clonal T-cell proliferation but never the
proof
Philippe et al. Acta Clinica Belgica 2009
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
T lymphomas
Diffusion à 90°
CD
45
Pe
rCP
CD19 APC CD4 PE-Cy7
CD16/56 PE CD8 APC-Cy7
CD
4 P
E-C
y7
CD
3 F
ITC
CD
3 F
ITC
C
D3
FIT
C
Mullier F. and al. Hématologie 2009
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
T lymphomas
CD10 FITC
CD10 FITC CD56 PE-Cy7
CD4 APC
CD
3 P
erC
P
CD
4 A
PC
C
D4 A
PC
CD
56 P
E-C
y7
Mullier F. and al. Hématologie 2009
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
• Quantification: Morphology > FCM (Paiva et al. Haematologica 2009/ Rawtron et al.
Haematologica 2008)
- Heterogenous distribution of plasmocytes in BM
- Contamination by peripheral blood 1ml= pure BM (Batinic BMT 1990)
- Selection of area riched in plasmocytes by the cytologist
- Adhesion to lipids (Nadav et al. BJH 2006)
- Quantification on biopsy: probably more accurate but standardization needed
- Weak plasmocyte
* Loss in the needle?
* Loss during RBC lysis (Smock et al. APLM 2007):
* Lowered CD138 expression by lysis (Smock et al. APLM 2007)
* Loss during centrifugations (Smock et al. APLM 2007)
Multiple myeloma
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS 23
• Plasmocytes gating
- CD38: unspecific, more expressed
- CD-138 (BB-4): very specific
- Characteristic Light scatter
23
San Miguel. European Journal of Cancer 2006
Multiple myeloma
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS 24
• Monoclonal gammapathies differentiation
- Myelomatous plasmocytes: CD38 ++, CD56+++, CD19- et CD45-
- Normal plasmocytes: CD38+++, CD56- CD19++ CD45+
24
San Miguel. European Journal of Cancer 2006
Myeloma diagnosis Myeloma post-transplantation
Multiple myeloma
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS 25 25
Paiva et al. Blood 2009
Multiple myeloma
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Patient Day 100 post ACT
Patient in stable CR Several years
Rawstron et al Haematologica 2008;93:431
CD19-:MM
CD19+:PC
CD19-:PC
CD19+:PC
Multiple myeloma
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Bruggeman et al Leukemia 2010;24(3):521-535
Minimal residual disease
Assessment of minimal residual disease (MRD) has acquired a prominent position
in European treatment protocols for patients with acute lymphoblastic leukemia
(ALL), on the basis of its high prognostic value for predicting outcome and the
possibilities for implementation of MRD diagnostics in treatment stratification.
Therefore, there is an increasing need for standardization of methodologies and
harmonization of terminology. For this purpose, a panel of representatives of all
major European study groups on childhood and adult ALL and of international
experts on PCR- and flow cytometry-based MRD assessment was built in the
context of the Second International Symposium on MRD assessment in Kiel,
Germany, 18-20 September 2008. The panel summarized the current state of MRD
diagnostics in ALL and developed recommendations on the minimal technical
requirements that should be fulfilled before implementation of MRD diagnostics into
clinical trials. Finally, a common terminology for a standard description of MRD
response and monitoring was established defining the terms 'complete MRD
response', 'MRD persistence' and 'MRD reappearance'. The proposed MRD
terminology may allow a refined and standardized assessment of response to
treatment in adult and childhood ALL, and provides a sound basis for the
comparison of MRD results between different treatment protocols
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Liquids: CSF
• Diagnosis leptomeningeal involvement: Great prognostic and therapeutic implications
(most common: myeloid and B lymphoid neoplasm)
• Immunopathogenesis of neuroinflammatory diseases (multiple
sclerosis,…)
• Cytology: High specificity but limited sensitivity (20 to 60%)
• FCM: Higher sensitivity
However: paucicellularity and rapidly decreasing viability
specific sample preparation protocols and analytical approaches
Kraan et al. Current protocols in Cytometry 2008; Unit 6.25:1-16
De Graaf et al. Cytometry Part B 2011; 80B:271–281
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Liquids: CSF
• Critical parameters:
- Rapidly decreasing viability (Cytotoxic effect on WBC)
Solution: Analyse within 1h after sampling
- Blood contamination
- Removal of free Ig otherwise binding of anti-kappa or anti-lambda
reduced or absent reactivity with anti-Hu-Ig reagents
solution: diluting the sample with an equal amount of
buffer during the first centrifugation/concentration step
- Rare events CV: 100*√n/n
solution: count large number of cells
-Carry-over of cells in the cytometer from previous experiments
solution: extensive cleaning and washing
Kraan et al. Current protocols in Cytometry 2008; Unit 6.25:1-16
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
BCR-ABL Qualitative detection
Healthy subject CML
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
:
Paroxysmal Nocturn Hemoglobinuria
Red Blood Cells Granulocytes and Monocytes
Healthy subject
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
:
Paroxysmal Nocturn Hemoglobinuria
Red Blood Cells Granulocytes and Monocytes
Positive PNH
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Paroxysmal Nocturn Hemoglobinuria, FLAER
Positive PNH FL
AE
R (
FIT
C)
Healthy subject
FL
AE
R (
FIT
C)
PNH clone
Ongoing standardization by the French Society of Flow Cytometry
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
:
Hereditary spherocytosis
Perrotta S and al. Lancet 2008; Vol 372:1411-1426 King MJ and al. BJH 2000; Vol 111(3):924-933
EMA binding
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
AIDS
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS 36
Platelet disorders diagnosis
Balduini C and al. Haematologica 2003 and 2004
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS 37
Healthy
subject
Pauline
Subject Mean Fluorescence
Intensity
Number of GpIb
sites/cell
Normal range
Healthy subject 20825 33850 27000-49000
Pauline 27927 43450
Platelet disorders diagnosis
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS 38
Healthy
Subject
Pauline
Sujet Mean
Fluorescence
Intensity
Number of
GpIa sites/cell
Normal range Ratio
GpIb/GpIa
Sujet sain 2576 4805 2200-7800 7,0
Pauline 8873 14937 2,9
Platelet disorders diagnosis
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS 39
Healthy subject
Pauline
Subject Mean
Fluorescence
Intensity
Number of
GpIIIa sites
GpIIIa/cell
Normal range Ratio
GpIb/GpIIIa
Healthy
subject
27020 43173 41000-65000 0,8
Pauline 77614 112567 0,4
Platelet disorders diagnosis
BSS
Heterozygous
GpIbβ:
Pro105Leu
Unpublished data (collaboration with KUL Kortrijk H. Deckmyn and K.Vanhoorelbeke)
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Current applications, Reticulated platelets
Decreased formation: CIT and aplastic anemia
Enhanced destruction: ITP
Anti-platelet antibody
TPO
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
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Current applications, 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:
1.1-6.1%
Autoimmune thrombocytopenia:
IPF=17-22%
Healthy subject ITP Aplastic Anemia
APPLICATIONS:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
Mepacrine testing
Capture 16μM
Release 16μM
QUALITY CONTROL:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
• Foundation: 2000
• Analysis on EDTA fresh samples:
- Leucocytosis (Absolute Number (AN)), Lymphocytes (%)
- CD3/CD4/CD8 (% and AN)
- CD19(%, AN), NK (%,AN)
- kappa/lambda/ratio (%)
- CD34 (blood)
• Perspectives
- CD34 (cytapheresis)
- Interpretation
:
Belgian quality control
QUALITY CONTROL:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
89
10
11
12
13
Year
CV
(%
)
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
CD4
89
10
11
12
13
Year
CV
(%
)
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
CD4
10
12
14
16
18
Year
CV
(%
)
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
CD19
10
12
14
16
18
Year
CV
(%
)
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
CD19
:
Belgian quality control
With permission of ISP/WIV (M. Van Blerk)
QUALITY CONTROL:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
:
Belgian quality control
With permission of ISP/WIV (M. Van Blerk)
2000/2009 Number of participants
CD3 CD4 CD8 CD19
1 colour 2/0 0/0 0/0 5/2
2 colours 12/2 15/2 15/2 15/1
3 colours 18/6 17/6 17/6 11/6
4 colours 6/20 6/20 6/20 5/19
5 colours 0/6 0/6 0/6 0/6
6 colours 0/15 0/15 0/15 0/15
QUALITY CONTROL:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
:
Belgian quality control
With permission of ISP/WIV (M. Van Blerk)
Belgian consensus recommendations:
27.1% of the participants in 2000→86.0% in 2009
Gating strategy % in 2002 % in 2009
FSC/SSC 44.2 10.0
CD14/CD45 11.6 2.0
CD45/SSC 44.2 88.0
CONCLUSION:
INTRODUCTION SAMPLE PREPARATION QUALITY CONTROL CONCLUSION APPLICATIONS
• Numerous hematologic applications due to
technologic improvements since 1934
• Progress in quality control since 2000 however
lack of standardization remains one pitfall
• Perspectives: - Standardization
- New applications: MRD,…
References
1. Guidelines for an integrated diagnostic approach of chronic lymphoproliferative disorders in the routine laboratory of haematology in Belgium.
Philippé J, Nollet F, Bakkus M, Meeus P, Demanet C, Schaaf-Lafontaine N, Franke S, Chatelain B, Vermeulen K, Boone E, El Housni H, Heimann P, Husson B, Lambert F, Vannuffel P, Saussoy P, Maes B, Deschouwer P.
Acta Clin Belg. 2009 Nov-Dec;64(6):494-504. 2. Developments in the immunophenotypic analysis of haematological malignancies. Heel K, Tabone T, Röhrig KJ, Maslen PG, Meehan K, Grimwade LF, Erber WN. Blood Rev. 2013 Jul;27(4):193-207. doi: 10.1016/j.blre.2013.06.005. Epub 2013 Jul 8. 3. Flow cytometric immunophenotyping for hematologic neoplasms. Craig FE, Foon KA. Blood. 2008 Apr 15;111(8):3941-67. doi: 10.1182/blood-2007-11-120535. Epub 2008
Jan 15. Review.