CENTRO DE INVESTIGACI CENTRO DE INVESTIGACIÓN DEL C N DEL CÁNCER, NCER, UNIVERSIDAD Y HOSPITAL UNIVERSITARIO UNIVERSIDAD Y HOSPITAL UNIVERSITARIO DE DE SALAMANCA SALAMANCA 69 69 º º Congreso Congreso Argentino Argentino de de Bioqu Bioqu í í mica mica Buenos Aires, 30 de mayo de 2011 Buenos Aires, 30 de mayo de 2011 PAPEL DE LA CITOMETRIA DE PAPEL DE LA CITOMETRIA DE FLUJO FLUJO EN EL AN EN EL ANÁLISIS DE LISIS DE CÉLULAS Y SUS PRODUCTOS LULAS Y SUS PRODUCTOS - Microbiology - Biochemistry - Molecular Biology - Pathology - Flow cytometry - Microbiology - Biochemistry - Molecular Biology - Pathology - Flow cytometry TECHNIQUES FOR THE ANALYSIS OF CELLS & CELL PRODUCTS TECHNIQUES FOR THE ANALYSIS OF CELLS & CELL PRODUCTS CELL ANALYSIS TECHNIQUES CELL ANALYSIS TECHNIQUES Image Flow Biochemistry/ Analysis Cytometry Molecular biology Information based on per cell per cell Mean sample value Localization Yes No No in the cell Speed Low High High Type of Qualitative Qualitative Qualitative information Quantitative Quantitative M-parametric Yes Yes No Image Flow Biochemistry/ Analysis Cytometry Molecular biology Information based on per cell per cell Mean sample value Localization Yes No No in the cell Speed Low High High Type of Qualitative Qualitative Qualitative information Quantitative Quantitative M-parametric Yes Yes No FLOW CYTOMETRY FLOW CYTOMETRY -. Qualitative and quantitative information -. Sensitivity -. Objective and reproducible -. High speed and statistically reliable -. Multiparametric information - 1953: The “Coulter” principle and instrument development - 1965/68: Multiparameter and multicolour flow cytometry Marv van Dilla H. Crissman Joe Gray 1970 Early developments of flow cytometry Wallace Coulter Wofgang Göhde PRINCIPLE: PRINCIPLE: Hydrodinamic Hydrodinamic focusing focusing Sheath Sheath fluid fluid Sheath Sheath fluid fluid FLOW CHAMBER FLOW CHAMBER Cell Cell sample sample THE FLUIDIC SYSTEM OF A FLOW CYTOMETER
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CENTRO DE INVESTIGACICENTRO DE INVESTIGACIÓÓN DEL CN DEL CÁÁNCER, NCER, UNIVERSIDAD Y HOSPITAL UNIVERSITARIO UNIVERSIDAD Y HOSPITAL UNIVERSITARIO
DE DE SALAMANCASALAMANCA6969ºº CongresoCongreso ArgentinoArgentino de de BioquBioquíímicamicaBuenos Aires, 30 de mayo de 2011Buenos Aires, 30 de mayo de 2011
PAPEL DE LA CITOMETRIA DE PAPEL DE LA CITOMETRIA DE FLUJOFLUJO EN EL ANEN EL ANÁÁLISIS DE LISIS DE CCÉÉLULAS Y SUS PRODUCTOS LULAS Y SUS PRODUCTOS
TECHNIQUES FOR THE ANALYSIS OF CELLS & CELL PRODUCTS
TECHNIQUES FOR THE ANALYSIS OF CELLS & CELL PRODUCTS
CELL ANALYSIS TECHNIQUESCELL ANALYSIS TECHNIQUES
Image Flow Biochemistry/ Analysis Cytometry Molecular biology Information based on per cell per cell Mean sample value Localization Yes No No in the cell Speed Low High High Type of Qualitative Qualitative Qualitative information Quantitative Quantitative M-parametric Yes Yes No
Image Flow Biochemistry/ Analysis Cytometry Molecular biology Information based on per cell per cell Mean sample value Localization Yes No No in the cell Speed Low High High Type of Qualitative Qualitative Qualitative information Quantitative Quantitative M-parametric Yes Yes No
FLOW CYTOMETRYFLOW CYTOMETRY
-. Qualitative and quantitative information
-. Sensitivity
-. Objective and reproducible
-. High speed and statistically reliable
-. Multiparametric information
- 1953: The “Coulter” principle and instrument development- 1965/68: Multiparameter and multicolour flow cytometry
Marv van DillaH. Crissman
Joe Gray1970
Early developments of flow cytometry
Wallace Coulter
Wofgang Göhde
PRINCIPLE:PRINCIPLE:
HydrodinamicHydrodinamicfocusingfocusing
SheathSheath fluidfluidSheathSheath fluidfluid
FLOW CHAMBERFLOW CHAMBER
CellCell samplesample
THE FLUIDIC SYSTEM OF A FLOW CYTOMETER
LASERS: THE SOURCE OF LIGHT OF A FLOW CYTOMETER
BLUE LASER (Aligned and focused)
LASER LIGHT CHANGES:- Direction (Scatter)- Colour (Fluorescence)
- 1953: The “Coulter” principle and instrument development- 1965/68: Multiparameter and multicolour flow cytometry- 1970:
LASER GEOMETRY AND FLUORESCENCELASER GEOMETRY AND FLUORESCENCE--EMISSION DETECTION EMISSION DETECTION SYSTEM OF A 19SYSTEM OF A 19--PARAMETER FLOW CYTOMETERPARAMETER FLOW CYTOMETER
Reproduced from:Perfetto et al, Nat Rev Immunol, 2004
SETS OF FLUOROCHROMES FOR FLOW CYTOMETRY IMMUNOPHENOTYPING
- 1953: The “Coulter” principle and instrument development- 1965/68: Multiparameter and multicolour flow cytometry- 1970: FACS: “fluorescence activated cell sorter”.
Len & LeeHerzenberg
Early developments of flow cytometry
FUNCTIONAL FEATURES OF A FLOW CYTOMETER
FUNCTIONAL FEATURES OF A FLOW CYTOMETER
Cell counter
Cell analyzer
Cell sorter
APPLICATIONS OF FLOWCYTOMETRY
APPLICATIONS OF FLOWCYTOMETRY
-. Immunophenotype of cells-. DNA, RNA, protein cell contents-. Enzyme analysis-. Ca++ and other ion measurements-. Intracellular pH-. Drug effects and drug resistance-. Apoptosis and cell death-. Phagocytosis-. Chromosome analysis-. Quantitation of soluble proteins-. Study of cell organelles-. Cell/chromosome sorting
-. Immunophenotype of cells-. DNA, RNA, protein cell contents-. Enzyme analysis-. Ca++ and other ion measurements-. Intracellular pH-. Drug effects and drug resistance-. Apoptosis and cell death-. Phagocytosis-. Chromosome analysis-. Quantitation of soluble proteins-. Study of cell organelles-. Cell/chromosome sorting
GG22MM GG00
GG11
ss
0 200 400 600 800 1000
GG00GG11
ss GG22MM
DNA AnalysisDNA Analysis
DNA content
C
ou
n
t
2N2N 4N4N
Cell Cycle Analysis by FCM PROGNOSTIC VALUE OF DNA STUDIES BY FCM
PROGNOSTIC VALUE OF DNA STUDIES BY FCM
Tumor type DNA variable Clinical impact Tumor type DNA variable Clinical impact
Bladder Aneuploid Agressive S-phase Agressive Bone Aneuploid Short survival Breast Aneuploid Short survival? S-phase Relapse, short surv Colorectal Aneuploid Short survival? S-phase Relapse, short surv Gastric Aneuploid Short survival Cervix Aneuploid Invasive, short surv Kidney Aneuploid Short survival Lung Aneuploid Short survival Prostate Aneuploid Short survival Neuroblastoma Aneuploid Long survival
Bladder Aneuploid Agressive S-phase Agressive Bone Aneuploid Short survival Breast Aneuploid Short survival? S-phase Relapse, short surv Colorectal Aneuploid Short survival? S-phase Relapse, short surv Gastric Aneuploid Short survival Cervix Aneuploid Invasive, short surv Kidney Aneuploid Short survival Lung Aneuploid Short survival Prostate Aneuploid Short survival Neuroblastoma Aneuploid Long survival
César MILSTEINHoward SHAPIRO
Gunter VALET
Kohler G, Milstein C. Continuous cultures offused cells secreting antibody of pre-definedspecificity. Nature 1975;256:495-497.
- 1953: The “Coulter” principle and instrument development- 1965/68: Multiparameter and multicolour flow cytometry- 1970: FACS: “fluorescence activated cell sorter”.- 1975: Production of monoclonal antibodies
Early developments of flow cytometry
0 256 512 768 1024
RM30243.100
FL2-Area ->
ALL: DNA PLOIDY & CELL CYCLE ANALYSISALL: DNA PLOIDY & CELL CYCLE ANALYSIS
NORMALB-CELLS
NEOPLASTIC B-CELLS
G0/G1 DNA synthesis G2/M
RESIDUAL NON-B CELLS RM30243.100
FL2-Area ->
0 256 512 768 1024
Dna cell contents
Dna cell contents
B-CE
LL M
ARK
ERS
FLOW CYTOMETRY:FLOW CYTOMETRY:TYPE OF INFORMATIONTYPE OF INFORMATION
IMMUNOPHENOTYPIC CHARACTERIZATION OF TNF-αααα+ T-CELLS
CCR7
APC
cGra
nzim
a B-
PE
100 101 102 103 104100
101
102
103
104
CD45 RA-FITC100 101 102 103 104
TNF-α α α α APC
100
101
102
103
104
Rodriguez-Caballero et al, Clin Cytometry, 2007
0
10
20
30
40
50
60
70
80
90
100
VB 3
VB 7.1
VB 5.3
VB 16
VB 17
VB 9
VB 20
VB 5.1
VB 18
VB 8
VB13.6
VB 13.1
VB 12
VB 2
VB 5.2
VB 21.3
VB 1
VB 23
VB 14
VB 22
VB 11
VB 7.2
VB 4
VB 13.2
CD4
CD8
VB 3
VB 7.1
VB 5.3
VB 16
VB 17
VB 9
VB 20
VB 5.1
VB 18
VB 8
VB13.6
VB 13.1
VB 12
VB 2
VB 5.2
VB 21.3
VB 1
VB 23
VB 14
VB 22
VB 11
VB 7.2
VB 4
VB 13.20
102030
40
506070
80
90
100
TNFα+ T-cells
VB 3
VB 7.1
VB 5.3
VB 16
VB 17
VB 9
VB 20
VB 5.1
VB 18
VB 8
VB13.6
VB 13.1
VB 12
VB 2
VB 5.2
VB 21.3
VB 1
VB 23
VB 14
VB 22
VB 11
VB 7.2
VB 4
VB 13.2
CD4
CD8
0
10
20
30
40
50
60
70
80
90
100
VB 3
VB 7.1
VB 5.3
VB 16
VB 17
VB 9
VB 20
VB 5.1
VB 18
VB 8
VB13.6
VB 13.1
VB 12
VB 2
VB 5.2
VB 21.3
VB 1
VB 23
VB 14
VB 22
VB 11
VB 7.2
VB 4
VB 13.20
10203040
506070
80
90
100
TNFα-T-cells
TNFα+ T-cells TNFα-T-cells
TCRVTCRVββββββββ REPERTOIRE OF HCMVREPERTOIRE OF HCMV--SPECIFIC SPECIFIC ACTIVATED ACTIVATED (TNF(TNFαααααααα++)) TT--LYMPHOCYTESLYMPHOCYTES
• hCMV-activated T-cells show a restricted TCRVβ repertoire.
TCR-Vββββ FAMILIES
% O
F CE
LLS
Rodriguez-Caballero et al, Clin Cytometry, 2007
hCMVhCMV--ASSOCIATED TCRVASSOCIATED TCRVββββββββ REPERTOIRE: REPERTOIRE: association with the HLA association with the HLA haplotypeshaplotypes
% of cases
HLA-DR0701+ HLA-DR0701-
100%100%
0
20
40
60
80
100
HLA-DQ0202+ HLA-DQ0202-
100%100%
0
20
40
60
80
100
TNFTNF--αααααααα++ CD8CD8+ + TT--cellscells
HLA-C1203+ HLA-C1203-0
20
40
60
80
100
100%100%
• The expansion of some TCRVβ families is associated with specific HLAhaplotypes.
23%23%
p=0.04
100%100%
HLA-C1203+ HLA-C1203-0
20
40
60
80
100TCRVββββ5.1
% of cases
TCRVββββ4
15%15%p=0.02
TCRVββββ13.1
p=0.001
0%0% 0%0%
TCRVββββ13.1
p=0.001
TNFTNF--αααααααα++ CD4CD4++ TT--cellscells
7%7%
100%100% TCRVββββ16
p=0.03
HLA-DQ0402+ HLA-DQ0402-0
20
40
60
80
100
Rodriguez-Caballero et al, Clin Cytometry, 2007
hCMV: HLADRB1*0701 COMPLEMENTARYPEPTIDES
LGL-T CD4+ AND HLADRB1*0701: RESPONSE AGAINST THE ”MQLIPDDYSNTHSTRYVTVK” PEPTIDE
10 10 10 10 100 1 2 3 4
B PEPTIDO.002
CD4 ->CD4
TNF-
αα αα
10 10 10 10 100 1 2 3 4
b peptido 6h+tapi-2.002
CD4 PerCP ->CD4
TNF-
αα αα
10 10 10 10 100 1 2 3 4
B PEPTIDO.002
CD56 APC ->CD4
TNF-
αα αα
10 10 10 10 100 1 2 3 4
D15989 6h TAPI PEPTIDO.004
CD4 PerCPCY5.5 ->CD4
TNF-
αα αα
10 10 10 10 100 1 2 3 4
b peptido .002
CD4 PerCP ->CD4
TNF-
αα αα
10 10 10 10 100 1 2 3 4
b peptido .002
CD3 FITC ->CD4
TNF-
αα αα
TCRVβ13.1+ (n=4)
TCRVβ22+
TCRVβ5.1+
RESPONSE OF CLONAL CD4+ T-LGL TO THE “MQLIPDDYSNTHSTRYVTVK”hCMV PEPTIDE AND hCMV WHOLE LYSATE: SECRETION OF IFN-γγγγ
Vββββ13.1+CD4+T-LGL(n=4)
0
5000
10000
15000
Concentration of soluble IFN-γ(pg/mL)
Non Vββββ13.1+CD4+
T-LGL(n=4)
Peptide
hCMV whole lysate
Rodriguez-Caballero et al, Blood, 2008
CD38:AF70
0 LOGIC
AL
CD27:APC LOGICAL
CD
10:P
E-C
y7
LO
GIC
AL
Immature
Naïve
Memory
Plasmablasts
Percentage Absolute count
(cells/ µL)
ImmatureImmature 5.4%±±±±3.7%
(1.8%-4.5%)
9±±±±9
(3-11)
NaiveNaive 64%±±±±12%
(57%-73%)
101±±±±57
(61-130)
MemoryMemory 31%±±±±12%
(21%-40%)
52±±±±39
(26-65)
Plasmablasts/Plasmablasts/
Plasma cellsPlasma cells
2.5%±±±±1.4%
(0.8%-2.3%)
3.0±±±± 6.8
(1.0-3.1)
Distribution of B-cell maturation subsets in peripheral blood from normal individuals (N=600)
Memory Plasmablasts/Plasma cells
sIgA+
(21% ±±±± 9%)
sIgG+
(23% ±±±± 10%)
sIgMD+
(52% ±±±± 15%)
sIgG+
(13% ±±±± 11%)
sIgM+
(18% ±±±± 12%)
sIgneg
(14% ±±±± 12%)sIgD+
(5% ±±±± 5%)
sIgA+
(49% ±±±± 18%)
Caraux A Haematologica 2010, Perez-Andres M Clinical Cytometry 2010
% d
e cé
lulasen
fas
eS+G
2/M
GGLL
0%
5%
10%
15%
20%
25%
30%
MO SP MO
*
* * *
*
*
*
*
CD45lo
CD19loCD45+
CD19hi
Precursores B
CD45hi
CD19+CD20+
CD23+CD20+
CD23-CD38lo
CD20+CD38+
CD20hiCD38hi
CD20loCD38hi
CD19+
Linfocitos B maduros Células Plasmáticas
TASA PROLIFERATIVA DE LAS CÉLULAS B NORMALES A LO LARGO DE LA DIFERENCIACIÓN B
*p<0.05
FCM ANALYSIS OF THE SPECIFICITY OF ANTI-PLATELET ANTIBODIES
FCM ANALYSIS OF THE SPECIFICITY OF ANTI-PLATELET ANTIBODIES
Patient's sera
+
Anti-CD41a-PE Anti-hu-Igs-FITC
+
HLA-I gp IIb/IIIa
Energy transfer
No energy transfer
CYTOMETRY BEAD ARRAYSCYTOMETRY BEAD ARRAYS
Direct Sandwich AssayDirect Sandwich Assay
Covalently-coupledcapture Ab
PE-labeled detector Ab
Analyte
BeadBead
MULTIPLEXED BEAD ARRAYSMULTIPLEXED BEAD ARRAYS
Bead-coupled with capture Ab
Fluorochrome-labeled detector Ab
AnalyteFusion protein
BeadBead AA
antianti--proteinprotein AA
Bead BBead B
antianti--protein Bprotein B
Bead NBead N
antianti--protein Nprotein N
AntiAnti--PtPt--A A
AntiAnti--PtPt--BB
AntiAnti--PtPt--NN
Simultaneous identification of activated cells and Simultaneous identification of activated cells and quantification of multiple cytokinesquantification of multiple cytokines
Cruz et al, Cruz et al, AmAm J J ClinClin PatholPathol, 2005, 2005
CK18+ BREAST CANCER CELLS
SERVICIO DE CITOMETRIA, DEPARTAMENTO DE MEDICINA Y SERVICIO DE CITOMETRIA, DEPARTAMENTO DE MEDICINA Y CENTRO DE INVESTIGACICENTRO DE INVESTIGACIÓÓN DEL CN DEL CÁÁNCER (IBMCC) NCER (IBMCC)