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
- 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 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:
Joe Gray1970
Early developments of flow cytometry
FLOW CYTOMETRY PARAMETERS
FLOW CYTOMETRY PARAMETERS
-. Light scatter: - Low angle light scatter - Side (90º) scatter -. Fluorescence emissions: * - FL1 - FL3 - FL2 - FL4 *artifitial or autofluorescence
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
Fluorochrome Excitation Emission LasersPeak (nm) Peak (nm) Argon Violet Helium-Neon
Marina Blue 365 460 +
FITC 495 520 +
Phycoerythrin (PE) 565 575 +
PE Texas Red 565 615 +
Per-CP 490 670 +
PE-Cy7 565 770 +
Pacific Blue 405 455 +
AmCyan 405 490 +
Pacific Orange 405 550 +
Allophycocyanin (APC)650 660 +
Alexa 700 635 720 +
APC-Cy7 650 770 +
- 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
--IdentificationIdentification ofof cellcell populationspopulations
--QuantificationQuantification ofof cellcell numbersnumbers
--CharacterizationCharacterization ofof cellcell populationspopulations
--IsolationIsolation//purificationpurification ofof cellscells
APPLICATIONS OF FCM TO TRANSFUSION MEDICINE
APPLICATIONS OF FCM TO TRANSFUSION MEDICINE
- Cell analysis: - Leucocyte contamination of transfusion products - Fetomaternal hemorrhage - Monitoring of transfused red cells /platelets - Red cell chimerism or mosaicism - Mutant/variant RBC - Platelet activation & functionality - Immunophenotypic analysis of blood cells - Specific protein studies: - Proteins bound to blood cells (i.e. plateled & red cell associated Igs /C3)
- Anti-platelet antibodies - Anti-leucocyte antibodies
10 10 10 10 100 1 2 3 4
ACM199005
CD8/CD19 LYMPHOGRAM ->
10 10 10 10 100 1 2 3 4
ACM199005
CD3/CD56 LOTE 2 ->
0 256 512 768 1024
ACM199005
FSC-Height ->
10 10 10 10 100 1 2 3 4
ACM199006
CD8/CD19 LYMPHOGRAM ->
CD56 / CD3 - PE
CD19 / CD8 - FITC
CD
56 /
CD
3 -
PE
CD19 / CD8 - FITC
Tra
nsf
orm
edS
SC
Tra
nsf
orm
edS
SC
Tra
nsf
orm
edS
SC
FSC - Height
PHENOTYPIC ANALYSIS OF PB LYMPHOCYTE PHENOTYPIC ANALYSIS OF PB LYMPHOCYTE
SUBSETS IN HIV: CURRENT APPROACHESSUBSETS IN HIV: CURRENT APPROACHES
PB +Mab
+ Red cell lysis
+ Referenceparticles BEADS
- Number of PB CD4+ T-cells
- Viral load measured in the serum
- Mean number of CD38 molecules expressed
by CD8+ T-cells
- Other lymphoid subsets/serologic markers:
CD8/CD45ro, CD8/CD28, CD95/CD4,
CD95/CD8, sCD95, sCD8,
- Number of PB CD4+ T-cells
- Viral load measured in the serum
- Mean number of CD38 molecules expressed
by CD8+ T-cells
- Other lymphoid subsets/serologic markers:
CD8/CD45ro, CD8/CD28, CD95/CD4,
CD95/CD8, sCD95, sCD8,
PROGNOSTIC MARKERS INHIV INFECTION
PROGNOSTIC MARKERS INHIV INFECTION
SINGLE PLATFORM METHODS
CD34 HPC ABSOLUTE COUNT FLOW CYTOMETRY
REFERENCE FLUORESCENCE BEADSREFERENCE FLUORESCENCE BEADS
ProCOUNTTM KIT
FLOWCOUNTTM BEADS
CD45-FITC
SID
E S
CA
TT
ER
R1
CD34-PE
SID
E S
CA
TT
ER
R2
CD45-FITC
SID
E S
CA
TT
ER
R3
FORWARD SCATTER
SID
E S
CA
TT
ER
R4
FORWARD SCATTER
FL
3-H
EIG
HT
R5
IMPACT OF THE COMPOSITION OF THE LEUKAPHERESIS PRODUCTS OIMPACT OF THE COMPOSITION OF THE LEUKAPHERESIS PRODUCTS ONN
THE THE HEMATOPOIETIC RECOVERYHEMATOPOIETIC RECOVERY AFTER AFTER AUTOLOGOUS TRANSPLANTAUTOLOGOUS TRANSPLANT
Variables without predictive value on the short-term hematopoietic recovery:
Immature CD34+ HPC, erythroid CD34+ HPC, Total
MNCs, Monocytes, CD4+ T-cells, B-cells, Dendritic cells, basophils
PREDICTIVE VARIABLE
MOBILIZATION REGIMEN
N. of TOTALCD34+ HPC/Kg
N. of MYELOID CD34+ HPC/Kg
N. of NEUTROPHILS /Kg
N. of Total LYMPHOCYTES /Kg
N. of CD3+ T-LYMPHOCYTES /Kg
N. of CD8+ T-LYMPHOCYTES/Kg
N. of NK Cells /Kg
ANC
>0.5x109/L
p-value
U- M-
ANC
>1x109/L
p-value
U- M-
PLT
>20x109/L
p-value
U- M-
PLT
>50x109/L
p-value
U- M-
* * * *
* * * *0.0060.006 0.0010.001
* * - -0.0070.007 0.0090.009
* * - -
* * - -* * - -* * - -* * - -
* p<0.05
MenendezMenendez et al, et al, TransfusionTransfusion, 2002, 2002
3210N. of CD4+ T-cells x106 infused /Kg
Dev
elo
pm
ent
of
infe
ctio
ns
aft
ertr
an
spla
nta
tio
n
NO
YES
N. of CD4+ T-cells x106 infused /Kg
YES NO
35±±±±10 100±±±±78
p=0.008
POST-TRANSPLANT
INFECTIONS
PREDICTIVE VARIABLE
MOBILIZATION REGIMEN
N. of TOTALCD34+/Kg
N. of TOTAL LYMPHOCYTES /Kg
N. of CD3+ T-LYMPHOCYTES/Kg
N. of CD4+ T-LYMPHOCYTES /Kg
N. of CD8+ T-LYMPHOCYTES /Kg
Transfusion Needs
RBC
p-value
U- M-
PLT
p-value
U- M-
Hospital
Stay (days)
p-value
U- M-
Incidence of
Infections
p-value
U- M-
- - * 0.020.02 -- - * -- - * *
- - - *
- - - * 0.0060.006
- - *
Variables without predictive value on the
the long-term hematopoietic recovery
Immature CD34+ HPC
Myeloid CD34+ HPC
Erythroid CD34+ HPC
Total MNCs
Neutrophils
Monocytes
CD4+ T-cells
B-cells
Dendritic cells
Basophils
* p<0.05*
IMPACT OF THE COMPOSITION OF THE LEUKAPHERESIS PRODUCTSIMPACT OF THE COMPOSITION OF THE LEUKAPHERESIS PRODUCTS
ON ON THE THE STABILITYSTABILITY OFOF THE TRANSPLANTTHE TRANSPLANT
MenendezMenendez et al, et al, TransfusionTransfusion, 2002, 2002
Days after transplantation
8006004002000
% ofpatientswho
developGVHD
Log-rank: p=0.013
CD34+ HPC ≥≥≥≥3.5x106 Myelomonocytic /Kg
0%
20%
40%
60%
80%
100%
CD34+ HPC<3.5x106 Myelomonocytic /Kg
N. of TOTAL CD34+ HPC/Kg
N. of MYELOMONOCyTIC
CD34+ HPC/Kg
GVHD NO GVHD
6.1±±±±3 4±±±±2
4.3±±±±2 2.3±±±±2
p=0.005
INFLUENCE OF THE COMPOSITION OF LEUKAPHERESIS PRODUCTS ON THEINFLUENCE OF THE COMPOSITION OF LEUKAPHERESIS PRODUCTS ON THE
INCIDENCE OF INCIDENCE OF GVHDGVHD AFTER A AFTER A NON MYELOABLATIVE ALLONON MYELOABLATIVE ALLO--TRANSPLANTTRANSPLANT
MenendezMenendez et al, Br J et al, Br J HaematolHaematol, 2002, 2002
10 10 10 10 100 1 2 3 4
8870.008
CK18PROGEN ->
10 10 10 10 100 1 2 3 4
8870.005
CK18PROGEN ->CK18
DILUTION 1/10000DILUTION 1/10000DILUTION 1/10000DILUTION 1/10000DILUTION 1/10000DILUTION 1/10000DILUTION 1/10000DILUTION 1/10000
CK18
FL2-A
0.025%0.025%0.025%0.025%
10 10 10 10 100 1 2 3 4
8870.010
CK18PROGEN ->
10 10 10 10 100 1 2 3 4
8870.009
CK18PROGEN ->
DILUTION 1/100000DILUTION 1/100000DILUTION 1/100000DILUTION 1/100000DILUTION 1/100000DILUTION 1/100000DILUTION 1/100000DILUTION 1/100000
FL2-A
CK18 CK18
0.001%0.001%0.001%0.001%
10 10 10 10 100 1 2 3 4
8870.004
CK18PROGEN ->CK18
FL2-A
DILUTION 1/1DILUTION 1/1DILUTION 1/1DILUTION 1/1DILUTION 1/1DILUTION 1/1DILUTION 1/1DILUTION 1/1
98%98%98%98%
10 10 10 10 100 1 2 3 4
8870.001
CK18PROGEN ->CK18
FL2-A
DILUTION 1/10DILUTION 1/10DILUTION 1/10DILUTION 1/10DILUTION 1/10DILUTION 1/10DILUTION 1/10DILUTION 1/10
8.39%8.39%8.39%8.39%
10 10 10 10 100 1 2 3 4
8870.002
CK18PROGEN ->CK18
FL2-A
DILUTION 1/100 DILUTION 1/100 DILUTION 1/100 DILUTION 1/100 DILUTION 1/100 DILUTION 1/100 DILUTION 1/100 DILUTION 1/100
1.24%1.24%1.24%1.24%
10 10 10 10 100 1 2 3 4
8870.005
CK18PROGEN ->CK18
FL2-A
DILUTION 1/1000DILUTION 1/1000DILUTION 1/1000DILUTION 1/1000DILUTION 1/1000DILUTION 1/1000DILUTION 1/1000DILUTION 1/1000
0.09%0.09%0.09%0.09%
10 10 10 10 100 1 2 3 4
8870.012
CK18PROGEN ->
10 10 10 10 100 1 2 3 4
8870.007
CK18PROGEN ->
DILUTION 1/1000000DILUTION 1/1000000DILUTION 1/1000000DILUTION 1/1000000DILUTION 1/1000000DILUTION 1/1000000DILUTION 1/1000000DILUTION 1/1000000
FL2-A
CK18 CK18
0.0002%0.0002%0.0002%0.0002%
10 10 10 10 100 1 2 3 4
8870.014
CK18PROGEN ->
10 10 10 10 100 1 2 3 4
8870.007
CK18PROGEN ->
DILUTION 1/10000000DILUTION 1/10000000DILUTION 1/10000000DILUTION 1/10000000DILUTION 1/10000000DILUTION 1/10000000DILUTION 1/10000000DILUTION 1/10000000
CK18 CK18
FL2-A
CK18 CK18
0.00002%0.00002%0.00002%0.00002%
DETECTION OF MICROMETASTASIS
IN BREAST CANCER
Cruz et al, Cruz et al, AmAm J J ClinClin PatholPathol, 2005, 2005
MONITORING OF THE IMMUNE SYSTEM AND VIRAL INFECTIONS
VERY EARLY: in vitro culture
EARLY: acute viral infection
LATE: chronic infection
ACTIVATED T-CELLS:Phenotypic changes
Relationship between CD20 expression andComplement-dependent cytotoxicity.
Bellosillo B, Villamor N et al. Blood. 2001;98: 2771.Golay J et al. Blood 2001; 98:3383
7000
6000
5000
4000
3000
2000
1000
0
CD56lowCD56highCD4-/CD8-/lowCD4+/CD8lowCD8+/CD4-CD4+/CD8-B-cellsPlasmacytoidCD16+BDCA3+
MyeloidBDCA3-
MyeloidEosinophilsNeutrophilsMonocytes
Dendritic Cells T-Cells NK Cells
CD
52 A
nti
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Bin
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ap
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EXPRESSION OF CD52 (CAMPATH-1) BY NORMAL PB LEUKOCYTES
HernHernáándezndez--Campo et al, 2005 (Campo et al, 2005 (submittedsubmitted) )
10 10 10 10 100 1 2 3 4
CD69 PE ->
CD123-APC
CD63-PE
EXPRESSION OF ALLERGEN-INDUCED ACTIVATION MARKERS ON BASOPHILS
10 10 10 10 100 1 2 3 4
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CD63-PE
RESTING BASOPHILS ALLERGEN-ACT BASOPHILS
CDs mieloidesCDs plasmocitoides
Identificación CDs y MONOCITOS de SP
CDs CD16+
CDs mieloidesCDs plasmocitoides CDs asociadas a monocito (CD16+)
10 10 10 10 100 1 2 3 4
CO
NT
RO
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AT
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CD33 APC
10 10 10 10 100 1 2 3 4
10
10
10
10
10
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4
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TN
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10 10 10 10 100 1 2 3 4
10
10
10
10
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PROD. ESPONTÁNEA
PRODUCCIÓN DE TNF-αααα por las CDs de SP en pacientes VIH-1+
PROD. ESTIMULADA
AllogeneicCD4+ T cells
Negative controlnon-stimulated CFDA,SE – stained PB T cells
Stage I pre-pDC
Stage II pre-pDC
Stage III pre-pDC
Positive control(PHA)
3.6% 6.9%
69%
6.2% 8.8%
71%
CFDA,SE - FITC
Functional characteristics of normal BM pre-pDC subsetsAbility to induce T-cell proliferation
AllogeneicCD8+ T cells
CFDA,SE - FITC
~ 0%
~ 0%
Martin et al, Transfusion, 2009
Time (Seconds)0 36 72 108 144 180
RA
TIO
[sh
ort
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]
0 2
00
400
600
800
1000
StimulationStimulation
Anti-CD3 induced T-cell activation(intracellular Ca++ mobilization)
Laso et al, Hepatology, 1997Laso et al, Alc Clin Exp Res, 1997
Laso et al, Alc Clin Exp Res 2007
Laso et al, Clin Cytometry, 2000
Laso et al,Alc Clin Exp Res 2010
SISTEMA INMUNE Y ALCOHOLISMO (Adaptado de Laso J )Adaptado de Laso J )
Linfocito T
citotóxico CD8+
Linfocitos T citotóxicos
CD8+ activados
Activación de célula NK
IL-12
Linfocito T
virgenLinfocito T CD4+
↓ Citocinaspatrón Th2
↑ Citocinaspatrón Th1
NECROSIS/APOPTOSIS
ETANOL
Intestino
ENDOTOXINA
IL-1IL-6TNF-alfa
CPA
NECROSIS/APOPTOSISINFLAMACIÓN
Laso et al, Alc Clin Exp Res 2007Laso et al, Clin Cytometry, 2007
Laso et al, J Hepatol, 1998
Laso et al, Alc Clin Exp Res 1999
Laso et al, Alc Clin Exp Res, 1996Laso et al, Clin Cytometry,1996
Linfocitos T
reguladores
Graves Disease: Effect of methimazole on PB CD69+ T-cells
Day 0 +7 +14+30 +90 +180Days of treatment with methimazole
% T-cells
20%
0%
15%
10%
5%
0%
ENFERMEDAD de GRAVES
100
80
60
40
20
0% I
L-6+
pDC
Controls DM no ATAT
#
*
30
20
10
0
ControlsDM no ATAT
N. mDC/
µµ µµl
# #
DIABETES MELLITUS tipo 2
% O
F C
YT
OK
INE
+ C
ELLS
POST-T126310
10
8
6
4
2
0CONTROLS
IL-6
IL6 Production by CD16+ DC
MONTH OF THERAPY
CONTROLS
DIABETIC MEN
PAPEL DE LAS CÉLULAS DENDRÍTICAS EN LA INFECCIÓN POR HIV
ABNORMAL CYTOKINE PRODUCTION BY CIRCULATING MONOCYTES AND DENDRITIC CELLS OF MYELOID ORIGIN IN ART-TREATED HIV-1+ PATIENTS RELATES TO CD4+ T-CELL
RECOVERY AND HCV CO-INFECTION
Maria Almeida1,2, Miguel Cordero1,3, Julia Almeida1,2, and Alberto Orfao* 1,2
1Department of Medicine, University of Salamanca. 2Service of Flow Cytometry and Center
for Cancer Research, University of Salamanca. 3Service of Internal Medicine-Unity of
Infectious Diseases, University Hospital of Salamanca. Salamanca. SPAIN.
CurrentCurrent HIV Res, 2007HIV Res, 2007
10 10 10 10 100 1 2 3 4
9621.003
vB3/vB7.1 FITC ->
10 10 10 10 100 1 2 3 4
9621.004
vB16/vB17 FITC ->
10 10 10 10 100 1 2 3 4
9621.005
vB20/vB5.1 FITC ->
10 10 10 10 100 1 2 3 4
9621.006
vB8/vB13.6 FITC ->
10 10 10 10 100 1 2 3 4
9621.007
vB12/vB2 FITC ->
10 10 10 10 100 1 2 3 4
9621.008
vB21.3/vB1 FITC ->
10 10 10 10 100 1 2 3 4
9621.009
vB14/vB22 FITC ->
10 10 10 10 100 1 2 3 4
9621.010
vB7.2/vB4 FITC ->
10 10 10 10 100 1 2 3 4
9621.014
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18
/
18
/
18
/
18
/ V
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1-P
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Vββββ20 / Vββββ5.15.15.15.1- FITC
Vββ ββ
23
/
23
/
23
/
23
/ V
ββ ββ1-
PE
Vββββ21.3 / Vββββ1111- FITC
Vββ ββ
5.2
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5.2
/
5.2
/
5.2
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Vββββ12 / Vββββ2222- FITC
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Vββ ββ
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/
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1
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PHENOTYPIC ASSESSMENT OF MONO- vs OLIGO- vs POLYCLONALITY
DISTRIBUTION OF THE NORMAL TCRVββββ REPERTOIRE
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Vb
2.1
Vb
3.1
Vb
5.1
Vb
5.2
Vb
5.3
Vb
6.1
Vb
6.7
Vb
7.1
Vb
8.1+
8.2
Vb
9.1
Vb
11.1
Vb
12.2
Vb
13.1
Vb
13.6
Vb
14.1
Vb
16.1
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17.1
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18.1
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21.3
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Vb
2.1
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3.1
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Vb
5.2
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6.1
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6.7
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8.2
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11.1
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16.1
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17.1
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18.1
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D8+
+
SYNTHESIS OF TETRAMERES OF HLA MOLECULES
Detection of influenza hemagglutinin (HA) specificCD4+ T cell clone (B16) using DR*0401 HA iTAg
Tetramers
A) Whole blood
(unspiked)
B) Whole blood spiked with
1% B16 cells
C) Whole blood spiked
with 5% B16 cells
A B C
Modified from Bekmann/Coulter brochureModified from Bekmann/Coulter brochure
10 10 10 10 100 1 2 3 4
CD69 FITC ->
Control EBV CMV
CD25
PE
->
10
10
10
0
1
2
103
104
24h
48h
10 10 10 10 100 1 2 3 4
CD69 FITC ->
CD25
PE
->
10
10
10
0
1
2
103
104
10 10 10 10 100 1 2 3 4
CD69 FITC ->
CD25
PE
->
10
10
10
0
1
2
103
104
10 10 10 10 100 1 2 3 4
CD69 FITC ->
CD25
PE
->
10
10
10
0
1
2
103
104
10 10 10 10 100 1 2 3 4
CD69 FITC ->
CD25
PE
->
10
10
10
0
1
2
103
104
10 10 10 10 100 1 2 3 4
CD69 FITC ->
CD25
PE
->
10
10
10
0
1
2
103
104
ANTIANTI--hCMV ThCMV T--CELL RESPONSE: CELL RESPONSE: expression of activation antigens on Texpression of activation antigens on T--cellscells
Rodriguez-Caballero et al, Blood, 2008
EBV
CMV
Control
NORMAL T-CELLS CLONAL T-CELLS
CD4-PerCP CD4-PerCPFSC-H
Sim
ulat
ed T
IME
Cy-I
FNγγ γγ-P
ECy
-IFN
γγ γγ-P
ECy
-IFN
γγ γγ-P
E
Cy-I
FNγγ γγ-P
ECy
-IFN
γγ γγ-P
ECy
-IFN
γγ γγ-P
E
VIRUSVIRUS--SPECIFIC TSPECIFIC T--CELL RESPONSES CELL RESPONSES (INFINICYT(INFINICYTTMTM EUROFLOWEUROFLOWTMTM Software)Software) IMMUNOPHENOTYPIC CHARACTERIZATION IMMUNOPHENOTYPIC CHARACTERIZATION
OF OF hCMVhCMV--SPECIFIC TSPECIFIC T--CELLSCELLS
Tra
nsfo
rmed
SSC
0
256
512
768
1024
P1
CD3 PE-Cy7100 101 102 103 104
P2
100 101 102 103 1040
256
512
768
1024
TNF-α α α α PE
P3P4
0
256
512
768
1024
100 101 102 103 104CD8 PerCP-Cy5.5
IDENTIFICATION OF TNF-αααα+ T-CELLS
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
PMA/ionomycin (3h)Stimuli:
-
100 101 102 103 104100
101
102
103
104
CD45-PC5/Bead fluorescence
Ant
i TNF-
αα αα-P
E
Ant
i cy
tokine
s-PE
+82±5% 82±11%
IFN-γγγγ
TNF-αααα IL-4
IL-2
100
101
102
103
104
100 101 102 103 104
CD45-PC5/Bead fluorescence
Ant
i TNF-
αα αα-P
E
Ant
i cy
tokine
s-PE
LymphocytesCD3+CD4- CD3+CD4+
100
101
102
103
104
100 101 102 103 104
CD4-APC/Bead fluorescence
CD3-
FITC
LPS(4h)
% of TNF-αααα+
monocytes 77±21%
IL-1ββββ (pg/mL) 4247±2368
IL-6 (pg/mL) 3390±762
IL-8 (pg/mL) 3664±1241
TNF-αααα (pg/mL) 273±112
PMA+Ionomycin(3h)
% of TNF-αααα+T-cells 82±11%
IFN-γγγγ (pg/mL) 3747±1727
TNF-αααα (pg/mL) 223±127
IL-4 (pg/mL) 50±22
IL-2 (pg/mL) 1163±381
Rodriguez-Caballero et al, Lab Invest, 2004
TYPES OF PROBES ON BEADS TYPES OF PROBES ON BEADS
-- NUCLEOTYDESNUCLEOTYDES::
-- MaximumMaximum ofof 1616--20 bases20 bases-- HybridizationHybridization decreaseddecreased withwith 1/15 1/15 missmatchmissmatch
-- ANTIBODIES & OTHER PROTEINSANTIBODIES & OTHER PROTEINS::
-- AllAll isotypesisotypes-- OrientedOriented oror notnot-- Variable Variable amountsamounts ((i.ei.e. . spacersspacers) )
APPLICATIONS OF FLOW CYTOMETRYAPPLICATIONS OF FLOW CYTOMETRYBASED BEADBASED BEAD--ARRAY SYSTEMSARRAY SYSTEMS
-- ProteinProtein analysisanalysis::-- CytokineCytokine measurementsmeasurements-- Viral Viral serologyserology-- AllergyAllergy teststests-- HormoneHormone measurementsmeasurements-- Cardiovascular Cardiovascular markersmarkers-- ImmunoglobulinImmunoglobulin isotypesisotypes-- PhosphorylatedPhosphorylated proteinsproteins-- MutatedMutated andand fusionfusion proteinsproteins
-- StudyStudy ofof oligonucleotydeoligonucleotyde sequencessequences::-- HLA HLA typingtyping-- SNP SNP analysisanalysis
DETECTION OF FUSION PROTEINS DERIVED FROM CHROMOSOMAL TRANSLOCATIONS BY FCM
5' gene A gene 3'B
fusion gene
B A
B
A
BA
B
transcription
translation
cell lysate
A
B
A
B
A
B
bead
beads coatedwith anti-Aantibody
bead
bead
A
B A
B
A
B
FITC-conjugatedanti-B antibody
A
Patents: US 6,610,498 B1 (26th of August 2003)US 6,686,165 B2 (3rd of February 2004)
Van Dongen et al
BCR-ABL RUO testing by EuroFlow
MFI values of the different cell samples (n=217)
135
repeated analysis for confirmationof weak positivity
negative
p210
p190
result of RQ-PCR
Verkampt et al, Leukemia 2009
100% K562 100% 697
10% K562/69710% 697/K562
R2 = green = BCR beads
R3 = pink = E2A beads
SIMULTANEOUS DETECTION OF BCR-ABL & E2A-PBX FUSION PROTEINS At this moment the technical developments for 7 well-
defined fusion proteins have (virtually) been completed:
● CML: − BCR-ABL : completed RUO kit launched and published
● precursor-B-ALL: BCR-ABL : completed
− TEL-AML : completed
− E2A-PBX2 : completed
− MLL-AF4 : completed
● AML: − AML-ETO : completed
− CBFB-MYH11: completed
− PML-RARA : completed RUO testing ongoing
Core-factor tubeMultiplex tube: testing ongoing
Precursor-B-ALL tubeMultiplex tube close to completion
BD Biosciences
PROTEOMICS: THE WORLD OF PROTEINS
G0/G1 checkpoint
Proteomics 2009
Size
Fractions:
Size exclusion chromatography 0.5-1h
Bead array
MINUTES
FACS
OVERNIGHT INCUBATION
IP+Western blots: weeksIP+ Mass-spectrometry: monthsELISA: lack of sandwich pairs
ARRAYS OF BEADS COUPLED TO DIFFERENT ANTIBODIES CAPTURING MOLECULES OF INTEREST
Anti-CD3
Anti-bcr
Anti-Akt
Anti-CD4
Anti-JNK
Anti-Abl
Anti-CD8
Anti-ERK
Anti-STAT5
Lund-Johansson et al, Cell Mol Proteomics, 2008 Pacific Orange mIgG PE
mIg
G P
E/C
y7
Pac
ific
Blu
e
G1(singlets)
Alexa-488
Ale
xa-6
47
Forward Scatter
Sid
e S
catt
er
BEAD ARRAY (n=1300 proteins)
BETA 2 MICROGLOBULIN /MHC CLASS I
Resolution of protein complexes with dual specificity
100
1000
10000
100000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
B2M-01
B2M-02
PROTEIN SIZE
Prot
ein/
bead
(PE
MFI
)
ββββ2m/HLAIαααα/X ββββ2m/HLAIαααα ββββ2m
10
100
1000
10000
v67
044
015
8 75 43
Cdk6 p19/INK4d Control Ig
QUIESCENT CELLS
Cdk6
p16
PROTEIN SIZE
Prot
ein/
bead
(PE
MFI
)
Go/G1 Cell cycle checkpointResolution of protein complexes with dual specificity
Lund-Johansson et al, Cell Mol Proteomics, 2008
Lucio et al, Leukemia, 1999
IMMUNOPHENOTYPIC CHARACTERISTICS OF NORMAL vs LEUKEMIC B-CELLS
CD19+ B-CELLS 8- COLOR flow cytometry: Bcp-ALL
EuroFlow panel (450 bivariate plots)
CD19-PECy7
CD45-PO
SSC
SSC
Precursor-B-ALL
LAIP:
CD45- Tdtlo
CD38
APC
CD15
FITC
CD10
APC
CD45
PO
CD16
FITC
CD11
7PE
CD11
bAPC
HLA
DRP
E
CD13
PECD
34Pe
rCP
Maturation stage of neutrophil precursors in normal BM
N-DIMENSIONAL NEUTROPHIL MATURATION IN NORMAL BM
Myeloblasts Promyelocytes
MyelocytesMeta-
myelocytes
Bands
Neutrophils
N-DIMENSIONAL NEUTROPHIL MATURATION IN NORMAL BM VS AML
CD15 26.6
CD117 19.4
SSC 19.2FSC 19.0
CD11b 6.0
Maturation stage of AML blasts
Aberrant phenotype of AML blasts
Aberrant markers
REFERENCE DATAFILES: NORMAL vs. CLL B-CELLS
Normal PB (n=8) CLL cases (n=6)
Case number
CD19
-PEC
y7
Normal PB CD19+ B-cells
Case number
CD19
-PEC
y7
CD19+ CLL B-cells
Case number
CD19
-PEC
y7
Normal PB B cells
CLL cases
LAIR1 9.3
CD5 8.6
CD79b 8.2
IgM 8.1
COMPARE A CASE VS NORMAL & CLL B-CELLS
CLL cases (n=6)Normal PB (n=8)
Case number
CD19
-PEC
y7
CD19+ CLL B-cells
SSC
CD19-PECy7 CD19-PECy7
SSC
Normal PB B cells
CLL cases
Case number
CD19
PEC
y7
CLLNormal
PB B-cells
FLOW CYTOMETRY:FLOW CYTOMETRY:TYPE OF INFORMATIONTYPE OF INFORMATION
--IdentificationIdentification ofof cellcell populationspopulations
--QuantificationQuantification ofof cellcell numbersnumbers
--CharacterizationCharacterization ofof cellcell populationspopulations
--IsolationIsolation//purificationpurification ofof cellscells
CK18+ BREAST CANCER CELLS
10 10 10 10 100 1 2 3 4
8870.008
CK18PROGEN ->
10 10 10 10 100 1 2 3 4
8870.005
CK18PROGEN ->CK18
DILUTION 1/10000DILUTION 1/10000DILUTION 1/10000DILUTION 1/10000DILUTION 1/10000DILUTION 1/10000DILUTION 1/10000DILUTION 1/10000
CK18
FL2-A
0.025%0.025%0.025%0.025%
10 10 10 10 100 1 2 3 4
8870.010
CK18PROGEN ->
10 10 10 10 100 1 2 3 4
8870.009
CK18PROGEN ->
DILUTION 1/100000DILUTION 1/100000DILUTION 1/100000DILUTION 1/100000DILUTION 1/100000DILUTION 1/100000DILUTION 1/100000DILUTION 1/100000
FL2-A
CK18 CK18
0.001%0.001%0.001%0.001%
10 10 10 10 100 1 2 3 4
8870.004
CK18PROGEN ->CK18
FL2-A
DILUTION 1/1DILUTION 1/1DILUTION 1/1DILUTION 1/1DILUTION 1/1DILUTION 1/1DILUTION 1/1DILUTION 1/1
98%98%98%98%
10 10 10 10 100 1 2 3 4
8870.001
CK18PROGEN ->CK18
FL2-A
DILUTION 1/10DILUTION 1/10DILUTION 1/10DILUTION 1/10DILUTION 1/10DILUTION 1/10DILUTION 1/10DILUTION 1/10
8.39%8.39%8.39%8.39%
10 10 10 10 100 1 2 3 4
8870.002
CK18PROGEN ->CK18
FL2-A
DILUTION 1/100 DILUTION 1/100 DILUTION 1/100 DILUTION 1/100 DILUTION 1/100 DILUTION 1/100 DILUTION 1/100 DILUTION 1/100
1.24%1.24%1.24%1.24%
10 10 10 10 100 1 2 3 4
8870.005
CK18PROGEN ->CK18
FL2-A
DILUTION 1/1000DILUTION 1/1000DILUTION 1/1000DILUTION 1/1000DILUTION 1/1000DILUTION 1/1000DILUTION 1/1000DILUTION 1/1000
0.09%0.09%0.09%0.09%
10 10 10 10 100 1 2 3 4
8870.012
CK18PROGEN ->
10 10 10 10 100 1 2 3 4
8870.007
CK18PROGEN ->
DILUTION 1/1000000DILUTION 1/1000000DILUTION 1/1000000DILUTION 1/1000000DILUTION 1/1000000DILUTION 1/1000000DILUTION 1/1000000DILUTION 1/1000000
FL2-A
CK18 CK18
0.0002%0.0002%0.0002%0.0002%
10 10 10 10 100 1 2 3 4
8870.014
CK18PROGEN ->
10 10 10 10 100 1 2 3 4
8870.007
CK18PROGEN ->
DILUTION 1/10000000DILUTION 1/10000000DILUTION 1/10000000DILUTION 1/10000000DILUTION 1/10000000DILUTION 1/10000000DILUTION 1/10000000DILUTION 1/10000000
CK18 CK18
FL2-A
CK18 CK18
0.00002%0.00002%0.00002%0.00002%
DETECTION OF MICROMETASTASIS
IN BREAST CANCER
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)
UNIVERSIDAD DE SALAMANCAUNIVERSIDAD DE SALAMANCA
EuroFlow consortium aims at
innovation in flow cytometry
MUCHAS MUCHAS GRACIASGRACIAS