CELL ANALYSIS TECHNIQUES FLOW CYTOMETRY

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


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


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


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



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 ->


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


98%98%98%98%

10 10 10 10 100 1 2 3 4

8870.001

CK18PROGEN ->CK18

FL2-A


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


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 ->


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 ->


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

gen

Bin

din

gC

ap

acit

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BC

)

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

FL2-Height ->

CD123-APC

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

L N

EG

AT

IVO

PE

CD33 APC

10 10 10 10 100 1 2 3 4

10

10

10

10

10

01

23

4

CD33 APC

TN

F- αα αα

PE

10 10 10 10 100 1 2 3 4

10

10

10

10

10

01

23

4

CD33 APC

TN

F- αα αα

PE

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

/lo

ng

]

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

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OK

INE

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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

CD5 APC ->

Vββ ββ

5.3

/

5.3

/

5.3

/

5.3

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1 -

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Vββ ββ

9 /

9

/

9 /

9

/ V

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Vββββ16 / Vββββ17171717- FITC

Vββ ββ

18

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18

/

18

/

18

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Vββββ20 / Vββββ5.15.15.15.1- FITC

Vββ ββ

23

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23

/

23

/

23

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PE

Vββββ21.3 / Vββββ1111- FITC

Vββ ββ

5.2

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5.2

/

5.2

/

5.2

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PE


Vββ ββ

13

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1

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Vββ ββ

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22

/

22

/

22

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Vββ ββ

13

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1

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13

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1

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PE

Vββββ7.2 / Vββββ4444- FITC

CD5 - APC

CD

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rCP

PHENOTYPIC ASSESSMENT OF MONO- vs OLIGO- vs POLYCLONALITY

DISTRIBUTION OF THE NORMAL TCRVββββ REPERTOIRE

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2.1

Vb

3.1

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5.1

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5.2

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5.3

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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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20.1

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21.3

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22.1

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2.1

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3.1

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5.2

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8.2

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16.1

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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


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


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


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 ->


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


98%98%98%98%

10 10 10 10 100 1 2 3 4

8870.001

CK18PROGEN ->CK18

FL2-A


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


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 ->


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 ->


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

CELL ANALYSIS TECHNIQUES FLOW CYTOMETRY

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