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23/10/2017

1

NORMAL vs LEUKEMIC B-CELL DEVELOPMENT THROUGH LIFE ASSESSED

BY FLOW CYTOMETRY

CANCER RESEARCH CENTER-IBSAL UNIVERSITY & UNIVERSITY HOSPITAL,

SALAMANCA (SPAIN)

ESCCA 2017 CourseThessaloniki (Greece), September 25th, 2017

DISCLOSURES

- I am co-author of patents that are property of USAL and EuroFlowand that the corresponding IP rights have been licensed toBecton/Dickinson Biosciences and Cytognos SL, and from which bothUniversity of Salamanca and EuroFlow receive royalties.

B-CELL PRODUCTION DURING LIFE

Generation of mature B lymphocytes

Embryo Adult

Yolk sacFetal liverFetal BM

Bone marrow

Individual life time (age)

Prod

ucci

ón l

info

cito

s

10 – 12 years

Refill of secondary lymphoid tissues Replacement

IMMUNOPHENOTYPIC CHARACTERIZATION OF “NORMAL” VS

NEOPLASTIC PRECURSORS:

THE B-CELL COMPARTMENT IN NORMAL BM

SCF

IL3

IL7

IL9 EPO

IL11TPO

M-CSF

G-CSF

GM-CSF

Hematopoietic Stem cell

Lymphoidprecursor

CFU-GMCFU-G

CFU-MCFU-Eo

CFU-Bs

Bone marrow Blood - tissues

Myeloid precursor

Neutrophil

Monocyte/Macrophage/DC

Eosinophil

Basophil

HEMATOPOIESIST/NK cell precursor

B-cell precursor

Erythrocytes

Platelets

BFU-E

CFU-Mk

pDC precursor Dendritic cell

Mast cell

CFU-MC

?

B lymphocyte

T lymphocyte

NK cell

IMMUNOPHENOTYPIC IDENTIFICATION OF LINEAGE COMMITMENT OF CD34+ BM CELLS

nTDT FITC

Cy

MP

O P

E

c

10 10 10 10 100 1 2 3 4

TR

AN

SF

OR

ME

D S

SC

CD34 APC

02

56

51

27

68

10

24

a

10 10 10 10 100 1 2 3 41024

TRANSFORMED SSC

0 256 512 768

bCD

45 P

ER

CP

b

Matarraz S et al. Leukemia 2008

Neutrophil precursors

B-cell precursors

23/10/2017

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GATED CD34+ HPC

B-LYMPHOID DIFFERENTIATION IN NORMAL BM

Menendez et al, 2002Earlier B-cell markers: NuTDT, CyCD79a, CD10 & CD19

IMMUNOPHENOTYPE OF LINEAGE COMMITTED CD34+HPC

CELL LINEAGE FREQUENCY

Erythroid 15% (5%-35%)

Megakaryocytic <1%

Neutrophil 31% (12-39%)

Eosinophil <1%

Basophil <1% (<1%-3%)

Monocytic 5% (3%-15%)

Mast cell <1% (<1%)

pDC 6% (1%-15%)

B-Lymphoid 23% (<1%-45%)

CD7+ (Early T-lymphoid?) 11% (5-18%)

Total: 92%

Molecular processes in precursor-B-cells

M.C. van Zelm, et al. J Immunol. 2005; 175: 5912-5922CD5:APC

CD10:PECy7

CD20:PB

CD10:PECy7

CD38:APC-H7

sIgD:PE

CD38:APC-H7

sIgM:FITC

sIgM:FITC

sIgD:PE

NORMAL B-CELL MATURATION PATHWAYS IN HUMAN BM (Gated CD19+ B-cells)

CD10

CD34

CD38

CD20

Tdt

sIg

CD9

CD81

NORMAL B-CELL MATURATION IN BMProtein expression profiles

Maturation stage

Amou

nt o

f pr

otein/c

ell

Design of antibody panel – phase 5

Theunissen P, et al. Blood 2017

23/10/2017

3

CD34+ BCP ALL

(non-supervised)

PC1

PC

2

Top ranked = 3 points

Second rank = 2 points

Third rank = 1 point

Other ranks = 0 points

Design of antibody panel – phase 1

EuroFlow Report on MRD in ALL, P. Theunissen, E. Mejstrikova,…. V.H.J. van der Velden, BLOOD, 2017

Diagnostic Genetic Aberrant immunophenotypeSubtype lesion

Null/BI 11q23 CD34+,CD10-,7.1+,CD15+

Common/BII t(9;22)* CD34hi,CD10+,CD38lo,CD13lo

Common/BII t(12;21) CD34het,CD10hi,CD20-,CD13lo

Pre-B/BIII t(1;19) CD34-, CyIgm+, CD10+, CD20-, CD9+hom

Common/BII t(5;14) CD10+ with associated eosinophilia

BCP-ALL: GENOTYPIC-PHENOTYPIC ASSOCIATIONS

Ortuño F, Orfao A, Cytometry B, 2004 (updated)

10 10 10 10 100 1 2 3 4

HG30672.002CD10 ->

10 10 10 10 100 1 2 3 4

HG30672.006CD34 ->

10 10 10 10 100 1 2 3 4

HG30672.008CD34 ->

10 10 10 10 100 1 2 3 4

HG30672.010CD13 ->

10 10 10 10 100 1 2 3 4

RFR7690.007CD34 ->

10 10 10 10 100 1 2 3 4

RFR7690.008CD10 ->

10 10 10 10 100 1 2 3 4

RFR7690.008CD13 ->

10 10 10 10 100 1 2 3 4

RFR7690.011CD34 ->

10 10 10 10 100 1 2 3 4

UVJ39869.002CD34 PE ->

10 10 10 10 100 1 2 3 4

UVJ39869.004CD13 PE ->

10 10 10 10 100 1 2 3 4

MO21489004CD34 ->

10 10 10 10 100 1 2 3 4

AP36452.003CD10 ->

ADULT PRECURSOR B-ALL: IMMUNOPHENOTYPE OF BCR/ABL+ CASES

Normal BM

CD34+ B-cells

DNA diploidBcr/abl+ ALL

DNA aneuploidBcr/abl+ ALL

CD10 CD13 CD34 CD34

CD10 CD13 CD34 CD34

CD10 CD13 CD34 CD34

CD19

CD19

CD19

CD45

CD45

CD45

CD38

CD38

CD38

CD19

CD19

CD19

PROTEIN EXPRESSION PROFILES IN BCP-ALL with t(v;11q23.3); KMT2A rearranged

Case 1

Case 2

PROTEIN EXPRESSION PROFILES IN BCP-ALL with t(12;21); ETV6-RUNX1

Case 2

Case 1

PROTEIN EXPRESSION PROFILES IN BCP-ALL with t(1;19); TCF3-PBX1

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Automated immunophenotypic classification of BCP-ALL based on aberrant blast cell maturation

Dissection of Precursor-B-cell differentiation

Identification and isolation of BM B-cell subsets

CD34+

lin–

SmIgM

CD

20

CD3/13/19/33/56

CD

34

CD34+

CD22

CD

19

CD10

CD

34

CD10

CD

20

CD10

CD

20

SmIgl

Sm

Igk

UCB bone marrow tonsil

pro-B pre-B-I pre-B-IIlarge

pre-B-IIsmall

immatureB

mature-BSmIg +k

mature-BSmIg +l

CD34+ CD19 + CD19 + CD19 + CD19 + CD19 +

CD34+

lin–

(defined as: CD3/13/19/33/56– )

CD34+

CD22+

CD19 –

CD34+

CD19 +

CD10 +

CD20–

CD34–

CD19 +

CD10 +

CD20dim

SmIgM–

CD34–

CD19 +

CD10 +

CD20–

CD34–

CD19 +

CD10 +

CD20hi

CD19 +

CD20+

SmIg + or SmIg +kl

M.C. van Zelm, et al. J Immunol. 2005; 175: 5912-5922.


Is there more than one parallel B-cell differentiationpathway?


CD20 Expression

CD34+

cyIgM-

smIgM-

CD34-

cyIgM+

smIgM-CD34-

cyIgM+

smIgM+

IgMExpression

ImmatureB-cells

PreB-cells

BM MATURATION PATHWAYS OF CD10+CD38+ B-CELL PRECURSORS

23/10/2017

5

Pro-B-cell

CD79a+, Tdt+, CD10-/lo

Pre-B I cellCD79a+ Tdt, CD10+ CD19+

Early pre-B IIa cell

CD79a+ Tdt+, CD10hi CD19+

CyIgm+

Early pre-B IIb cell

CD79a+ Tdt+, CD10lo CD19+

CyIgm+

CyIgm-Early pre-B

IIa cellCD34+ CD79a+

Tdt+, CD10hi

CD19+

CyIgm-Early pre-B

IIb cellCD34- Tdt+ CD20+ CD10lo

CD19+ CD20lo

B-CELL MATURATION PATHWAYS IN HUMAN BONE MARROW

1-2%

5-10%

0.5-1%

10-20%

4-5%

0.5-1%

Delayed pre-B II cell

CD34lo CD79a+

Tdtlo, CD10+CD20+ CD19+

CyIgm+

1%

Immature sIgM+ B lymphocytes

CD34- CD79a+ Tdt-, CD10+ CD20++ CD19+ sIgM+/++

10-15%

Pre-B II cellsCD34- CD79a+ Tdt-, CD10+

CD20++ CD19+ CyIgm+ sIgM-

30-40%IGL inframe

IGL in frame

IGL in frame

IGH inframe

CD20

BM MATURATION PATHWAYS OF CD10+CD38+ B-CELL PRECURSORS

t(1;19)(Pink)

t(9;22)Brown/Blue

IMMUNOPHENOTYPIC CHARACTERIZATION OF “NORMAL” VS

NEOPLASTIC PRECURSORS:

THE NORMAL PERIPHERAL BLOOD B-CELL COMPARTMENT

CIRCULATING TRANSITIONAL/IMMATURE B-CELLS PHENOTYPE OVERLAPS WITH BM IMMATURE B-CELLS

Peripheral Blood

Naïve

Immature

Immature

Naïve

ProB/PreB

Perez-Andres et al, Cytometry B, 2010

CD10-PECy7 CD38-APCH7 CD5-APC sIgM-FITC

CD10-PECy7 CD38-APCH7 CD5-APC sIgM-FITC

CD10-PE

Cy7

CD38-APC

H7

sIgD

-PE

CD20-Pa

cBlue

CD10-PE

Cy7

CD38-APC

H7

sIgD

-PE

CD20-Pa

cBlue

Bone marrow

IMMATURE: CD38++ CD5+ CD21-/+ CD24++ smIgM++D+

PreGC B-cells (CD27-):

NAIVE CD5+: CD38-/+ CD5+ CD21+ CD24+ smIgM+D++

NAIVE CD5-: CD38-/+ CD5- CD21+(-) CD24+ smIgM+D++

MEMORY USwt: CD38++ CD5+ CD21-/+ CD24++ smIgM++D+

PostGC B-cells (CD27+):

MEMORY Swt: CD38-/+ CD5+ CD21+(-) CD24+/- smIgMD-

Plasma cells: CD38+ CD5- CD21+(-) CD24+ smIgMD+/-

PB B-CELL SUBSETS IDENTIFICATION OF PB B-CELL SUBSETS IN 5-D PLOT

PC1

PCA2

IMMATURE

NAIVE CD5+

NAIVE CD5-

MEMORY USwt

MEMORY Swt

Plasma cells

Discrete maturation

23/10/2017

6

CLL vs normal PB B-cells

CLL cells

Marker PC1 Marker PC2

IgM 31 CD5 48

CD27 30 IgM 20

IgD 27 CD38 18

CD38 8.9 CD27 9.1

APS1 for the Backbone markers

Plasma cellsexcluded

SwitchedMemory B-cells

ImmatureB-cells

UnswitchedMemory B-cells

NaiveB-cells

IgM

* **

CD27

*

*

CD22

** * *

CD21*

*

CD38

*

ROR-1

** * *

CD5

*

*

**

IgD

* *

*

**

CD24

*

CD79b

**

*

*

CD81* * *

*

Mean

Fluor

esc

enc

eInt

ens

ity

(Log

sca

le)

CLL vs Normal B Cell Subsets

• p<0.05

CLL cells

Immature B cells

Naive B cells

Unswitchedmemory B Cells

Switched memoryB cells

Plasmablasts

CLL MRD: Multiple Patient samplesPathological vs each subset of normal B cells in PB

CD200

* *

* *

ImmatureB-cells

NaiveB-cells

MemorySwitchedB-cells

MemoryUnswitched

B-cells

CLL

CLL vs Immature B-cells CLL vs Naive B-cells CLL vs unswitched memory CLL vs Switched memory

Phenotypic differences between CLL cells and distinct maturation-associated subsets of

normal PB B-cells

CLL CLL

CLL

Marker PC1

IgM 23

CD5 13

CD38 11

CD200 9.5

ROR-1 7.5

Marker PC1

CD5 22

CD200 15

CD27 15

ROR-1 13

CD38 9.3

CD180 5.9

© Dept. of Immunology, Erasmus MC, Rotterdam

Molecular processes in precursor-B-cells and

peripheral B-cells

PB LYMPHOID TISSUES PB BM/TISSUES

PHENOTYPIC PROFILES OF PB B CELLS

Caraux et al, Haematologica 2010

Perez-Andres et al, Clin Cytometry 2010

BCR-associated

signaling

molecules

T cell-associated

molecules

involved in B-T

cell interactions

CD5

CD19

CD20

CD21

CD22

CD45

CD53

CD81

sIgH

CD23

CD25

CD27

CD40

Immature Naive PC

Other cell

surface

molecules

CD86

CD95

CD200

HLA-DR

++

+/++

-

+ +d

+ -

-

+ -

++ +/++

+d

++ +

++ +d

-/++ -

--

- ++

+

Memory

-/+ -

+ +

+ +

++ ++

+ +

++ ++

+ ++

+ ++++

-/++ -

- +

- +

++ ++ ++ +

- - - +

- - -/+ +

+ -

++ ++ ++ +/++

CCR6

CD138

+ + + -

- - - -/+d

CD10

CD24

CD38

CD43

CD53

+

+

+

+

-

- - -

+d + -

-/+d -/+d +++

- - +

+ ++ +d

N.A. N.A.

IMMUNOPHENOTYPE OF B-CELL SUBSETS IN LYMPHOID TISSUES

CD20 CD38 CD10 CD5 CD23 SmIg Bcl2 Ki67

Mantle zone B-cell + + - -/+ -/+ + + -

GC B-cell + + + - - + + +

Marginal zone B-cell + -/+ - - - + + -

PEREZ-ANDRES et al, Clin Cytometry, 2010

23/10/2017

7

B-CELL MATURATION TO EFFECTOR PLASMA CELLS & MEMORY B-CELLS AT LYMPHOID TISSUES

Plasma cells

T-cell stimulation

CD3-FITC

B-cells

CD20-PB

T-B cell doublets

T-cells

GC B-cells

CD44-PerCPCy5.5

CD10-PECy7

Naïve B-cells

Plasma cells

CD38-AF700

CD27-APC

Memory B-cells

GC

B-cells

Cent

roblast

s

Cent

rocy

tes

CXCR4-PEN.o

f ce

lls

Cell subset % B-cells

Naïve B-cells 48%+10%Centrocytes 13%+8%Centroblasts 10%+5%Memory B-cells 26%+10%Plasmablasts 1.9%+1.1%

Memory B-cells

Ag-recognition

Clonal expansion

Maturation

TH

FDC

IGH class switch

Somatic hypermutation

Naïve B-cell

LN: PHENOTYPIC CHANGES DURING B CELL MATURATION

103

102

101

Small Mantle Non-cleaved Centrocyte Centroblast Marginal zone PlasmablastLymphocyte cell small cell B-lymphocyte

CD20

CD79a

Bcl2 Bcl2

CD38

CD5

CD23 CD10

Bcl6

CD43

CD43

CLL MCL Burkitt ABC FL & GC MZL LPL Lymphoma DLBCL DLBCL MALT

Antigen profiles of B-CLPD classified per diagnostic category

BL CLL DLBCL FL HCL LPL MCL MZL N



© Dept. of Immunology, Erasmus MC, Rotterdam

Molecular processes in precursor-B-cells and

peripheral B-cells

1400

1000

30

200

<0.01

600

*

δδ

N. of

cells/

uLPB

Age (years)

Immature B-lymphocytes

Memory B-cells400

200

100

30

<0.01

*#

#

*

Plasma cells100

60

20

3

<0.01

#

## #

##

*

3000

2000

100

400

<0.01

1000

Naïve B-lymphocytes

*

δ

δ

Blanco et al. Manuscript in preparation

DISTRIBUTION OF NORMAL PB B-CELL SUBSETS THROUGH LIFE: MATURATION-ASSOCIATED SUBSETS

*p <0.05; # p <0.01; δ p <0.001. vs. previous age group

1-5 mo

1-11 mo

6-23 mo

2-4 y

23/10/2017

8

SIMULTANEOUS ANALYSIS OF PLASMA CELLS FROM TONSILS, PB AND BM

Tonsillar PC

PB PC

BM PC

Tonsillar PC

PB PC

BM PC

PB vs BM PLASMA CELL PHENOTYPES

BM plasma cells (PC)

BM-Clonal PC

PC 2

PC 1

BM-Normal PC

PB plasma cells (PC)

PB-Normal PC

PB-Clonal PC

PC 2

PC 1

• BM: 36% normal PCs; 64% clonal PCs

1.487 clonal PCs

• PB: 97% normal PC; 3% clonal PC

2,01 PC/μL (1,94 nPC/ μL; 0,067 cPC/μL)41 clonal PCs

nPC: normal plasma cells cPC: clonal plasma cells Other unclassified cells

PB vs BM PLASMA CELL PHENOTYPES EuroFlow-IMF NGF-MRD DESIGN

1. IDENTIFICATION OF MOST EFFICIENT MRD DISCRIMINATING MARKERS (n=94)

N. Marker selectedTimes within

top 8

1 CD19:PE Cy7 62

2 CD45:PB 57

3 CD56:PE 56

4 CD81:APC H7 56

5 CyIgλ:APC H7 47

6 CD27:PerCP Cy5.5 46

7 CD117:APC 38

8 CyIgκ:APC 36

9 B2micro:PerCP Cy5.5 36

10 CD38:FITC 32

11 CD138:PO 20

12 CD28:PE 19

nPC reference populations

(n= 31 merged normal/reactive

BM samples)

cPC populations from MM patients

(n= 63 merged MM patients BM

samples)

23/10/2017

9

IgA1

IgA2

IgD

IgG1

IgG2

IgG3

IgG4

IgM

IgA1

IgA2

IgD

IgG1

IgG2

IgG3

IgG4

Plasma

cells

CD27- and CD27+

Memory B-cells

Dissection of the blood B-cell compartment

PB LYMPHOID TISSUES PB BM/TISSUES

IgM

IgG1+

IgG2+

IgG3+

IgG4+

IgA1+

IgA2+

Only-IgD+

IgM(D)+

IgMD+

IgA1+

IgA2+

Only-IgD+

IgG1+

IgG2+

IgG3+

IgG4+

IgM+ IgA1+

IgA2+

Only-IgD+

IgG1+

IgG2+

IgG3+IgG4+

IGH isotypesSµ Sγ3 Sγ1 Sγ4Sγ2Sα1 Sα2Sε

V D J Cµ Cδ Cγ3 Cγ1 Cα1 Cγ2 Cγ4 Cε Cα2

PC1

PC

2

Immature

Naive CD5+

Naive CD5-

Memory Non-Switched

Memory Swt

(n=45 subsets)

Plasma cells

(n=64 subsets)

IGH class switch analysis in memory B-cells and plasma cells

Responsible scientists: Alberto Orfao , Martin Perez, Elena Blanco

Identification of 115 functional B-cell populations in normal peripheral blood



%of

S+G2/M

cells

LN

0%

5%

10%

15%

20%

25%

30%

BM PB BM

*

** *

*

*

*

*

CD45lo

CD19lo

CD45+

CD19hi

B-cell precursors

CD45hi

CD19+

CD20+

CD23+

CD20+

CD23-

CD38lo

CD20+

CD38+

CD20hiCD38hi

CD20lo

CD38hi

CD19+

Mature B-lymphocytes Plasma cells

PROLIFERATIE RATE OF NORMAL B-CELLS ALONG THEIR MATURATION

*p<0.05Quijano et al, Blood 2008

20

4

1

<0.01

10

30

*

#

# #


IgM+

50

30

10

<0.01

3

IgA1+

**

#

#

*

Age (years)

4

2

1

<0.01

0.3

IgG2+

*

**

4

2

1

<0.01

0.3

IgG3+

#

#

#

IgG1+20

5

1

<0.01

10*

*

**

#2

1

<0.01

0.4*

IgD+

0.2

0.1

<0.01

0.4 IgG4+*

<0.01

10

5

2

0.30

IgA2+*

*

#


DISTRIBUTION OF NORMAL PB B-CELL SUBSETS THROUGH LIFE: PLASMA CELLS

*

Sµ Sγ3 Sγ1 Sγ4Sγ2Sα1 Sα2Sε


6-11 mo 6-11 mo

6-11mo

6-11mo

12-23mo

12-23mo

5-9 y2-4 y

N. of

cells/

uLPB



10

6

0.3

<0.01

2

IgA2+

*

#

200

150

100

50

<0.01

IgMD+

*

# #

#

#

6

4

2

<0.01

10.4

IgD+*

δ

#

150

100

50

10

<0.01

IgG1+*

#

##

30

20

10

4

<0.01

1

IgA1+

# #

3

2

1

<0.01

0.4

IgG4+

15

10

5

<0.01

1

IgG2+

*

*20

10

5

<0.01

1

IgG3+

*

**#


DISTRIBUTION OF NORMAL PB B-CELL SUBSETS THROUGH LIFE: MEMORY B-CELLS


2-4 y

2-4 y

5-9 y

2-4 y

2-4 y

20-39 y

20-39 y

20-39 y

N. of

cells/

uLPB

Age (years)

23/10/2017

10

100

80

60

40

LOD

20

IgG3+

*

100

60

20

LOD

3

**

*

*#

δ


10

6

2

LOD

0.6

IgD+

#

δ

500

300

100

30

LOD

*

*

IgA1+ IgA2+

200

100

40

10

LOD

IgM+

*#

δ

1200

800

400

LOD

IgG1+

δ

300

150

50

LOD

10

IgG4+

*

#

δ

600

400

200

LOD

IgG2+

*

*δ

δ

δ

Plasm

a a

ntibod

yleve

ls(m

g/dl)

Age (years)

DISTRIBUTION OF NORMAL PB B-CELL SUBSETS THROUGH LIFE: SOLUBLE Ig LEVELS IN PLASMA




20-39 years

2-4 years

>80 years

40-59 years

>80 years

40-59 years

>80 years

20-39 years

0

20

40

60

80

100

1 2 3 4 5 6 7 8 9 10 11

IgM+

0

20

40

60

80

100

1 2 3 4 5 6 7 8 9 10 11

IgG3+

LODLOD

0

20

40

60

80

100

1 2 3 4 5 6 7 8 9 10 11

IgD+

LOD 0

20

40

60

80

100

1 2 3 4 5 6 7 8 9 10 11

IgG1+

LOD 0

20

40

60

80

100

1 2 3 4 5 6 7 8 9 10 11

IgG4+

LOD

0

20

40

60

80

100

1 2 3 4 5 6 7 8 9 10 11

IgA1+

LOD 0

20

40

60

80

100

1 2 3 4 5 6 7 8 9 10 11

IgA2+

LOD

Nor

malize

dCell

Num

bers

and

Ig

values

IgH SUBCLASS PLASMA CELL vs. MEMORY B-CELL SUBSETS vs. SOLUBLE IG LEVELS THROUGH LIFE

Plasma cells

Memory B cells

Soluble levels

Blanco et al. Manuscript in preparationAge (years)

6-11 mo 6-11 mo

6-11 mo

6-11 mo

12-23 mo

12-23 mo

5-9 y

2-4 y

2-4 y

2-4 y

5-9 y

2-4 y

2-4 y

20-39 y

20-39 y

20-39 y

20-39 y

2-4 y

>80 y 40-59 y

>80 y

40-59 y>80 y

20-39 y0

20

40

60

80

100

1 2 3 4 5 6 7 8 9 10 11

LOD

IgG2+

CONCLUDING REMARKS:

- Optimized multi-color antibody combinations have been proposed whichfacilitate assessment of the normal B-cell maturation compartments inhuman BM, PB and lymphoid tissues.

- Important advances have been made in the understanding of thephenotypic profiles of normal B cell maturation in different tissuecompartments.

- All the above has highlighted the potential existence of distinct B-cellmaturation pathways in the bone marrow.

- Important age-related differences are confirmed, which point out thedramatic changes which occur in PB in the first months of life, alsocontributing to a better understanding of B-cell homeostasis.

- Such increased knowledge about the normal B-cell maturation pathwaysprovides the basis for a comprehensive identification and classificationof B-cell leukemias and lymphomas as well as PID.

THE CIC/USAL-IBSAL TEAM

AKNOWLEDGEMENTS

Euroflow is an independent scientific consortium, which aims at innovation in flow cytometry for improving diagnostic patient care

www.euroflow.org

EuroFlow is a working group of EHA (European Hematology Association)

THANK YOU

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