Post on 14-Dec-2015
CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN)
Multicolor Immunophenotyping: Standardization and ApplicationsMarch 9-11, 2012 TMH, Mumbay (India)
B-CELL NEOPLASMS (PRECURSOR AND MATURE)
1. Making the diagnosisNormal reactive/regenerating malignant
Annually > 300,000 new patients with a hematological malignancy in developed countries
2. Classification of hematopoietic malignancies- relation with prognosis- relevance of risk-group definition in treatment protocols
Based on differentiation characteristics and particularly on chromosome aberrations, resulting in fusion gene transcripts or aberrantly (over) expressed genes
3. Evaluation of treatment effectivenessDetection of minimal residual disease (MRD):
MRD-based risk-group stratification (treatment reduction or treatment intensification)
Annually > 400,000 follow-up samples in leukemia patients (ALL, AML, CML)
DIAGNOSTICS IN HEMATO-ONCOLOGY
Prepared by JJM van Dongen
- B-cell precursor (immature) derived acute lymphoblastic leukemia/lymphoblastic
lymphoma
- Mature (peripheral) B-cell neoplasms
WHO CLASSIFICATION OF LYMPHOID MALIGNANCIES
(2002-2008)
B-cell precursor acute lymphoblastic leukemia/lymphoma
(B-ALL)• B Lymphoblastic Leukemia/Lymphoma, not
otherwise specified
• B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities
– BCP-ALL/LL with t(9:22) (q34;q11.2); BCR/ABL
– BCP-ALL/LL with t(v;11q23); MLL rearranged
– BCP-ALL/LL with t(12;21) (p13;q22); TEL/AML1 (ETV6-RUNX1)
– BCP-ALL/LL with hyperdiploidy
– BCP-ALL/LL with hypodiploidy (Hypodiploid ALL)
– BCP-ALL with t(5;14)(q31;q32)(IL3-IGH)
– BCP-ALL with t(1;19)(Q23;P13.3); (E2A-PBX1; TCF3/PBX1)
ALOT
BCP-ALL T-ALL AML/MDS
4 tubes 4 tubes 4 to 7 tubes
1 tube
ALOT: B-cell precursor ALL
BM stained with ALOT 8-color tube
CyCD3CD7sCD3CD19
CyCD79aCyMPOCD45CD34
Responsible scientist: Ludovic Lhermitte
ALOT (Acute Leukemia Orientation Tube)
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3
Designed for assessment of the nature of immature blast cell
populations in acute leukemia samples
Designed to choose appropriate immunophenotypic panel(s)
Acute Leukemia Orientation Tube (AL0T)
Target Antigen Fluorochrome conjugateGating markers
(first level)Gating Markers (second level) Immaturity markers Lineage markers
cyMPO FITC X MycyCD79a PE X B, T
CD34 PerCP Cy5.5 X X -CD19 PE CY7 X B, MyCD7 APC X X T, My
smCD3 APC H7 X TcyCD3 Pacific Blue X TCD45 PO X X -
ALOT
EuroFlow ALOT: assessment of blast cell lineage
Van Dongen et al: EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia 2012 (under revision)
Precursor-B ALL:
- B I (CD19+/cCD79a+/CD10-)- B II (CD10+/Ig-)- B III (cIgm+)- B IV (sIg+)
T-ALL:
- T I (CD7+,cCD3+)- T II(CD2+ &/or CD5+ &/or CD8+- T III (CD1a+)- T IV (CD1a-,CD3+)
CLASSIFICATION OF PRECURSOR-B ALL & T-ALL
BCP-ALL panel
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
smIgK CD45 cyIgM CD33 CD34 CD19smIgM
+CD117 smIgL
CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24
CD21 CD45CD15
+CDw65 NG2 CD34 CD19 CD123 CD81
BCP-ALL
BC
P-A
LL
BCP-ALL panel
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
smIgK CD45 cyIgM CD33 CD34 CD19smIgM
+CD117 smIgL
CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24
CD21 CD45CD15
+CDw65 NG2 CD34 CD19 CD123 CD81
BC
P-A
LL
BCP-ALL panel
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
smIgK CD45 cyIgM CD33 CD34 CD19smIgM
+CD117 smIgL
CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24
CD21 CD45CD15
+CDw65 NG2 CD34 CD19 CD123 CD81
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3ALO
TB
CP
-ALL
BCP-ALL panel
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3
CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
smIgK CD45 cyIgM CD33 CD34 CD19smIgM
+CD117 smIgL
CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24
CD21 CD45CD15
+CDw65 NG2 CD34 CD19 CD123 CD81
ALO
TB
CP
-ALL
BCP-ALL panel
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3
CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
smIgK CD45 cyIgM CD33 CD34 CD19smIgM
+CD117 smIgL
CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24
CD21 CD45CD15
+CDw65 NG2 CD34 CD19 CD123 CD81
ALO
TB
CP
-ALL
BCP-ALL panel
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3
CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
smIgK CD45 cyIgM CD33 CD34 CD19smIgM
+CD117 smIgL
CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24
CD21 CD45CD15
+CDw65 NG2 CD34 CD19 CD123 CD81
Positive Diagnosis
ALO
TB
CP
-ALL
BCP-ALL panel
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3
CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
smIgK CD45 cyIgM CD33 CD34 CD19smIgM
+CD117 smIgL
CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24
CD21 CD45CD15
+CDw65 NG2 CD34 CD19 CD123 CD81
Differential Diagnosis&
Ambiguous lineage acute leukemia
ALO
TB
CP
-ALL
BCP-ALL panel
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3
CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
smIgK CD45 cyIgM CD33 CD34 CD19smIgM
+CD117 smIgL
CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24
CD21 CD45CD15
+CDw65 NG2 CD34 CD19 CD123 CD81
Maturation stage (EGIL)
ALO
TB
CP
-ALL
BCP-ALL panel
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3
CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
smIgK CD45 cyIgM CD33 CD34 CD19smIgM
+CD117 smIgL
CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24
CD21 CD45CD15
+CDw65 NG2 CD34 CD19 CD123 CD81
Alternative classification
Immunophenotypic features associated with well-defined molecular aberrations
ALO
TB
CP
-ALL
BCP-ALL panel
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3
CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
smIgK CD45 cyIgM CD33 CD34 CD19smIgM
+CD117 smIgL
CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24
CD21 CD45CD15
+CDw65 NG2 CD34 CD19 CD123 CD81
Prognosis markers
ALO
TB
CP
-ALL
BCP-ALL panel
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3
CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
smIgK CD45 cyIgM CD33 CD34 CD19smIgM
+CD117 smIgL
CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24
CD21 CD45CD15
+CDw65 NG2 CD34 CD19 CD123 CD81
LAP markers
ALO
TB
CP
-ALL
BCP-ALL panel
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3
CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
smIgK CD45 cyIgM CD33 CD34 CD19smIgM
+CD117 smIgL
CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24
CD21 CD45CD15
+CDw65 NG2 CD34 CD19 CD123 CD81
markers present in the virtual merged
and calculated tube Panel
orie
ntatio
n
Positiv
e dia
gnosis
Differ
entia
l dia
gnosis
Ambig
uous lin
eage
acute
leuke
mia
scr
eenin
g
Mat
uratio
n of a
rrest
sta
ging
Prognosi
s
Phenoty
pic fe
ature
s as
soci
ated
with c
hrom
osam
al a
bnormal
ity
MRD L
AP det
ectio
n
ALOT X X X XBCP-ALL - Tube 1 X X X X XBCP-ALL - Tube 2 X X X X XBCP-ALL - Tube 3 X X X X XBCP-ALL - Tube 4 X X X X X
ALO
TB
CP
-ALL
BCP-ALL panel
Pac Blue Pac Orange FITC PE PerCP Cy5.5 PE Cy7 APC APC H7
cyCD3 CD45 cyMPO cyCD79a CD34 CD19 CD7 smCD3
CD20 CD45 CD58 CD66c CD34 CD19 CD10 CD38
smIgK CD45 cyIgM CD33 CD34 CD19smIgM
+CD117 smIgL
CD9 CD45 TdT CD13 CD34 CD19 CD22 CD24
CD21 CD45CD15
+CDw65 NG2 CD34 CD19 CD123 CD81
LS CD45 CD19 CD34 cyCD3 cyMPO cyCD79a CD7 smCD3 CD20 CD58 CD66C CD10 CD38 smIgK cyIgM CD33 smIgM+CD117 smIgL CD9 TdT CD13 CD22 CD24 CD21 CD15+65 NG2 CD123 CD81
31 parameters
ALO
TB
CP
-ALL
BCP-ALL:DETECTION OF ABERRANT PHENOTYPES
Mix of 3 different regenerating B cell populations (Haematogones)
BCP-ALL blast cells
0 256 512 768 1024
RM30243.100FL2-Area ->
PRECURSOR B-ALL: DNA ANEUPLOIDY
NORMALB-CELLS
NEOPLASTIC
B-CELLS
RESIDUAL NON-B-CELLS RM30243.100
FL2-Area ->0 256 512 768 1024
B-CELL PRECURSOR ALL: DNA ANEUPLOIDY
BCP-ALL Phenotype vs cytogenetics:
• t(9;22)* Sensitivity 100%, Specificity >90%
CD19+ CD10+ CD34++ CD38-/d CD13d
• 11q23 Sensitivity 95%, Specificity 85%CD19+ CD10- CD20-CD34+CD15+CD65+ 7.1+
• t(12;21) Sensitivity 86%, Specificity 100%
CD19+ CD10+ CD34d CD45-/d DR++
*Leukemia 15:406 Am J Clin Pathol 111:467
Leukemia 14:1225
IMMUNOPHENOTYPE OF NEOPLASTIC B-CELL
PRECURSORS
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 diploid Bcr/abl+ ALL
DNA aneuploid Bcr/abl+ ALL
CD10 CD13 CD34 CD34
CD10 CD13 CD34 CD34
CD10 CD13 CD34 CD34
CD
19
CD
19
CD
19
CD
45
CD
45
CD
45
CD
38
CD
38
CD
38
CD
19
CD
19
CD
19
Bead-based flow cytometric assay for detection of fusion proteins
5' gene A gene 3 'B fusion gene
B A
B
A
B A
B
transcrip tion
translation
cell lysate
A
B
A
B
A
B
bead
beads coatedw ith anti-Aantibody
bead
bead
AB A B
AB
FITC -conjugatedanti-B antibody
A
Patents: US 6,610,498 B1 (26 August 2003)US 6,686,165 B2 (3 February 2004)
Kindly provided by JJM Van Dongen on behalf of the Euroflow group
BCR-ABL RUO testing by EuroFlowMFI values of the different cell samples (n=217)
135
repeated analysis for confirm ationof w eak positiv itynegativep210
p190
result o f R Q -PC R
Weerkamp et al, Leukemia, 2009
1. High specificity of BCR-ABL RUO High concordance (100%; 145/145) between BCR-ABL PCR results and the BCR-ABL RUO results; Results: - 17/78 precursor-B-ALL were BCR-ABL positive in both
assays (mainly adults) - 19/19 CML were BCR-ABL positive in both assays (with
borderline positivity in one CML case; MFI of 144)- 0/48 of other (acute) leukemias were BCR-ABL
positive
2. Mean Fluorescence Intensity (MFI) values two main groups of positive patient samples were seen:
● high level positivity: MFI values ≥ 1,000● lower level positivity: MFI values ≥ 135, but < 1,000
negative samples were defined as MFI values < 135
3. Different MFI values in precursor-B-ALL and CMLPrecursor-B-ALL: 88% (15/17) high level positivityCML: 84% (16/19) low level positivity (true low expressionor remaining protease activity?)
Results concerning BCR-ABL RUO kitWeerkamp et al, Leukemia, 2009
Immunobead flow cytometry with BD™ CBA
Flex beads BCR-ABL t(9;22) fusion protein:
Specificity
Black: 697 (t(1;19), neg. control)Blue: TOM-1, BCR-ABL+ (p190)Green: LAMA-84 BCR-ABL+ (p210)Purple: AR230, BCR-ABL+ (p230)
Catching antibody: anti-BCR (clone 3E2C10)Bead: BD-Flex bead (A7)Detection antibody: biotinylated anti-ABL (clone 8E9) + SA-PE
Final Statement about the BCR-ABL RUO testing
The main advantages of the immunobead assay are: 1. Not dependent of the breakpoint position in the fusion gene;
2. No need for special laboratory facilities other then a routine flow cytometer;
3. Providing results within several hours;
4. The possibility to run in parallel to routine immunophenotyping: no extra technician time needed !!;
5. Allowing multiplexing with differently-labeled beads, that can detect different fusion proteins within the same disease category
PANELS OF IMMUNOBEADS FOR THE CLASSIFICATION OF ACUTE LEUKAEMIAS
Precursor-B-ALL AML ‘MLL’ T-ALL
BCR-ABL PML-RARA MLL-AF4 CALM-AF10
TEL-AML1 AML1-ETO MLL-AF9 LMO2
E2A-PBX1 CBFB-MYH11 MLL-AF10 HOX11L2
( MLL-AF4) MLL-ENL TAL1
MLL-AF6
Precursor B-ALL multiplex tube: E2A-PBX1 t(1;19)
Sensitivity for detection on cell line <10%
Specificity E2A-PBX1 (Dec 12, 2008)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
RS(4,11) K562 697 RCH-ACV Kasumi NB4 REH
cell line
MF
I
0
100
200
300
400
500
600
700
800
med
ian
sign
al
*Exp. Performed on April 27th 2007Neg. patientsPos patients
Specificity TEL-AML (July 30th 2008)
0
10000
20000
30000
40000
50000
60000
70000
ME1 RS4,11 K562 697 RCH-ACV
Kasumi NB4 REH
cell line
MF
I
Sensitivity for detection REH cell line in:– WBC : 10-50%– PBMC : 1-10%
TEL-AML in patients
0
10000
20000
30000
40000
50000
60000
70000
dono
r 1
dono
r 2
dono
r 3
dono
r 4
dono
r 5
TE
L-A
ML
patie
nt 1
TE
L-A
ML
patie
nt 2
TE
L-A
ML
patie
nt 3
TE
L-A
ML
patie
nt 4
TE
L-A
ML
patie
nt 5
B-A
LL c
ontr
ol 1
B-A
LL c
ontr
ol 2
B-A
LL c
ontr
ol 3
B-A
LL c
ontr
ol 4
B-A
LL c
ontr
ol 5
healthy control TEL-AML precursor B-ALL w ithouttranslocation
MF
I
Precursor B-ALL multiplex tube: TEL-AML1 t(12;21)
Sensitivity for detection MV4;11 line in:– 697 cell line: 10%– WBC : 10-50% (close to 10%)– PBMC: idem 10%
Specificity MLL-AF4
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
ME1
RS(
4;11
)
MV(
4;11
)
ALL-
PO
K562 69
7
RC
H-A
CV
Kasu
mi
NB4
REH
MFI
patients in MLL-AF4
0
1000
2000
3000
4000
5000
6000
7000
8000
do
no
r
do
no
r
do
no
r
do
no
r
do
no
r
ML
L-
ML
L-
ML
L-
ML
L-
ML
L-
B-A
LL
B-A
LL
B-A
LL
B-A
LL
B-A
LL
MLL-AF4 precursor B-ALL control
MF
I
Precursor B-ALL multiplex tube: MLL-AF4 t(4;11)
100% K562 100% 697
10% K562/697
10% 697/K562
R2 = green = BCR beads
R3 = pink = E2A beads
SIMULTANEOUS DETECTION OF THE BCR-ABL AND E2A-PBX FUSION PROTEINS
CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN)
Multicolor Immunophenotyping: Standardization and ApplicationsMarch 9-11, 2012 TMH, Mumbay (India)
B-CELL NEOPLASMS (PRECURSOR AND MATURE)
WHO: Mature B-cell Neoplasms
B CELL CHRONIC LYMPHOPROLIFERATIVE DISORDERSHeterogeneous group of diseases typically characterized by
a monoclonal expansion of a mature-appearing neoplastic B-lymphocyte
Mature/peripheral B cell chronic lymphoid leukemias: Chronic lymphocytic leukemia/Small B cell lymphocytic lymphoma Prolymphocytic leukemia Hairy cell leukemiaMature/pheripheral B-cell lymphomas: Lymphoplasmacytic lymphoma Splenic marginal zone lymphoma Extranodal marginal zone lymphoma (MALT-type) Nodal marginal zone lymphoma Follicular lymphoma Mantle cell lymphoma Diffuse large B-cell lymphoma Burkitt lymphomaPlasma cell neoplasias: Multiple myeloma/plasmacytoma
WHO CLASSIFICATION OF B-CLPD
DIAGNOSIS OF CLONAL HAEMATOLOGICAL DISORDERS
Clinical symptoms Laboratory and signs findings
Morphology + cytochemistry
Cytogenetics
Immunophenotyping
Molecular biology/FISH
IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT
TYPES OF B-CLPD (Orfao et al, In: “B-CLL”.Humana Press, 2004) sIg CD5 CD10 CD20 CD11c CD23 CD24 CD25 CD38 CD43 CD79b CD103 FMC7
B-CLL d + - d -/+ ++ + + -/+ + d - -
PLL + -/+ - + -/+ -/+ + -/+ -/+ -/+ + - +
HCL + - - ++ ++ - -/+ ++ - - + + +
SMZL + -/+ - + + - + -/+ - - + -/+ +
LPL + - - + - - + + -/+ - + - -/+
MCL + + - + -/+ - + -/+ - + + - -/+
FL + - + + -/+ -/d + -/+ + - + - +
LDBCL + - - + -/+ - -/+ - + - + - +
BL -/+ - + + - - + - ++ -/+ -/+ - +
MATUTES et al SCORE FOR B-CLL
MARKER PATTERN SCORE
CD5 positive 1CD23 positive 1CD22 dim 1sIg dim 1FMC7 negative 1(CD79b) dim 1
Diagnosis of CLL requires a score > 3 (4)
Matutes et al, Leukemia 1994
WHO: B-cell malignancies
Histology &cytology
DLBCLB-PLL
Cytogenetics
MCLBL
Immunophenotype
CLLHCL
Clinic
MALT
The EuroFlow comprehensive approach
Clinical question
Screening tube
Diagnostic panel
MRD
The EuroFlow comprehensive approach
Monoclonalcomponent
Monoclonalcomponent
non-IgM,Bone lesions
BM plasmacytosisSustained
monocytosisUnexplainedEosinophilia
High suspicion ofacute leukemia
e.g. blast cells observedUnexplained
cytopenia
Atypical lymphocytesSplenomegalyLymphocytosis
LN enlargement
Highmonoclonalcomponent
non-IgM
Suspicion oflymphoma
localization in“small cell number”samples e.g. CSF,
vitreous
Clinical question
Screening tube
Diagnostic panel
MRD
Monoclonalcomponent
Monoclonalcomponent
non-IgM,Bone lesions
BM plasmacytosis
ALOT LST PCSTfirst tube of PCD
SST
Sustainedmonocytosis
UnexplainedEosinophilia
reactive/polyclonal
other ? B-CLPD
High suspicion ofacute leukemia
e.g. blast cells observedUnexplained
cytopenia
Atypical lymphocytesSplenomegalyLymphocytosis
LN enlargement
clonal
reactive/polyclonal
clonal/aberrant
Highmonoclonalcomponent
non-IgM
Suspicion oflymphoma
localization in“small cell number”samples e.g. CSF,
vitreous
ALOT – acute leukemia orientation tubeLST – lymphocytosis screening tubePCD – Plasma cell discrasia screening tubeSST – small sample tube
Clinical question
Screening tube
Diagnostic panel
MRD
The EuroFlow comprehensive approach
Monoclonalcomponent
Monoclonalcomponent
non-IgM,Bone lesions
BM plasmacytosis
ALOT LST PCSTfirst tube of PCD
SST
BCP-ALL T-ALL AML/MDS B-CLPDlimited T-CLPD NK-CLPD
Sustainedmonocytosis
UnexplainedEosinophilia
reactive/polyclonal
other ? B-CLPD
High suspicion ofacute leukemia
e.g. blast cells observedUnexplained
cytopenia
Atypical lymphocytesSplenomegalyLymphocytosis
LN enlargement
clonal
reactive/polyclonal
clonal/aberrant
first4 tubes
PCD
Highmonoclonalcomponent
non-IgM
Suspicion oflymphoma
localization in“small cell number”samples e.g. CSF,
vitreous
first4 tubes
B-CLPDcomplete
Comprehensive network of panels aiming the diagnosis and characterization of the major WHO
entities
Clinical question
Screening tube
Diagnostic panel
MRD
The EuroFlow comprehensive approach
Clinical question
Screening tube
Diagnostic panel
MRD
The EuroFlow comprehensive approach
Monoclonalcomponent
Monoclonalcomponent
non-IgM,Bone lesions
BM plasmacytosis
ALOT LST PCSTfirst tube of PCD
SST
BCP-ALLL T-ALL AML/MDS B-CLPDlimited T-CLPD NK-CLPD
Sustainedmonocytosis
UnexplainedEosinophilia
reactive/polyclonal
other ? B-CLPD
CLL
non-CLL
CLL
MCL
FCL
HCL
other clonal B
reactive
aberrantab+
reactive
aberrantNK cellsvarious
subtypesofBCP-ALL
MDS
PNH
CML
CML-BC
other MPD
High suspicion ofacute leukemia
e.g. blast cells observedUnexplained
cytopenia
Atypical lymphocytesSplenomegalyLymphocytosis
LN enlargement
clonal
reactive/polyclonal
clonal/aberrant
first4 tubes
PCD
various subtypesof PCD
Highmonoclonalcomponent
non-IgM
Suspicion oflymphoma
localization in“small cell number”samples e.g. CSF,
vitreous
first4 tubes
varioussubtypesof T-ALL
varioussubtypesofAML
B-CLPDcomplete
aberrantgd+
THE EUROFLOW APPROACH TO LEUKEMIA/LYMPHOMA IMMUNOPHENOTYPING
Clinical question
Diagnostic screening tube
“Diagnostic classification”
panel
MRD monitoring
Evaluation
Majority of diseases?
Majority of
cases?
New disease entities?
Knowledge
14 Major groups
154 Nosologic entities
Experience
Reference profiles
CONSTRUCTION OF EUROFLOW LEUKEMIA/ LYMPHOMA IMMUNOPHENOTYPING ANTIBODY
PANELClinical
request/need
Medical indication
Design of MAb panels (Medical indication-oriented) & immuno-phenotyping
strategy
TechniquesPanel evaluation vs conventional in-use
panels
Panel optimization (re-design)
Panel evaluatio
n
Proposed
strategy
Panel optimization (re-design)
2-8 cycles
Panel construction
Selection of:
• Fluorochromes• Antibodies• Fluorochrome + antibody combinations• 8-color combinations• Panels
Selection of:
• Fluorochromes• Antibodies• Fluorochrome + antibody combinations• 8-color combinations• Panels
Reagent stability
Brightness
Low fluorescence background levels
No spectral overlap between fluorochrome emissions
Panel construction
Initial selection
FL Chann
elLaser
Commonly available
fluorochromes
1 Violet Pacific Blue
HORIZON V450
2* Violet AmCyan
Pacific OrangeHORIZON V500
3 BlueFITC
Alexa Fluor 488
4 Blue PE
5 Blue PE-TxRed
6 BluePerCP Cy5.5
PerCP
7 Blue PE Cy7
8 Red APC
Alexa Fluor 647
9 RedAPC Cy7APC H7
Alexa Fluor 700
Further comparisons
FL Chann
elLaser
Commonly available
fluorochromes
1 Violet Pacific Blue
HORIZON V450
2 Violet AmCyan*
Pacific OrangeHORIZON V500
3 BlueFITC
Alexa Fluor 488
4 Blue PE
5 Blue PE-TxRed
6 BluePerCP Cy5.5
PerCP
7 Blue PE Cy7
8 Red APC
Alexa Fluor 647
9 RedAPC Cy7APC H7
Alexa Fluor 700
FLUOROCHROME SELECTION
*Alternative new additional fluorochromes currently under evaluation
Responsible scientists: T.Kalina, J.Flores
Panel construction – Antibodies
Selection of:
• Fluorochromes• Antibodies• Fluorochrome + antibody combinations• 8-color combinations• Panels
Backbone antibodies
The same antibodies in every tube of the panel
Essential for merge-calculation function
Characterization antibodies
- Lineage assessment, - Differentiation lineage
markers,- Maturation stage, - Aberrant markers,- Relation to cytogenetic
abnormality, - LAP, - MRD …
Panel construction – Antibodies
Backbone antibodies
The same Ab in every tube of the panel
Essential for merge-calculation function
Backbone markers:
Should identify all cells belonging to the target lineage, either normal or malignant
Backbone candidates for B-CLPD: CD19? CD20? CD22? CD37?
- Aberrant underexpression of CD19 and/or CD20 frequently observed- κ/CD37/λ/CD19/CD22/CD20 tested in 69 B-NHL cases
Responsible scientist: S. Böttcher
Panel construction – Antibodies
Panel Construction: selection of backbone B cell markers
Responsible scientist: S. Böttcher
Conclusion: CD37 & CD22 redundant, as CD20 PacB plus CD19 PE-Cy7 were sufficient to identify all malignant B cells in all cases
Panel construction
Selection of:
• Fluorochromes• Antibodies• Fluorochrome + antibody combinations• 8-color combinations• Panels
Panel Pac Blue
Pac Orange FITC PE
PerCP
Cy5.5
PE Cy7 APC APC
H7
BCP-ALL CD45 CD34 CD19
T-ALL cyCD3 CD45 CD3
AML/MDS HLADR CD45 CD34 CD11
7
B-CLPD CD20 CD45 CD19
T-CLPD CD4 CD45 CD3 CD8
NK-CLPD CD45 CD3 CD56 CD19
PCD CD45 CD138 CD38 CD19
Panel construction – Fluorochrome + antibody BB combinations
Monoclonalcomponent
Monoclonalcomponent
non-IgM,Bone lesions
BM plasmacytosis
ALOT LST PCSTfirst tube of PCD
SST
Sustainedmonocytosis
UnexplainedEosinophilia
High suspicion ofacute leukemia
e.g. blast cells observedUnexplained
cytopenia
Atypical lymphocytesSplenomegalyLymphocytosis
LN enlargement
Highmonoclonalcomponent
non-IgM
Suspicion oflymphoma
localization in“small cell number”samples e.g. CSF,
vitreous
Pac Blue
Pac Orang
eFITC PE
PerCP Cy5.5
PE Cy7 APCAPC H7
CD20CD4
CD45Lambd
aCD8
KappaCD56
CD5CD19
TCR gdCD3 CD38
LST – Lymphocytosis screening tube
Responsible scientist: J. Flores Montero
Pac Blue
Pac Orang
eFITC PE
PerCP Cy5.5
PE Cy7 APCAPC H7
CD20CD4
CD45Lambd
aCD8
Kappa
CD56CD5
CD19TCR gδ
CD3 CD38
Responsible scientist: J. Flores Montero
Able to identify all the sample major populations:
Non-hematopoietic cells
LST – Lymphocytosis screening tube
Pac Blue
Pac Orang
eFITC PE
PerCP Cy5.5
PE Cy7 APCAPC H7
CD20CD4
CD45Lambd
aCD8
KappaCD56
CD5CD19
TCR gδCD3 CD38
Responsible scientist: J. Flores Montero
Able to identify all the sample major populations:
Non-hematopoietic cells
T lymphocytes
LST – Lymphocytosis screening tube
Pac Blue
Pac Orang
eFITC PE
PerCP Cy5.5
PE Cy7 APCAPC H7
CD20CD4
CD45Lambd
aCD8
KappaCD56
CD5CD19TCR gδ
CD3 CD38
Responsible scientist: J. Flores Montero
Able to identify all the sample major populations:
Non-hematopoietic cells
T lymphocytes
B lymphocytes
LST – Lymphocytosis screening tube
Pac Blue
Pac Orang
eFITC PE
PerCP Cy5.5
PE Cy7 APCAPC H7
CD20CD4
CD45Lambd
aCD8
Kappa
CD56CD5
CD19TCR gδ
CD3 CD38
Responsible scientist: J. Flores Montero
Able to identify all the sample major populations:
Non-hematopoietic cells
T lymphocytes
B lymphocytes
NK cells
LST – Lymphocytosis screening tube
Pac Blue
Pac Orang
eFITC PE
PerCP Cy5.5
PE Cy7 APCAPC H7
CD20CD4
CD45Lambd
aCD8
KappaCD56
CD5CD19
TCR gδCD3 CD38
Responsible scientist: J. Flores Montero
Able to identify all the sample major populations:
Non-hematopoietic cells
T lymphocytes
B lymphocytes
NK cells
Plasma cells
LST – Lymphocytosis screening tube
Pac Blue
Pac Orang
eFITC PE
PerCP Cy5.5
PE Cy7 APCAPC H7
CD20
CD4CD45
Lambda
CD8
Kappa
CD56CD5
CD19
TCR gδ
CD3 CD38
Responsible scientist: J. Flores Montero
Able to identify all the sample major populations:
Non-hematopoietic cells
T lymphocytes
B lymphocytes
NK cells
Plasma cells
(T-cell subpopulations)
LST – Lymphocytosis screening tube
Pac Blue
Pac Orang
eFITC PE
PerCP Cy5.5
PE Cy7 APCAPC H7
CD20CD4
CD45Lambd
aCD8
KappaCD56
CD5CD19
TCR gδCD3 CD38
Responsible scientist: J. Flores Montero
Able to identify all the sample major populations:
Non-hematopoietic cells
T lymphocytes
B lymphocytes
NK cells
Plasma cells
(T-cell subpopulations)
(B-cell subsets & Ig light chain restriction)
LST – Lymphocytosis screening tube
Pac Blue
Pac Orang
eFITC PE
PerCP Cy5.5
PE Cy7 APCAPC H7
CD20CD4
CD45Lambd
aCD8
KappaCD56
CD5CD19TCR gδ
CD3 CD38
Responsible scientist: J. Flores Montero
Able to identify all the sample major populations:
Non-hematopoietic cells
T lymphocytes
B lymphocytes
NK cells
Plasma cells
(T-cell subpopulations)
(B-cell light chain restriction)
B-NHL panel backbone
LST – Lymphocytosis screening tube
Panel construction
Selection of:
• Fluorochromes• Antibodies• Fluorochrome + antibody combinations• 8-color combinations• Panels
Characterization markers
CD10, CD20, CD22 CD24, CD27, CD38 CD39, CD43, CD63
CD81, CD95, CD138 Bcl-2, HLA-
DR, IgM
Normal B lymphopoiesi
s
CD11a, CD11c, CD31, CD49d, CD62L, CXCR5,
CCR6, LAIR1
B cell homing
Known to differentiate
CD5, CD23, CD25 FMC7, CD79b,
CD103, CD200, sIg
Responsible scientist: Sebastian Bottcher
Characterization markers
CD10, CD20, CD22 CD24, CD27,
CD38 CD39, CD43, CD63 CD81, CD95, CD138 Bcl-2,
HLA-DR, IgM
Normal B lymphopoiesi
s
CD11a, CD11c, CD31, CD49d, CD62L, CXCR5,
CCR6, LAIR1
B cell homing
Known to differentiate
CD5, CD23, CD25 FMC7, CD79b,
CD103, CD200,sIg
Responsible scientist: Sebastian Bottcher
Tested markers (n=66):
• Backbone markers (e.g. CD19, CD20, CD22, CD37, CD45).• Lineage assignment and maturation stage (e.g. Bcl-2,
HLA-DR, IgM, CD10, CD43, CD24, CD27, CD38, CD39, CD63, CD81, CD95, CD138).
• Disease specific (e.g. CD5, CD23, CD25, CD79b, CD103, CD200).
• Integrins and chemokine receptors (e.g. CD11a, CD11c, CD31, CD49d CD62L, CXCR5, LAIR1).
150 cases of B-Lymphoproliferative disorders tested; aim:
• Improve differential classification of B-NHL• Avoid markers with redundant information
FINAL: 4 tube 8-color panel (20 antibodies)
X
XX
X – univariate analysis
X – multivariate analysis
X
XX
Panel construction – characterization markers
Responsible scientist: S. Böttcher
LST + BCLPD classification panel
CD49dCXCR5CD19CD22CD95CD103CD45CD204
CD27CD19HLA-DRCD39CD62LCD45CD205R
CD81sIgMCD19CD11cLAIRCD31CD45CD203
CD43CD200CD19CD79bCD10CD23 CD45CD202
CD38CD3CD19/
TCRgd
CD5sIgK /CD56
sIgl/CD8
CD45CD20 /CD4
1=LST
APC-H7APCPECy7PerCP-Cy5.5
PEFITCPacOrang
e
PacBlue
CD20/CD4/CD45/sIgl/sIgK/CD8/CD56/CD5/CD19/CD38/CD23/CD10/CD79b/CD200/CD43/CD31/LAIR1/CD11c/sIgM/CD81/CD103/CD95/CD22/CXCR5/CD49d/CD62L/CD39/HLA-DR/
CD19/CD27
30-colors flow cytometry !
Responsible scientist: Sebastian Bottcher
CD20/CD4/CD45/sIgl/sIgK/CD8/CD56/CD5/CD19/CD38/CD23/CD10/CD79b/CD200/CD43/CD31/LAIR1/CD11c/sIgM/CD81/CD103/CD95/CD22/CXCR5/CD49d/CD62L/CD39/HLA-DR/
CD19/CD27
30-colors flow cytometry !
LST + BCLPD classification panel
CD49dCXCR5CD19CD22CD95CD103CD45CD204
CD27CD19HLA-DRCD39CD62LCD45CD205R
CD81sIgMCD19CD11cLAIRCD31CD45CD203
CD43CD200CD19CD79bCD10CD23 CD45CD202
CD38CD3CD19/
TCRgd
CD5sIgK /CD56
sIgl/CD8
CD45CD20 /CD4
1=LST
APC-H7APCPECy7PerCP-Cy5.5
PEFITCPacOrang
e
PacBlue
Responsible scientist: Sebastian Bottcher
B-CLPD: 4-COLOUR STAINING PANEL
- FITC PE PerCP/Cy5.5 APC
- Control Control CD19 Control - sIgk sIgl CD19 CD5 - CD22 CD23 CD19 CD20
- Fmc7 CD24 CD19 CD34- CD43 CD79b CD19
CD49d- CyBcl2 CD10 CD19 CD38- CD103 CD25 CD19 CD11c- sIgM CD27 CD19 CCR6- CD3 CyZap70 CD19
CD5
REFERENCE DATAFILES FOR CLL B-CELLS
CD19+ CLL B-cells
Case number
C
D1
9-P
EC
y7
CLL case 2
CLL case 3
CLL case 4
Merge Calculated Data files
Responsible scientist: Sebastian Bottcher
CLL case 1
Characterization markers:
CD200
Responsible scientist: Sebastian Bottcher
MZLMCLHCL LPLFLCLL DLBCLBL
Characterization markers:
LAIR1(CD305)
MZLMCLHCL LPLFLCLL DLBCLBL
Responsible scientist: Sebastian Bottcher
Characterization marker: CD5
MZL MCLHCL LPL FL CLL DLBCLBL
Parameter Significance
1 IgM 14.02
2 CD200 13.76
3 CD79b 11.94
4 CD23 8.57
5 CD38 7.73
CLL immunophenotypic diagnosis
CD10+ CD10-
Best 5 markers for the DD between CLL and
MCL according to EuroFlow analysis
Responsible scientist: S. Böttcher
PCA of total immunophenotype
MCL
CLL
Principal component 1 →
Pri
nci
pal co
mpon
en
t 2
→
1 SD 2 SD
Responsible scientist: Sebastian Bottcher
PCA of total immunophenotype
MCL
CLL
Principal component 1 →
Pri
nci
pal co
mp
on
en
t 2
→
1 SD 2 SD
Responsible scientist: Sebastian Bottcher
PC1
1 IgM 14.09
2 CD200 14.06
3 CD79b 13.39
4 CD23 8.60
5 CD20 6.43
…
MZL MCLHCL LPL FL CLL DLBCLBLCD10+ CD10-
Parameter Significance
1 IgM 14.02
2 CD200 13.76
3 CD79b 11.94
4 CD23 8.57
5 CD38 7.73
Best 5 markers for the DD between CLL and
MCL according to EuroFlow analysis
Responsible scientist: S. Böttcher
Characterization marker: smIgM
CLL immunophenotypic diagnosis
MZL MCLHCL LPL FL CLL DLBCLBLCD10+ CD10-
Parameter Significance
1 IgM 14.02
2 CD200 13.76
3 CD79b 11.94
4 CD23 8.57
5 CD38 7.73
Best 5 markers for the DD between CLL and
MCL according to EuroFlow analysis
Responsible scientist: S. Böttcher
Characterization marker: CD200
CLL immunophenotypic diagnosis
Major consecutive development steps in the design of the EuroFlow B-CPLD panelVersion Tube Fluorescence channel
PacB PacO FITC PE PECy5 PECy7 APC APCCy7
Backbone 1 1 SmIg CD37 SmIg CD19 CD22 CD20
Backbone 2 1 CD20 SmIg CD37 SmIg CD19 CD22
1 CD20 CD45 Ig Ig PerCPCy5.5 CD19 IgM AF700CD5 CD22
2 CD20 CD45 CD103 CD10 CD5 CD19 CD43 CD22
Panel 1 3 CD20 CD45 CD81 CD79b CD5 CD19 CD23 CD22
4 CD20 CD45 CD31 CD63 CD5 CD19 CXCR5 CD22
5 CD20 CD45 CD24 LAIR1 CD5 CD19 CD11a CD22
6 CD20 CD45 CD38 CD25 CD138 CD19 CD11c CD22
1 CD20 CD45 Ig Ig CD22 CD19 CD23 APCH7CD81
Panel 2 2 CD20 CD45 CD103 CD25 CD11c CD19 IgM CD24
3 CD20 CD45 CD31 LAIR1 CD5 CD19 CD43 CXCR5
4 CD20 CD45 bcl2 CD10 CD79b CD19 CD38 CD49b
PacB: Pacific Blue; FITC: Fluorescein isothiocyanate; PE: Phycoerythrin; Cy5: cyanin5; Cy7: Cyanin7; APC: Allophycocyanin; PerCP: Peridinin-chlorophyll-protein; AF700: Alexa Fluor 700; H7: Hilite7
Responsible scientist: S. Böttcher
Major consecutive development steps in the design of the EuroFlow B-CPLD panel (continued)Version Tube Fluorescence channel
PacB PacO FITC PE PECy5 PECy7 APC APCCy7
1 CD20 CD45 Ig Ig CD22 CD19 CD23 CD81
2 CD20 CD45 CD103 CD25 CD11c CD19 IgM
Panel 3 3 CD20 CD45 CD31 LAIR1 CD5 CD19 CD43
4 CD20 CD45 bcl2 CD10 CD79b CD19 CD38 CD49d
5 CD20 CD45 CD24 CD95 CD19 CD200
1 CD20 CD45 Ig Ig CD22 CD19 CD23 CD81
2 CD20 CD45 CD103 CD25 CD11c CD19 IgM CD43
Panel 4 3 CD20 CD45 CD31 LAIR1 CD5 CD19 CD43 CD24
4 CD20 CD45 bcl2 CD10 CD79b CD19 CD83 CD49d
5 CD20 CD45 CD24 CD95 CD19 CD200 CD31
6 CD20 CD45 CD19 CXCR5 CD103
1=LST CD20 CD45 CD8 + CD56 + CD5 CD19 + CD3 CD38Ig Ig TCR
2 CD20 CD45 CD23 CD10 CD79b CD19 CD200 CD43
Panel 5 3 CD20 CD45 CD31 LAIR CD11c CD19 IgM CD81
4 CD20 CD45 CD103 CD95 CD22 CD19 CXCR5 CD49d
5 CD20 CD45 CD62L CD39 HLADR CD19 CD27
B-CLPD: Diagnostic work-flow
Monoclonalcomponent
ALOT LST
B-CLPDlimited
B-CLPDbroad
Eosinophilia
reactive/non-aberrant
CLL
non-CLL
CLL
MCL
FCL
HCL
other clonal B
Acute leukemia CytopeniaLymphocytosis
LN involvement
> 90% pure by BB
Responsible scientists: Juan Flores and Sebastian Bottcher
CLL MCL
MCLCLL MCL
Full
pane
lTu
bes
1 &
2 o
nly
BCLPD classification panel: modular design
CLL HCL
CLL HCL
CLL MZL
CLL MZL
CLL FL
CLL FL
CLL DLBCL
CLL DLBCL
Responsible scientist: Sebastian Bottcher
MZL HCL MCL CLL FL CLL
PCA of total immunophenotype: clearly separated diseases
Responsible scientist: S. Böttcher
PC1
1 CD38 9.87
2 CD10 8.92
3 IgM 8.48
4 CD200 8.33
5 CD81 7.37
…
PC1
1 IgM 14.09
2 CD200 14.06
3 CD79b 13.39
4 CD23 8.60
5 CD20 6.43
…
MCL CLLCD10+DLBCL BL
2 SD separated 1 SD separated
Responsible scientist: Sebastian Bottcher
PCA of total immunophenotype
FL CD10-DLBCL
Overlap of 1st SD
PCA of B-CLPD panel
Responsible scientist: S. Böttcher Designed by: Q Lecrevisse
BL vs. DLBCL CD10-
BL vs. DLBCL CD10+
BL vs. CLL
BL vs. FL
BL vs. HCL
BL vs. LPL
BL vs. MCL
BL vs. MZL
BL vs.Normal
Separation power of different types of BCLPD
CLL DLBCL CD10+
DLBCLCD10-
FL HCL LPL MCL MZL
BL
CLL
DLBCL CD10 +
DLBCL CD10 -
FL
HCL
LPL
MCL
1 x 1comparison n = 150
2 SD separated
1 SD separated
Overlap of 1st SD
Responsible scientist: S. Böttcher
EuroFlow B-CLPD panel: summary
• B-CLPD panel allows unequivocal classification of most mature B-cell malignancies according to WHO
• Most differential diagnoses achieved (n=32/36) efficiently except for the following 1 vs 1 comparisons:
FL vs DLBC MZL vs LPL LPL vs DLBCL MZL vs DLBCL
Responsible scientist: Sebastian Bottcher
Expert pathologist agreement with the consensus diagnosis
The NHL Classification Project, Bood 1997;89:3909-3918 Kindly provided by Raul Braylan
MZL
LPL
5 expert hematopathologists~1,400 lymphoma cases
B-CLPD: Comparative analysis of “our case” vs multiple reference groups
Responsible scientists: Sebastian Bottcher
Costa et al, Leukemia, 2010
B-CLPD:Comparative analysis of “our case” vs multiple reference groups
Responsible scientist: Sebastian Bottcher
Costa et al, Leukemia, 2010
IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT
TYPES OF B-CLPD CD5 CD19 CD38 CD20 CD23 CD10 CD79b CD43 CD11c IGM CD103 CD22
B-CLL + lo hi lo + lo lo
MCL +
HCL hi hi -/+ -/+ hi hi hi
MZL lo -/+
LPL lo -/+
FL lo +
LDBCL + -/+
LDBCL -/+ -/+
BL hi lo + + hi
IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT
TYPES OF B-CLPD CD200 CD31 CD305 CD81 CD95 CXCR5 CD49d CD62L CD39 CD27
B-CLL hi hi -/+ lo -
MCL lo -/+ -
HCL hi hi lo + -
MZL lo
LPL
FL lo lo lo - -
LDBCL lo
LDBCL + +
BL lo lo hi - - -
• How to get optimal and
comparable measurements?
• Which are the most
appropriate fluorochromes?
• What is the optimal sample
preparation protocol?
REPRODUCIBLE & OBJECTIVE RESULTS
UTILITY OF THE NEW SOFTWARE TOOLSStandardization of FCM immunophenotyping
EuroFlow participantsUniversity Institutes / Medical SchoolsErasmus MC, Rotterdam, NL J.J.M. van Dongen, V.H.J. van der Velden…
USAL, Salamanca, ES A. Orfao, J. Flores, J. Almeida, Q. Lecrevisse…
IMM, Lisbon, PT P. Lucio, A. Mendonça, A. Parreira a.o…
UNIKIEL, Kiel, DE M. Kneba, S. Böttcher, M. Ritgen, M. Brüggemann …
AP-HP, Paris, FR E. Macintyre, L. Lhermitte, V. Asnafi …
UNIVLEEDS, Leeds, GB S. Richards, A.C. Rawstron. P. Evans …
DPH/O, Prague, CZ O. Hrusak, T. Kalina, E. Mesjstrikova …
SAM, Zabrze, PL T. Szczepanski, L. Sedek …
DCOG, The Hague, NL E. Sonneveld, A. van der Sluijs-Gelling ...
KUL, Leuven, BE N. Boeckx …
HGSA, Porto, PT M. Lima, AH Santos
UFRJ, Rio de Janeiro, BR C. Pedreira, E.S. Costa
Companies (SME’s)DYNOMICS, Rotterdam, NL E. Dekking, F. Weerkamp …
CYTOGNOS, Salamanca, ES M. Martin, J. Bensadon, J. Hernandez, M. Muñoz …
www.euroflow.org
Contributors - Acknowledgements
Elizabeth MacIntyreVahid AsnafiLudovic Lhermitte
Juan Flores-MonteroJulia AlmeidaQuentin Lecrevisse
Jacques van DongenVincent van der VeldenJeroen Te MarveldeAlita van der Sluijs
Michael KnebaMonika BrüggemannSebastian BöttcherStephen RichardsPaul EvansMatt CullensRuth de TuteAndy Rawstron
Tomek SzczepanskiLukasz Sedek
Paulo LucioAndreia MendonçaEvan Jensen
Ondrej HrusakTomas KalinaEster Mejstrikova
Photo Lukasz Sedek
THANK YOU