1
UTILIDAD DE LA CITOMETRIA DE FLUJO EN EL DIAGNÓSTICO, CLASIFICACIÓN Y
MONITORIZACIÓN DE HEMOPATÍAS MALIGNAS
CANCER RESEARCH CENTER, UNIVERSITY & CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) SALAMANCA (SPAIN)
CursoCurso AvanzadoAvanzado de de ActualizaciActualizacióónn en en OncohematologOncohematologííaa porpor citometriacitometria de de flujoflujoBuenos Aires, 30 de mayo de 2011Buenos Aires, 30 de mayo de 2011
IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES
- 1953/1994: From the development of the instruments& techniques to the WHO classificationof haematological malignancies.
- 1994/2006: The ability to specifically identifyleukaemic cells: from normal phenotypesto aberrant phenotypic profiles.
- 2006/-: Recent contributions of immuno-phenotyping of haematologicalmalignancies: pointing to the future.
- 1953: The “Coulter” principle and instrument development- 1965/68: Multiparameter and multicolour flow cytometry
Marv van DillaH. Crissman
Joe Gray1970
IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES
Wallace Coulter
Wofgang Göhde
- 1953: The “Coulter” principle and instrument development- 1965/68: Multiparameter and multicolour flow cytometry- 1970: FACS: “fluorescence activated cell sorter”.
IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES
L. Herzenberg & FACS II (1976)
- 1953: The “Coulter” principle and instrument development- 1965/68: Multiparameter and multicolour flow cytometry- 1970: FACS: “fluorescence activated cell sorter”.
Len & LeeHerzenberg
IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES FluorescenceFluorescence activatedactivated cellcell sortingsorting
Prepared by A.Salvador
2
¿WHERE CAN I APPLY FLOW CYTOMETRY?
César MILSTEIN
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
IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES
Tabla 2.- Immunological classification
of ALL
Phenotype
B-ALL Ig+
T-ALL SER+
non-T non-B ALL cALLA+
cALLA- 10 10 10 10 100 1 2 3 4
nTDT
CyC
D3
- 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- 1978/80: Immunophenotyping of leukemia cells
Immunophenotypic classification of ALL- 1981: Definition of aberrant marker expression- 1985: Use of Immunophenotyping to classify FAB M7
IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES
David MASON
- 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- 1978/80: Immunophenotyping of leukemia cells
Immunophenotypic classification of ALL- 1981: Definition of aberrant marker expression- 1985: Use of Immunophenotyping to classify FAB M7- 1985: APAAP technique
IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES
Loken et al, Blood, 1987 Loken et al, Blood, 1987
IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES
- 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- 1978/80: Immunophenotyping of leukemia cells
Immunophenotypic classification of ALL- 1981: Definition of aberrant marker expression- 1985: Use of Immunophenotyping to classify FAB M7- 1985: APAAP technique- 1986: Benchtop 3-color flow cytometers- 1987: Immunophenotypic analysis of normal hematopoiesis
- 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- 1978/80: Immunophenotyping of leukemia cells
Immunophenotypic classification of ALL- 1981: Definition of aberrant marker expression- 1985: Use of Immunophenotyping to classify FAB M7- 1985: APAAP technique- 1986: Benchtop 3-color flow cytometers- 1987: Immunophenotypic analysis of normal hematopoiesis- 1988: Second MIC classification of haematological malignancies- 1993: CD45-based blast cell gating/identification- 1994: REAL classification of lymphoid neoplasias- 1997: WHO classification of lymphoid neoplasias
IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES
3
FCM IMMUNOPHENOTYPING IN THE 80`S: FCM IMMUNOPHENOTYPING IN THE 80`S: PANELS OF REAGENTS AND TECHNIQUESPANELS OF REAGENTS AND TECHNIQUES
PANELS OF REAGENTS:PANELS OF REAGENTS:
-- PanelsPanels ofof relevantrelevant markersmarkers forfor thethe diagnosticdiagnosticclassificationclassification ofof patientspatients suspectedsuspected ofof::
-- AML, ALLAML, ALL-- BB--CLPD, TCLPD, T--CLPDCLPD
TECHNIQUES:TECHNIQUES:
-- IsolationIsolation ofof MNC MNC -- DifficultDifficult toto distinguishdistinguish normal/normal/leukemicleukemic cellscells
-- IndirectIndirect andand directdirect IF IF –– FewFew fluorochromefluorochrome conjugatedconjugated MAbMAb availableavailable
-- Single Single stainingsstainings –– FewFew fluorochromefluorochrome availableavailable
DIAGNOSIS OF HAEMATOLOGICAL MALIGNANCIESDIAGNOSIS OF HAEMATOLOGICAL MALIGNANCIES
ClinicalClinical symptomssymptoms LaboratoryLaboratoryandand signssigns findingsfindings
MorphologyMorphology + + cytochemistrycytochemistry
CMDCMDMDS MDS ImmunophenotypingImmunophenotyping MRDMRDMGMG
AcuteAcute leukemiasleukemias ChronicChronic lymphoidlymphoidleukemiasleukemias
CLINICAL UTILITY OF IMMUNOPHENOTYPING OF HAEMATOLOGICAL MALIGNANCIES
- Acute leukaemias:- Lineage assignment (myeloid vs lymphoid -B or T-)
- Diagnosis of biphenotypic leukaemias/mixed lymphoid/myeloid- Phenotypic classification of B-cell precursor ALL- Phenotypic classification of T-ALL- Lineage subclassification of AML (e.g: AML with monocytic maturation)
- Chronic lymphoproliferative disorders:- Diagnosis of T & B-cell clonality- Phenotypic classification of T/NK-CLPD- Phenotypic classification of B-cell CLPD
EGIL: DEFINITION OF BAL
Score
c/mCD3
B-Lineage T-Lineage Myeloid lineage
2 cCD79a MPO
cIgM TCR Lisozyme
cCD22
1 CD2, CD5 CD13, CD33
CD20 CD8, CD10 CD117, CDw65
0.5 Tdt, CD24 Tdt, CD7 CD14, CD15
CD1a CD64
Criteria: > 2 points
IMMUNOPHENOTYPIC CLASSIFICATION OF ACUTE LEUKAEMIAS
IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT TYPES OF BTYPES OF B--CLPD CLPD ((Orfao et al, In: “B-CLL”.Humana Press, 2004)
sIgsIg CD5 CD10 CD20 CD5 CD10 CD20 CD11cCD11c CD23 CD24 CD25 CD38 CD43 CD23 CD24 CD25 CD38 CD43 CD79bCD79b CD103 FMC7CD103 FMC7
BB--CLLCLL d d ++ -- d d --/+ /+ ++++ + + + + --/+ /+ ++ d d -- --
PLL + PLL + --/+ /+ -- + + --/+ /+ --/+ + /+ + --/+ /+ --/+ /+ --/+ + /+ + -- ++
HCL + HCL + -- -- ++ ++ ++++ -- --/+ /+ ++++ -- -- + + + + ++
SMZLSMZL + + --/+ /+ -- + + + + -- + + --/+ /+ -- -- + + --/+ +/+ +
LPLLPL + + -- -- + + -- -- + + + + --/+ /+ -- + + -- --/+ /+
MCL + MCL + ++ -- + + --/+ /+ -- + + --/+ /+ -- ++ + + -- --/+/+
FL + FL + -- ++ + + --/+ /+ --/d + /d + --/+ /+ ++ -- + + -- ++
LDBCL + LDBCL + -- -- + + --/+ /+ -- --/+ /+ -- + + -- + + -- ++
BL BL --/+ /+ -- ++ + + -- -- + + -- ++++ --/+ /+ --/+ /+ -- ++
0 256 512 768 1024
10112.001
FSC-Height ->
TR
AN
SF
OR
ME
D S
SC
FSC Height
MNCMNC
10 10 10 10 100 1 2 3 4
EGV47955.003
CD4 APC ->CD4 APC
CD
8 P
erC
P
EGV47955.003
CD3 FITC ->
10 10 10 10 100 1 2 3 4
CD3 APC
TRADITIONAL FCM PHENOTYPING
MNC
MNC
Ficoll
Blood
15%15%
CriteriaCriteria forfor positivitypositivity: >20%: >20%
4
10 10 10 10 100 1 2 3 4
EGV47955.003
CD4 APC ->CD4 APC
CD8
PerC
P
MULTICOLOR FLOW CYTOMETRY vsSINGLE-STAININGS IN ONE TUBE
10 10 10 10 100 1 2 3 4
65295.003CD19 PerCPCY5.5 ->
CD3
APC
CD19 PerCP/Cy5.5
- 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- 1978/80: Immunophenotyping of leukemia cells
Immunophenotypic classification of ALL- 1981: Definition of aberrant marker expression- 1985: Use of Immunophenotyping to classify FAB M7- 1985: APAAP technique- 1986: Benchtop 3-color flow cytometers- 1987: Immunophenotypic analysis of normal hematopoiesis- 1988: Second MIC classification of haematological malignancies- 1993: CD45-based blast cell gating/identification- 1994: REAL classification of lymphoid neoplasias- 1997: WHO classification of lymphoid neoplasias
IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES
IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES
- 1953/1994: From the development of the instruments& techniques to the WHO classificationof haematological malignancies.
- 1994/2006: The ability to specifically identifyleukaemic cells: from normal phenotypesto aberrant phenotypic profiles.
- 2006/-: Recent contributions of immuno-phenotyping of haematologicalmalignancies: pointing to the future.
FLOW CYTOMETRY:FLOW CYTOMETRY:TYPE OF INFORMATIONTYPE OF INFORMATION
-- IdentificationIdentification ofof cellcell populationspopulations
-- EnumerationEnumeration ofof cellcell numbersnumbers
-- CharacterizationCharacterization ofof cellcell populationspopulations
Identification of different granulocytic subpopulations in childhood BM
E.G. van Lochem et al., Cytometry Part B 2004; 60B: 1-13.
FSC
SS
C
CD11b-APC
CD
13
-PE
CD16-FITC
CD
13-P
E
CD13-PE
SS
C
staining 5CD16/CD13/CD45/CD11b
CD16-FITC
SS
C
CD16-FITC
CD
11
b- A
PC
Myelo/monoblast Promyelocyte MetamyelocyteMyelocyte Neutrophill
IMMUNOPHENOTYPIC IDENTIFICATION OF LINEAGE IMMUNOPHENOTYPIC IDENTIFICATION OF LINEAGE COMMITMENT OF CD34COMMITMENT OF CD34+ + BM CELLSBM CELLS
nTDTnTDT FITCFITC
Cy
MP
OC
yM
PO
PE
PE
10
01
01
10
21
03
10
4
c
10 10 10 10 100 1 2 3 4
TR
AN
SF
OR
ME
D S
SC
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 SSCTRANSFORMED SSC
0 256 512 768
bCD
45
PE
RC
PC
D45
PE
RC
P1
00
10
11
02
10
31
04
b
Matarraz S et al. Leukemia 2008
Neutrophil precursors
B-cell precursors
5
10 10 10 10 100 1 2 3 4
CD38 FITC ->
TR
AN
SF
OR
ME
D S
SC
->
10 10 10 10 100 1 2 3 4
CD38 FITC ->
CD
138 P
erC
P/C
y5 -
>
CD38-FITC
CD38-FITC gated PC
T-S
SC
CD13
8-Pe
rCP/
Cy5.
5
MONOCLONAL GAMMOPATHIES: IDENTIFICATION OF CLONAL PLASMA CELLS
ClonalPC
Normal PC
CD
56
PE
9
1
D
C
CP
A
54
DC
CD19
-Pcp
Cy5
CD56-PE CD45-A
PC
Perez-Andres, J Biol Reg, 2004
DIAGNOSIS OF HAEMATOLOGICAL MALIGNANCIESDIAGNOSIS OF HAEMATOLOGICAL MALIGNANCIES
ClinicalClinical symptomssymptoms LaboratoryLaboratoryandand signssigns findingsfindings
MorphologyMorphology + + cytochemistrycytochemistry
CytogeneticsCytogenetics
ImmunophenotypingImmunophenotyping
Molecular Molecular biologybiology/FISH/FISH
*0.35% of all B-cells & O.O3% of all leucocytes
Clonal B cells
Normal B cells
ImmunophenotypicImmunophenotypic identificationidentification ofof PB PB BB--cellscells withwith a CLLa CLL--likelike phenotypephenotype
Nieto et al, Blood 2009
MINIMAL RESIDUAL DISEASE IN B-CLL
Brugiatelli M et al.(Cancer 1989)
Robertson LE et al.(Blood 1992)
Leonormand B et al.(Leukemia 1994)Cabezudo E et al.(Leukemia, 1997)
García-Vela A et al.(Leukemia, 1999)
Rawstron AC et al.(Blood 2001)
Maloum K et al.(Br J Haematol 2002)
Gupta R et al.(Am J Clin Pathol 2004)
Bottcher S et al.(Leukemia 2004)Moreton P et al.
(J Clin Oncol 2005)Montillo M et al.
(Cancer Invest 2005)
Sensitivity
10-2
10-2
10-3
10-3
10-4
10-4
10-4
10-3
10-4
10-5
10-5
Aberrant criteria
sIgκ+/sIgλ+ ratio
sIgκ+/sIgλ+ ratio
CD19+/CD5+
CD19+/CD5+
CD19+/CD79b+d
CD19+/CD20+d/CD5+/CD79b+d
CD19+/CD20+d/CD5+/CD79b+d
CD19+/CD5+
CD19+/CD5+/CD43+/CD20+d
CD19+/CD20+d/CD5+/CD79b+d
CD19+/CD20+d/CD5+/CD79b+d
Prognostic value
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Not analyzed
Not analyzed
Yes
Yes
METHODS FOR MRD METHODS FOR MRD INVESTIGATIONINVESTIGATION
101000
101011
101022
101033
101044
101055
101066
101077
101088
101099
10101010
10101111
NeoplasticNeoplastic cellscellsSensitivitySensitivitySensitivity MorphologyMorphology, , CytogeneticsCytogenetics, ,
SouthernSouthern--BlotBlot,,
FCM DNA FCM DNA aneuploidyaneuploidy
F.I.S.HF.I.S.H
FlowFlow cytometrycytometry
P.C.RP.C.R..1010--66
1010--55
1010--44
1010--33
1010--22
FCM IMMUNOPHENOTYPING IN THE 90`S: FCM IMMUNOPHENOTYPING IN THE 90`S: PANELS OF REAGENTS AND TECHNIQUESPANELS OF REAGENTS AND TECHNIQUES
PANELS OF REAGENTS:PANELS OF REAGENTS:
-- PanelsPanels ofof informativeinformative combinationscombinations ofof reagentsreagents forfor::
-- AML, ALL, BALAML, ALL, BAL-- MM, WM, MGUSMM, WM, MGUS-- BB--CLPD, TCLPD, T--CLPDCLPD-- MDSMDS
TECHNIQUES:TECHNIQUES:
-- NonNon--NRBC NRBC lysislysis -- DistinctDistinct normal normal vsvs leukemicleukemic phenotypesphenotypes
-- DirectDirect IF IF –– ManyMany fluorochromefluorochrome conjugatedconjugated MAbMAb availableavailable
-- MultipleMultiple stainingsstainings –– IncreasedIncreased numbernumber ofof fluorochromefluorochrome availableavailable
Diagnosis & follow-up ofMRD in acute
leukaemias, CLPD & MM
6
1. Making the diagnosis
Normal ↔ reactive/regenerating ↔ malignantAnnually > 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 effectiveness
Detection 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
JJM van Dongen
What problems are we experiencing?What problems are we experiencing?
-- ManyMany reagentsreagents: : costlycostly andand complexcomplex
-- NeedNeed expertiseexpertise in normal (& in normal (& referencereference) ) cellcell populationspopulations
-- TimeTime consumingconsuming
-- TechnicalTechnical limitationslimitations
-- ManyMany (my) (my) strategiesstrategies toto reachreach a similar a similar resultresult butbutsuboptimalsuboptimal
-- NotNot standardizedstandardized: : reproduciblyreproducibly harmonizedharmonized? ?
-- PartialPartial andand more more limitedlimited clinicalclinical utilityutility thanthan expectedexpected
STANDARDIZATION EFFORTS FORSTANDARDIZATION EFFORTS FORIMMUNOPHENOTYPIC STUDIESIMMUNOPHENOTYPIC STUDIES
-- CLSI CLSI ((ClinicalClinical LaboratoryLaboratory Standards Standards InstituteInstitute):):-- StetlerStetler--Stevenson et al.: Stevenson et al.: ClinicalClinical flowflow cytometriccytometric analysisanalysis ofofneoplasticneoplastic hematolymphoidhematolymphoid cellscells; ; ApprovedApproved guidelineguideline. CLSI . CLSI documentdocument H43H43--A2. CLSI, 2007A2. CLSI, 2007
-- CCSCCS ((ClinicalClinical CytometryCytometry SocietySociety):):-- Davis et al: 2006 Bethesda International Davis et al: 2006 Bethesda International ConsensusConsensus
recommendationsrecommendations onon thethe flowflow cytometriccytometric immunophenotypicimmunophenotypicanalysisanalysis ofof hematolymphoidhematolymphoid neoplasias. neoplasias. ClinClin CytometryCytometry, 72B, , 72B, 2007.2007.
-- ESCCAESCCA (European (European SocietySociety forfor ClinicalClinical CellCell AnalysisAnalysis: : www.escca.euwww.escca.eu))
-- European European LeukemiaLeukemia NetNet ((www.leukemiawww.leukemia--net.orgnet.org))
-- Consenso LatinoamericanoConsenso Latinoamericano ((ClinClin CytometryCytometry, 1998 y 2006), 1998 y 2006)
Standardization in diagnostic flow cytometry
HOWEVER: Standardization according to GLP guidelines demands for much higher levels of standardization
EuroFlow standardization aims at:– usage of comparable flow cytometers (3 lasers and ≥ 8 colors)
– full standardization of instrument settings (e.g. based on standard beads)– standardized laboratory protocols and immunostaining procedures (SOP’s)
– careful selection of optimal antibody clones per marker/CD code– selection of optimal 8-color antibody combinations and fluorochromes– design of combinations of multiple 8-color tubes: estimation and APS view
– new software for fast and easy data analysis with automated pattern recognition– recognition of normal and abnormal leukocyte subsets (complete differentiation
pathways) with the same immunostaining protocols– mapping of new patient samples against large data base of earlier collected
patient samples, analyzed with the same immunostaining protocol
Standardization according to literature generally refers to:– lists of CD codes and markers per disease category
– rarely a specific antibody is recommended and (almost) never a fluorochrome is proposed
CLINICAL APPLICATIONS OF FLOW CYTOMETRY
Microscopy Flow cytometry
Hybridoma technology
Monoclonal antibodies
Fluorochrome-conjugates
From research laboratories to clinical diagnostics
Digital instruments
>4 color flow cytometers
Higher analytical speed
Exponentially growing amount of complexinformation/data
70s-90s
XXI century
IMMUNOPHENOTYPING OFHAEMATOLOGICAL MALIGNANCIES
- 1953/1994: From the development of the instruments& techniques to the WHO classificationof haematological malignancies.
- 1994/2006: The ability to specifically identifyleukaemic cells: from normal phenotypesto aberrant phenotypic profiles.
- 2006/-: Recent contributions of immuno-phenotyping of haematologicalmalignancies: pointing to the future.
7
GUEST EDITORIAL: The Continuing Evolution of Hematology
BEREND HOUWEN
Laboratory Hematology 8:204
© 2002 Carden Jennings Publishing Co., Ltd.
EXCERPT:
The evolution of laboratory hematology is a continual process, as
the papers in the following Special Section demonstrate. These papers
cover a variety of topics related to the practice of laboratory
hematology. Application of extended differential capabilities decreases
the need for routine microscopic intervention for samples with
nucleated red blood cells. Similarly, as careful examination of flagging
performance shows, increased productivity can be achieved by fewer
morphology reviews. In most laboratories, rules for review of
hematology results are applied by the technologists. Many times these
rules are quite informal, but when formalized and embedded in a
computer-based algorithm, they can form a powerful management
tool, as illustrated in one of the papers.
THANK THANK YOUYOU
RECENT CONTRIBUTIONS OF IMMUNO-PHENOTYPING IN THE DIAGNOSIS OF
HAEMATOLOGICAL MALIGNANCIES
-- StandardizationStandardization ofof immunophenotypicimmunophenotypic analysesanalyses
-- DiagnosisDiagnosis ofof clonalclonal multilineagemultilineage diseasedisease
-- RapidRapid screeningscreening ofof relevantrelevant cytogeneticcytogenetic subgroupssubgroups ofofacuteacute leukaemiasleukaemias
-- OntogenicOntogenic characterizationcharacterization ofof CLPDCLPD
CONSTRUCTION OF EUROFLOW LEUKEMIA/ LYMPHOMA IMMUNOPHENOTYPING ANTIBODY PANEL
Clinical request/need
Medical indication
Design of MAb panels (Medical
indication-oriented) & immuno-phenotyping strategy
TechniquesPanel evaluation vsconventional in-use
panels
Panel optimization (re-design)
Panel evaluation
Proposed strategy
Panel optimization (re-design)
2-8 cycles
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 PANELS: MEDICAL INDICATION ORIENTATION/SCREENING & CLASSIFICATION PANELS
Van Dongen et al: EuroFlow antibody panels for standardized n-dimensional flow cytometricimmunophenotyping of normal, reactive and malignant leukocytes.
Synchronized light scatter experiments
Normal PB samples processed according to
the standardized EuroFlow sample preparation protocol
7 different normal PB samples acquired in 7 different centers
“Local” settings EuroFlow settings
8
BCLPD panel: classification of an atypical case vs the reference WHO diagnostic groups
Responsible scientist: Sebastian Bottcher
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
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-PECy7CD19-PECy7
SSC
Normal PB B cells
CLL cases
Case number
CD19
PEC
y7
CLLNormal
PB B-cells
RECENT CONTRIBUTIONS OF IMMUNO-PHENOTYPING IN THE DIAGNOSIS OF
HAEMATOLOGICAL MALIGNANCIES
-- StandardizationStandardization ofof immunophenotypicimmunophenotypic analysesanalyses
-- DiagnosisDiagnosis ofof clonalclonal multilineagemultilineage diseasedisease
-- RapidRapid screeningscreening ofof relevantrelevant cytogeneticcytogenetic subgroupssubgroups ofofacuteacute leukaemiasleukaemias
-- OntogenicOntogenic characterizationcharacterization ofof CLPDCLPD
9
MAST CELLS
MONOCYTES
LYMPHOCYTES
EOSINOPHILS
NEUTROPHILS
CD34+ HPC
PURIFICATION OF BM MAST CELLS IN MASTOCYTOSIS
Case 1 Case 2
KIT MUTATION IN SM: PATTERN OF CELLULAR INVOLVEMENT
Eos Eos
NeutNeut
Mo Mo
LymphsLymphsNRC
NRC
CD34+HPC
CD34+HPC
MastCells
MastCells
KIT mutation
FREQUENCY OF MULTILINEAL KIT MUTATION IN MASTOCYTOSIS (n=202)
MC & CD34+ HPC
Only MC
Myeloid lineages
Myeloid & lymphoid lineages
% O
F CA
SES
SUBTYPE OF MASTOCYTOSISTeodosio et al, J Alllergy Clin Immunol 2010
SM: PROGNOSTIC FACTORS FOR SM: PROGNOSTIC FACTORS FOR PROGRESSIONPROGRESSION--FREE SURVIVALFREE SURVIVAL
VARIABLE UNIVARIATE MULTIVARIATE
ANALYSIS ANALYSIS
RR (IC 95%) p-value RR (IC 95%) p-value
Age >60 13.2 (1.2-150) 0.03 NS
Cytopenias 16.6 (1.5-179) 0.02 NS
High β2-microglobulin 1.65 (1.2-2.3) 0.003 1.90 (1.2-2.9) 0.003
Germinal KIT mutation 8.01 (1.3-49) 0.02 13.3 (1.4-124) 0.02
Escribano et al, J Allergy Clin Immunol, 2009
RECENT CONTRIBUTIONS OF IMMUNO-PHENOTYPING IN THE DIAGNOSIS OF
HAEMATOLOGICAL MALIGNANCIES
-- StandardizationStandardization ofof immunophenotypicimmunophenotypic analysesanalyses ((sessionsession V)V)
-- Diagnosis Diagnosis ofof clonalclonal multilineagemultilineage diseasedisease ((sessionsession I)I)
-- RapidRapid screeningscreening ofof relevantrelevant cytogeneticcytogenetic subgroupssubgroups ofofacuteacute leukaemiasleukaemias ((sessionsession III)III)
-- OntogenicOntogenic characterizationcharacterization ofof CLPD CLPD ((SessionSession III)III)
Percentage of cases
ag
e(y
ea
rs)
0 10 20 30 40 50 60 70 80 90 100
5.7%
5.2%
15.5%
17.8%
21.5%
50%
40
45
50
55
60
65
70
75
80
85
90
95
FrequencyFrequency ofof CLLCLL--likelike MBL MBL cellscells in in healthyhealthy individualsindividuals
MBL: MBL: 80/639 (12.5%)80/639 (12.5%)
10
0% (0/45)0% (0/45)8% (4/45)36% (16/45)0.5Population
(Salamanca)
(6)*
0% (0/10)0% (0/21)18% (4/22)39% (15/38)9Population (Leeds) (8)
3% (1/33)6% (2/33)21% (7/33)58% (19/33)3141Clinic (Leeds)
(8)
3% (4/126)2% (2/126)18% (21/126)44% (56/126)2757Clinic
(Mayo) (12,13)
7% (23/325)18% (58/235)16% (53/325)54%
(178/238)>5,000/uLCLL (17)
17p deletion11q deletionTrisomy 1213q14
deletionMedian
CLL count
Rawstron et al, Cytometry B, 2010 (in press)
CLL vs CLL-LIKE MBL:Genetic features of clonal B-lymphocytes
NoNo CLL-
associated2/7 (29%)0.4%0.5
Population
(Salamanca)
(6)
No4-59/6136/51 (70%)7%1.0Population
(Italy) (4)
Yes3-07, 3-23, 4-3418/20 (90%)80%9Population
(Leeds) (8)
Yes3-07, 4-3412/16 (75%)25%26Familial
(Duke) (24)
Yes3-07, 1-69, 4-34,
3-2384/109 (77%)>95%2757
Clinic (Mayo)
(12,13)
Yes3-07, 3-23, 4-3418/20 (90%)>95%3141Clinic (Leeds)
(8)
-3-07, 1-69, 4-34,
3-23
534/927
(57.6%)>95%>5,000CLL (25)
Similar to CLL?
Predominant
CLL cell IGHV
gene
Cases with
<98% IGHV
homology
CLL-like B-
cells (median
%)
Median
CLL cell
count
Source
CLL vs CLL-LIKE MBL:Biological features of clonal B-lymphocytes
Rawstron et al, Cytometry B, 2010 (in press)
100%
50%
0%10.10.01 5 10 20 40 5030
Whole series
10.10.01 5 10 20 40 5030
40 - 59 years
10.10.01 5 10 20 40 5030
60 - 69 years
100%
50%
0%
100%
50%
0%
100%
50%
0%
≥≥≥≥ 70 years
PB Volume (mL)10.10.01 10 20 40 5030
% ofcases witha CLL-likeclone
5
70%
100% 100%
18%
46%32%
88%
62%36%
100%100%100%
DETECTED FREQUENCY
FREQUENCY OF CLL-like MBL IN HEALTHY ADULTS
100%
50%
0%10.10.01 5 10 20 40 5030
Whole series
10.10.01 5 10 20 40 5030
40 - 59 years
10.10.01 5 10 20 40 5030
60 - 69 years
100%
50%
0%
100%
50%
0%
100%
50%
0%
≥≥≥≥ 70 years
PB Volume (mL)10.10.01 10 20 40 5030
% ofcases witha CLL-likeclone
5
70%
100% 100%
18%
46%32%
88%
62%36%
100%100%100%
CALCULATED FREQUENCYDETECTED FREQUENCY
FREQUENCY OF CLL-like MBL IN HEALTHY ADULTS
Volume of PB analyzed: 1mL
Volume of PB analyzed: 50mL
% of CLL-like cells: 0.02% of the whole B-cell population
ImmunophenotypicImmunophenotypic identificationidentification ofof CLLCLL--likelike clonalclonal BB--cellscells in in healthyhealthy adultsadults
FREQUENCY OF CLLFREQUENCY OF CLL--LIKE MBL IN HEALTHY LIKE MBL IN HEALTHY ADULTS: ANALYSIS OF 50 ADULTS: ANALYSIS OF 50 mLmL OF PB OF PB
Number of positive cases: 8/9
- Frequency: < 0.01%- Count: < 0.12 CLL-like B-cells/µµµµL
0,032 cel/uL0,018 cel/uL
0,016%0,009%
0,49%0,27%
BiclonalK+dL+d
M73CASE 9
0,009 cel/uL0,013%0,14%MonoclonalK+dM88CASE 8
0,009 cel/uL0,004 cel/uL
0,02%0,01%
0,18%0,095%
BiclonalK+dL+d
F82CASE 7
0,112 cel/uL0,08%1,3%MonoclonalK+dM77CASE 6
0,036 cel/uL0,05%0,73%MonoclonalK+dM73CASE 5
NDM87CASE 4
0,009 cel/uL0,007 cel/uL
0,008%0,006%
0,11%0,08%
BiclonalK+dL+d
F72CASE 3
0,0018 cel/uL0,0009 cel/uL
0,002%0,001%
0,28%0,14%
BiclonalK+dL+d
F77CASE 2
0,066 cel/uL0,041%0,8%MonoclonalL+dM78CASE 1
ABSOLUTE COUNT
% FROM B-CELLS
% OF WBC (x10-3)
MONOCLONALsIgGENDER AGECASE ID
11
CIC/UNIVERSITY OF CIC/UNIVERSITY OF SALAMANCA SALAMANCA
L Escribano L Escribano I I AlvarezAlvarez TwoseTwoseL Sanchez MuL Sanchez MuññozozI SI Sáánchez Matasnchez MatasA MatitoA Matito
MAST CELL UNIT,MAST CELL UNIT,HOSPITAL VIRGEN del VALLEHOSPITAL VIRGEN del VALLE
TOLEDOTOLEDO
EuroFlow consortium aims at innovation in flow cytometry
www.euroflow.org