FLOW CYTOMETRY
IMMUNOPHENOTYPING IN INTEGRATED
DIAGNOSTICS OF PATIENTS WITH
NEWLY DIAGNOSED CYTOPENIA
Anna Porwit
Department of Laboratory Medicine and Pathobiology,
University Health Network , Toronto, ON, Canada
Disclosures
• Novartis: speaker and consultation fees
• Beckman Coulter: travel support to Harmonemia working
group meetings
Marked cytopenia
• Hemoglobin < 110 g/L
• Neutrophils < 1.5x109/L
• Platelets <100x109/L
• Mild cytopenia: lower than reference for lab but
higher than above
Causes of new onset cytopenia
Adults: AML 31% ALL 6% MDS or MDS/MPN 47.2% MPN 3.2% lymphoma 9.6%
Children: acute leukemia 95% (B-ALL 85%)
Non-neoplastic causes of cytopenia (adults)
• Multifactorial 32%
• Idiopathic aplastic anemia 22%
• Unknown 13%
• Drug induced 10%
• Infection 10%
• Other systemic disease 7.5%
• Hypersplenism 4.5%
Diagnosis of
myelodysplastic
syndrome
Diagnostic tool Diagnostic value Priority
Peripheral blood smear • Evaluation of dysplasia in one or more cell lines
• Enumeration of blasts Mandatory
Bone marrow aspirate
• Evaluation of dysplasia in one or more myeloid cell
lines
• Enumeration of blasts
• Enumeration of ring sideroblasts
Mandatory
Bone marrow biopsy • Assessment of cellularity, CD34+ cells, and fibrosis Mandatory
Cytogenetic analysis
• Detection of acquired clonal chromosomal
abnormalities that can allow a conclusive diagnosis and
also prognostic assessment
Mandatory
FISH
• Detection of targeted chromosomal abnormalities in
interphase nuclei following failure of standard G-
banding
Recommended
Flow cytometry
immunophenotype
• Detection of abnormalities in erythroid, immature
myeloid, maturing granulocytes, monocytes,
immature lymphoid compartments
Recommended*
If according to ELN
guidelines
SNP-array • Detection of chromosomal defects at a high resolution
in combination with metaphase cytogenetics
Suggested (likely to
become a diagnostic
tool in the near
future)
Mutation analysis of
candidate genes
• Detection of somatic mutations that can allow a
conclusive diagnosis and also reliable prognostic
evaluation
Suggested (likely to
become a diagnostic
tool in the near
future)
FCM as a part of diagnostic approach to MDS 2015
Malcovati L, et al., ELN guidelines. Blood 2013;122:2943-64; Greenberg P et al., J Nat Compr Netw
Canc 2013;11:838-74; *Westers TM, et al., Leukemia 2012;26:1730-41
WHO 2016 update, R. Hasserjian , USCAP 2015
• Use FCM application for MDS diagnostics
• Follow International MDS Flow recommendations (ELN/NCCN)
• For screening purposes
• Follow a mini-panel based on the so-called Ogata score
• For extended analysis: perform FCM in all cell compartments
• Myeloid and lymphoid progenitor cells
• Maturing myelomonocytic cells
• Immature and mature erythroid cells
Malcovati L, et al., ELN guidelines 2013: Blood 2013;122:2943-64; Greenberg P, et al., J Nat Compr Netw Canc
2013;11:838-74; Westers TM, et al., Leukemia 2012;26:1730-41; Van de Loosdrecht AA, Westers TM. J Natl Comp Canc
Netw 2013;11:892-902;
Why the new ST?
SCREENING TUBE 10 COLORS, 14 ANTIBODIES
• Enumerate Major Populations: • Blasts
• B- lymphocytes, T- lymphocytes,
• NK cells
• Monocytes
• Neutrophils
• Enumerate Sub-populations: • T-Helper cells, T- Suppressor cells
• CD34+/CD19+ B- cell progenitors
• CD34+/CD33+ myeloid progenitors
• CD33+/CD10+ mature granulocytes
• B-cell clonality status: Kappa Lambda
• B-cell maturation status CD20/CD10
expression.
Rajab A, Porwit A. Screening bone marrow samples for abnormal lymphoid populations and myelodysplasia-related
features with one 10-color 14-antibody screening tube. Cytometry B Clin Cytom. 2015 Feb 9, E-pub
Similar approach has been applied by other groups in 4-
color, 6-color, 8-color and 10-color settings :
mainly for screening of lymphoid populations
• Quijano S, …etc. Spanish Group for the Study of CNS Disease in NHL.Identification of leptomeningeal disease in aggressive B-cell non-Hodgkin's lymphoma: improved sensitivity of flow cytometry. J Clin Oncol. 2009 Mar 20;27(9):1462-9.
• Preijers FW…etc. B. OMIP-010: a new 10-color monoclonal antibody panel for polychromatic immunophenotyping of small hematopoietic cell samples. Cytometry A. 2012 Jun;81(6):453-5.
• Costa ES, …etc. A new automated flow cytometry data analysis approach for the diagnostic screening of neoplastic B-cell disorders in peripheral blood samples with absolute lymphocytosis. Leukemia. 2006 Jul;20(7):1221-30
• van Dongen JJ,…etc. EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708). EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia. 2012 Sep;26(9):1908-75.
ANALYSIS NORMAL BM SAMPLE
Living Cells = NOT Debris
NORMAL BM SAMPLE
(BLAST ANALYSIS)
Dim CD45+ CD45-ve
NORMAL BM SAMPLE (CD45-/dim ANALYSIS)
NORMAL BM SAMPLE
(B-CELL ANALYSIS)
NORMAL BM SAMPLE
(T-CELL/ NK ANALYSIS)
NK = Lymphs AND (NOT CD19+)
NORMAL BM SAMPLE
(Grans & Monos analysis)
SCREENING TUBE REPORT
Lymphoproliferative neoplasms
B- CLL
FCL
T-LPD
VALIDATION FOR SCEENING OF LYMPHOID POPULATION
SUMMARY
• During August 2012 – December 2013 we have analyzed 1025 samples with query cytopenia and no lymphocytosis using ST.
• In 94% of the cases we could render a final FCM report and 6% of cases required further immunophenotyping.
The Screening Tube
• can detect aberrant antigen expression on B-cells (CD5, CD10, CD20,)
• establish B-cell clonality status (Kappa/Lambda)
• establish B-cell maturation pattern (CD10/CD20)
• detect some aberrant expressions on T-cells (CD3, CD5, CD4, CD8, & CD10)
Ogata score
FCM score > 2 was significantly associated with MDS diagnosis
High values were associated with multilineage dysplasia,
transfusion dependency and high-risk cytogenetics
Della Porta MG, Haematologica. 2012 Aug;97(8):1209-17
FCM- score for MDS using the screening tube
(NORMAL BM)
Gated Myeloblasts in all nucleated cells: 1.6% (If ≥2%, FCM score= 1)
Gated B-cell progenitor in CD34+ cells: 5.3% (If ≤5%, FCM score=1)
Lymphocyte to myeloblast CD45 MFI ratio= 6.4 (If ≤4 or ≥7.5, FCM score=1)
Granulocyte to lymphocyte SSC mode ratio: 8 (If ≤6, FCM score=1)
Total FCM score= 0
FCM- score for MDS (abnormal findings)
Gated Myeloblasts in all nucleated cells: 3.3 (If ≥2, FCM score= 1)
Gated B-cell progenitor in CD34+ cells: 0.4 (If ≤5, FCM score=1)
Lymphocyte to myeloblast CD45 MFI ratio: 12.5 (If ≤4 or ≥7.5, FCM score=1)
Granulocyte to lymphocyte SSC mode ratio: 4.7 (If ≤6, FCM score=1)
Total FCM score= 4
Validation of FCM score at UHN
Differences in scores between the control group and the MDS & MDS/MPN
and the MPN groups were statistically significant (p<0.001).
Scores above 2 had high predictive value for MDS and MDS/MPN diagnosis
Rajab A, Porwit A. Cytometry B Clin Cytom. 2015 Feb 9
International/European LeukemiaNet Working Group for Flow Cytometry in MDS. Porwit et al. Leukemia. 2014 Sep;28(9):1793-8.
AML 1 AML 2
AML 3
FITC CD65 CD36 CD71
PE CD13 CD64 CD11c
ECD CD14 CD56 CD4
PC5.5 CD33 CD33 CD33
PC7 CD34 CD34 CD34
APC CD117 CD123 CD2
APC_AlexaF700 CD7 CD19 CD10
APC_AlexaF750 CD11b CD38 CD235a
Pacific_BLUE CD16 HLA-DR CD15
Krome Orange CD45 CD45 CD45
10 color acute leukemia/MDS panel
at UHN
Summary of MDS-related aberrant features in myelomonocytic compartments that can be evaluated using 3 –tube panel
Myeloid progenitor cell population
(SSClow/CD45dim)
Differentiating granulopoietic
population (SSC high/CD45+)
Monocytic population
(SSCintermediate/CD45intermediate/bright) Marker/pattern Aberrant feature Marker/pattern Aberrant feature Marker/pattern Aberrant feature
CD45dim Increased CD45dim population Scatter Decreased SSC due to
low granularity
CD13+/CD33+ Increased number of CD33+/CD13-
or CD33-/CD13+ cells
CD34+ Increased number in BM CD13+/CD33+ Increased number of
CD33+/CD13- or CD33-
/CD13+ cells
HLA-DR Decreased expression
CD34++ Increased number of
CD34bright cells
CD13/CD11b Aberrant maturation
pattern
CD11b Decreased expression
CD34+/CD117+ Lack of CD34+ population or
decreased CD117+ population
CD13/CD16 Aberrant maturation
pattern
CD14 Decreased expression
CD34+/CD38dim/neg Increased proportion of CD38
negative/dim CD34 cells
HLA-DR Increased expression CD16 Aberrant expression pattern
CD34+/CD45- Increased number of CD45-
CD34 cells
CD117 Increased expression CD56 High expression
CD34+/CD19- Increased in proportion to
CD34+/CD19+ lymphoid
progenitors
CD10 Lack on mature
granulocytes
CD2, CD5, CD7 Aberrant expression
CD34+/HLA-DR- Increased proportion in
CD34+ cells
CD34+/CD15+ Asynchronous
expression
CD64 Decreased expression
CD34+/CD123+ Aberrant expression of
CD123 on CD34+ cells
CD2, CD5, CD7 Aberrant expression CD34 Asynchronous expression
CD13+/CD33+ Increased number of
CD33+/CD13- or CD33-/CD13+
cells
CD56 Aberrant expression
CD2, CD5, CD4,
CD7**
Aberrant expression on
CD34+ and or CD117+ cells
CD64 Decreased expression
CD56* Aberrant expression of CD56
on CD34+ and or CD117+ cells
CD36 Increased expression
CD11b* High expression on CD34+
cells
Examples of aberrant findings
Expression of CD7 and CD11b in CD34+ cells Low SSC Increased CD56
Decreased CD4 and CD11c Increased CD56
• A:
FCM analysis: NO MDS-related features
• B:
FCM analysis: some changes often seen in MDS
• C:
FCM analysis: consistent with MDS
How to report FCM findings?
Guidelines of the IMDSflow WG on FCM in MDS 2015
Loosdrecht AA van de, Westers TM. J Nat Compr Cancer Netw 2013;11:892-902
Westers TM, et al., Leukemia 2012;26:1730-41; Porwit A, et al., Leukemia 2014;28:1793-98
Diagnostic flow score
(Ogata et al.)
<2 <2 <2 <2 ≥2 ≥2 ≥2 ≥2
Dysplasia by FC
myeloid progenitors
- - + + - - + +
Dysplasia by FC
- Neutrophils (SSC or two or
more other aberrancies)
- Monocytes (CD56 or two or
more other aberrancies)
- + - + - + - +
Conclusion A A/B A/B C A/B B/C B/C C
Loosdrecht AA van de, Westers TM. J Natl Comp Canc Netw 2013;11:892-902;
Porwit A, Loosdrecht AA van de, et al., Leukemia 2014;28:1793-98
Integrated Flow Cytometric diagnostic approach
Scoring system score combined with FCSS (Wells)
parameters
>20% blasts: acute leukemia
Leukemia analysis: standardized analysis mall
“Empty spaces”
AML at diagnosis
Normal bone marrow
Blasts and
CD34bright/CD117dim
And
CD33-/CD13+
<0.01%
Sequential and boolean gating strategy to be applied in follow-up samples
AL panel: information also on other cells
Cytoplasmic tube for MPAL
B-lineage:
CD19
T-lineage: CD3 Myeloid lineage:
myeloperoxidase
Monocytic lineage:
CD14, CDD11c,
CD14, CD64, lysozyme,
nonspecific esterase
Reporting flow cytometry findings in AL:
Interpretative comment
• Quality of the sample, in terms of viability;
• General description of the gating procedure;
• Immunophenotype of blast cells;
• Description of cells surrounding blasts;
• Diagnostic conclusions;
• (Definition of an antigen panel for the
detection of minimal residual disease).
Haematologica Vecchio et al 2004;89:594-598
Take home message
• FCM should be integrated in the work-up of patients with
pacytopenia, as it can increase diagnostic accuracy by
excluding reactive conditions, lymphoproliferative
disorders and by providing additional evidence of
dysplasia in MDS work-up.
• Standard methods for FCM diagnostics in MDS have been
established by the International Flow Cytometry Working
Group within the European Leukemia Network
• Use of 10 color FCM assay improves qualitative
information on leukemia phenotype and allows use of the
same panels for diagnosis and follow-up (MRD) in acute
leukemia