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