“Cellular phenotyping and application of cytometry for diagnostics purposes”

Part II

Karolina Bukowska-Straková

Minimal Residual Disease – MRD

Most hematopoietic malignant cells resemble

normal lymphoid cells.

Yet, hematopoietic malignancies usually display aberrant or unusual antigen expression → detection of minimal residual disease (MRD) is still possible.

Multiparameter flow cytometry allows reliable detection of MRD in most lymphoid malignancies, thereby providing a better insight into treatment

effectiveness.

Aberrant immunophenotypes as targets for MRD detection

Aberrant or unusual immunophenotypes are the result of:- cross-lineage antigen expression, - maturational asynchronous expression of antigens,- antigen over- or under-expression, - the absence of antigen expression, - ectopic antigen expression

Aberrant immunophenotyp bringing the malignant blasts into the ‘empty spaces’ between normal lymphoid differentiation

Detection MRD is based on leukemia-specific immunophenotypes of malignant cells.

Day of diagnosis

33 day of treatment

1. Use muliticolour panel of mAbs (6≥mAbs) for optimal characteristics of leukemia-specific immunophenotypes

2. Select fluorochrome: - the brightest fluorochromes for weakly expressed antigens - the dim fluorochromes for strongly expressed antigens

3. Select best clone of mAbs

4. Take into consideration:- how rare are your cells- the sensitivity of FC

An approach to flow cytometric analysis of residual cells

Six-color panel

1) Syto16/-/CD19-APC/-/-/CD45-APC-H72) CD20-FITC/CD38-PE/CD34-PerCP-Cy5.5/CD10-PC7/CD19-APC/CD45-APC-H73) CD58-FITC/CD66c-PE/CD34-PerCP-Cy5.5/CD10-PC7/CD19-APC/CD45-APC-H7

Day of diagnosis

15 day of treatment

1. Use muliticolour panel of mAbs (6≥mAbs) for optimal characteristics of leukemia-specific immunophenotypes

2. Select fluorochrome: - the brightest fluorochromes for weakly expressed antigens - the dim fluorochromes for strongly expressed antigens

3. Select best clone of mAbs

4. Take into consideration:- how rare are your cells- the sensitivity of FC

An approach to flow cytometric analysis of residual cells

Optimal choice of fluorochromes according to the antigens and the flow cytometer

1. Use muliticolour panel of mAbs (6≥mAbs) for optimal characteristics of leukemia-specific immunophenotypes

2. Select fluorochrome: - the brightest fluorochromes for weakly expressed antigens - the dim fluorochromes for strongly expressed antigens

3. Select best clone of mAbs

4. Take into consideration:- how rare are your cells- the sensitivity of FC

An approach to flow cytometric analysis of residual cells

Choice of optimal clone of mAbs

José M. Casasnovas, Mykol Larvie and Thilo StehleThe EMBO Journal (1999) 18, 2911 - 2922

Compensation and calibration

The use of multiple fluorochromes requires an appropriate instrument set-up to compensate for spectral spillover.

In a standardized setting, with a calibrated flow cytometer and using the same mAbs clones, the staining patterns for all leukocyte subsets are stable

1. Use muliticolour panel of mAbs (6≥mAbs) for optimal characteristics of leukemia-specific immunophenotypes

2. Select fluorochrome: - the brightest fluorochromes for weakly expressed antigens - the dim fluorochromes for strongly expressed antigens

3. Select best clone of mAbs

4. Take into consideration:- how rare are your cells- the sensitivity of FC

An approach to flow cytometric analysis of residual cells

SENSITIVITY of FC10-3 - 10-4

(1 leukemic cell per 1.000 – 10.000)

At least 10-20 cells with leukemia-specific immunophenotype to be sure we see real cells

Positive MRD > 0,01% of BM cells

minimal number of cells to acquire 20 cells – 0,01%200.000 cells – 100%

yet, there are usually debris is sample (sometimes even more then 20%)

minimal number of events to collect > 300.000

Day of diagnosis

33 day of treatment

Day of diagnosis

ConclusionThe story of MRD is one of the most exciting examples of translational research, where basic research was transferred into high-technology laboratory diagnostics

International collaborative efforts ensure that all diagnostic MRD laboratories speak the same ‘MRD language’.

Flow-cytometric immunophenotyping is the sole technique that fulfils the requirements of high speed, broad applicability at diagnosis and during follow-up of immunological and hematological disorders with accurate focusing on the cell population of interest.

CBA(Cytometric Bead Array)

CBA(Cytometric Bead Array)

CBA(Cytometric Bead Array)

CBA(Cytometric Bead Array)

CBA(Cytometric Bead Array)

The CBA Human Soluble Protein Flex Set system provides several advantages when compared with conventional ELISA:

The CBA Human Soluble Protein Flex Set assays allow for multiplexed analysis of multiple proteins from a single sample

The CBA Human Soluble Protein Flex Set assays have a wider dynamic range than conventional ELISAs.

Lower amount of sample is needed.

Luminex system in our laboratory (FlexMap3D)

+Monensin

Surface Ag staining

Cytoplasmaticstaining

Cytoplamatic detection of cytokinesCytoplamatic detection of cytokines

Detection of Ag specific lymphocytes

using HLA-I pentamers

Cell sorting

Cell sorted MoFlo-XDP used in our laboratory

87%

3%

7%

99%

98%

84%

Magnetic cell sorting

1

2

3

AutoMacs used in our laboratory

Image Stream System – used in our laboratory

objective20x, 40x, 60x

ImageStream - applications

Stem cell analysisStem cell analysis

Co-localizataionCo-localizataion

Co-localization in cell subsets

Phagocytosis

Thank you for your attention =)