A 19-Color Multiparameter White Blood Cell Panel Designed ...

A 19-Color Multiparameter White Blood Cell Panel

Designed for the Immunophenotyping of Normal

and Malignant Leukocytes by Flow Cytometry

Michelle Scott, Chris Brampton PhD, Kelly Kroeger PhD

Bio-Rad Laboratories, Life Science Group, 2000 Alfred Nobel Dr., Hercules, CA 94547

Cell Analysis

Bulletin 6993

Abstract

Flow cytometry is an important tool for the diagnosis and classification of leukemia. While

many leukocyte markers have been identified and are used to determine the exact subtypes of

leukemia a patient may have, high-dimensional multicolor analysis has been limited by instrument

capabilities. This has often resulted in splitting a valuable limited sample into multiple independent

tests to fully define the patient¡¯s phenotype.

A 19-antibody white blood cell panel was developed using the ZE5? Cell Analyzer from Bio-Rad

Laboratories. With up to five lasers and 30 analysis parameters (of which 27 channels are for

fluorescence detection), all the markers necessary to classify chronic/acute lymphoproliferative

leukemia disorders can be combined into a single tube. Using this panel, a patient with chronic

B-cell lymphocytic leukemia was successfully identified from a normal PBMC sample.

Introduction

With the advent of flow cytometry into the clinical arena as

a diagnostic tool, high-dimensional multicolor analysis has

become increasingly recognized as invaluable for the diagnosis

and classification of leukemia. In the past, clinicians were

restricted by the number of channels available to construct

multicolor panels of leukocyte markers. As a result, defining

populations of normal and aberrant blood cells often required

splitting valuable samples into multiple tubes to achieve the

needed resolution.

The ZE5 Cell Analyzer, when configured with five lasers and

27 channels of fluorescence detection, allows all the markers

necessary to classify chronic/acute lymphoproliferative

leukemia disorders (CLL/ALL) to be mixed into a single tube.

This panel combines 19 surface membrane markers of both

cluster of differentiation and Ig species with TCR expression to

successfully dissect aberrant leukocyte populations present in

a patient with chronic B-cell lymphocytic leukemia (B-CLL).

?

Materials and Methods

Antibodies

CD3 BUV496, CD4 BUV661, CD19 BUV737, TCRgd BUV395,

CD8 BV711, CD45 VioGreen, CD56 BV785, Kappa Light Chain

BV421, CD7-BV650, HLADR VioBlue, CD117 BV605, CD33

FITC, CD34 PE-Vio770, CD64 PE-CF594, CD11b PE, CD14

PE-Cy5, CD20 APC-Vio770, CD38 APC-R700,

Lambda Light Chain APC

Blocking Buffer

PBS + 50 ?g/ml mouse, rat, rabbit IgG + 1% BSA + 1% glucose

+ 20 mM EDTA + 0.1% azide

Washing Buffer

PBS + 1% BSA + 1% glucose + 20 mM EDTA + 0.1% azide

Beads

AbC Total Antibody Compensation Bead Kit

(Thermo Fisher Scientific)

Media

RPMI 1640 or DMEM + 10% FBS

Cell preparation

Normal PBMCs and B-CLL patient PBMCs were thawed

and stained using standard procedures. Thawed samples

were placed into 5 ml of prewarmed cell media in 50 ml

polypropylene centrifuge tubes. Cells were counted and

assessed for viability using the TC20? Automated Cell Counter

A 19-Color Multiparameter White Blood Cell Panel Designed for the Immunophenotyping of Normal and Malignant Leukocytes by Flow Cytometry

(Bio-Rad Laboratories) to obtain an accurate cell count and

viability measurement for each sample. PBMCs were allowed

to recover in culture media in a 37¡ãC, 5% CO2 incubator for at

least an hour.

Cell Staining

The normal PBMC sample and the B-CLL PBMC patient

sample (each sample contained 1 x 106 cells ) were treated

with blocking buffer for 10 min and then stained by adding

a prepared cocktail containing all 19 antibodies. Antibody

concentrations used were based on manufacturer

recommendations. Samples were incubated on ice, protected

from light, for 45 min to an hour. Cells were washed twice

and resuspended into 1 ml of ice cold wash buffer. They were

stored on ice and protected from light until acquisition on

the ZE5 Cell Analyzer. Gating was carried out using FlowJo

Software (Tree Star, Inc.). Stained compensation beads were

used as single-stained controls (data not shown).

Table 1. Nineteen-Antibody White Blood Cell Panel

Marker

Cell Distribution

Description

CD3

Mature T-cells and thymocytes

T-cell activation signaling and regulation of TCR expression

CD4

T-helper cells, regulatory T-cells, monocytes, and macrophages

T-cell activation, thymic differentiation, and receptor for HIV

CD19

B-cells (but not plasma cells) and follicular dendritic cells

Regulator of B-cell development, activation, and differentiation

TCRgd

T subset

Antigen recognition

CD8

Thymocyte subsets and cytotoxic T-cells

Coreceptor for MHC class I molecules

CD45

Hematopoietic cells (not erythrocytes and platelets)

Critical for B- and T-cell receptor¨Cmediated activation. Also required for

thymic selection

CD56

NK, T subset, neurons, some large granular lymphocyte leukemias,

myeloid leukemias

Adhesion

Kappa

light chain

Immunoglobulin light chain

Antibodies are produced by B lymphocytes, each expressing only one

class of light chain. Once set, light chain class remains fixed for the life

of the B lymphocyte. In a healthy individual, the total kappa to lambda

ratio is roughly 3:1

CD7

Thymocytes, T-cells, natural killer cells, and pluripotent hematopoietic

stem cells

T-cell costimulation. Interacts with SECTM1

HLA-DR

MHC class II cell surface receptor

Presentation of peptides to CD4+ T lymphocytes

CD117

Hematopoietic stem cells and progenitors

Receptor for stem cell factor or c-kit ligand

CD33

Monocytes, granulocytes, mast cells, and myeloid progenitors

Lectin activity and adhesion. A receptor that inhibits the proliferation of

normal and leukemic myeloid cells

CD34

Hematopoietic stem cells and progenitors and capillary endothelial cells

Cell adhesion (via L-selectin). Possible role in early hematopoiesis

through mediation of the attachment of stem cells to the bone marrow

extracellular matrix or directly to stromal cells

CD64

IgG Fc receptor monocytes and macrophages

Binds to the Fc region of IgG with high affinity

CD11b

Granulocytes, monocytes, natural killer cells, T- and B-cells, and

dendritic cells

Integrin alpha-M

CD14

Monocytes, macrophages (myelomonocytic cells), Langerhans cells,

and granulocytes

Receptor of complex of LPS and LBP

CD20

B-lymphocyte surface antigen B1 and T- and B-cell subsets

B-cell activation and proliferation

CD38

Variable expression levels on most hematopoietic and some

nonhematopoietic cells. High levels on plasma cells, early

T- and B-cells, activated T-cells and germinal center B-cells

Regulator of cell activation, proliferation, and adhesion

Lambda light

chain

B-cells

Antibodies are produced by B lymphocytes, each expressing only one

class of light chain. Once set, light chain class remains fixed for the life

of the B lymphocyte. In a healthy individual, the total kappa to lambda

ratio is roughly 3:1

Results

PBMCs from a healthy normal donor were stained using a

19-color panel and run on the ZE5 Cell Analyzer. Results

demonstrated that all of the common markers were easily

identifiable with no need for FMO controls.

PBMCs from a chronic lymphocytic leukemia patient were

then stained with the same 19-color panel. Though the

majority of cell populations remained unchanged in this

panel, they could be useful for identification of other leukemia

subtypes in additional samples. The panel easily identified the

patient sample as a chronic B-cell leukemia based on:

? 2017 Bio-Rad Laboratories, Inc.

1. T

 he expression of CD19 along with an aberrant

expression of surface kappa light chain and lambda light

chain, indicating a monoclonal B-cell population (100%

expression of only one type light chain).

2. Lowered expression of CD38 on CD20-expressing cells.

Bulletin 6993

A 19-Color Multiparameter White Blood Cell Panel Designed for the Immunophenotyping of Normal and Malignant Leukocytes by Flow Cytometry

Normal White Blood Cell Immunophenotype

General Subset Identification

00

Lymphocytes

68.9%

Lymphocytes

68.9%

0

0

50K

100K

50K

150K

2

10102

00

2

-10-102

0

10

200K

10K488150K

FSC

200K

0

1

10

2

10

T-Cells

70.3%

3

10

0

10

1

10

10 0

CD3 BUV496

2

10

Th Cells

Th

Cells

00

00

101 CD3 BUV496

102

103

FSC 488

T-Cells

70.3%

33

1010

103103

CD8 BV711

3

10103

Tctl Cells

Tctl Cells

44

1010

CD8 BV711

50K50K

CD19 BUV737

100K

100K

NK-Cells

NK-Cells

10.8%

10.8%

10104 4

CD56 BV786

CD56 BV785

SSC 488

SSC 488

150K

150K

CD19 BUV737

10105 5

200K

200K

3

10

0

101 CD3 BUV496

102

103

0

3

10

4

10

10BUV66110

CD4

3

CD3 BUV496

5

10

105

4

CD4 BUV661

Fig. 1. Normal peripheral blood subset analysis shows typical distribution of all common markers. These panels show NK-cell, B-cell,

and T-cell sets, along with T-cell subsets of T-helper (Th) and cytotoxic T (Tctl) cells.

0

10

1

10

2

10

102102

00

-10-102 2

3

10

3

-10

101 CD3 BUV496

102

103

CD3 BUV496

3

10

0

-103

4

10

103103

00

-10-103 3

5

10

0

10

104

Lambda

LC3 APC

B-Cells

B-Cells

104104

105

10104 4

CD34 PE-Vio770

103103

10105 5

CD34 PE-Vio770

104104

CD38 APC-R700

CD38 APC-R700

00

Kappa light chain BV421

Kappa LC BV421

103103

10 0

105105

105105

B-Cells

B-Cells

11.0%

11.0%

104104

CD19 BUV737

CD19 BUV737

B-Cell Subset Identification

Lymphocytes

Lymphocytes

10103 3

00

-10-103 3

3

-10

0

-103

Lambda LC APC

3

10

4

10

5

10

0CD20

103APC-Vio770

104

0

105

0

CD20 APC-Vio770

3

10

4

10

10 BUV737

10

CD19

3

5

10

105

4

CD19 BUV737

Fig. 2. Normal peripheral blood subset analysis of B-cell subset. These panels show the distribution of surface membrane kappa and

lambda light chains, CD20 with CD38 co-expression, and CD19 with CD34 co-expression.

Myeloid-Cell Subset Identification

00

-10-102 2

0

0

0

3

10

4

10

3

4

10 PE-Cy5

10

CD14

5

10

3

10

0

103

0

CD14 PE-Cy5

4

10

5

10

103 PE 104

CD11b

103

Monocytes

Monocytes

103103

Monocytes

Monocytes

150K

150K

SSC 488

4104

SSC 488

102102

10

CD 64 PE-CF 594

50K50K

3103

CD33 FITC

CD33 FITC

100K

100K

10

CD64 PE-CF594

Monocytes

Monocytes

150K

150K

0

200K

200K

105105

Monocytes

Monocytes

SSC 488

SSC 488

200K

200K

100K

100K

50K50K

102102

00

-10-102 2

3

10

0

0

CD11b PE

4

10

5

10

103PE-Cy5

104

CD14

105

00

4

10

5

10

3

10

CD11b

PE10 4

3

10

105

0

0

CD14 PE-Cy5

CD11b PE

Fig. 3. Normal peripheral blood subset analysis of myeloid-cell subset. These panels show the expected distribution of CD14, CD11b,

CD64, and CD33 antibodies.

Diseased White Blood Cell Immunophenotype

Sample Name Count

B-CLL

25,603

PBMC

34,623

0

0

0

10

1

10

10 0 101

2

10

3

10

4

10

10 VioGreen

10

10

CD45

2

3

4

5

10

105

CD45 VioGreen

Overlay of total population of

normal PBMCs with B-CLL

sample, showing almost

complete dominance of

lymphocyte population based on

CD45 and side scatter.

10

3

3

10

10

2

2

10

10

1

1

10

10

Sample Name

B-CLL

PBMC

0

0

10

0

10

1

10

10 0 101

2

10

3

10

4

10

5

10

2 10 3

10

104 105

CD3 BUV496

CD3 BUV496

Same total population now

overlaid with CD3 and CD19,

showing nearly complete

dominance of CD19-positive

B-cell population in the

B-CLL sample.

104104

103103

102102

00

-10-102 2

Count

22,113

3,735

105105

CD38 APC-R700

50K50K

4

4

10

Kappa light chain BV421

100K

100K

10

Count

22,113

3,735

105105

Kappa LC BV421

150K

150K

CD19 BUV737

CD19 BUV737

105105

SSC 488

SSC 488

200K

200K

Sample Name

B-CLL

PBMC

104104

CD38 APC-R700

Sample Name Count

B-CLL

25,603

PBMC

34,623

103103

102102

101101

0

3

-10

0

-103 0

3

10

4

10

10 LC APC

10

Lambda

3

4

5

10

105

Lambda LC APC

Overlay of B-cell gated populations

from normal PBMCs and B-CLL

sample. Normal PBMC B-cells

will display a ratio of surface

membrane Ig light chain of kappa

to lambda. The B-CLL sample

shows a single chain expression,

indicating a monoclonal population

of B-cells.

10 010

0

10

0

1

10

2

10

3

10

4

10

5

10

101 CD20

102 APC-Vio770

103 104 105

CD20 APC-Vio770

Overlay of B-cell gated

populations from normal PBMCs

and B-CLL sample. Normal

PBMC B-cells will have a relatively

high expression of CD20 and

CD38. The same gated population

from a B-CLL patient shows a

diminished CD38 expression.

Fig. 4. Aberrant expression of CD19 and surface membrane lg light chain lambda and reduced expression of CD38 on

CD20-positive B-cells are indicative of chronic B-cell lymphocytic leukemia.

? 2017 Bio-Rad Laboratories, Inc.

Bulletin 6993

A 19-Color Multiparameter White Blood Cell Panel Designed for the Immunophenotyping of Normal and Malignant Leukocytes by Flow Cytometry

Diseased White Blood Cell Immunophenotype

B-Cell Lymphocytic Leukemia

0

10

1

10

2

10

102102

00

-10-102 2

3

-10

3

10

10 0 101 CD3 BUV496

102

103

0

-103

CD3 BUV496

3

10

4

10

0

10

10

Lambda

LC APC

3

4

T-Cells

T-Cells

1.98%

1.98%

10103 3

00

0

10

5

10

105

1

10

10 0

101

Lambda LC APC

2

10

10

CD3 BUV496

2

Kappa light chain BV421

103103

CD19 BUV737

104104

105105

B-Cells

B-Cells

89.6%

89.6%

10104 4

Kappa LC BV421

00

CD19 BUV737

Kappa LC BV421

T-Cells

T-Cells

70.3%

70.3%

33

1010

Kappa light chain BV421

105105

B-Cells

B-Cells

11.0%

11.0%

44

1010

CD19 BUV737

CD19 BUV737

Normal Blood

104104

103103

102102

00

-10-102 2

3

10

103

3

-10

0

4

10

5

10

-103

Lambda

LC3APC10 4

0

10

105

CD3 BUV496

3

10

Lambda LC APC

Fig. 5. Diseased peripheral blood subset analysis comparing the profile of normal blood to that of a patient with chronic B-cell

lymphocytic leukemia. Note the increase in the B-cell subset along with single light chain distribution in the disease state.

B-Cell Lymphocytic Leukemia

105105

103103

00

104104

CD38 APC-R700

B-Cells

B-Cells

CD34 PE-Vio770

00

CD38 APC-R700

103103

105105

105105

104104

CD34 PE-Vio770

CD34 PE-Vio770

104104

CD38 APC-R700

CD38 APC-R700

105105

103103

00

-10-103 3

3

-10

-103

0

3

10

4

10

0 CD20

103APC-Vio770

104

5

10

105

CD20 APC-Vio770

104104

CD34 PE-Vio770

Normal Blood

B-Cells

B-Cells

103103

00

-10-103 3

0

0

3

10

10BUV737

CD19

3

4

10

104

CD19 BUV737

3

-10

-103

0

3

10

4

10

0 CD20

103APC-Vio770

104

5

10

105

CD20 APC-Vio770

0

0

3

10

10

CD19 BUV737

3

4

10

104

CD19 BUV737

Fig. 6. Diseased peripheral blood subset analysis comparing the profile of normal blood to that of a patient with chronic B-cell

lymphocytic leukemia. Note the loss of CD38 expression along with an increase in overall B-cell population in the disease state.

Conclusions

¡ö¡ö 

Large-panel multiparameter design and analysis is straightforward using the ZE5 Cell Analyzer¡¯s 27

fluorescent detection channels

¡ö¡ö

 19-color panel, designed from existing literature, could allow classification of CLL/ALL disorders

A

utilizing a single tube with the combined antibodies on the ZE5 Cell Analyzer

Visit ZE5 for more information.

Data were originally presented as Poster #B86 at the 2017 32nd Congress of the International Society for Advancement of Cytometry

(CYTO) meeting (Boston, MA).

AbC is a trademark of Thermo Fisher Scientific. Cy is a trademark of GE Healthcare. FlowJo is a trademark of FlowJo, LLC. Vio,

VioBlue, and VioGreen are trademarks of Miltenyi Biotec GmbH.

For Research Use Only. Not for use in diagnostic procedures.

Bio-Rad

Laboratories, Inc.

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