a 19-color multiparameter white blood cell panel …...2017 bio-rad laboratories, inc. bulletin 6993...

IntroductionWith 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 MethodsAntibodies

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 µg/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

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.

Cell Analysis Bulletin 6993

Michelle Scott, Chris Brampton PhD, Kelly Kroeger PhD Bio-Rad Laboratories, Life Science Group, 2000 Alfred Nobel Dr., Hercules, CA 94547

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

© 2017 Bio-Rad Laboratories, Inc. Bulletin 6993


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).

1. The 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.

(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°C, 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

ResultsPBMCs 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:

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–mediated 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

© 2017 Bio-Rad Laboratories, Inc. Bulletin 6993


CD

8 B

V71

1 103

0

0 103 104 105

CD4 BUV661

0

103

CD

8 BV

711

Tctl Cells

Th Cells

0 103 104 105

CD4 BUV661

104

103

0

100 101 102 103

CD3 BUV496

0

103

104

CD

19 B

UV7

37

70.3%T-Cells

100 101 102 103

CD3 BUV496

Tctl Cells

Th CellsT-Cells70.3%

SS

C 4

88

200K

150K

100K

50K

0

Lymphocytes68.9%

0 50K 100K 150K 200K

FSC 488

0

50K

100K

150K

200K

SSC

488

0 50K 10K 150K 200K

FSC 488

CD

19 B

UV

737

CD

56 B

V78

5

105

104

103

102

0-102

NK-Cells10.8%

100 101 102 103

CD3 BUV496

0

-10 2

102

103

104

105

CD

56 B

V786

0 101 102 103

CD3 BUV496

General Subset Identification

NK-Cells10.8%

Lymphocytes68.9%

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.

CD

38 A

PC

-R70

0

105

104

103

0

-103

0-103 103 104 105

CD20 APC-Vio770

0

-10 3

103

104

105

CD

38 A

PC-R

700 B-Cells

-103 0 103 104 105

CD20 APC-Vio770

B-Cells

CD

34 P

E-V

io77

0

105

104

103

0

-103

0 103 104 105

CD19 BUV737

0

-10 3

103

104

105

CD

34 P

E-Vi

o770

Lymphocytes

0 103 104 105

CD19 BUV737

Lymphocytes

CD

19 B

UV

737 104

103

0

B-Cells11.0%

100 101 102 103

CD3 BUV496

0

103

104

CD

19 B

UV7

37

100 101 102 103

CD3 BUV496

B-Cell Subset Identification

B-Cells11.0%

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.

Kap

pa

LC B

V421

105

104

103

102

0-102

0-103 103 104 105

Lambda LC APC

0

-10 2

102

103

104

105

Kapp

a lig

ht c

hain

BV4

21

-103 0 103 104 105

Lambda LC APC

SS

C 4

88

200K

150K

100K

50K

00 103 104 105

CD14 PE-Cy5

0

50K

100K

150K

200K

SSC

488

Monocytes

0 103 104 103

CD14 PE-Cy5

CD

33 F

ITC 103

102

0

-102

0 103 104 105

CD11b PE

0

-10 2

102

103

CD

33 F

ITC

Monocytes

0 103 104 103

CD11b PE

CD

64 P

E-C

F594

105

104

103

102

0-102

0 103 104 105

CD14 PE-Cy5

0

-10 2

102

103

104

105

CD

64

PE-C

F 59

4

Monocytes

0 103 104 105

CD14 PE-Cy5

SS

C 4

88200K

150K

100K

50K

00 103 104 105

CD11b PE

0

50K

100K

150K

200K

SSC

488

Monocytes

0 103 104 105

CD11b PE

Myeloid-Cell Subset Identification

MonocytesMonocytes

Fig. 3. Normal peripheral blood subset analysis of myeloid-cell subset. These panels show the expected distribution of CD14, CD11b, CD64, and CD33 antibodies.

Monocytes Monocytes

Normal White Blood Cell Immunophenotype

Sample Name CountB-CLL 25,603PBMC 34,623




SS

C 4

88

200K

150K

100K

50K

0100 101 102 103 104 105

CD45 VioGreen

0

50K

100K

150K

200K

SSC

488

100 101 102 103 104 105

CD45 VioGreen

CD

19 B

UV

737

105

104

103

102

101

100

100 101 102 103 104 105

CD3 BUV496

100

101

102

103

104

105

CD

19 B

UV7

37

100 101 102 103 104 105

CD3 BUV496

Kap

pa

LC B

V421

105

104

103

102

0

-102

0-103 103 104 105

Lambda LC APC

0

-10 2

102

103

104

105

Kapp

a lig

ht c

hain

BV4

21

-103 0 103 104 105

Lambda LC APC

CD

38 A

PC

-R70

0

105

104

103

102

101

100

100 101 102 103 104 105

CD20 APC-Vio770

100

101

102

103

104

105

CD

38 A

PC-R

700

0 101 102 103 104 105

CD20 APC-Vio770

Overlay of total population of normal PBMCs with B-CLL sample, showing almost complete dominance of lymphocyte population based on CD45 and side scatter.

Same total population now overlaid with CD3 and CD19, showing nearly complete dominance of CD19-positive B-cell population in the B-CLL sample.

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.

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.

Diseased White Blood Cell Immunophenotype


Bulletin 6993 Ver A US/EG 17-0752 0917 Sig 1216

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Bio-Rad Laboratories, Inc.

Life ScienceGroup

CD

19 B

UV

737 104

103

0

100 101 102 103

CD3 BUV496

0

103

104

CD

19 B

UV7

37

B-Cells11.0%

T-Cells70.3%

100 101 102 103

CD3 BUV496

T-Cells70.3%

CD

19 B

UV

737 104

103

0

B-Cells89.6%

T-Cells1.98%

100 101 102 103

CD3 BUV496

0

103

104

CD

19 B

UV7

37

100 101 102 103

CD3 BUV496

B-Cells89.6%

T-Cells1.98%

Kap

pa

LC B

V421

105

104

103

102

0-102

0-103 103 104 105

Lambda LC APC

0

-10 2

102

103

104

105

Kapp

a lig

ht c

hain

BV4

21

-103 0 103 104 105

Lambda LC APC

Kap

pa

LC B

V421

105

104

103

102

0-102

0-103 103 104 105

Lambda LC APC

0

-10 2

102

103

104

105

Kapp

a lig

ht c

hain

BV4

21

-103 0 103 104 105

Lambda LC APC

Normal Blood B-Cell Lymphocytic Leukemia

CD

38 A

PC

-R70

00-103 103 104 105

CD20 APC-Vio770

0

103

104

105

CD

38 A

PC-R

700

Normal Blood B-Cell Lymphocytic Leukemia

CD

34 P

E-V

io77

0

105

104

103

0

-103

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.

CD

38 A

PC

-R70

0

105

104

103

0

105

104

103

0CD

34 P

E-V

io77

0

105

104

103

0

-103

0 103 104

CD19 BUV737

0

-10 3

103

104

105

CD

34 P

E-Vi

o770 B-Cells

0 103 104

CD19 BUV737

0

-10 3

103

104

105

CD

34 P

E-Vi

o770

B-Cells

0 103 104

CD19 BUV737 0 103 104

CD19 BUV737

0-103 103 104 105

CD20 APC-Vio770

0

103

104

105

CD

38 A

PC-R

700

-103 0 103 104 105

CD20 APC-Vio770 -103 0 103 104 105

CD20 APC-Vio770

B-Cells

B-Cells

B-Cells11.0%

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.

Conclusions■■ Large-panel multiparameter design and analysis is straightforward using the ZE5 Cell Analyzer’s 27

fluorescent detection channels■■ A 19-color panel, designed from existing literature, could allow classification of CLL/ALL disorders

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

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.

Diseased White Blood Cell Immunophenotype

Visit bio-rad.com/ZE5 for more information.

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a 19-color multiparameter white blood cell panel …...2017 bio-rad laboratories, inc. bulletin 6993...

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