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 Materials and Methods With the advent of flow cytometry into the clinical arena as Antibodies a diagnostic tool, high-dimensional multicolor analysis has CD3 BUV496, CD4 BUV661, CD19 BUV737, TCRgd BUV395, become increasingly recognized as invaluable for the diagnosis CD8 BV711, CD45 VioGreen, CD56 BV785, Kappa Light Chain and classification of leukemia. In the past, clinicians were BV421, CD7-BV650, HLADR VioBlue, CD117 BV605, CD33 restricted by the number of channels available to construct FITC, CD34 PE-Vio770, CD64 PE-CF594, CD11b PE, CD14 multicolor panels of leukocyte markers. As a result, defining PE-Cy5, CD20 APC-Vio770, CD38 APC-R700, populations of normal and aberrant blood cells often required Lambda Light Chain APC splitting valuable samples into multiple tubes to achieve the Blocking Buffer needed resolution. PBS + 50 µg/ml mouse, rat, rabbit IgG + 1% BSA + 1% glucose The ZE5™ Cell Analyzer, when configured with five lasers and + 20 mM EDTA + 0.1% azide 27 channels of fluorescence detection, allows all the markers Washing Buffer necessary to classify chronic/acute lymphoproliferative PBS + 1% BSA + 1% glucose + 20 mM EDTA + 0.1% azide leukemia disorders (CLL/ALL) to be mixed into a single tube. Beads This panel combines 19 surface membrane markers of both AbC Total Antibody Compensation Bead Kit cluster of differentiation and Ig species with TCR expression to (Thermo Fisher Scientific) successfully dissect aberrant leukocyte populations present in a patient with chronic B-cell lymphocytic leukemia (B-CLL). 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 a prepared cocktail containing all 19 antibodies. Antibody viability measurement for each sample. PBMCs were allowed concentrations used were based on manufacturer to recover in culture media in a 37°C, 5% CO2 incubator for at recommendations. Samples were incubated on ice, protected least an hour. from light, for 45 min to an hour. Cells were washed twice Cell Staining and resuspended into 1 ml of ice cold wash buffer. They were The normal PBMC sample and the B-CLL PBMC patient stored on ice and protected from light until acquisition on sample (each sample contained 1 x 106 cells ) were treated the ZE5 Cell Analyzer. Gating was carried out using FlowJo with blocking buffer for 10 min and then stained by adding 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–mediated activation. Also required for thymic selection CD56 NK, T subset, neurons, some large granular lymphocyte leukemias, Adhesion myeloid leukemias Kappa Immunoglobulin light chain Antibodies are produced by B lymphocytes, each expressing only one light chain 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 T-cell costimulation. Interacts with SECTM1 stem cells 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 Integrin alpha-M dendritic cells CD14 Monocytes, macrophages (myelomonocytic cells), Langerhans cells, Receptor of complex of LPS and LBP and granulocytes 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 Regulator of cell activation, proliferation, and adhesion nonhematopoietic cells. High levels on plasma cells, early T- and B-cells, activated T-cells and germinal center B-cells Lambda light B-cells Antibodies are produced by B lymphocytes, each expressing only one chain 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 1. The expression of CD19 along with an aberrant 19-color panel and run on the ZE5 Cell Analyzer. Results expression of surface kappa light chain and lambda light demonstrated that all of the common markers were easily chain, indicating a monoclonal B-cell population (100% identifiable with no need for FMO controls. expression of only one type light chain). PBMCs from a chronic lymphocytic leukemia patient were 2. Lowered expression of CD38 on CD20-expressing cells. 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:

Normal White Blood Cell Immunophenotype

General Subset Identification

5 5 200K200K 1010 NK-CellsNK-Cells 10.8% TctlTctl Cells Cells 4 4 10.8% 1010 4 4 150K 10 150K 10 3 3 10 10 T-Cells 3 3 ThTh Cells Cells 3 3 1010 T-Cells 100K100K 1010 70.3%70.3% SSC 488 CD8 BV711 CD56 BV786 CD19 BUV737 SSC 488

2 2 BV711 CD8 0 CD56 BV785 1010 0 50K50K CD19 BUV737 Lymphocytes 00 00 Lymphocytes 2 2 68.9%68.9% -10-10 0 0 0 1 2 3 0 1 2 3 3 4 5 0 50K 100K 150K 200K 10 10 10 10 10 10 10 10 0 10 10 10 1 2 3 0 1 2 3 3 4 5 0 50K FSC10K 488 150K 200K 0 10 CD3 BUV49610 10 10 10 CD3 BUV49610 10 0 CD410 BUV661 10 10 FSC 488 CD3 BUV496 CD3 BUV496 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.

B-Cell Subset Identification

5 5 5 5 5 10 10 10 10 5 B-CellsB-Cells 10 10 11.0% 4 4 10 10 11.0% 4 4 4 B-Cells 4 10 10 B-Cells 4 4 10 10 1010 Lymphocytes 3 3 Lymphocytes 10 10 3 3 3 3 10 10 10 10 3 3 1010 CD19 BUV737 CD38 APC-R700 CD34 PE-Vio770 0

CD19 BUV737 2 0 Kappa light chain BV421 2 00 0 10 10 0 CD34 PE-Vio770 CD38 APC-R700 Kappa BV421 LC 00 3 2 2 3 3 -10 3 -10-10 -10-10 -10 0 1 2 3 3 3 4 5 3 3 4 5 3 4 5 10 10 10 10 -10 0 10 10 10 -10 0 10 10 10 0 10 10 10 0 1 2 3 3 3 4 5 3 3 4 5 3 4 5 10 10 CD3 BUV49610 10 -10 Lambda0 10 LC APC10 10 -10 0CD20 10 APC-Vio770 10 10 0 CD1910 BUV737 10 10 CD3 BUV496 Lambda LC APC CD20 APC-Vio770 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

200K 5 5 200K 10 10 200K200K MonocytesMonocytes MonocytesMonocytes

3 3 4 4 MonocytesMonocytes 150K150K 10 10 150K150K 10 10 MonocytesMonocytes

100K100K 3 3 100K 10 10 100K SSC 488 SSC 488 CD33 FITC

SSC 488 2 2 SSC 488 10 10 CD 64 PE-CF 594 CD33 FITC CD33 2 50K50K 10 2 50K50K 00 10 CD64 PE-CF594 CD64 00 2 2 -10-10 2 2 0 0 -10-10 0 0 3 4 5 3 4 5 3 4 5 3 4 5 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 3 4 3 3 4 3 3 4 5 3 4 5 0 CD1410 PE-Cy5 10 10 0 CD11b10 PE10 10 0 CD1410 PE-Cy5 10 10 0 10CD11b PE10 10 CD14 PE-Cy5 CD11b PE 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 Sample Name Count Sample Name Count Sample Name Count B-CLL 25,603 B-CLL 25,603 B-CLL 22,113 B-CLL 22,113 PBMC 34,623 PBMC 34,623 PBMC 3,735 PBMC 3,735

5 5 5 5 200K 10 5 5 10 200K 10 10 10 10

4 4 4 4 1010 10 10 150K 4 4 150K 10 10

3 3 3 3 10 10 10 10

100K100K 3 3 10 10 SSC 488 2 2 2 2 1010 10 10 CD19 BUV737 SSC 488 CD38 APC-R700

50K CD19 BUV737 2 50K Kappa light chain BV421 2 1 1 10 10 1 1 1010 10 10 Kappa BV421 LC CD38 APC-R700 00

0 2 2 0 0 0 10010 -10-10 10010 0 1 2 3 4 5 0 1 2 3 4 5 3 3 4 5 0 1 2 3 4 5 10 10 10 10 10 10 10 10 10 10 10 10 -10 0 10 10 10 10 10 10 10 10 10 0 1 2 3 4 5 0 1 2 3 4 5 3 3 4 5 1 2 3 4 5 10 10 CD4510 VioGreen10 10 10 10 10 10CD3 BUV49610 10 10 -10 0 Lambda10 LC APC10 10 0 10 CD2010 APC-Vio77010 10 10 CD45 VioGreen CD3 BUV496 Lambda LC APC CD20 APC-Vio770 Overlay of total population of Same total population now Overlay of B-cell gated populations Overlay of B-cell gated normal PBMCs with B-CLL overlaid with CD3 and CD19, from normal PBMCs and B-CLL populations from normal PBMCs sample, showing almost showing nearly complete sample. Normal PBMC B-cells and B-CLL sample. Normal complete dominance of dominance of CD19-positive will display a ratio of surface PBMC B-cells will have a relatively lymphocyte population based on B-cell population in the membrane Ig light chain of kappa high expression of CD20 and CD45 and side scatter. B-CLL sample. to lambda. The B-CLL sample CD38. The same gated population shows a single chain expression, from a B-CLL patient shows a indicating a monoclonal population diminished CD38 expression. of B-cells.

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

Normal Blood B-Cell Lymphocytic Leukemia

5 5 5 5 10 10 10 10 B-Cells B-CellsB-Cells 4 B-Cells 4 10 4 10 4 89.6% 10 11.0%11.0% 10 89.6% 4 4 4 4 10 10 10 10

3 T-CellsT-Cells 3 3 10 3 1010 T-Cells 10 3 3 T-Cells 3 3 70.3%70.3% 10 10 10 10 1.98%1.98% CD19 BUV737 CD19 BUV737 CD19 BUV737 CD19 BUV737 Kappa light chain BV421 2 Kappa light chain BV421 2 2 2 10 10 00 10 00 10 Kappa BV421 LC Kappa BV421 LC 00 00

2 2 2 2 -10-10 -10-10 0 1 2 3 3 3 4 5 0 1 2 3 3 3 4 5 10 10 10 10 -10 0 10 10 10 10 10 10 10 -10 0 10 10 10 100 101 102 103 -10 3 0 103 104 105 100 101 102 103 -10 3 0 103 104 105 CD3 BUV496 Lambda LC APC CD3 BUV496 Lambda LC APC CD3 BUV496 Lambda LC APC CD3 BUV496 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.

Normal Blood B-Cell Lymphocytic Leukemia

5 5 5 5 5 5 5 5 10 10 10 10 10 10 10 10

4 4 4 4 4 4 10 10 4 10 10 10 4 10 10 B-CellsB-Cells 10

B-CellsB-Cells 3 3 3 3 10 10 3 3 10 10 3 3 1010 10 10 CD38 APC-R700 CD34 PE-Vio770 CD34 PE-Vio770 CD38 APC-R700

00 00 0 00 CD34 PE-Vio770 0 CD34 PE-Vio770 CD38 APC-R700 CD38 APC-R700 3 3 3 3 -10-10 -10-10

3 3 4 5 3 4 3 3 4 5 3 4 -10 0 10 10 10 0 10 10 -10 0 10 10 10 0 10 10 3 3 4 5 3 4 3 3 4 5 3 4 -10 0 CD2010 APC-Vio770 10 10 0 CD1910 BUV737 10 -10 0 CD2010 APC-Vio770 10 10 0 CD19 BUV73710 10 CD20 APC-Vio770 CD19 BUV737 CD20 APC-Vio770 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 ■■ 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

Visit bio-rad.com/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.

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