OMIP - 081: A new 21-monoclonal antibody 10-color panel for diagnostic polychromatic immunophenotyping.

The 10-color panel consisting of 21 monoclonal antibodies (mAbs) is developed as a one-tube panel to detect leukemia and lymphoma cells in all hematopoietic cell lineages. In particular, this tube is mentioned for a fast screening to identify aberrant cells in samples suspected for malignant cell localization and to enable comprehensive immunophenotyping of samples with low cell counts. The panel contains mAbs for selection of the populations and mAbs against target antigens on the various hematopoietic maturation stages. Due to the limited number of PMTs in most used flow cytometers for clinical purposes, stacking of conjugates in one color is needed to include all relevant markers for simultaneous analysis of the aberrant cells. The 21-mAb panel is tested on peripheral blood (PB), and bone marrow (BM) samples and enables an efficient and correct identification of hematological malignancies. This panel improves the diagnostic potential.

PURPOSE AND APPROPRIATE SAMPLE TYPES

The 10‐color panel consisting of 21 monoclonal antibodies (mAbs) is developed as a one‐tube panel to detect leukemia and lymphoma cells in all hematopoietic cell lineages. In particular, this tube is mentioned for a fast screening to identify aberrant cells in samples suspected for malignant cell localization and to enable comprehensive immunophenotyping of samples with low cell counts. The panel contains mAbs for selection of the populations and mAbs against target antigens on the various hematopoietic maturation stages. Due to the limited number of PMTs in most used flow cytometers for clinical purposes, stacking of conjugates in one color is needed to include all relevant markers for simultaneous analysis of the aberrant cells. The 21‐mAb panel is tested on peripheral blood (PB), and bone marrow (BM) samples and enables an efficient and correct identification of hematological malignancies. This panel improves the diagnostic potential.

BACKGROUND

The rapid and sensitive multiparameter detection, even at a single cell level, renders flow cytometry (FCM) to a powerful tool to distinguish malignant hematopoietic cells from normal cells. Due to the technical aspects of the FCMs for diagnostics, currently immunophenotypic analyses often consist of up to 10‐mAb panels and oblige the use of multiple tubes that contain the specific combinations of mAbs to characterize the aberrant cell population. However, this approach hampers the evaluation of co‐expression patterns from mAbs that are present in different tubes. In particular, determinations like minimal residual disease (MRD) or characterization of very small cell populations need high‐level multicolor analyses [1, 2, 3, 4, 5]. Furthermore, the multiple color analysis using multiple tubes is time consuming and cost ineffective. Therefore, a one‐tube approach with 21 mAbs is more sensitive and adequate to detect aberrant cells. Besides the need of a subsequent second step and more targeted immunophenotyping will be reduced (Table 1).

TABLE 1

Summary table for application of the OMIP

Purpose	Improvement of immunophenotyping of leukemia and lymphoma by a fast one‐tube screening panel consisting of 10 colors and 21 mAbs
Species	Human
Cell type	Bone marrow, peripheral blood
Cross‐references	OMIP 10 (see Reference [2])

At present, the application of panels with more than 10 colors is restricted technically for diagnostic purposes. In order of improve immunophenotyping by applying more than 10 mAbs in one tube, stacking of antibodies within one fluorescence channel is therefore needed. We developed a screening panel using stacking up to three antibodies resulting in up to 23 evaluable parameters (FS, SS, and mAbs). Similar approaches of stacking have been applied by other groups [6, 7, 8, 9, 10]. This 21‐mAb in 10‐colors panel can be a reliable alternative for the use of 23‐PMT containing FCMs that are presently hardly available.

After selection of the target antigens and search for suitable mAb clones and fluorochromes (Table 2), all mAbs were titrated to find the optimal concentration. Subsequently, compensation of spectral overlap and extensive balancing of the panel was performed to establish suitable and correct combinations of stacked mAbs with preservation of their capacity to separate and identify the different hematopoietic cell populations and maturation stages.

TABLE 2

Reagents used for the OMIP

Specificity	Clone	Fluorochrome	Purpose
CD45	J33	KO	WBC subpopulations
CD3	UCHT1	APC‐AF750	Mature T cells
CD4	SFCI12T4B11	PE‐Cy7	Helper T cells, Monocyte lineage
CD8	B9.11	PacB	Cytotoxic T cells, NK‐cell subset
CD2	39C15	APC‐AF700	T‐cell lineage, NK‐cell subset
CD5	BL1a	PE‐Cy5.5	T‐cell lineage, B‐cell subset
CD7	8H8.1	FITC	T‐cell lineage, T‐cell activation, NK‐cell subsets
CD16	B73.1	PE	T‐cell subsets, NK cells, monocyte differentiation, eosinophil exclusion, myeloid lineage
CD56	NCAM16.2	PE	NK cells, plasma cell subset
CD19	J3.119	ECD	B‐cell lineage
CD20	B9E9	PacB	Mature B cells
Igkappa	Rb‐anti‐Hu a	APC	B‐cell subset, B‐cell lineage clonality
Iglambda	Rb‐anti‐Hu a	PE	B‐cell subset, B‐cell lineage clonality
CD138	B‐B4	FITC	Plasma cells
CD14	RM052	ECD	Mature monocytes
CD36	FA6.152	APC‐AF700	Monocyte lineage, erythroid lineage, megakaryocytes
CD33	D3HL60.251	PE‐Cy5.5	Monocyte lineage, myeloid lineage
CD15	80H5	PacB	Myeloid lineage
CD34	581	PE‐Cy7	Progenitor cells
CD117	104D2D1	APC	Myeloid precursors
CD10	ALB1	APC‐AF700	B‐cell precursors, mature neutrophils

Abbreviations: AF, AlexaFluor; APC, allophycocyanin; Cy, cyanin; ECD, energy‐coupled dye (PE coupled to Texas Red); FITC, fluorescein isothiocyanate; KO, Krome Orange; PacB, Pacific Blue; PE, R‐phycoerythrin; WBC, white blood cells.

Polyclonal rabbit‐anti‐human antibody.

Testing of the panel was performed on a Navios (10‐color/3‐laser instrument; Beckman Coulter; see Table S1). KALUZA™ analysis software was used to establish a suitable analysis protocol (Table 3) with Boolean gating to separate expression patterns of the various stacked mAbs in the different channels. Thereby, we could identify the main subpopulations of all hematopoietic lineages and malignant aberrancies in this one‐tube panel (Figure 1).

TABLE 3

Gating strategy of the 21‐mAb 10‐color panel to identify and characterize the relevant cell populations adequately

Population of interest	mAbs to select populations	Relevant Abs in respective stacked combination	Identification purpose	Used Abs to detect subset in selected population
Lymphocytes	CD45	CD45 KO
Monocytes	CD45	CD45 KO
Myeloid cells	CD45	CD45 KO
Precursors	CD45	CD45 KO
Plasma cells	CD138	CD7 CD138 FITC
T cells	CD3	CD3 APC‐AF750	T‐cell activation	CD2 CD5 CD7
Within lymphocytes and precursors		CD7 CD138 FITC	Helper T cell	CD4+ CD8‐
		CD5 CD33 PE‐Cy5.5	Cytotoxic T cell	CD4‐ CD8+
		CD4 CD34 PE‐Cy7	T‐cell subset	CD4+CD8+
		CD2 CD10 CD36 APC‐AF700	T‐cell subset	CD4‐CD8‐
		CD8 CD15 CD20 PacB
B cells	CD19	CD16 CD56 Iglambda PE	Clonality in B cells	Igkappa/Iglambda
Within lymphocytes and precursors		CD117 Igkappa APC	B‐cell precursor	CD10+ CD20− CD20dim
		CD2 CD10 CD36 APC‐AF700	Mature B cell	CD10− CD20+
		CD8 CD15 CD20 PacB
NK cells	CD3− CD16/CD56+	CD3 APC‐AF750
Within lymphocytes		CD16 CD56 Iglambda PE
NK‐T cells	CD3+ CD16/CD56+	CD3 APC‐AF750
Within lymphocytes		CD16 CD56 Iglambda PE
Precursors	CD34 CD117	CD14 CD19 ECD	B‐cell precursor	CD34+ CD117− CD19+
Within CD45dim/SSdim		CD5 CD33 PE‐Cy5.5	Myeloid Precursor	CD34+ CD117+ CD33+
		CD4 CD34 PE‐Cy7
		CD117 Igkappa APC
Myeloid cells	CD15+	CD5 CD33 PE‐Cy5.5	Granulocytes	CD33+ CD15+
Within CD45dim/SS+		CD8 CD15 CD20 PacB
Monocytes	CD14+	CD14 CD19 ECD	Monocytes	CD33++ CD36+
Within CD45+/SSdim		CD5 CD33 PE‐Cy5.5
		CD2 CD10 CD36 APC‐AF700
Plasma cells	CD138+	CD16 CD56 Iglambda PE	Plasma cells	CD138+ CD19+
Within CD138+		CD16 CD56 Iglambda PE	Clonality	Igkappa/Iglambda
		CD14 CD19 ECD	Abarrant markers	CD56+ CD117+
		CD5 CD33 PE‐Cy5.5	Exclusion of non‐plasma cells	CD2 CD10 CD5
		CD117 Igkappa APC
		CD117 Igkappa APC
		CD2 CD10 CD36 APC‐AF700

Note: The gating procedure of the relevant cell populations is shown in Figure 1. Color‐marked populations and antibodies are corresponding with the colors in Figure 1.

FIGURE 1

Staining and evaluation protocol of the new 21‐mAb 10‐color panel. By using the established gating procedure, the combination of 21 mAbs in 10 colors provides optimal information about lymphocyte subsets, monocytes, myeloid cells, precursor cells, and aberrant cell populations. A normal bone marrow was used to establish the efficacy of the 21‐mAb panel. A, Monocytes are excluded from gated cells; B, Lymphocytes, monocytes, granulocytes, and plasma cells are excluded from gated cells; C, T cells, B cells, and NK‐T cells are excluded from gated cells

Designing such a high‐level powerful screening tool relies on the proper balancing of the panel. Furthermore, the use of correct analysis protocols generates more accurate results by creating more co‐expression pattern and to maximize the evaluation possibilities of samples with low cell counts.

CONFLICT OF INTEREST

All authors confirm that they have no conflicts of interest with the subjects mentioned in the paper. The authors alone are responsible for the content and writing of the paper.

AUTHOR CONTRIBUTIONS

Erik H. L. P. G. Huys: Conceptualization (equal); data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); resources (equal); software (equal); supervision (equal); validation (equal); visualization (equal); writing—original draft (equal); writing—review and editing (equal). Willemijn Hobo: Writing—review and editing (equal). Frank W. M. B. Preijers: Conceptualization (lead); data curation (equal); funding acquisition (lead); investigation (equal); methodology (equal); project administration (equal); resources (equal); software (equal); supervision (lead); validation (equal); visualization (equal); writing—original draft (equal); writing—review and editing (lead).

PEER REVIEW

The peer review history for this article is available at https://publons.com/publon/10.1002/cyto.a.24511.

MIFlowCyt: MIFlowCyt‐Compliant Items.

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Appendix S1: Supporting Information

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FIGURE S1 A and B Normal BM was spiked with BM of an AML patient having a patient‐specific leukemia‐associated immunophenotype (LAIP) to obtain 4.5% AML cells in the ultimately sample. Subsequently 105 (Online Figure 1A.) and 103 (Online Figure 1B.) WBC of this mixed sample were flow cytometric analyzed. In the CD45/SS plot the lymphocytes, monocytes and granulocytes and the location of the AML cells to detect the LAIP (gate CD45 + ‐) were gated. Each gate is separately further analyzed using plots with the relevant mAb combinations.

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FIGURE S2 Representative analysis of a BM sample with the new 21‐mAbs 10‐color panel with suspicion of a lympho‐proliferative process. The lymphoid population (bright CD45 expression) was gated in the CD45/SS plot and lymphoid subpopulations were further defined as T cells (CD3+), NK cells (CD3‐/CD56+), NK‐T cells (CD3+/CD56+) and B cells (CD19+). Within the T cells the CD4/CD8 ratio was normal. The B cells expressed a normal Igkappa / Iglambda ratio and displayed a normal maturation pattern (CD20/CD10). No plasma cell (CD138+) aberrancies were found. No aberrant immature cells were detected in the CD45/SS plot and CD34/CD117 plot.

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FIGURE S3 Representative analysis of a BM sample with the new 21‐mAbs 10‐color panel with suspicion of acute myeloid leukemia (AML). The lymphoid population (bright CD45 expression) was identified within the CD45/SS plot. Lymphoid subpopulations were further classified as T cells (CD3+), NK cells (CD3‐/CD56+), NK‐T cells (CD3+/CD56+) and B cells (CD19). Within the T‐cells the CD4/CD8 ratio was normal. The B cells expressed a normal Igkappa/Iglambda ratio, though only mature B cell (CD20) were present. No aberrant Plasma cells (CD138+) were found. A large population aberrant immature cells was seen in the CD45/SS plot and CD34/CD117 plots. These aberrant cells also expressed CD33 + .

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FIGURE S4 Representative analysis of a PB sample with the new 21‐mAbs 10‐color panel with suspicion of evidence of acute lymphoblastic leukemia (ALL). The lymphoid population (bright CD45 expression) was identified within the CD45/SS plot. Lymphoid subpopulations were further classified as T cells (CD3+), NK cells (CD3‐/CD56+), NK‐T cells (CD3+/CD56+) and B cells (CD19). Within the T cells the CD4/CD8 ratio was normal. The B cells expressed a normal Igkappa/ Iglambda ratio and only mature B cells (CD20) were present. No aberrant plasma cells (CD138+) were found. A population of immature cells (CD45+/− SS and CD34‐ CD117+/− plots) was seen expressing CD19+ CD10+ and CD20+/−.

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FIGURE S5 Representative analysis of a BM sample with the new 21‐mAbs 10‐color panel with suspicion of evidence of a monoclonal B cell neoplasia. The lymphoid population (CD45[+]) was identified within the CD45/SS plot. The lymphoid subpopulations were further identified as T cells (CD3+), NK cells (CD3‐/CD56+), NK‐T cells (CD3+/CD56+) and B cells (CD19+).The CD4/CD8 ratio was normal. In contrast, the B‐cell population was aberrant and displayed a restricted Igkappa, CD10‐ and CD20+ expression. Furthermore, a small population of plasma cells (CD138+), CD19+ was identified expressing CD117 and Igkappa. Aberrant immature cells were not detected in the CD45 + ‐ gating of CD45/SS plot in combination with the CD34/CD117 plots.

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FIGURE S6 Representative analysis of a BM sample with the new 21‐mAbs 10‐color panel with suspicion of evidence of a monoclonal B cell neoplasia. The lymphoid population (bright CD45 expression) was identified within the CD45/SS plot. Lymphoid subpopulations were further identified as T cells (CD3+), NK cells (CD3‐/CD56+), NK‐T cells (CD3+/CD56+) and B cells (CD19+). Within the T cells the CD4/CD8 ratio was normal. The B cells expressed almost only Igkappa and CD20. CD19 + CD5+ B‐lineage cells could not be detected. Only normal plasma cells (CD138+) and immature cells were present in the CD45+/− SS plot and CD34/CD117 plot.

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