Cytometry Part B (Clinical Cytometry) 00B:00–00 (2014)

Brief Communication

Unexpected Interference in Cell Surface Staining by Monoclonal Antibodies to Unrelated Antigens Martina De Vita,1 Valentina Catzola,1 Alexia Buzzonetti,1 Marco Fossati,1 Alessandra Battaglia,1 Loris Zamai,2 and Andrea Fattorossi1* 1

Laboratorio di Immunologia dei tumori, Unita di Ginecologia Oncologica, Universita Cattolica del Sacro Cuore, Roma, Italia 2 Dipartimento di Scienze della Terra, della Vita e dell’Ambiente, Universita di Urbino ‘‘Carlo Bo,’’ Urbino, Italia

Background: The possible occurrence of an erroneous immunophenotyping due to interference between monoclonal antibodies (MoAbs) is often overlooked when the epitopes are assumed to be not close to each other. This is particularly important when exploring immune cell populations whose identification is still investigational. The commonly held view is that myeloid derived suppressor cells can be identified as either HLA-DRneg/dim cells or interleukin-4 receptor-a (CD124)1 cells among peripheral blood monocytes. We made the serendipitous observation that the fluorescence signal provided by the PE-CD124 MoAb was attenuated when the PE-CF594-HLA-DR MoAb was added to the staining tube. Methods: Peripheral blood mononuclear cells from healthy donors were stained with the PE-CD124 MoAb and, as control, PE -CD40, -CD4 and -CD14, and either the PE-CF594-HLA-DR MoAb or its unlabeled form. B cells, which also express CD124, were analyzed for comparison. Results: The PE-CF594-HLA-DR MoAb but not its unlabeled form reduced PE-CD124 MoAb staining on monocytes and B cells. No other monocyte and B cell surface marker staining was affected by the PECF594-HLA-DR MoAb. The PE-CF594-HLA-DR MoAb interfered with the PE-CD124 MoAb likely because of steric hindrance by bulky fluorochromes, although a quenching due to fluorescence resonance energy transfer might also cooperate to the PE-CD124 MoAb staining attenuation. Conclusions: Present observations highlight the importance of interference between MoAbs as a C 2014 International Clinical Cytometry Society source of error when analyzing multicolor flow cytometry data. V Key terms: immunophenotyping; steric hindrance; quenching; flow cytometry

How to cite this article: De Vita M, Catzola V, Buzzonetti A, Fossati M, Battaglia A, Zamai L, and Fattorossi A. Unexpected Interference in Cell Surface Staining by Monoclonal Antibodies to Unrelated Antigens. Cytometry Part B 2014; 00B: 000–000.

Continuous improvements in instrumentation and the possibility to perform multicolor immunophenotypic evaluation of immune cell subsets have made flow cytometry an essential tool in the research and clinical diagnostic laboratory. In the last decade, the development of new fluorescent dyes and tandems has increased dramatically the number of parameters that can be simultaneously scored through a variety of excitation and emission spectra. Consequently, when creating reagent panels the focus of attention is on how ensure consistency of reagents, balance expression level of the target molecule versus fluorochrome brightness and set proper compensation, whereas the impact of a possible

C 2014 International Clinical Cytometry Society V

interference between monoclonal antibodies (MoAbs) on immunophenotyping is generally not adequately tested. When we scanned the literature on routine M. De Vita and V. Catzola contributed equally to this work. *Correspondence to: Andrea Fattorossi, Laboratorio di Immunologia dei tumori, Unit a di Ginecologia Oncologica, Universit a Cattolica del Sacro Cuore, L.go Agostino Gemelli, 8, 00168, Roma, Italia. E-mail: [email protected] Received 12 May 2014; Revised 1 September 2014; Accepted 29 September 2014 Published online 00 Month 2014 in Wiley Online Library ( DOI: 10.1002/cyto.b.21197



immunophenotyping, we were able to find reference to this issue in only two papers (1,2). The Purdue Cytometry Discussion List at includes comments on MoAbs interference but does not pay attention to epitopes that are supposed to be far away from each other, the discussion being essentially restricted to molecules that are known to be physiologically related to each other, for example, ab T cell receptor and CD3. Our flow cytometry laboratory is involved in the immunomonitoring of cancer patients enrolled in clinical trials. Among the various cell populations regarded as detrimental for an effective antitumor immune response, myeloid derived suppressor cells (MDSCs), a heterogeneous immune cell population that includes immature macrophages, granulocytes, dendritic cells, and other progenitor stage myeloid cells (3), are commonly investigated. In the context of tumor immunology, MDSC have been enumerated as either HLA-DRneg/dim cells (4) or interleukin-4 receptor-a (CD124)1 cells among monocytes (5). In this kind of studies changing the compilation of markers to delineate the immune cell populations of interest during the follow up precludes comparison of consecutive data. Thus, to maximize our chances of identifying MDSC changes occurring during a combinatorial immunotherapy clinical trial for Oregovomab (6) in ovarian cancer patients, we decided to identify MDSC among peripheral blood mononuclear cells (PBMC) by MoAbs to HLA-DR and CD124. While selecting MoAb pairs to these antigens, we serendipitously observed that the fluorescence signal provided by the PE-CD124 MoAb (clone 25463, R&D) was reduced when the PE- aminooxyacetamide (CF594)-HLA-DR MoAb (clone G46-6, BD Biosciences) was added to the staining tube (Fig. 1A). Errors in compensation were excluded, as we routinely use BDTM CompBead (BD Biosciences) that provides the brightest reagent possible for each compensation, and further check compensation appropriateness for each MoAb panel using single color stained cells. Errors in MoAb amount were also excluded, as in preparation of our reagents we routinely titer the reagent according to the saturating concentration and the minimum amount of reagent needed to achieve good separation between positive and negative populations. Finally, an unwanted binding of the MoAbs to Fc receptors on monocytes was not an issue, as we routinely use the FcR Blocking Reagent (Miltenyi). Thus, we set about investigating the reasons for the phenomenon and performed a series of staining experiments in PBMC. We found that the PE-CD124 MoAb staining was not affected by the unlabeled form of the HLA-DR MoAb (Fig. 1A). We also found that the PECF594-HLA-DR MoAb interference with the PE-CD124 MoAb staining was selective, as the PE-CF594-HLA-DR MoAb did not interfere with the staining of the PE-CD40 MoAb (clone MAB89, Beckman Coulter) (Fig. 1A), as well as of two additional PE-labeled MoAbs to monocyte surface markers, namely CD4 (clone SK3, BD Biosciences)

FIG. 1. Exemplificative data analysis strategy to assess PE-CD124 MoAb staining modulation induced by the PE-CF594-HLA-DR MoAb in A, monocytes and B, B cells. Gray histograms, PE-CD124 MoAb 1 PECF594-HLA-DR MoAb; light gray histograms, PE-CD124 MoAb 1 unlabeled HLA-DR MoAb; deep gray histograms, PE-CD124 MoAb alone. The mean fluorescence channel number was taken as a measure of the mean fluorescence intensity (MFI) of PE-CD124 MoAb staining and is shown in each panel. A, monocytes were gated according to their forward (FS) and side scatter (SS) characteristics. B, a lymphocyte gate was initially set according to FS and SS characteristics (not shown), and B cells were further gated by a FITC-labeled CD19 MoAb (clone J3–19, Beckman Coulter).

and CD14 (clone RM052, Beckman Coulter) (data not shown). Identical results were obtained in B cells, the only additional cell population that expresses CD124 among PBMC (7) (Fig. 1B). These experiments allowed us to infer that the HLA-DR and CD124 molecules must be close to each other on the cell surface. Moreover, the observation that the PE-CF594 labeled form of the HLADR MobAb and not its unlabeled form hampered the PECD124 MoAb staining indicated that the proximity of the binding sites of the two MoAbs caused a steric hindrance because of the cumulative size of the antibodies and fluorochromes. However, we observed that the PE-CF594HLA-DR MoAb reduced the PE-CD124 MoAb staining also when the PE-CD124 MoAb was given a binding advantage by adding it first to the staining tube (not shown).

Cytometry Part B: Clinical Cytometry



Thus, a steric hindrance cannot be the entire explanation for the reduced PE-CD124 MoAb staining in the presence of the PE-CF594-HLA-DR MoAb, as it is highly unlikely that the latter competes with the PE-CD124 MoAb already bound to its cognate antigen. An additional reason for the reduced PE-CD124 MoAb staining relates to fluorescence resonance energy transfer (FRET). Because FRET results in the quenching of donor intensity (8), it is possible the PE-CD124 MoAb staining be attenuated by PE-CF594 tandem adsorbing a portion of PE-CD124-derived energy. Unfortunately, we had not the possibility to address FRET-induced donor attenuation directly by performing formal FRET experiments on the surface colocalization of the epitopes recognized by the two MoAbs being restricted by hardware. It is clear that more work needs to be done in clarifying in full the mechanisms underlying the interference of the PE-CF594-HLA-DR MoAb with the PE-CD124 MoAb staining as well as in understanding the possible role of molecular interaction of CD124 and HLA-DR molecules on monocyte and B cell functionality, but this is beyond the scope of our report. The purpose of this report is to highlight a technical variable due to epitope proximity that should be considered in formulating schemes for immune cell analysis. Knowing which MoAbs may interfere with the staining of other MoAbs in multicolor panels should be assessed beforehand, especially when the distance among epitopes is

Cytometry Part B: Clinical Cytometry

unknown and reagent panels are aimed to explore immune cell populations for which there are no definite reference intervals and in studies concerning patients that may be lost to follow-up.

LITERATURE CITED 1. Kanakoudi-Tsakalidou F, Debonera F, Drossou-Agakidou V, Sarafidis K, Tzimouli V, Taparkou A, Kremenopoulos G. Flow cytometric measurement of HLA-DR expression on circulating monocytes in healthy and sick neonates using monocyte negative selection. Clin Exp Immunol 2001;123:402–407. 2. D’hautcourt J-L, Isaac J. Mean fluorescence intensity of dual stained cells. Cytometry 1999;38:44–45. 3. Haile LA, Greten TF, Korangy F. Immune suppression: The hallmark of myeloid derived suppressor cells. Immunol Invest 2012;4:581– 594. 4. Filipazzi, P, Valenti R, Huber V, Pilla L, Canese P, Iero M, Castelli C, Mariani L, Parmiani G, Rivoltini. Identification of a new subset of myeloid suppressor cells in peripheral blood of melanoma patients with modulation by a granulocyte-macrophage colony-stimulation factor-based antitumor vaccine. J Clin Oncol 2007;25:2546–2553. 5. Mandruzzato S, Solito S, Falisi E, Francescato S, Chiarion-Sileni V, Mocellin S, Zanon A, Rossi CR, Nitti D, Bronte V, Zanovello P. IL4Ralpha1 myeloid-derived suppressor cell expansion in cancer patients. J Immunol 2009;182:6562–6568. 6. Schultes BC, Baum RP, Niesen A, Noujaim AA, Madiyalakan R. Antiidiotype induction therapy: anti-CA125 antibodies (Ab3) mediated tumor killing in patients treated with Ovarex mAb B43.13 (Ab1). Cancer Immunol Immunother 1998;46:201–212. 7. Galanaud P, Karray S, Llorente L. Regulatory effects of IL-4 on human B-cell response to IL-2. Eur Cytokine Netw 1990;1:57–64. 8. Batard P, Szollosi J, Luescher I, Cerottini J-C, MacDonald R, Romero P. Use of phycoerythrin and allophycocyanin for fluorescence resonance energy transfer analyzed by flow cytometry: advantages and limitations. Cytometry 2002;48:97–105.

Unexpected interference in cell surface staining by monoclonal antibodies to unrelated antigens.

The possible occurrence of an erroneous immunophenotyping due to interference between monoclonal antibodies (MoAbs) is often overlooked when the epito...
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