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Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative
BACKGROUND: The Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validat...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8874145/ https://www.ncbi.nlm.nih.gov/pubmed/35222411 http://dx.doi.org/10.3389/fimmu.2022.827898 |
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author | Kužílková, Daniela Puñet-Ortiz, Joan Aui, Pei M. Fernández, Javier Fišer, Karel Engel, Pablo van Zelm, Menno C. Kalina, Tomáš |
author_facet | Kužílková, Daniela Puñet-Ortiz, Joan Aui, Pei M. Fernández, Javier Fišer, Karel Engel, Pablo van Zelm, Menno C. Kalina, Tomáš |
author_sort | Kužílková, Daniela |
collection | PubMed |
description | BACKGROUND: The Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking. OBJECTIVE: To develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories. METHODS: A high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ(+), regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation. RESULTS: Using automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen. CONCLUSION: We optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops. |
format | Online Article Text |
id | pubmed-8874145 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-88741452022-02-26 Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative Kužílková, Daniela Puñet-Ortiz, Joan Aui, Pei M. Fernández, Javier Fišer, Karel Engel, Pablo van Zelm, Menno C. Kalina, Tomáš Front Immunol Immunology BACKGROUND: The Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking. OBJECTIVE: To develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories. METHODS: A high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ(+), regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation. RESULTS: Using automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen. CONCLUSION: We optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops. Frontiers Media S.A. 2022-02-11 /pmc/articles/PMC8874145/ /pubmed/35222411 http://dx.doi.org/10.3389/fimmu.2022.827898 Text en Copyright © 2022 Kužílková, Puñet-Ortiz, Aui, Fernández, Fišer, Engel, van Zelm and Kalina https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Immunology Kužílková, Daniela Puñet-Ortiz, Joan Aui, Pei M. Fernández, Javier Fišer, Karel Engel, Pablo van Zelm, Menno C. Kalina, Tomáš Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative |
title | Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative |
title_full | Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative |
title_fullStr | Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative |
title_full_unstemmed | Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative |
title_short | Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative |
title_sort | standardization of workflow and flow cytometry panels for quantitative expression profiling of surface antigens on blood leukocyte subsets: an hcdm cdmaps initiative |
topic | Immunology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8874145/ https://www.ncbi.nlm.nih.gov/pubmed/35222411 http://dx.doi.org/10.3389/fimmu.2022.827898 |
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