Editorial
Stardust Memories Attila Tarnok1,2*
SEVERAL sources of error can hamper compatibility of flow cytometry data for specimens from different individuals or when comparing different treatments of samples, for example, in drug discovery or in revealing signal transduction cascades on the single cell level. The major sources of errors are preanalytics (1), instrument instability (2) and inter instrument differences (2,3), and data analysis. These technically introduced errors reduce the sensitivity of any assay. An elegant way to overcome some of these obstacles is cellular barcoding (4) by which each sample is receiving a unique (color) code. All samples are pooled together and then stained, measured, and analyzed in a single run. Barcoding is possible with cocktails of different dyes that permanently label cells (4), by genetic barcoding (5) or monoclonal antibodies (this issue, Lai et al. page 369; Maiga et al., page 285). Barcoding is also advantageous when several precious samples with minimal sample volumes are handled. And not to forget, barcoding is also a cost saver and reduces both preparation time and reagent cost which is of particular relevance for high-content analysis by polychromatic flow cytometry and highly multiplexed mass cytometry. Here two new approaches for cellular barcoding are proposed. Maiga and coworkers from France communicate (this issue, page 285) for polychromatic analysis of different cancer cell lines to take advantage of different HLA class I antigens of these cells and use of specific HLA I antibodies for barcoding. In mass-cytometry or CyTOF of intracellular cytokine expression in peripheral blood mononuclear cells (PBMC) after stimulation, Lai and coworkers from Singapore (this issue, page 369) propose to use anti CD45 antibodies for cell bar-
coding prior to cell stimulation and intracellular labeling. Each individual sample is labeled with a cocktail of CD45 antibodies conjugated to different Lanthanide isotopes, enabling their unequivocal authentication. For a different assay, namely immunophenotyping by surface antigen staining, a comparable barcoding approach was presented independently last month by others (6) showing the broad field of application of the method. This brings me to the title I have chosen for this editorial. It intends to resonate with my first editorial on masscytometry in 2012 (7). In this issue, we have a nice collection of three papers on CyTOF from three continents. Since its beginning just a few years ago and with the earliest publication in this journal less than three years ago (8), the technology has matured and become common in many advanced laboratories around the world. Here, Han and coworkers from USA and Canada (this issue, page 346) report on the analysis of complex intracellular signaling using surface antigen labeling in combination with phospho-flow. They demonstrate that flow and mass cytometry produce comparable results and are in agreement with Western blot. Of course, as shown by the authors mass cytometric analysis can be much more complex than that by conventional flow cytometry. The broadening field of applications and users makes it sensible to start thinking about issues of standardization and quality control and aspects of instrument sensitivity and specificity (1–3) also in mass cytometry. In its early, primordial beginnings setting up mass cytometry experiments and designing complex staining cocktails is a simple
Published online in Wiley Online Library (wileyonlinelibrary.com) 1
2
Department of Pediatric Cardiology, Heart Centre Leipzig, Leipzig, Germany Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Leipzig, Germany
Received 12 February 2015; Accepted 2 March 2015 Grant sponsor: German Federal Ministry of Education and Research, Grant number: BMBF, AT: PtJ-Bio, 1315883 *Correspondence to: Prof. Attila T arnok, Department of Pediatric Cardiology, Heart Centre Leipzig, University of Leipzig, Str€ umpellstr. 39, 04289 Leipzig, Germany. E-mail: [email protected]
Cytometry Part A 87A: 283 284, 2015
DOI: 10.1002/cyto.a.22660 C 2015 International Society for Advancement of Cytometry V
Editorial straightforward task and any antibody with any isotope goes. For me an eye opener was a talk of Antonio Cosma at an INSERM conference in Warsaw at the Flow Cytometry Workshop back in April 2013. Here it became clear to me that designing the correct and best combination of antibodies and Lanthanide isotopes is not as simple as I thought. CyTOF detection sensitivity depends on isotope mass so it matters which conjugate is used as an infrequent and a frequent antigen. I also thought that a mass is a mass is a mass. If I measure an isotope with the mass, let’s say, of 141 I will see its signal only in the mass channel 141 and not left or right of it. But this is not essentially the case and can produce spillover into adjacent channels resembling spectral spillover in a traditional flow. Antonio and I discussed that this information should be provided to a broader audience. This, to my knowledge, is the first manuscript on cross-platform evaluation (Tricot and coworkers from France and Italy; this issue, page 357.) and other important issues on sensitivity of mass-cytometry and I am happy that it is published in Cytometry Part A. I particularly find the conclusion of the authors important stating that mass specific sensitivity patterns and additional aspects
284
should be taken into account in future panel design. This is a call for development and submission OMIPs for mass cytometry.
LITERATURE CITED 1. Bocsi J, Melzer S, D€ahnert I, Tarnok A. OMIP-023: 10-color, 13 antibody panel for in-depth phenotyping of human peripheral blood leukocytes. Cytometry A 2014; 85A:781–784. 2. Kalina T, Flores-Montero J, Lecrevisse Q, Pedreira CE, van der Velden VH, Novakova M, Mejstrikova E, Hrusak O, B€ ottcher S, Karsch D, et al. Quality assessment program for EuroFlow protocols: Summary results of four-year (2010–2013) quality assurance rounds. Cytometry A 2015;87A:145–156. 3. Solly F, Rigollet L, Baseggio L, Guy J, Borgeot J, Guerin E, Debliquis A, Drenou B, Campos L, Lacombe F, et al. Comparable flow cytometry data can be obtained with two types of instruments, canto II, and navios. A GEIL study. Cytometry A 2013; 83A:1066–1072. 4. Krutzik PO, Nolan GP. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods 2006;3: 361–368. 5. Smurthwaite CA, Hilton BJ, O’Hanlon R, Stolp ZD, Hancock BM, Abbadessa D, Stotland A, Sklar LA, Wolkowicz R. Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry. Cytometry A 2014; 85A:105–113. 6. Mei HE, Leipold MD, Schulz AR, Chester C, Maecker HT. Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry. J Immunol 2015;194:2022–2031. 7. Tarnok A. Cosmic chemistry for cytometry. Cytometry A 2012;81A:541–543. 8. Fienberg HG, Simonds EF, Fantl WJ, Nolan GP, Bodenmiller B. A platinum-based covalent viability reagent for single-cell mass cytometry. Cytometry A 2012;81A:467–475.
Editorial
Stardust Memories Attila Tarnok1,2*
SEVERAL sources of error can hamper compatibility of flow cytometry data for specimens from different individuals or when comparing different treatments of samples, for example, in drug discovery or in revealing signal transduction cascades on the single cell level. The major sources of errors are preanalytics (1), instrument instability (2) and inter instrument differences (2,3), and data analysis. These technically introduced errors reduce the sensitivity of any assay. An elegant way to overcome some of these obstacles is cellular barcoding (4) by which each sample is receiving a unique (color) code. All samples are pooled together and then stained, measured, and analyzed in a single run. Barcoding is possible with cocktails of different dyes that permanently label cells (4), by genetic barcoding (5) or monoclonal antibodies (this issue, Lai et al. page 369; Maiga et al., page 285). Barcoding is also advantageous when several precious samples with minimal sample volumes are handled. And not to forget, barcoding is also a cost saver and reduces both preparation time and reagent cost which is of particular relevance for high-content analysis by polychromatic flow cytometry and highly multiplexed mass cytometry. Here two new approaches for cellular barcoding are proposed. Maiga and coworkers from France communicate (this issue, page 285) for polychromatic analysis of different cancer cell lines to take advantage of different HLA class I antigens of these cells and use of specific HLA I antibodies for barcoding. In mass-cytometry or CyTOF of intracellular cytokine expression in peripheral blood mononuclear cells (PBMC) after stimulation, Lai and coworkers from Singapore (this issue, page 369) propose to use anti CD45 antibodies for cell bar-
coding prior to cell stimulation and intracellular labeling. Each individual sample is labeled with a cocktail of CD45 antibodies conjugated to different Lanthanide isotopes, enabling their unequivocal authentication. For a different assay, namely immunophenotyping by surface antigen staining, a comparable barcoding approach was presented independently last month by others (6) showing the broad field of application of the method. This brings me to the title I have chosen for this editorial. It intends to resonate with my first editorial on masscytometry in 2012 (7). In this issue, we have a nice collection of three papers on CyTOF from three continents. Since its beginning just a few years ago and with the earliest publication in this journal less than three years ago (8), the technology has matured and become common in many advanced laboratories around the world. Here, Han and coworkers from USA and Canada (this issue, page 346) report on the analysis of complex intracellular signaling using surface antigen labeling in combination with phospho-flow. They demonstrate that flow and mass cytometry produce comparable results and are in agreement with Western blot. Of course, as shown by the authors mass cytometric analysis can be much more complex than that by conventional flow cytometry. The broadening field of applications and users makes it sensible to start thinking about issues of standardization and quality control and aspects of instrument sensitivity and specificity (1–3) also in mass cytometry. In its early, primordial beginnings setting up mass cytometry experiments and designing complex staining cocktails is a simple
Published online in Wiley Online Library (wileyonlinelibrary.com) 1
2
Department of Pediatric Cardiology, Heart Centre Leipzig, Leipzig, Germany Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Leipzig, Germany
Received 12 February 2015; Accepted 2 March 2015 Grant sponsor: German Federal Ministry of Education and Research, Grant number: BMBF, AT: PtJ-Bio, 1315883 *Correspondence to: Prof. Attila T arnok, Department of Pediatric Cardiology, Heart Centre Leipzig, University of Leipzig, Str€ umpellstr. 39, 04289 Leipzig, Germany. E-mail: [email protected]
Cytometry Part A 87A: 283 284, 2015
DOI: 10.1002/cyto.a.22660 C 2015 International Society for Advancement of Cytometry V
Editorial straightforward task and any antibody with any isotope goes. For me an eye opener was a talk of Antonio Cosma at an INSERM conference in Warsaw at the Flow Cytometry Workshop back in April 2013. Here it became clear to me that designing the correct and best combination of antibodies and Lanthanide isotopes is not as simple as I thought. CyTOF detection sensitivity depends on isotope mass so it matters which conjugate is used as an infrequent and a frequent antigen. I also thought that a mass is a mass is a mass. If I measure an isotope with the mass, let’s say, of 141 I will see its signal only in the mass channel 141 and not left or right of it. But this is not essentially the case and can produce spillover into adjacent channels resembling spectral spillover in a traditional flow. Antonio and I discussed that this information should be provided to a broader audience. This, to my knowledge, is the first manuscript on cross-platform evaluation (Tricot and coworkers from France and Italy; this issue, page 357.) and other important issues on sensitivity of mass-cytometry and I am happy that it is published in Cytometry Part A. I particularly find the conclusion of the authors important stating that mass specific sensitivity patterns and additional aspects
284
should be taken into account in future panel design. This is a call for development and submission OMIPs for mass cytometry.
LITERATURE CITED 1. Bocsi J, Melzer S, D€ahnert I, Tarnok A. OMIP-023: 10-color, 13 antibody panel for in-depth phenotyping of human peripheral blood leukocytes. Cytometry A 2014; 85A:781–784. 2. Kalina T, Flores-Montero J, Lecrevisse Q, Pedreira CE, van der Velden VH, Novakova M, Mejstrikova E, Hrusak O, B€ ottcher S, Karsch D, et al. Quality assessment program for EuroFlow protocols: Summary results of four-year (2010–2013) quality assurance rounds. Cytometry A 2015;87A:145–156. 3. Solly F, Rigollet L, Baseggio L, Guy J, Borgeot J, Guerin E, Debliquis A, Drenou B, Campos L, Lacombe F, et al. Comparable flow cytometry data can be obtained with two types of instruments, canto II, and navios. A GEIL study. Cytometry A 2013; 83A:1066–1072. 4. Krutzik PO, Nolan GP. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods 2006;3: 361–368. 5. Smurthwaite CA, Hilton BJ, O’Hanlon R, Stolp ZD, Hancock BM, Abbadessa D, Stotland A, Sklar LA, Wolkowicz R. Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry. Cytometry A 2014; 85A:105–113. 6. Mei HE, Leipold MD, Schulz AR, Chester C, Maecker HT. Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry. J Immunol 2015;194:2022–2031. 7. Tarnok A. Cosmic chemistry for cytometry. Cytometry A 2012;81A:541–543. 8. Fienberg HG, Simonds EF, Fantl WJ, Nolan GP, Bodenmiller B. A platinum-based covalent viability reagent for single-cell mass cytometry. Cytometry A 2012;81A:467–475.
Editorial
