Stardust memories

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 barcoding 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