Advancing Cytometry for Immunology
Cytometry is a key technology for immunology. It allows researchers to scrutinize the cells of the immune system in molecular detail, and to assess phenotype and function at the level of individual cells, no matter how rare these cells may be. The International Society for the Advancement of Cytometry, ISAC, by way of its meetings, online resources and publications (e.g. Cytometry Part A and Current Protocols in Cytometry, which are all published by Wiley) track the ever advancing developments regarding cytometry instrumentation and reagents, and the analysis of complex data sets. In June this year in Leipzig, Germany, ISAC held its annual conference “CYTO 2012”, a marketplace of innovation in cytometry.
Introduction into the field
The first flow cytometry applications were not focused on immunology, but rather on the general measurement of cellular DNA content using fluorescent intercalating dyes. Later, dyes indicating biochem-ical reactions were used to study cell physiology. It was not until the introduction of fluorescent antibodies, and more specifically, monoclonal antibodies, that the technology of flow cytometry became a unique tool for the analysis of the immune system in the mid-1970s. Cells of the immune system often look alike under the microscope, but are revealed to be very diverse when analyzed using specific markers for cell surface proteins. Today, immuno-fluorescence has advanced to allow detection of even subtle molecular differences inside the cell, on its surface or the molecules secreted by it, and to analyze signaling processes, cellular biochemistry, proliferation and differentiation, as well as cell-cell interactions. Moreover, the cells of interest can be isolated with precision by cell sorting. State-of-the-art flow cytometers are more user-friendly than ever, no longer requiring the involvement of dedicated operators. Today's challenges mostly have to do with the handling of cells, the experimental design, and the evaluation of data. In other words, we are no longer limited by complex instrumentation, but by our creativity to ask the critical questions. It´s up to us, the scientists! The marriage between flow cytometry and immunofluorescence has come of age; most of the technical limitations of the combination of these technologies have been resolved; however, we now realize that there is more to come; since exciting new developments are emerging, including new tools to push forward immunological research. Here we report on the recent CYTO 2012 conference in Leipzig, organized by the International Society for the Advancement of Cytometry (ISAC). Apart from providing a platform for education, standardization and scientific communication, this conference offered a comprehensive overview of the latest technology.
Tools and toys for the immunologist
Hot topics discussed at CYTO 2012 give an idea of the directions in which cyto-metric technology is heading; many of these topics open up new avenues for immunological research and clinical developments. One such hot topic is the recent introduction of single cell mass spectrometry (mass cytometry) by Scott Tanner and his group from the University of Toronto. CyTOF (cytometry by time-of-flight) mass spectrometry detects rare earth isotopes, in particular lanthanide chelates and, when conjugated to antibodies, these isotopes can then be used to stain cells. Mass cytometry overcomes the limitations of the optical spectrum and immunofluorescence, expanding the limits of the parameters which can be measured simultaneously on one cell, from about a dozen to more than 30. By avoiding the need for (tricky) fluorescence compensation, CyTOF makes a step toward the global assessment of the proteome of individual cells. At CYTO 2012, Garry Nolan (Stanford University) described his lab's efforts to exploit mass cytometry. At present, the logistics of antibody-conjugates and the bioinformatics of data evaluation still present a major challenge but despite this, CyTOF is still capable of dissecting heterogeneous cell populations such as blood cells with unprecedented resolution .
While the introduction of mass spectrometry for single cell analysis has great potential, optical measurements still have their advantages. These advantages are being extended by the development of spectral flow cytometry, which provides new opportunities for multiparameter analysis. Spectral flow cytometry uses dispersive optics (prisms or gratings) and array detectors to measure signals, rather than the conventional approach of bandpass filters and photomultiplier tubes. Measurement of the complete optical spectrum of individual cells enables a new approach to multiparameter analysis of fluorescence  and surface enhanced Raman scattering (SERS)  signals. CYTO 2012 showcased recent advances in spectral flow cytometry instrumentation and data analysis, as well as an exhibition floor display of a 32-channel spectral flow cytometer from the Sony Corporation. The advent of commercial instruments for spectral cytometry, in particular with cell sorting options, will definitely spur the future of immunofluorescence: In mass cyto-metry, the cells analyzed cannot be recovered or sorted as they are destroyed in the process which is a major limitation.
Beyond immunofluorescence and immuno-mass-spectrometry, another new dimension of cytometry is opened up by the use of fluorescent in situ amplification technologies for the multiparametric, specific detection of mRNAs expressed in individual cells. Conventional fluorescent in situ hybridization technology cannot detect mRNAs below a copy number of 50 per cell, but many mRNAs of physiological relevance have a much lower copy number. Emily Park of the group of Vernon Maino from Becton-Dickinson Research, San Jose, presented a thrilling first set of convincing data on the establishment, calibration and multiparametric labeling of lymphocytes according to mRNA molecules as rare as one copy per cell. Her group has used the RNAScope technology, originally developed for histological in situ hybridization, and showed that RNAScope works just as well in flow cytometry. These initial data open up not only an entirely new dimension of cytometry, i.e. analyzing gene expression at the level of mRNA instead of protein, with highly specific nucleotide probes instead of antibodies, but moreover offer a perspective on the level of regulatory RNAs. Unfortunately, at this time, labeling is achieved with either oligomeric probes or antibodies, but not both, since the cells have to be fixed for hybridization in a way that destroys the protein epitopes, however, it is just a matter of time until this limitation will be overcome.
The strive for “more parameters per cell” is also evident in image cytometry. Kristina Micheva (Stanford University) presented an intriguing illustration of multiparameter image cytometry, using “array tomography”  to determine different parameters in sequential, adjacent sections of one neural tissue specimen, then aligning those tomographs to provide a multiparametric map of cellular heterogeneity in the nervous system. The beauty of this approach is its strategic simplicity.
The last technological station at the cytometry playground to be reported here is the use of acoustics for cell sorting, i.e. the alignment and positioning of cells along standing acoustic waves. While Steven Graves and his colleagues from the University of New Mexico are using this technology to focus particles into 300 parallel streams, i.e. to enhance throughput by parallel cell sorting, Thomas Laurell and his colleagues from Lund University are using acoustic cell sorting to isolate cells for microchip-based lab-on-chip (LOC) applications. Imaging-based systems are also being developed, using microsystems to analyze and isolate, paradoxically, large particles, e.g. embryoid bodies, as reported by David Buschke from the University of Wisconsin . Microchips to collect and present extremely rare cells, such as rare leukocyte subsets or circulating tumor cells, for image analysis are also advancing towards clinical application .
Innovation drives discovery
In recent years, the introduction of more sophisticated cytometric technologies and the standardization of staining panels between clinical labs have allowed the identification of novel biological mechanisms and new roles for “old” molecules. Several reports covered this topic with a particular emphasis on advancements in understanding the biology and defining a better recognition of immune cells in pathophysiology.
The Euroflow consortium, a group of European scientists headed by Alberto Orfao (Salamanca, Spain), has established staining panels for the in-depth diagnosis of T-cell chronic lymphoproliferative disorders, using 4 mAbs as a backbone-labeling panel, and 20 additional markers for fine-tuning. A similar approach has been used to classify NK-cell proliferative diseases, to distinguish between monoclonal and polyclonal expansions, and to establish quality control schemes for creating standardized staining panels for a number of hematologic malignancies [7, 8].
In functional terms, Harris Fienberg from Stanford University reported on how mass cytometry enables the combinatorial measurement of total levels, conformations, cleavage states and phosphorylation states of 10 downstream effectors of TRAIL and 14 factors involved in regulatory signals and the control of the cell cycle. His presentation was a fine example of how the new technologies can serve to visualize molecular networks in single cells.
The cytometry of cells in the context of the organism/tissue is still largely restricted to imaging, in particular intra-vital microscopic approaches, and in vivo flow cytometry remains only on the horizon . Nevertheless, flow cytometry and sorting of individualized mesenchymal cells is being used and has yielded interesting results. An example was given by Bruno Peault from the University of California, who reported that mesenchymal stem cells, as defined by their differentiation and immunomodulation in vitro, are derived exclusively from CD34−CD31−CD146+CD45− pericytes or from CD34+CD31−CD146−CD45− adventitial cells, i.e. from cells sitting on the blood vessels.
Finally, imaging cytometry is uncovering the molecular machinery of cellular movement. Till Bretschneider from the University of Warwick reported on the formation of blebs, an essential step in cellular movement, by myosin-induced detachment of actin from the membrane cortex. Iva Tolic-Norrelykke from the Max Planck Institute in Dresden reported on how dynein is assembled and oriented on microtubules, with the switch to directed movement coming when dynein binds to the cortex. Although these results were obtained with Dictyostelium and yeast, their implications for immunology are obvious. Space limitations prevent us from listing all the exciting examples of cutting-edge applications of cytometry presented at CYTO 2012 but this Congress gave ample evidence for the adage that necessity is the mother of innovation.
More than you ever wanted to know — systems cytometry
Cytometry offers the unique option of studying systems biology of individual cells (Cytomics), rather than cell populations, and of cell populations at the single cell level. At present, several thousand parameters can already be analyzed per sample by multiplexed immunofluorescence . Mass cytometry will push this limit even further.
Several groups have published both supervised and unsupervised methods for the analysis of complex cytometric data [11-14]. At CYTO 2012, Ryan Brinkman from the University of British Columbia introduced the flowBin, a fully automated method for analyzing multitube flow experiments. Paul Robinson from Purdue University introduced Plate Analyzer, an interactive and intuitive software that operates in real time even with huge data sets , and “Spanning-tree progression analysis of density-normalized events” (SPADE), an algorithm that already has been used for the immunofluorescent cytometric analysis of mouse bone marrow and the mass cytometric analysis of human bone marrow .
CYTO 2012 was also a forum for the discussion of how data should be stored and presented. With regard to interpretation and presentation, this is still an unmet challenge for most journals, and the source of many misconceptions. Often, gating is not documented, in particular scatter gates are omitted, and statistical evaluations are orientated around dubious “isotype controls” or 1% thresholds. To meet these challenges, ISAC published their criteria for Minimal Informationon on Flow Cytometric data, MIFlowCyt, in 2008 , and, at the same time, implemented them for the journal, Cytometry Part A. As outlined by Filby and colleagues  most of these criteria would also be appropriate for image flow cytometric data. It has to be wondered when the immunological journals will follow these standards.
To address the problem of availability and storage, a repository for original flow cytometric list-mode data has recently been established under the auspices of ISAC and the NIH . Finally, in order to ease the establishment and use of highly multiplexed staining panels, Cytometry Part A has introduced a unique manuscript format for the publication of OMIPs (optimized multicolor immunofluorescence panels) . Several newly developed OMIPs were presented and discussed at CYTO 2012 and will be published soon. OMIPs enable the community to establish complex polychromatic (10 or more color) flow cytometry panels for unequivocal identification of leukocyte subsets and their functions (antigen expression, cytokine production, or signaling) from the shelf. There is no more need for time consuming, labor intensive and costly establishment in other laboratories because this has already been performed by the OMIP authors.
This year's annual meeting of ISAC, CYTO 2012, attracted over 1,500 attendants to Leipzig, Germany. With 100 speakers, 400 posters and over 60 exhibitors, including manufacturers and reagent and service providers, it offered a comprehensive overview of the state-of-the-art in cytometry. The CYTO 2013 conference will be held May 19–23, 2013 in San Diego, with lots of good news for immunologists.
The work presented in this paper (editorial) was made possible by funding from the German Federal Ministry of Education and Research (BMBF, PtJ-Bio, 0315883) and the Saxonian Ministry for Science and Education (SMWK).
Declaration of interest
Attila Tárnok is Editor-in-Chief of Cytometry Part A.