Cyto 2011—Overview of the XXVI ISAC Congress Proceedings Issue of Cytometry

This issue of Cytometry presents the third occasion on which a call has been issued to encourage authors to submit their work to the journal for publication as part of a special Congress Proceedings Issue of Cytometry. An incentive for submission was that accepted papers would be guaranteed an oral presentation at the XXVI ISAC Congress in Baltimore—CYTO 2011. The topics of the accepted papers range from proteome-wide modeling to CD4 testing in resource-limited settings. The research articles do not speak to a given theme, rather they provide a snap-shot of the diversity of interest in cytometry with proffered examples of some of the latest trends in the field of cytometry. The Proceedings Issue of Cytometry also incorporates two reviews. The first review, by Darzynkiewicz et al., has been scheduled for publication in alignment with the conference, as it speaks to a topical area—“click chemistry”—an increasingly used conjugation and assembly technology with attendant advantages of biocompatibility, selectivity, and yield (1). The second review, presented at CYTO 2011, provides an update on cytometric challenges in veterinary andrology. In parallel with the discovery of the structure of DNA, 1953 also saw the publication of a study by Howard and Pelc (2) on the patterns of radiolabel incorporation by meristematic cells of Vicia faba. This landmark paper proposed the existence of a cell division cycle demarcated by pre- and post-gaps for DNA synthesis. Their discovery set in process the widespread application of labeling methods for the analysis of the eukaryotic cell cycle, leading to the recognition of the universality of its major regulators (3) and exploitation in studies on anticancer drug-induced perturbations of cell cycle kinetics. In this issue, Darzynkiewicz et al. overview the Cytometry of DNA replication and RNA synthesis from its historical backdrop to recent advances based on “Click Chemistry.” The review covers fluorescence quenching methods, immunocytochemical detection through to the recent incorporation of EdU for the click chemistry detection of DNA damage via histone H2AX phosphorylation analyzed by flow or laser scanning cytometry. K.B. Sharpless, the 2001 Nobel Prize winner for Chemistry, introduced the concept of click chemistry for reactions that fulfill useful criteria including being modular and providing very high yields with favored physiologically stable, single reaction products (4). The concept drives applications for addition reaction conditions using preferred solvents and simple routes for product isolation—key issues for the cytometrist. Since the first reports of sexed sperm, flow technology efforts have been largely focused on improving sample throughput resulting in routine high-purity sorting of X- or Y-chromosome-bearing sperm (5). However, a more refined assessment of fertilizing potential of veterinary semen samples by flow cytometry has yet to exploit the full potential of the technology. Petrunkina and Harrison in Cambridge, UK have worked on the determinants of sperm quality and fertility in domestic species and recently provided an exact mathematical analysis of the systematic mis-estimation of semen-derived cell subpopulations by flow cytometry (6). The current review addresses cytometric solutions presenting recent developments, advantages and limitations together with the aspects of sperm physiology that could benefit from multicolor flow cytometric procedures. Advantageous biophotonic characteristics of molecular probes underpin many cytometric studies. The detection of luminescence-labeled microorganisms by time-gated long-lifetime signatures using excitation conditions that facilitate the suppression of nontarget emission has proved a highly attractive option for exploration in flow cytometry (7). In a previous review in Cytometry, the chemical and physical properties of lanthanide ion complexes and of other narrow-emitting species have been highlighted with particular reference to their utility as biolabels (8). However, lanthanide complexes emit far fewer photons per unit time than conventional fluorescent probes, presenting a challenge for the production of high quality images at probe concentrations that are relevant for live cell experiments (9). In this issue, Dayong Jin, working at Macquarie University in Australia, describes a low-cost demonstration of true-color high-contrast microorganism imaging for terbium bioprobes. The authors describe the use of a commercial terbium complex for high-contrast imaging of the environmental microorganism, Cryptosporidium parvum. The system demonstrates practical high-contrast bioimaging, based on a compact 315 nm pulsed LED with a modified microscopy set up but further exploiting the auto-fluorescence suppression in time domain. Also in this issue, a multicontinent collaboration is reported by Yiqing Lu et al. describing an approach for the automated detection of rare-event pathogens through time-gated luminescence scanning microscopy. It has been recognized that luminescence microscopes operating in the time-domain can improve the detection of signals from long fluorescence lifetimes (tau > 100 microseconds) in a background of short-lived autofluorescence lifetime noise frequently observed in micro-organisms (10). The team addressed the need to improve methods for the rapid detection of infectious disease, in particular monitoring waterborne pathogenic microorganisms in recreational waters (typically, Giardia lamblia cysts and Cryptosporidium parvum oocysts). The aim was to develop an approach that avoided unacceptable false positives and negatives when dealing with the issue of accurate counting of rare-event cells. Their approach details the strategy of time-gated luminescent scanning, which exploits the large difference in luminescence lifetimes between the lanthanide biolabels to significantly reduce bioimaged areas to areas of interest for the accurate mapping of all the potential Giardia cysts using automated microscopy. The combination of optics and microfluidics to create novel functionalities on small chip-based systems holds significant promise in healthcare and laboratory-based technologies (11). Creativity and technology access in this area drives advances in cytometry. However, the application of microfluidic options in cytometry still faces the problems of design and prototyping. Furthermore, in many research and biomedical laboratories, access to appropriate fabrication facilities and expertise is frequently limited. In this issue, in the paper by a Canadian-based group, Islam et al. describe rapid and cheap prototyping of a microfluidic cell sorter to exemplify a low cost approach. The approach describes fabrication using poly(ethylene-co-methacrylic acid)ionomers (Surlyn) and optical fiber coupling of a 532-nm diode laser for in-channel sorting of beads based on the level of the photodiode detected signal. The approach described permits prototype preparation even if no state-of-the-art micro-fabrication facility is available. Urothelial carcinoma (UC) is the second most common cause of death in urinary tract malignancies and the early detection of high grade tumors—cystoscopy and voided urine cytology remain diagnostic gold standards. Following on from their previous study on genetically aberrant cells analyzed by the UroVysion test (11), the current paper by Pajor et al., with collaborators in Hungary, Germany, and USA, presents an evaluation of a promising tool for the detection of bladder cancer—a multitarget fluorescence in situ hybridization (FISH) assay. The paper speaks to considerations for modifying the current evaluation scheme with the aim of raising the diagnostic efficiency of Urovysion. Samples from 42 patients with high clinical suspicion for UC were subjected to Urovysion-alone and “targeted-Urovysion” (cytokeratin 7 immunophenotyping followed by Urovysion). The group has addressed the lack of consensus regarding abnormal signal pattern, cut-off level, and sought to explore the diagnostic benefits of a recently introduced method featuring target-specific genotyping. The authors conclude that CK7 targeting raises diagnostic efficiency of Urovysion and could be an ideal tool for identifying tumor cells in ambiguous cases or when other tumors are present. The hope is that this work will facilitate interlaboratory comparisons. CD4+ T cell counts, both absolute and percentage of lymphocytes, inform decisions about initiating antiretroviral therapy. It is widely recognized that patient care in sub-Saharan Africa operates under conditions where HIV quantification is frequently unsustainable for the great majority of those affected in the region resulting in a focus on CD4+ T cell testing (12). In this issue, the paper by Marinucci et al. reports on the experience of decentralization of CD4 testing in resource limited settings—relating 7 years of experience in six African countries. Focused on supporting accessibility to reliable CD4 counting—the group report on a model developed at the University of Maryland School for site assessment/improvement, technology selection with capacity building and quality management with monitoring. The paper describes the need to balance country-specific factors with the location of the clinic, number of patients, and the expected workload, for decentralizing CD4. The paper by Sugár et al. from New York's Mount Sinai School of Medicine describes an approach for compensation in flow cytometry by multivariable optimization. Conventional compensation of flow cytometry data. In this article, a new, optimization based, compensation method is described that proposes the use of not only single-stained but also multistained samples to seek to improve estimates of the spillover coefficients. The authors suggest that this approach is practical and may lead to significant improvements in flow cytometric compensation. Understanding the distributions of proteins within wide macromolecular structures is clearly of importance in refining our models of systems-level cellular functions. Drawing such information from images representing subcellular patterns and quantitative levels of a protein are significant challenges (13). In this issue, the paper by Tao Peng and Robert Murphy, based at Carnegie Mellon University, outlines one approach to proteome-wide modeling using image-derived, three-dimensional generative models of cellular organization. The underlying principle driving the study was that through 3D-generative models, protein subcellular locations can be described more accurately. The models developed comprise submodels representing features such as nuclear shape, cell shape, and organelle size relative to nuclear and cell boundaries. The authors underline the potential for generating synthetic multichannel images containing as many proteins as desired. Such work not only providing a basis for tracking emergent behavior within systems but also has immediate practical applications in high-content screening and imaging flow cytometry. We hope that the articles in this special issue will provide a useful complement to the oral and poster presentations at the XXVI ISAC Congress.

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