Year: 2017
Type: article
Abstract: Who would have been able to predict how the field of cytometry would take off when Mack Fulwyler 1 at Los Alamos modified a Coulter counter to sort cells based on differences in volume? The Coulter counter had been developed by Wallace Coulter and has become a common sight in clinical laboratories for rapid counting of cells or particles that could be discriminated based on their electrical resistance as they passed through a detector in a liquid stream, as their electrical resistance was proportional to their volume. In a clinical setting, this allows accurate counting of platelets, erythrocytes, and leukocytes. A new era was born when Leonard Herzenberg modified Fulwyler's cell sorter 2 so that cells stained with fluorochrome-labeled antibodies could be identified based on their fluorescence emission rather than cell volume, establishing a technical basis for systematic cellular phenotyping. Now looking back it is amazing how the field of cytometry has developed over time and how our “phenotyping tools” have evolved into a stellar cutting-edge technology. In this Special Section, we compiled a selection of four articles presenting different developments of cytometric phenotyping technologies, which nicely demonstrate current trends and possibilities. We would like to draw your attention to the articles from Basso-Ricci et al. (this issue, page 952) and Smolen et al. (this issue, page 966), which both introduce new holistic approaches for whole blood phenotyping assays. The work from Elhmouzi-Younes et al. (this issue, page 969) represents a reference publication comparing innate myeloid leukocytes from healthy humans and macaques using time-of-flight cytometry (CyTOF), and is a great example for high-end multiparametric phenotypic analysis. The fourth article by Song et al. (this issue, page 983) proposes a general workflow for phenotypic assays based on automated fluorescence microscopy. We hope you enjoy reading this Special Section as much as we did putting it together. The first article of our Special Section by Luca Basso-Ricci et al. (this issue, page 952) describes a novel comprehensive immune phenotyping tool. The authors named their flow cytometry protocol whole blood dissection (WBD), as it was capable of analyzing hematopoietic progenitors and all major mature bone marrow- or peripheral blood (PB)-derived cell lineages in a single test tube through a multiparametric flow cytometry analysis. This new protocol permits unambiguous identification, in the same tube, of up to 23 different blood cell types. Notably, this includes not only all the major myeloid and lymphoid lineage compartments at different stages of maturation but also hematopoietic stem/progenitor cell subtypes, thanks to a combination of 17 surface and 1 viability cell markers. Overall, the authors showed that WBD allows a streamlined identification of >99% of cell subpopulations found in blood samples in a standardized and reproducible manner. Cost and time efficiency are additional points in favor of this new protocol. WBD has been shown to possess a wide range of potential applications making this protocol a possible asset in both preclinical and clinical settings. The OMIP from Smolen et al. (this issue, page 966) presents another whole blood panel in this special section, adding novel aspects of the logistics of immune phenotyping. This 14-color flow cytometry panel focuses on the assessment of the innate immune response in whole blood after Toll-like receptor ligand or cytokine stimulation and captures most of the major innate immune subsets in PB and allows the detection of their key intracellular cytokines. This article presents a strategy to analyze Toll-like receptor ligand and cytokine stimulated whole blood in combination with a fixation and freezing procedure to allow the flow cytometric analysis at the same time thereby minimizing technical artifacts. Mass spectrometry-based flow cytometry is a cutting-edge technology, which aims to increase the resolution of multiparametric analyses and allows to simultaneously study a large number of different cell markers 3. CyTOF mass cytometry utilizes heavy metal ion-conjugated antibodies and mass spectrometry combining single-cell analysis typical of flow cytometry with the high resolution and sensitivity of TOF-mass spectrometry, thereby offering a novel ultrahigh-content phenotyping tool 4, 5. The article by Elhmouzi-Younes and colleagues (this issue, page 969) used this technique to compare the immunophenotypic profile of circulating innate myeloid cells in humans and nonhuman primates. This is the first work analyzing simultaneous expression of 30 antigens on innate myeloid cells. The study identified major similarities and some differences between human and macaque innate cells and supports that these nonhuman primates are a suitable preclinical model to help transit basic research findings from bench to bedside. The article by Song et al. (this issue, page 983) presents a cytometric phenotyping tool from a completely different perspective. The authors present an image analysis pipeline to perform functional phenotyping as a way to follow the dynamic markers and active processes in live cells. They concentrate on multiparametric analysis to study the infection of host macrophages by Mycobacterium tuberculosis, specifically, investigating the disease propagation and fate of the host macrophages. To achieve this, modern bio-imaging techniques were used in viable cells to follow active intracellular events. They proposed this pipeline of combined methods in order to visualize intracellular biochemical and metabolic processes together. Processes such as zinc intoxication, phagosomal acidification, lipid bodies formation, and phagosomal vacuole rupture could then be visualized together with analysis of intracellular mycobacterial replication, followed by high-quality image analysis including segmentation process. Importantly, this way of functional cellular phenotyping has a broader perspective and has potential to be easily transferred to studies of other infectious diseases and pathogens, in order to better investigate the host–pathogen interplay. While the selected articles in this Special Section highlight current technical advances in multiparametric fluorescence and mass cytometry advancing our portfolio of phenotyping tools, the era of -omics in flow cytometry is just starting. Indeed, this is an exciting time in which rapid technical progress spearheads major breakthroughs in cytometry, expanding our understanding of biology in an unprecedented manner.
Source: Cytometry Part A
DOI: 10.1002/cyto.a.23262
Language: English
Subfield: Biomedical Engineering
Field: Engineering
Domain: Physical Sciences
Open Access status: bronze
OpenAlex ID: W2765801054