OBJECTIVE: We assessed the utility of red blood cell (RBC) CD105 and side scatter (SSC) parameters by flow cytometry for the detection of low-grade myelodysplastic neoplasms (MDS) in bone marrow specimens.
METHODS: Ten RBC parameters incorporating CD105 or SSC combined with the Meyerson-Alayed scoring system (MASS) metrics were retrospectively evaluated by flow cytometry for utility in detecting low-grade MDS (n = 56) compared with cytopenic controls (n = 86).
RESULTS: Myelodysplastic neoplasms were associated with 7 of the RBC parameters in univariate analysis. Multivariate analysis using cutoff values based on optimal and 95% specificity levels of the RBC metrics and the MASS parameters revealed the SSC ratio of CD105-positive and CD105-negative RBC fractions (CD105+/- SSC); the percentage and coefficient of variation of the CD105-positive fraction of RBCs (CD105%, CD105+CV) emerged as significant RBC variables. Two simple scoring schemes using these RBC values along with MASS parameters were identified: 1 using CD105+/- SSC, CD105%, and CD105+CV combined with the percentage of CD177-positive granulocytes (CD177%), myeloblast percentage (CD34%), and granulocyte SSC (GranSSC), and the other incorporating CD105+/- SSC, CD105+CV, CD177%, CD34%, GranSSC, and B-cell progenitor percentage. Both demonstrated a sensitivity of approximately 80%, with a specificity of roughly 90% for the detection of MDS compared with cytopenic controls.
CONCLUSIONS: The red blood cell parameter, CD105+/- SSC, appears to be beneficial in the evaluation of low-grade MDS by flow cytometry.

The diagnosis of myelodysplastic syndrome (MDS) is complex. Flow cytometric analysis of the myelomonocytic compartment can be helpful, but it is highly subjective and reproducibility by non-specialized groups is unclear. Analysis of the erythroid lineage by flow cytometry is emerging as potentially more reproducible and easier to conduct, while keeping a high diagnostic performance.
We review the evidence in this area, including 1) the use of well-established markers - CD71 and CD36 - and other less well-established markers and parameters; 2) the use of flow cytometric scores for the erythroid lineage; and 3) additional aspects, including the emergence of computational tools and the roles of flow cytometry beyond diagnosis. Finally, we discuss the limitations with the current evidence, including 1) the impact of the sample processing protocol and reagents on the results, 2) the lack of a standard gating strategy, and 3) conceptualization and design issues in the available publications.
We end by offering our recommendations for the current use - and our personal take on the value - of the analysis of erythroid lineage by flow cytometry.

In keeping with the rule of \"form follows function\", morphological aspects of a cell can reflect its role. Here, it is shown that the cellular granularity of a lymphocyte, represented by its intrinsic side scatter (SSC), is a potent indicator of its cell state and function. The granularity of a lymphocyte increases from naïve to terminal effector state. High-throughput cell-sorting yields a SSChigh population that can mediate immediate effector functions, and a highly prolific SSClow population that can give rise to the replenishment of the memory pool. CAR-T cells derived from the younger SSClow population possess desirable attributes for immunotherapy, manifested by increased naïve-like cells and stem cell memory (TSCM )-like cells together with a balanced CD4/CD8 ratio, as well as enhanced target-killing in vitro and in vivo. Altogether, lymphocyte segregation based on biophysical properties is an effective approach for label-free selection of cells that share collective functions and can have important applications for cell-based immunotherapies.

Evaluation of the potential hazard of man-made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. A FACSCalibur™ flow cytometer and a Stratedigm S-1000 flow cytometer were used to measure changes in light scatter from cells after incubation with either silver nanoparticles (AgNP) or TiO2 nanoparticles. Within the range of between 0.1 μg/mL and 30 μg/mL the nanoparticles caused a proportional increase of the side scatter and decrease of the forward scatter intensity signals. At the lowest concentrations of TiO2 (ranging between 0.1 μg/mL and 0.3 μg/mL), the flow cytometer can detect as few as 5-10 nanoparticles per cell. The influence of nanoparticles on the cell cycle was detected by nonionic detergent lysis of nanoparticle incubated cells that were stained with DAPI or propidium iodide (PI). Viability of nanoparticle treated cells was determined by PI exclusion. Surface plasmonic resonance (SPR) was detected primarily in the far-red fluorescence detection channels after excitation with a 488 nm laser.Our results suggest that the uptake of nanoparticles within cells can be monitored using flow cytometry. This uptake of nanoparticle data was confirmed by viewing the nanoparticles in the cells using dark-field microscopy. The flow cytometry detection of nanoparticles approach may help fill a critical need to assess the relationship between nanoparticle dose and cellular toxicity. Such experiments using nanoparticles could potentially be performed quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health.

Diagnosis of myelodysplastic syndromes (MDS) is not straightforward when objective data, such as blast excess and abnormal cytogenetics, are lacking. Expert laboratories use flow cytometry (FCM) to help diagnose MDS. However, most of FCM protocols for MDS are complex, requiring a high level of expertise and high cost. We have reported a FCM mini-panel consisting of four FCM parameters (so-called Ogata score), which is simple to conduct and inexpensive. In this paper, to refine this mini-panel, we have introduced a new FCM parameter, which quantifies CD33 expression on CD34+ cells (called Granulocyte/CD34 cell CD33 ratio). Bone marrow cells from MDS without blast excess (low-grade MDS) and controls were stained with CD34, CD45, and CD33 and analyzed for five parameters (\"Granulocyte/CD34 cell CD33 ratio\" plus four parameters in the Ogata score). By a multivariate logistic regression model, only three parameters, including \"Granulocyte/CD34 cell CD33 ratio\" had statistically significant power for diagnosing low-grade MDS. Based on the results, we constructed a new scoring system, which showed approximately 50% sensitivity and more than 95% specificity in diagnosing low-grade MDS. Our revised mini-panel is suitable for screening samples suspected for MDS and provides a basis for further improvement in diagnostic FCM protocols for MDS.

It is important to understand the ecology and physiology of microbes in activated sludge of wastewater treatment plants. Recently, molecular based approaches such as 16S rRNA genes and environmental genomics have illuminated black boxes in nutrient removal process and expanded our knowledge. However, most microbes responsible for the removal of phosphate and nitrogen such as Accumulibacter and Nitrospira remain uncultured. This is because optimum methodologies to concentrate these uncultured microbes and to obtain pure cultures have not been established. Here, we report a novel approach for physical enrichment of uncultured Accumulibacter and Nitrospira from microbial communities in activated sludge by a cell sorting system. Two scattering signatures representing forward scatter and side scatter of this system allowed morphological characterization of microbial particles in activated sludge. The distribution and size of microbial particles consisting of single cells, microcolonies, and aggregates depended on the levels of scattering signatures. Next generation sequencer and principal component analysis revealed each microbial population fractionated according to the levels of scattering signatures, resulting that uncultured Accumulibacter and Nitrospira could be sorted as single cells or microcolonies. Finally, quantitative fluorescence in situ hybridization analysis determined optimum fractions to collect sufficiently these target microbes from activated sludge. Consequently, this method would be very useful as an enrichment technique prior to isolation, genomic analysis, and physiological investigation of uncultured bacteria.

There is a need to accurately detect, characterize, and quantify nanoparticles in suspensions. This study helps to understand the complex interactions between similar types of nanoparticles. Before initiating a study of metal nanoparticles, five submicron PS beads with sizes between 200 nm and 1 µm were used to derive a reference scale that was useful in evaluating the flow cytometer for functionality, sensitivity, resolution, and reproducibility. Side scatter intensity (SSC) from metal nanoparticles was obtained simultaneously from 405 nm and 488 nm lasers. The 405 nm laser generally yielded histogram distributions with smaller CVs, less side scatter intensity, better separation indices between beads and decreased scatter differences between different sized particles compared with the 488 nm laser. Submicron particles must be diluted to 10(6) and 10(7) particles/mL before flow cytometer analysis to avoid coincidence counting artifacts. When particles were too concentrated the following occurred: swarm, electronic overload, coincidence counting, activation of doublet discrimination and rejection circuitry, increase of mean SSC histogram distributions, alterations of SSC and pulse width histogram shape, decrease and fluctuations in counting rate and decrease or elimination of particulate water noise and 1 µm reference bead. To insure that the concentrations were in the proper counting range, the nanoparticle samples were mixed with a known concentration of 1 µm counting beads. Sequential dilutions of metal nanoparticles in a 1 µm counting bead suspension helped determine the diluted concentration needed for flow cytometer analysis. It was found that the original concentrated nanoparticle samples had to be diluted, between 1:10,000 and 1:100,000, before characterization by flow cytometry. The concentration of silver or gold nanoparticles in the undiluted sample were determined by comparing them with a known concentration (1.9 × 10(6) beads/mL) of 1 µm polystyrene reference beads.

Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEON(LA)) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEON(LA) with an additional protein corona formed by bovine serum albumin (SEON(LA-BSA)) and commercially available Rienso(®) particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products.

Cellular aggregation is a physiological response of lymphocytes to various extracellular stimuli. Currently, lymphocytes aggregation is only evaluated qualitatively or by semiquantitative methods. In this study, we assessed the capacity of flow cytometry to measure lymphocytes aggregation in a quantitative, accurate, and reproducible manner, and examined the significance of aggregation responses in various lymphoproliferative diseases.
Extracellular triggers such as anti-CD19 antibodies or phorbol ester were utilized to induce lymphoid cells aggregation in a concentration dependent manner. Aggregation was quantified by flow cytometry based on the forward or side scatter (SSC), or by dark-field SSC of aggregates measured by ImageStreamX. Accuracy, reproducibility, and limitations of the methodology were evaluated. Aggregation responses were measured in various types of lymphoproliferative diseases, and correlated with immunophenotyping and IGHV mutational status in chronic lymphocytic leukemia.
Lymphoid aggregates provoked by extracellular stimuli elevate the forward and SSC signals relatively to the number of cells in each event. Aggregation responses vary among different types of lymphoproliferative diseases. Moreover, elevated levels of CD19-induced aggregation are associated with aberrant chronic lymphocytic leukemia characteristics, but not with IGHV mutational status of the disease
We have demonstrated that flow cytometry can provide accurate and reproducible measurement of both primary as well as T and B cell lines aggregation in response to extracellular stimuli. The use of quantitative evaluation of activation driven or other cellular aggregation may provide an analytical tool to elucidate biochemical and molecular mechanisms associated with lymphoproliferative diseases. © 2015 International Clinical Cytometry Society.

To screen a high-producing recombinant Chinese hamster ovary (CHO) cell from transfected cells is generally laborious and time-consuming. We developed an efficient enrichment strategy for high-producing cell screening using flow cytometry (FCM). A stable pool that had possibly shown a huge variety of monoclonal antibody (mAb) expression levels was prepared by transfection of an expression vector for mAb production to a CHO cell. To enrich high-producing cells derived from a stable pool stained with a fluorescent-labeled antibody that binds to mAb presented on the cell surface, we set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and collected the brightest 5% of fluorescein isothiocyanate (FITC)-positive cells from each group by FCM. The final product concentration in a fed-batch culture of cells sorted without FSC and SSC gates was 1.2-1.3-times higher than that of unsorted cells, whereas that of cells gated by FSC and SSC was 3.4-4.7-fold higher than unsorted cells. Surprisingly, the fraction with the highest final product concentration indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all fractionated cells. Our results showed that our new screening strategy by FCM based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells with the smallest value of FSC and SSC.