Mass cytometry enables an unprecedented number of parameters to be measured in individual cells at a high throughput, but the large dimensionality of the resulting data severely limits approaches ...relying on manual “gating.” Clustering cells based on phenotypic similarity comes at a loss of single-cell resolution and often the number of subpopulations is unknown a priori. Here we describe ACCENSE, a tool that combines nonlinear dimensionality reduction with density-based partitioning, and displays multivariate cellular phenotypes on a 2D plot. We apply ACCENSE to 35-parameter mass cytometry data from CD8 ⁺ T cells derived from specific pathogen-free and germ-free mice, and stratify cells into phenotypic subpopulations. Our results show significant heterogeneity within the known CD8 ⁺ T-cell subpopulations, and of particular note is that we find a large novel subpopulation in both specific pathogen-free and germ-free mice that has not been described previously. This subpopulation possesses a phenotypic signature that is distinct from conventional naive and memory subpopulations when analyzed by ACCENSE, but is not distinguishable on a biaxial plot of standard markers. We are able to automatically identify cellular subpopulations based on all proteins analyzed, thus aiding the full utilization of powerful new single-cell technologies such as mass cytometry.
Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made ...for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.
Recent work has demonstrated that automated fluorescence flow cytometry (FLC) is a potential alternative for the detection and quantification of Plasmodium parasites. The objective of this study was ...to apply this novel FLC method to detect and quantify Babesia parasites in venous blood and compare results to light microscopy and polymerase chain reaction methods.
An automated hematology/malaria analyzer (XN-31; Sysmex) was used to detect and quantify B microti-infected red blood cells from residual venous blood samples (n = 250: Babesia positive, n = 170; Babesia negative, n = 80). As no instrument software currently exists for Babesia, qualitative and quantitative machine learning (ML) algorithms were developed to facilitate analysis.
Performance of the ML models was verified against the XN-31 software using P falciparum-infected samples. When applied to Babesia-infected samples, the qualitative ML model demonstrated an area under the curve (AUC) of 0.956 (sensitivity, 95.9%; specificity, 83.3%) relative to polymerase chain reaction. For valid scattergrams, the qualitive model achieved an AUC of 1.0 (sensitivity and specificity, 100%), while the quantitative model demonstrated an AUC of 0.986 (sensitivity, 94.4%; specificity, 100%).
This investigation demonstrates that Babesia parasites can be detected and quantified directly from venous blood using FLC. Although promising, opportunities remain to improve the general applicability of the method.
Recent developments in microflow cytometry have concentrated on advancing technology in four main areas: (1) focusing the particles to be analyzed in the microfluidic channel, (2) miniaturization of ...the fluid-handling components, (3) miniaturization of the optics, and (4) integration and applications development. Strategies for focusing particles in a narrow path as they pass through the detection region include the use of focusing fluids, nozzles, and dielectrophoresis. Strategies for optics range from the use of microscope objectives to polymer waveguides or optical fibers embedded on-chip. While most investigators use off-chip fluidic control, there are a few examples of integrated valves and pumps. To date, demonstrations of applications are primarily used to establish that the microflow systems provide data of the same quality as laboratory systems, but new capabilities--such as automated sample staining--are beginning to emerge. Each of these four areas is discussed in detail in terms of the progress of development, the continuing limitations, and potential future directions for microflow cytometers.
B-lineage acute lymphoblastic leukemias (B-ALL) harboring rearrangements of the histone lysine K-Methyltransferase 2A (
) gene on chromosome 11q23 (
) represent a category with dismal prognosis. The ...prompt identification of these cases represents an urgent clinical need. Considering the correlation between rat neuron glial-antigen 2 (NG2) chondroitin-sulfate-proteoglycan molecule expression and
, we aimed to identify an optimized cytofluorimetric diagnostic panel to predict the presence of
.
We evaluated 88 NG2+ B-ALL cases identified with an NG2 positivity threshold >10% from a cohort of 1382 newly diagnosed B-ALLs referred to the Division of Hematology of 'Sapienza' University of Rome.
Eighty-five of 88 (96.6%) NG2+ B-ALLs harbored
and were mainly pro-B ALL (77/85; 91%). Only 2 B-ALLs with
showed NG2 expression below 10%, probably due to the steroid therapy administered prior to cytofluorimetric analysis.Compared to
cases,
B-ALLs showed a higher blast percentage, significantly higher mean fluorescence intensity (MFI) of CD45, CD38, and CD58, and significantly lower MFI of CD34, CD22, TdT, and CD123.The study confirmed differences in CD45, CD34, CD22, and TdT MFI within the same immunologic EGIL group (European Group for the immunological classification of leukemias), indicating no influence of the B-ALLs EGIL subtype on the
B-ALLs immunophenotype.
Our data demonstrate the association between NG2 and
in B-ALLs identify a distinctive immunophenotypic pattern, useful for rapid identification in diagnostic routines of these subtypes of B-ALLs with a poor prognosis that benefits from a specific therapeutic approach.
Frequency-domain flow cytometry techniques are combined with modifications to the digital signal-processing capabilities of the open reconfigurable cytometric acquisition system (ORCAS) to analyze ...fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency (RF)-modulated detector signals, implementing Fourier analysis programming with ORCAS' digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5-25 ns simulated lifetime), pulse widths ranging from 2 to 15 μs, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142° to 1.6°. The lowest coefficients of variation (<1%) were found for 10-MHz modulated waveforms having pulse widths of 6 μs and simulated lifetimes of 4 ns. Direct comparison of the digital analysis system to a previous analog phase-sensitive flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells, and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a RF-modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to ~98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to simply and inexpensively implement this system on a commercial flow sorter will allow both better dissemination of this technology and better exploitation of the traditionally underutilized parameter of fluorescence lifetime. Published 2010 Wiley-Liss, Inc.
Evaluation of the potential hazard of man‐made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. In this study, different concentrations of TiO2 ...nanoparticles were suspended in cell culture medium. The suspension was then sonicated and characterized by dynamic light scattering and microscopy. Cultured human‐derived retinal pigment epithelial cells (ARPE‐19) were incubated with TiO2 nanoparticles at 0, 0.1, 0.3, 1, 3, 10, and 30 μg/ml for 24 hours. Cellular reactions to nanoparticles were evaluated using flow cytometry and dark field microscopy. A FACSCalibur™ flow cytometer was used to measure changes in light scatter after nanoparticle incubation. Both the side scatter and forward scatter changed substantially in response to the TiO2. From 0.1 to 30 μg/ml TiO2, the side scatter increased sequentially while the forward scatter decreased, presumably due to substantial light reflection by the TiO2 particles. Based on the parameters of morphology and the calcein‐AM/propidium iodide viability assay, TiO2 concentrations below 30 μg/ml TiO2 caused minimal cytotoxicity. Microscopic analysis was done on the same cells using an E‐800 Nikon microscope containing a xenon light source and special dark field objectives. At the lowest concentrations of TiO2 (0.1–0.3 μg/ml), the flow cytometer could detect as few as 5–10 nanoparticles per cell due to intense light scattering by TiO2. Rings of concentrated nanoparticles were observed around the nuclei in the vicinity of the endoplasmic reticulum at higher concentrations. These data suggest that the uptake of nanoparticles within cells can be monitored with flow cytometry and confirmed by dark field microscopy. This approach may help fulfill a critical need for the scientific community to assess the relationship between nanoparticle dose and cellular toxicity Such experiments could potentially be performed more quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health. Published 2010 Wiley‐Liss, Inc.
The anatomy of single cell mass cytometry data Olsen, Lars R.; Leipold, Michael D.; Pedersen, Christina B. ...
Cytometry. Part A,
February 2019, 2019-02-00, 20190201, Letnik:
95, Številka:
2
Journal Article
In this work, we studied the hydrolytic and photochemical degradation of three low-density polyethylene (LDPE) materials, within the size range of microplastics (MP). The MPs were exposed to ...mechanical agitation and UV irradiation equivalent to one year of solar UVB + UVA in a stirred photoreactor. Flow cytometry was used to track the formation of small (1–25 μm) MPs by applying Mie's theory to derive the size of MP particles from scattering intensity readings. The calculation was based on a calibration with polystyrene (PS) beads. The results showed that the generation of 1–5 μm MP reached 104-105 MPs in the 1–25 μm range per gram of LDPE. ATR-FTIR and micro-FTIR measurements evidenced the formation of oxygenated moieties, namely hydroxyl, carbonyl, and carbon-oxygen bonds, which increased with irradiation time. We also found evidence of the production of a high number of nanoplastics (<1 μm, NPs). The Dynamic Light Scattering size of secondary NPs was in the hundreds of nm range and might represent up to 1010 NPs per gram of LDPE. Our results allowed the unambiguous spectroscopic assessment of the generation of NPs from LDPE under conditions simulating environmental exposure to UV irradiation and used flow cytometry for the first-time to track the formation of secondary MPs.
Display omitted
•The mechanical fragmentation of LDPE produces high number of secondary MPs.•Solar photochemical ageing of LDPE produces NPs from secondary MPs.•Secondary MPs/NPs showed oxygenated moieties that increased upon irradiation.•High number of secondary MPs in the 1–25 μm range representing 104-105 items/g LDPE.•Estimation of the concentration of NPs in the order of 1010 NPs/g LDPE.
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