Cytometry Part A最新文献

Extracellular vesicles (EVs) are potential disease biomarkers released by cells into body fluids. Flow cytometers measure EV concentrations within their size detection range. These size ranges can be quantified by relating the arbitrary units of measured light scattering signals to the optical diameter of EVs with Mie theory. However, Mie theory requires input parameters, including the refractive index (RI) of the suspension liquid surrounding the measured particles, which is not accurately known. Here we measure traceable RIs of suspension liquids, including Dulbecco's phosphate buffered saline (DPBS) and human blood plasma dilutions with an uncertainty of $1.4\times {10}^{-5}$ for relevant wavelengths between 405 and 644 nm. We show that differences between traceably measured and currently used RIs for suspension liquids lead to differences in applied optical diameter gates and reported EV concentrations. The currently assumed RI of DPBS in Rosetta Calibration leads to a difference of 9% to 35% between reported and actual EV concentrations in diluted plasma, depending on the lower detection limit of the flow cytometer. A traceably measured RI of the suspension liquid should be used to calibrate light scattering signals with Mie theory to improve accuracy of optical diameter and concentration measurements by EV flow cytometry.

Cell lineage detection refers to the inference of differentiation pathways from immature cells to distinct mature cell types. We developed TimeFlow 2, a new method for lineage inference in large flow cytometry datasets. It uses a single static snapshot of unordered cells and does not require prior knowledge of the number of pathways, cell types or temporal labels. TimeFlow 2 uses the cell orderings from TimeFlow and defines coarse cell states along pseudotime segments. By connecting these states, it constructs paths at the cell state level. To approximate the trajectory structure, it further groups the paths based on an optimal transport-based cost function. We used TimeFlow 2 on three healthy bone marrow samples and accurately assigned monocytes, neutrophils, erythrocytes and B-cells of different maturation stages to four distinct pathways. Marker dynamics across the inferred pathways showed highly correlated patterns for the corresponding lineages in all three patients. We compared the performance of TimeFlow 2 and three other established methods using standard classification and correlation metrics. TimeFlow 2 outperformed the others on flow cytometry datasets and remained competitive on the challenging mass cytometry datasets. Overall, TimeFlow 2 detects biologically informative pathways, allowing bioinformaticians to model and compare marker dynamics across cell lineages in a data-driven way. Source code in Python and tutorials are available at https://github.com/MargaritaLiarou1/TimeFlow2.

The increasing uptake of adoptive CAR-T and TCR-T cell therapies into clinical practice has intensified the need for robust and detailed immunophenotyping of ex vivo expanded T cells. We have developed and optimized an extended 36-color full-spectrum flow cytometry panel suitable for in-depth immunophenotyping of both peripheral blood and ex vivo expanded human T cells. Our panel allows for analysis of CD4⁺ and CD8⁺ memory subpopulations (Tn/Tscm, Tcm, Tem, and Temra), Treg subsets (CD45RA⁺ and CD39⁺), and helper T cell subsets (Tfh, Th1, Th2, and Th17). Intracellular staining facilitates the evaluation of functional markers granzyme B (cytotoxicity) and Ki-67 (proliferation). Our panel allows detailed investigation of T cell activation using markers carrying a range of expression kinetics. Exhaustion and senescence features, known to correlate with resistance to immunotherapies, can also be thoroughly evaluated. TCR staining with pHLA-dextramer or anti-TCR antibody is also included to allow evaluation of TCR specificity and/or transduction efficiency. A PD-1 receptor occupancy component enables quantification of T cells bound to antibodies from immune checkpoint inhibitor (ICI) therapies. Overall, we have generated a fully optimized and fit-for-purpose extended T cell profiling panel with particular relevance to the field of immunotherapies including ICIs and adoptive T cell therapies.

Inherent cross-reactivity for shared epitopes is thought to be critical for vaccines, since this is likely the only way in which a vaccine can be anticipatory, similar to immune responses induced by natural infections. However, measuring antigen-specific and cross-reactive B cells remains technically challenging due to low abundance of these cells and background binding. Here, we have assessed different antigen-labeling methods to optimize the measurement of B cell specificity, cross-reactivity, heterocliticity, and average affinity for antigen by flow cytometry. Antigens covalently labeled with NHS ester amine-reactive dyes gave reliable results compared to other antigen labeling methods: namely conjugation of His-tagged antigen on fluorescent streptavidin nanoparticles, or on soluble streptavidin tetramers, bridged via the biomolecular adapter biotin-trisNTA(Ni). Blocking with unlabeled H1 or H7 clearly outcompeted H1- and/or H7-specific cells, indicating that cross-reactive antibodies possess lower affinities compared to the mono-reactive or the rare heteroclitic antibodies. Overall, these studies suggest that evaluating the antigen specificity and breadth of candidate vaccines, including multi-season or universal vaccines, may be best accomplished using flow cytometric quantification of clonally distributed B lineage cells using antigens covalently derivatized with fluorescent dyes, which is also analogous with the standard flow cytometry approach of quantifying antigen biomarkers on cells using fluorescent monoclonal antibodies (mAbs).

Spectral flow cytometry has evolved from a contentious idea into a mainstay of high-parameter single-cell analysis, yet its vocabulary (and the statistical reasoning behind it) remains a patchwork of overlapping, sometimes contradictory terms. This position paper highlights the terminological fragmentation and translation gap, and aims to harmonize the field's lexicon while providing a cohesive information-theoretic framework for panel optimization. We expose the limits of popular ad hoc heuristics, matrix condition numbers, and pairwise cosine similarities, and promote stronger surrogates: effective rank and information efficiency, which together capture spectral independence and numerical stability, and the Cramér-Rao lower bound (CRLB) matrix, which directly predicts fluorochrome-specific spreading error. Building on this foundation, we revive the optimal-design criteria: D-optimality maximizes total information; A-optimality minimizes the average parameter variance; E-optimality constrains worst-direction inflation. By aligning precise definitions with actionable design rules, we provide a roadmap for consistent terminology, next-generation panel construction, and objective instrument benchmarking.

Flow cytometry, a complex and important methodology, is exacerbating the systemic reproducibility crisis in biological research. Our systematic review of 100 published manuscripts on PD-1 flow cytometry data found systemic deficiencies in the clarity and completeness of methodological documentation across top-tier journals. We urgently request that the International Society for Advancement of Cytometry (ISAC) and dedicated journal editors from the scientific publishing community lead a global initiative mandating a unique common standard for all submissions (MIFlowCyt). This adoption is critical for enforcing transparency, enabling rigorous peer review, and fundamentally strengthening the scientific integrity of published cytometry data.

Spectral flow cytometry and high-parameter panels provide a powerful tool to meet the growing demands for deep immunophenotyping. This study aimed to adapt a published 40-color spectral flow cytometry panel for whole blood analysis, evaluate the impact of cryopreservation, and explore the potential benefits of overnight staining on data quality and cost-effectiveness. The optimization process revealed several artifacts, including loss of IgG signal and spectral profile changes in PerCP-eFluor710, which were addressed through protocol and reagent modifications. Cryopreservation resulted in significant alterations to granulocyte morphology and viability, limiting direct comparisons with fresh blood samples. Preservation also led to altered distributions in 30% of gated populations, particularly affecting T cell, dendritic cell, and monocyte subsets. Overnight staining with reduced antibody concentrations enhanced resolution for key markers while allowing for 5- to 20-fold reductions in antibody usage, substantially lowering panel costs. Contrary, extended incubation times also decreased the detection of markers critical for identification of NKT-like and regulatory T cell subsets, including CD2 and CD39. This underscores the importance of fluorochrome interference, preservation effects, and extended incubation times when optimizing high-dimensional panels, suggesting opportunities to improve staining protocols in clinical and epidemiological research, particularly where resolution or cost-effectiveness are primary concerns.