Potential and challenges of clinical high‐dimensional flow cytometry: A call to action

Cytometry Part A, Год журнала: 2024, Номер 105(11), С. 829 - 837

Опубликована: Окт. 23, 2024

Clinical biomarker strategies increasingly integrate translational research to gain new insights into disease mechanisms or define better biomarkers in clinical trials. High-dimensional flow cytometry (HDFCM) holds the promise enhance exploratory potential beyond traditional, targeted strategies. However, increased complexity of HDFCM poses several challenges, which need be addressed order fully leverage its and align with current regulatory requirements cytometry. These challenges include among others extended timelines for assay development validation, necessity extensive knowledge HDFCM, sophisticated data analysis no guidelines exist on how manage such adopting HDFCM. Our CYTO 2024 workshop "Potential high-dimensional cytometry" aimed find consensus across pharmaceutical industry broader scientific community overall benefits most urgent Here, we summarize gained from our workshop. While this report does not provide a blueprint, it is first step defining summarizing pressing implementing Furthermore, compile efforts goal overcome some these challenges. As bring health authorities together build solutions, will accelerate simplify full adoption

Язык: Английский

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Setting the gold standard: Commentary on designing and optimizing high‐parameter flow cytometry panels

Cytometry Part A, Год журнала: 2024, Номер 105(6), С. 428 - 429

Опубликована: Апрель 18, 2024

Technological advances in flow cytometry have greatly expanded its capabilities. These occurred gradually over time, but there also been several key that more markedly affected the technology and how it is used. A prime feature of ability to characterize cell marker expression at single-cell level as high-throughput high volume. The number markers could be measured simultaneously was initially very low has increasing almost exponentially time. There are many reasons for this increase including hardware instrumentation, introduction new types fluorescent dyes beyond those found nature, analysis tools not only enable efficient data elegantly allowed insights into dye/detector interactions provide theoretical bases optimal staining panel design dimensions. OMIP-102 [1] published here represents a milestone makes use expands upon all cumulative advances. Simply demonstration "50-color" remarkable, addition, approach careful methodical description process both main text online material definitive syllabus integrating best practices date. Because wide breadth information building so these major advances, worthwhile break down some digestible pieces highlight significance achievement. One advance optimization instrument optics relatively weak signal subdivided most efficiently across large arrays detectors. Instrument manufacturers developed their own methods achieve goal results successful with current routine capabilities detect separate signals from up 28 dyes, expanding. multiple Optimized Multicolor Immunofluorescence Panel (OMIP) publications demonstrating panels scale. Likely significant representing paradigm spectral optics. This brilliant concept retrospect seems obvious likely approach. While full spectrum first demonstrated 2004 [2, 3], required further software implementation by user base. potential exploiting light completely interrogating fluorescently labeled biological specimens directly called development order make high-parameter reality. As discovery natural fluorochromes appropriate brightness characteristics limiting, luckily, custom-designed became available—examples quantum dots [4] organic polymers [5, 6] (and tandem combinations conventional dyes), which stemmed discoveries earned Nobel prizes. Those us designing can remember sudden shift brought about bright dyes. Perhaps receiving same recognition game-changing impact on alternative approaches representation enabled extraction meaningful increasingly complex datasets. bi-exponential scaling [7, 8] may now seem trivial, just another tool universally used, "tool" does simply aesthetically pleasing representation. Rather, necessary properly interpret discern artifacts affecting negative space. dimensionality increased, hidden complicated. Purely empirical no longer feasible. It apparent "spillover spreading" important take account, metric measure display format spillover spreading matrix [9] an integral design. Other metrics, such complexity similarity indices, recently equally effective streamlining based considerations. authors [1], issue, showcase 50-color OMIP, where thoughtful selection marker-fluorescence clearly contribute panel's effectiveness analyzing visualizing cellular components. After existing aid endeavor, they metric, unmixing error, elucidates impacts rise noise cells per fluorochrome, enabling systematic immunofluorescence panel. exemplifies ideal optimization. everyone need create techniques, when confronted novel challenges stemming expanding possibilities stretch tools, essential adapt our procedures embrace innovative thinking. Stephen C. De Rosa: Conceptualization; writing – original draft. Yolanda D. Mahnke: declare conflict interest. peer review history article available https://www.webofscience.com/api/gateway/wos/peer-review/10.1002/cyto.a.24844.

Язык: Английский

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Predictive Value of Pretreatment Neutrophil to Albumin Ratio in Response to Neoadjuvant Chemotherapy of Breast Cancer

Breast Cancer Targets and Therapy, Год журнала: 2024, Номер Volume 16, С. 393 - 402

Опубликована: Июль 1, 2024

Background: The immune system appears to play a crucial role in how breast cancer responds chemotherapy. In this study, we investigated peripheral marker of and inflammation named the neutrophil albumin ratio (NAR) explore its potential relationship with pathological complete response (pCR) locally advanced patients who underwent neoadjuvant chemotherapy (NAC). Methods: We conducted retrospective analysis 212 consecutive received NAC. NAR was calculated by examining blood cell count level before starting Through ROC curve analysis, determined optimal cutoff value for as 0.0877. used Pearson's chi-square test or Fisher's exact evaluate between pCR, well other clinical characteristics. Logistic regression models were employed univariate multivariate analyses. Results: results both logistic analyses showed that associated tumor regression. high group had higher pCR rate compared low (OR 3.127 [95% CI 1.545– 6.328]; p = 0.002). Conclusion: According it observed levels more likely achieve when undergoing Keywords: cancer, chemotherapy, response, ratio, predictive factors

Язык: Английский

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The consequences of mismatched buffers in spectral cell sorting

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Авг. 19, 2024

ABSTRACT Although spectral flow cytometry has become a ubiquitous tool for cell analysis, the use of on sorters requires additional considerations arising from unique requirements sorting workflows. Here, we show that care should be taken when ascertaining purity sort sorter, as mismatch buffers used initial sample suspension and collection can affect unmixing data, potentially giving rise to erroneous check results.

Язык: Английский

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Using Spectral Flow Cytometry for CAR T-Cell Clinical Trials: Game Changing Technologies Enabling Novel Therapies

International Journal of Molecular Sciences, Год журнала: 2024, Номер 25(19), С. 10263 - 10263

Опубликована: Сен. 24, 2024

Monitoring chimeric antigen redirected (CAR) T-cells post-infusion in clinical trials is a specialized application of flow cytometry. Unlike the CAR T-cell monitoring for individual patients conducted laboratories, data generated during trial will be used not only to monitor therapeutic response single patient, but determine success therapy itself, or even an entire class compounds. The data, typically acquired at multiple testing compiled into database. may also mathematical modeling cellular kinetics identify predictive biomarkers. With expanded context use, robust, standardized assay mandatory order generate valuable and reliable set. Hence, requirements validation, traceable calibration, technology transfer, cross-instrument standardization regulatory compliance are high.

Язык: Английский

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The consequence of mismatched buffers in purity checks when spectral cell sorting

Cytometry Part A, Год журнала: 2024, Номер unknown

Опубликована: Ноя. 30, 2024

Abstract Although spectral flow cytometry has become a ubiquitous tool for cell analysis, the use of on sorters requires additional considerations arising from unique requirements sorting workflows. Here, we show that care should be taken when ascertaining purity sort sorter, as mismatch buffers used initial sample suspension and collection can affect unmixing data, potentially giving rise to erroneous check results.

Язык: Английский

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CAFE: An Integrated Web App for High-Dimensional Analysis and Visualization in Spectral Flow Cytometry

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Дек. 7, 2024

Abstract Spectral flow cytometry provides greater insights into cellular heterogeneity by simultaneous measurement of up to 50 markers. However, analyzing such high-dimensional (HD) data is complex through traditional manual gating strategy. To address this gap, we developed CAFE as an open-source Python-based web application with a graphical user interface. Built Streamlit, incorporates libraries Scanpy for single-cell analysis, Pandas and PyArrow efficient handling, Matplotlib, Seaborn, Plotly creating customizable figures. Its robust toolset includes density-based down-sampling, dimensionality reduction, batch correction, Leiden-based clustering, cluster merging annotation. Using CAFE, demonstrated analysis human PBMC dataset 350,000 cells identifying 16 distinct cell clusters. can generate publication-ready figures in real time via interactive slider controls dropdown menus, eliminating the need coding expertise making HD accessible all. licensed under MIT freely available at https://github.com/mhbsiam/cafe .

Язык: Английский

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Flow Cytometry Balances Complexity and Accessibility

Genetic Engineering & Biotechnology News, Год журнала: 2024, Номер 44(11), С. 36 - 38, 40, 41

Опубликована: Ноя. 1, 2024

Язык: Английский

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Protocol for murine multi-tissue deep immunophenotyping using a 40-color full-spectrum flow cytometry panel

STAR Protocols, Год журнала: 2024, Номер 5(4), С. 103492 - 103492

Опубликована: Дек. 1, 2024

The innate and adaptive immune systems, though often studied separately, interact deeply respond to stimuli simultaneously, with leukocytes displaying a range of pro- anti-inflammatory phenotypes. This protocol details procedure for characterizing murine phenotypes using 40-color full-spectral flow cytometry panel. We describe steps organ collection, sample preparation, immunofluorescent staining, acquisition reproducibly cost-effectively study tissue-resident leukocytes, their subpopulations, inflammatory status in various organs.

Язык: Английский

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