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Titrating Complex Mass Cytometry Panels.

ABSTRACT: We describe here a simple and efficient antibody titration approach for cell-surface markers and intracellular cell signaling targets for mass cytometry. The iterative approach builds upon a well-characterized backbone panel of antibodies and analysis using bioinformatic tools such as SPADE. Healthy peripheral blood and bone marrow cells are stained with a pre-optimized "backbone" antibody panel in addition to the progressively diluted (titrated) antibodies. Clustering based on the backbone panel enables the titration of each antibody against a rich hematopoietic background and assures that nonspecific binding and signal spillover can be quantified accurately. Using a slightly expanded backbone panel, antibodies quantifying changes in transcription factors and phosphorylated antigens are titrated on ex vivo stimulated cells to optimize sensitivity and evaluate baseline expression. Based on this information, complex panels of antibodies can be thoroughly optimized for use on healthy whole blood and bone marrow and are easily adaptable to the investigation of samples from for example clinical studies. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

SUBMITTER: Gullaksen SE

PROVIDER: S-EPMC6766997 | biostudies-literature |

REPOSITORIES: biostudies-literature

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CyTOFmerge: integrating mass cytometry data across multiple panels.

Project description:MotivationHigh-dimensional mass cytometry (CyTOF) allows the simultaneous measurement of multiple cellular markers at single-cell level, providing a comprehensive view of cell compositions. However, the power of CyTOF to explore the full heterogeneity of a biological sample at the single-cell level is currently limited by the number of markers measured simultaneously on a single panel.ResultsTo extend the number of markers per cell, we propose an in silico method to integrate CyTOF datasets measured using multiple panels that share a set of markers. Additionally, we present an approach to select the most informative markers from an existing CyTOF dataset to be used as a shared marker set between panels. We demonstrate the feasibility of our methods by evaluating the quality of clustering and neighborhood preservation of the integrated dataset, on two public CyTOF datasets. We illustrate that by computationally extending the number of markers we can further untangle the heterogeneity of mass cytometry data, including rare cell-population detection.Availability and implementationImplementation is available on GitHub (https://github.com/tabdelaal/CyTOFmerge).Supplementary informationSupplementary data are available at Bioinformatics online.

| S-EPMC6792069 | biostudies-literature

Human Bone Marrow Assessment by Single Cell RNA Sequencing, Mass Cytometry and Flow Cytometry

Project description:This SuperSeries is composed of the SubSeries listed below.

2018-09-26 | GSE120446 | GEO

Human Bone Marrow Assessment by Single Cell RNA Sequencing, Mass Cytometry and Flow Cytometry [bulk]

Project description:Bulk RNA Sequencing of Healthy Bone Marrow Mononuclear Cells

2018-09-26 | GSE120444 | GEO

Human Bone Marrow Assessment by Single Cell RNA Sequencing, Mass Cytometry and Flow Cytometry [scRNA]

Project description:New techniques for single-cell analysis have led to insights into hematopoiesis and the immune system, but the ability of these techniques to cross-validate and reproducibly identify the biological variation in diverse human samples is currently unproven. We therefore performed a comprehensive assessment of human bone marrow cells using both single-cell RNA sequencing and multiparameter flow cytometry from twenty healthy adult human donors across a broad age range. These data characterize variation between healthy donors as well as age-associated changes in cell population frequencies. Direct comparison of techniques revealed discrepancy in the quantification of T lymphocyte and natural killer cell populations. Orthogonal validation of immunophenotyping using mass cytometry demonstrated good correlation with flow cytometry. Technical replicates using single-cell RNA sequencing matched robustly, while biological replicates showed variation. Given the increasing use of single-cell technologies in translational research, this resource serves as an important reference dataset and highlights opportunities for further refinement. [Funding source] Project Number: 1ZIAHL006163-05 Contact PI / Project Leader: HOURIGAN, CHRISTOPHER Title: DETECTION, PREVENTION AND TREATMENT OF ACUTE MYELOID LEUKEMIA (AML) RELAPSE. Awardee Organization: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE

2018-09-21 | GSE120221 | GEO

Human Monocyte Heterogeneity as Revealed by High-Dimensional Mass Cytometry

Project description:We used the high dimensionality of mass cytometry together with the FlowSOM clustering algorithm to accurately identify and define monocyte subsets in blood of healthy human subjects and those with coronary artery disease (CAD). To study the behavior and functionality of the newly defined monocyte subsets, we performed RNA sequencing, transwell migration, and efferocytosis assays. Here, we identify 8 human monocyte subsets based on their surface marker phenotype. We found that 3 of these subsets fall within the CD16+ nonclassical monocyte population and 4 subsets belong to the CD14+ classical monocytes, illustrating significant monocyte heterogeneity in humans. As nonclassical monocytes are important in modulating atherosclerosis in mice, we studied the functions of our 3 newly identified nonclassical monocytes in subjects with CAD. We found a marked expansion of a Slan+CXCR6+ nonclassical monocyte subset in CAD subjects, which was positively correlated with CAD severity. This nonclassical subset can migrate towards CXCL16 and shows an increased efferocytosis capacity, indicating it may play an atheroprotective role.

2020-03-06 | GSE146447 | GEO

Polymeric dipicolylamine based mass tags for mass cytometry.

Project description:Mass cytometry is an emerging powerful bioanalytical technique for high-dimensional single-cell analysis. In this technique, cells are stained with metal-isotope-tagged antibodies and are analyzed by an inductively coupled plasma time-of-flight mass spectrometer. While there are more than 100 stable isotopes available in the m/z 75 to 209 detection range of the instrument, only about 50 parameters can be measured per cell because current reagents are metal-chelating polymers with pendant aminocarboxylate chelators that only bind hard metal ions such as the rare earths and Bi3+. Here we describe the synthesis and characterization of a new type of metal-chelating polymer with pendant dipicolylamine chelators suited to binding intermediate to soft metals such as rhenium and platinum. We introduce two different conjugation strategies, a thiol-maleimide reaction that works well for rhenium, and a DBCO-azide click reaction designed to avoid potential complications of Pt and other heavy metals interacting with thiol groups. We show that these polymers can serve as new elemental mass tags for mass cytometry. Antibody-polymer conjugates of CD20 and CD8a prepared by both coupling reactions were employed in conjunction with commercial metal-conjugated antibodies for multi-parameter single-cell immunoassays.

| S-EPMC8926288 | biostudies-literature

Mass Cytometry Analysis Reveals Complex Cell-State Modifications of Blood Myeloid Cells During HIV Infection.

Project description:Dendritic cells (DC), which are involved in orchestrating early immune responses against pathogens, are dysregulated in their function by HIV infection. This dysregulation likely contributes to tip the balance toward viral persistence. Different DC subpopulations, including classical (cDCs) and plasmacytoid (pDCs) dendritic cells, are subjected to concomitant inflammatory and immunoregulatory events during HIV infection, which hampers the precise characterization of their regulation through classical approaches. Here, we carried out mass cytometry analysis of blood samples from early HIV-infected patients that were longitudinally collected before and after 1 year of effective combination antiretroviral therapy (cART). Blood samples from HIV controller patients who naturally control the infection were also included. Our data revealed that plasma HIV RNA level was positively associated with a loss of cDC and pDC subpopulations that display high expression of LILR immunomodulatory receptors. Conversely, specific monocyte populations co-expressing high levels of HLA-I, 3 immunomodulatory receptors, CD64, LILRA2, and LILRB4, and the restriction factor CD317 (also known as BST2/Tetherin), were more abundant in early HIV-infection. Finally, our analysis revealed that the blood of HIV controller patients contained in a higher abundance a particular subtype of CD1c+ cDCs, characterized by elevated co-expression of CD32b inhibitory receptor and HLA-DR antigen-presentation molecules. Overall, this study unravels the modifications induced in DC and monocyte subpopulations in different HIV+ conditions, and provides a better comprehension of the immune regulation/dysregulation mechanisms induced during this viral infection.

| S-EPMC6882910 | biostudies-literature

Updating recommendations of the Brazilian Group of Flow Cytometry (GBCFLUX) for diagnosis of acute leukemias using four-color flow cytometry panels.

Project description:IntroductionFlow cytometry has become an increasingly important tool in the clinical laboratory for the diagnosis and monitoring of many hematopoietic neoplasms. This method is ideal for immunophenotypic identification of cellular subpopulations in complex samples, such as bone marrow and peripheral blood. In general, 4-color panels appear to be adequate, depending on the assay. In acute leukemias (ALs), it is necessary identify and characterize the population of abnormal cells in order to recognize the compromised lineage and classify leukemia according to the WHO criteria. Although the use of eight- to ten-color immunophenotyping panels is wellestablished, many laboratories do not have access to this technology.Objective and methodIn 2015, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) proposed antibody panels designed to allow the precise diagnosis and characterization of AL within available resources. As many Brazilian flow cytometry laboratories use four-color immunophenotyping, the GBCFLUX has updated that document, according to current leukemia knowledge and after a forum of discussion and validation of antibody panels.ResultsRecommendations for morphological analysis of bone marrow smears and performing screening panel for lineage (s) identification of AL were maintained from the previous publication. The lineage-oriented proposed panels for B and T cell acute lymphoblastic leukemia (ALL) and for acute myeloid leukemia (AML) were constructed for an appropriate leukemia classification.ConclusionThree levels of recommendations (i.e., mandatory, recommended, and optional) were established to enable an accurate diagnosis with some flexibility, considering local laboratory resources and patient-specific needs.

| S-EPMC8573049 | biostudies-literature

Single-cell mass cytometry reveals complex myeloid cell composition in active lesions of progressive multiple sclerosis.

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| S-EPMC7437178 | biostudies-literature

Lanthanide-Coordinated Semiconducting Polymer Dots Used for Flow Cytometry and Mass Cytometry.

Project description:Simultaneous monitoring of biomarkers as well as single-cell analyses based on flow cytometry and mass cytometry are important for investigations of disease mechanisms, drug discovery, and signaling-network studies. Flow cytometry and mass cytometry are complementary to each other; however, probes that can satisfy all the requirements for these two advanced technologies are limited. In this study, we report a probe of lanthanide-coordinated semiconducting polymer dots (Pdots), which possess fluorescence and mass signals. We demonstrated the usage of this dual-functionality probe for both flow cytometry and mass cytometry in a mimetic cell mixture and human peripheral blood mononuclear cells as model systems. The probes not only offer high fluorescence signal for use in flow cytometry, but also show better performance in mass cytometry than the commercially available counterparts.

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