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Infinicyt v2

Manufactured by Cytognos
Sourced in Spain

Infinicyt v2.0 is a software application designed for the analysis and interpretation of flow cytometry data. It provides a comprehensive platform for the visualization, gating, and processing of multiparametric flow cytometry data.

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7 protocols using infinicyt v2

1

Sensitive NGF Methodology for Plasma Cell Detection

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Follow-up samples were processed within 24 h after collection. Analysis was carried out using the recently developed NGF methodology (Table S1), following the EuroFlow guidelines, as described elsewere20 (link),22 (link). Events from two eight-color tubes (per sample) were merged using the merge function of the INFINICYT™ v2.0 software (Cytognos S.L. Salamanca, Spain). A sample was considered positive when at least 20 aberrant plasma cells were detected. Hemodilution of bone marrow samples for NGF evaluation was assessed through the identification of a significant decrease in non-plasma cell populations: mast cells (CD117hi), erythroblasts (CD45/sideward-scatterlo) and B-cell precursors (CD19+/CD38hi/CD45lo). The complete analysis of NGF performance for the GEM2012 trial has already been published28 (link).
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2

Multiparametric Flow Cytometry for Plasma Cell Characterization

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Patients were routinely characterized at diagnosis by multiparametric flow cytometry. Bone marrow samples were processed within 48 h after collection. Analysis was performed using different assays, following the EuroClonality/EuroFlow consortium guidelines: six-color panel for six markers (CD38/CD138/CD45/CD19/CD56/CD117) in 78 patients, eight-color panel for eight markers (CD38/CD138/CD45/CD19/CD56/CD117/CD27/CD81) in the remaining 335 patients.
For data analysis, the values of all parameters measured per tube were mathematically calculated for the individual plasma-cell events using the merge and calculation functions of the INFINICYT™ v2.0 software (Cytognos S.L. Salamanca, Spain). Plasma cells were identified based on the characteristic pattern of expression of CD38, CD138, CD45 and light scatter features.
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3

Correcting Signal Spillover in Cytometry Data

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Signal spillover between channels was corrected using the CATALYST R package as described previously.44 (link) Compensated fcs files were further processed using FlowJo (v10.5, FlowJo LLC) or Infinicyt v2.0 (Cytognos) software (Online Supplementary Figure S1). Heatmaps were created in MeV software45 (link) by displaying arcsinh-transformed absolute values of median intensities. The fold change was calculated by subtracting the arcsinh-transformed median of unstimulated samples from the arcsinh-transformed median of stimulated samples.
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4

Multicolor Flow Cytometric Analysis

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Bone marrow samples were routinely examined by eight-color labelling method. During the examined period, the combination of antibodies was slightly changed in accordance with the new guideline (Supplementary Figure S1) [29 (link)]. Conventional FC analysis was performed by FACS Diva software v 8.0.3 (Becton Dickinson, San Jose, CA, USA). The raw data from the bi-dimensional FC examination was reanalyzed with novel protocols based on multidimensional dot-plots of Kaluza v 2.1 (Beckman Coulter, Brea, CA, USA) and Infinicyt v 2.0 (Cytognos, Salamanca, Spain) software.
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5

Automated Immunophenotypic Data Deconvolution

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Data were analyzed using FlowCT, a semi-automated workflow developed for deconvolution of immunophenotypic data and objective reporting on large datasets [33 ]. Briefly, FlowCT starts by creating a matrix with expression data generating a SingleCellExperiment object and correcting possible discrepancies in markers’ nomenclature. Subsequently, it performs internal data quality control and normalization, and automated clustering followed by dimensionality reduction to visualize clusters’ identity before manual annotation. This step is completed using the Infinicyt software (Infinicyt v2.0; Cytognos SL, Salamanca, Spain). Afterwards, sub-clustering of APC, T, and B cells was performed. Comma-separated value files with population abundances were finally exported to evaluate statistical correlations and differences across groups.
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6

EF AML/MDS Antibody Panel Staining Protocol

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All samples were stained using the first three tubes of the EF AML/MDS antibody panel (7 (link)). Sample preparation and acquisition were performed according to the EF standard operating procedure and using the recommended EF instrument settings (8 (link)).
Appropriate instrument performance was confirmed by performing FranceFlow and EuroFlow quality assessments (9 (link), 10 (link)).
At least 100,000 BM cells/tube were acquired using a three-laser, eight-color BD FACSCanto-II™ flow cytometer (BD Bioscience, San José, CA, USA) at each study site. Acquired cells were then analyzed using Infinicyt V2.0 (Cytognos, Salamanca, Spain).
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7

Immune Profiling of Multiple Myeloma

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Subsequently, we applied FlowCT [21 (link)], an R-based package, to identify possible differences in the immune microenvironment among multiple myeloma patients with low or high ferritin levels. FlowSOM and Seurat clustering approaches were used for automated clustering. The median expression of each marker on multi-uniform manifold approximation and projection (UMAP) and the Infinicyt software (Infinicyt v2.0; Cytognos SL, Salamanca, Spain) were used to characterize each cluster.
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