No normalization was implemented in the flow cytometry data analysis. All raw data was analyzed with either FlowJo™ v10.7 Software (BD Life Sciences) or Infinicyt 2.0 (Cytognos) software. Statistical analyses were performed with GraphPad Prism v9 and SPSS 26. We applied unpaired two-sided Mann–Whitney U-tests to assess statistical significance between two groups of unpaired samples. We applied paired two-sided Wilcoxcon-matched-paired test to asses statistical significance between two groups of paired samples. AUC was calculated on days 5, 7, 11, 14 and 20 and peak CAR-T expansion was calculated only when data was obtained from at least three timepoints. For dPCR data analysis, “QuantStudio Absolute Q Digital PCR Software 6” (Applied Biosystems) with automatic Poisson correction was used. Normalization across samples was implemented by assuming there were two copies of the reference RNase P gen per cell. Results were expressed in CAR-T copies per cell. OS and EFS were estimated with the Kaplan-Meier method using survival in GraphPad Prism v9. EFS was computed from the date of infusion to the date of event, with both progression and death scored as event. To assess correlation, we calculated Spearman’s rank correlation coefficient.
Infinicyt 2
Manufactured by Cytognos
Sourced in Spain
Infinicyt 2.0 is a software application developed by Cytognos for the analysis of flow cytometry data. The software provides a comprehensive suite of tools for data visualization, gating, and statistical analysis. Infinicyt 2.0 supports a wide range of flow cytometry instruments and file formats, enabling researchers to efficiently process and interpret their experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (3 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Prism v9, by GraphPad (1 mentions)
Facscanto 2, by BD (1 mentions)
Trucount tube, by BD (1 mentions)
Omnicyt flow cytometer, by Cytognos (1 mentions)
Multitest, by BD (1 mentions)
Cd19 pe cy7 clone sj25c1, by BD (1 mentions)
Facs lysing solution, by BD (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
5 protocols using infinicyt 2
1
Comprehensive Cytometry Data Analysis
2
Flow Cytometry Standardized Leukemia Panel
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Flow cytometry staining was performed with the standardized EuroFlow ALOT (acute leukemia orientation tube) antibody panel (CD45-OC515, CD3-Pacific Blue, CyMPO-FITC, CyCD79a-PE, CD34-PerCP/Cy5.5, CD19-PE/Cy7, CD7-APC, SmCD3, APC/C750) and further tubes 1-3,27 (link) samples were acquired by using a FACSCanto II (Becton Dickinson), and data were analyzed by using Infinicyt 2.0 (Cytognos).
3
Comprehensive Immunophenotyping of Peripheral Blood
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For immunophenotyping characterization, peripheral blood samples collected into EDTA tubes were analyzed within 24 h after collection. In brief, cells were incubated with a prevalidated panel of monoclonal antibodies for 15 min, lysed, washed, and acquired in an 8-color BD FACS Canto II Flow Cytometer. The panel of monoclonal antibodies included CD3, CD4, CD8, CD16, CD19, CD20, CD27, CD38, CD45, CD45RA, HLA-DR, IgD, IgM and TCR gamma delta, assuring the identification of several leukocyte populations, and particularly, distinct subsets of T cells (i.e., naïve, memory and activated T cells within CD4, CD8 and gamma delta T cells) and B cells (i.e., naïve, unswitched and switched memory, and plasmablasts). A single-platform strategy with BD Trucount tubes was also used to assure absolute cell counts for each subset addressed.
BD FACS Diva software was used for acquisition purposes, and Infinicyt 2.0 software (Cytognos, Salamanca, Spain) was used for data analysis. All gating strategies are presented in (Appendix A.2 —Gating strategy for identifying immune subsets).
BD FACS Diva software was used for acquisition purposes, and Infinicyt 2.0 software (Cytognos, Salamanca, Spain) was used for data analysis. All gating strategies are presented in (
4
Multiparameter Flow Cytometry Analysis
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Cytometric experiments were carried out with a FACSCanto II flow cytometer (BD, Franklin Lakes, NJ, USA) equipped with an argon laser (Blue, Excitation 488 nm), a helium-neon laser (Red, Excitation 633 nm), a solid-state diode laser (Violet, Excitation 405 nm), and Omnicyt flow cytometer (Cytognos SL, Salamanca, Spain). Analyses were performed with the FACSDivaTM software (BD) and the Infinicyt 2.0 software (Cytognos SL). Approximately 10,000 cell events were acquired for each sample.
5
Multiparametric Flow Cytometry for Immune Profiling
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The multiparametric flow cytometry (MFC) was performed in all patients at the beginning of their care (day one to day three). Absolute leukocyte counts were performed using the Sysmex XN-3000 counter at the time of the MFC analysis. The MFC was conducted using whole blood lysed with the BD FACS Lysing Solution (1:10). The Multitest® (BD Biosciences, San Jose, USA) was used to search for subsets of T-cells. This reagent is composed of the cell surface markers CD3 FITC (clone SK3/Leu3a), CD4 APC (clone 2D1), CD8 PE (clone SK7/Leu-4) and CD45 PerCP (clone SK1). In addition, the CD19 PE-Cy7 (clone SJ25C1, BD Biosciences) were used to identify B-cells. A total of 50,000 cell/events per tube were acquired using the FACSCanto II® cytometer (BD Biosciences) and FACS Diva software (BD Biosciences). The performance, compensation and daily MFI control were performed according to the manufacturer's instructions and the analysis was performed on the Infinicyt™ 2.0 software (Cytognos, Salamanca, Spain). The analysis protocol included the removal of threshold debris and lymphocytes were initially identified based on low frontal (FSC) and side scatter (SSC) and strong CD45 staining, followed by the discrimination of helper and cytotoxic T-cells using CD3, CD4 and CD8, as widely known. The B-cells were identified by the CD19 positive staining.
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