Written informed consent was acquired at admission. Peripheral blood samples were collected in heparinized tubes before treatment and after treatment. The average time interval from blood collection to surgery is three days while post-operative blood samples were also collected before discharge, usually one week after the operation. Blood cells were treated with red blood cell lysis buffer (Beyotime, China), labelled with fluorochrome-conjugated monoclonal antibodies and incubated at 4 °C for 20 min. Detailed flow procedures cytometry using BD Multitest 6-colour TBNK reagent (BD Pharmingen, USA) have been described in a previous publication from our institution [13 (link)].
Multitest 6 colour tbnk reagent
Manufactured by BD
Sourced in United States
The BD Multitest 6-colour TBNK reagent is a laboratory product designed for the identification and enumeration of T cells, B cells, and natural killer cells in human whole blood samples using flow cytometry. The reagent contains a combination of fluorochrome-conjugated monoclonal antibodies that bind to specific cell surface markers, allowing the detection and quantification of these lymphocyte subsets.
Automatically generated - may contain errors
Lab products found in correlation
Trucount tube, by BD (4 mentions)
Facscanto 2, by BD (2 mentions)
Red blood cell lysis buffer, by Beyotime (1 mentions)
Cobas 8000, by Roche (1 mentions)
Facsaria fusion sorp flow cytometer, by BD (1 mentions)
Facs lysing solution, by BD (1 mentions)
Facs symphony a5 flow cytometer, by BD (1 mentions)
Facscanto, by BD (1 mentions)
7 protocols using multitest 6 colour tbnk reagent
1
Immunophenotyping of Peripheral Blood
2
Comprehensive Immune Cell Profiling
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Differential blood count was assessed by automatic cell counting. Leucocyte subsets were assessed in a central laboratory (CD19+ B cells, CD3+ T cells, CD3+CD4+ T helper cells, CD3+CD8+ cytotoxic T cells and CD56+CD16+ NK cells) using flow cytometry (FC). Blood samples were prepared using the BD Multitest 6-Colour TBNK Reagent (BD Biosciences) according to the manufacturer’s instructions. A BD Canto (BD Biosciences) was used to acquire and analyse data. Serum IgA, IgM and IgG levels were measured by turbidimetry using a Cobas 8000 (C701, Roche Diagnostics).
3
Multiparametric Immune Cell Enumeration
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BD multitest 6 colour TBNK reagent and BD Trucount TM tubes were used to enumerate absolute count of T cells, CD4 T cells, CD8 T cells, B cells, and Natural killer (NK) cells using 50 μl of fresh EDTA stabilised blood following stain/lyse/no-wash protocol. Data acquisition was performed on BD FACS Aria Fusion (SORP) Flow cytometer (BD Biosciences) where 5000 events gated on beads count were acquired. Data analysis was carried out using FlowJo software (BD biosciences)
4
Flow Cytometry for CD4+ and CD8+ T Cells
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CD4+T and CD8+T counts in fresh whole blood were obtained by flow cytometry on BD FACScantoII automatic flow cytometry (Becton Dickinson Immunocytometry System) within eight hours. The reagent is BD Multitest 6‐colour TBNK Reagent produced by Becton, Dickinson and Company, BD Biosciences.
5
Multiparametric Flow Cytometry for T Cell Subsets
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T lymphocyte subset absolute counts were obtained after labelling whole blood samples with BD Multitest 6-Colour TBNK Reagent using BD Trucount™ tubes (Becton Dickinson, San Jose, CA, USA). A lyse-no-wash protocol was used. Briefly, whole blood was stained with the Multitest 6-colour TBNK reagent (containing a cocktail of the following antibodies: CD3 FITC, CD16 and CD56 PE, CD45 PerCp Cy5.5, CD4 PE-Cy7, CD19 APC, and CD8 APC-Cy7). After the first incubation (15 min at room temperature in the dark), BD FACS lysing solution (Becton Dickinson, San Jose, CA, USA) was added to the tubes. After a second incubation (15 min at room temperature in the dark), samples were run on a BD FACSCanto II and analysed with the BD FACSCanto clinical software (Becton Dickinson, San Jose, CA, USA).
6
Multiparameter Flow Cytometry for TBNK Cell Counts
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Absolute numbers of B cells and T cells in peripheral blood were measured using BD Multitest™ 6‐colour TBNK reagents and BD Trucount™ Tubes (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Briefly, exactly 50 µL of heparinised anti‐coagulated blood was added to Trucount tubes within 3 h after extraction and stained for 15 min at RT in the dark. Samples were then fixed with 1X BD FACS lysing solution before acquiring data on a FACSSymphony A5 flow cytometer (BD Biosciences).
7
Immunophenotyping Analysis of Lymphocyte Subpopulations
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Immunophenotyping analysis was conducted on data from the intent-to-treat population. Whole blood was collected into BD TruCount tubes (BD Biosciences, San Jose, CA), and all testing was conducted within a centralized laboratory network with harmonized protocols. Lymphocyte subpopulations (CD3+ T cells, CD3+CD4+ T-helper cells, CD3+CD8+ cytotoxic T cells, CD19+ B cells, and CD16+CD56+ natural-killer [NK] cells) were quantified at BL, weeks 1, 2, 4, 8, and 12 by flow cytometry using the BD MultiTEST 6 Colour TBNK reagents on a BD FACSCanto. The time points for flow cytometry were conducted as part of the hematology and clinical chemistry laboratory assessments and were therefore more frequent than the biomarker collections.
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