Computational protocol: T Cell Response in Patients with Implanted Biological and Mechanical Prosthetic Heart Valves

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Protocol publication

[…] Stained samples were analyzed by running a four-color flow cytometry with FACSCalibur (Becton-Dickinson, USA) and Navios (Beckman Coulter, USA). Mathematical processing of the flow cytometry data was performed by using Kaluza™ v.1.2 (Beckman Coulter, USA) software.A threshold was set to a forward-scatter (FSC) parameter to exclude cell debris. The SSC and FSC settings were done with linear amplification and the logarithmic amplification scale was used for the fluorescence channels and dot plot analysis. Isotype-matched control abs. and “fluorescence-minus-one” gating techniques were used to evaluate thresholds for positivity of individual antibodies. The following CD4 and CD8 T cell subsets were analyzed: naïve (N, CD45RA+CD62L+), central memory (CM, CD45RA−CD62L+), effector memory (EM, CD45RA−CD62L−), and terminally differentiated CD45RA-positive effector memory (TEMRA, CD45RA+CD62L−) according to CD45RA and CD62L expression.Lymphocytic cells were gated according to forward-scatter (FSC) and side-scatter (SSC) properties. Representative bivariate dot plots of the isolated lymphocyte populations from human peripheral blood samples are presented. CD3+CD4+ T helper and CD3+CD8+ cytotoxic T cells are shown on dot plots, respectively. Immunophenotyping was done by analyzing flow cytometry data after costaining with CD3 and CD4 or CD3 and CD8 cell surface markers on FSC/SSC gated lymphocytes, respectively (). [...] Statistical analysis of the data was done by using Statistica 7.0 and GraphPad Prism software. The data are presented as median (Ме) values and interquartile range (25; 75%). Significance of differences between data was evaluated by applying nonparametric Mann-Whitney U test and W-Wilcoxon test. A correlation analysis was done by using Spearman r-test. Significance level was set at p < 0.05.Multivariate comparison was done by using discriminate analysis. A stepwise analysis enumerating steps, p value significance level, and F-test were performed. A discrimination level was evaluated by assessing Wilks' lambda. Significance of an identifying criterion was determined after drawing scatterplots of canonical values and calculating classification value and Mahalanobis squared distance. […]

Pipeline specifications

Software tools Kaluza, Statistica
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens