Computational protocol: Improved monitoring of clinical response in Systemic Lupus Erythematosus by longitudinal trend in soluble vascular cell adhesion molecule-1

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Protocol publication

[…] Fresh peripheral blood mononuclear cells were isolated from blood obtained from 34 SLE individuals. Cells were stained with LIVE/DEAD Fixable Blue Dead cell stain (Invitrogen, Paisley, UK) to exclude dead cells, Fc receptor blocked (Human TruStain FcX; BioLegend, Oxford, UK) and surface stained using the following markers: IgD-BrilliantViolet(BV)421 (IA6-2), CD19-BV510 (HIB19), CD27-BV650 (O323), CD138-FITC or CD138-PE-Cy7 (MI15), CD24-PerCP-Cy5.5 (ML5), CD95-PE-Cy7 (DX2), CD38-APC (HB7) and CD20-APC-H7 (2H7) from BioLegend or BD. Cells were fixed with BD stabilising fixative reagent. Freshly stained cells were acquired on a 5 laser BD SORP LSRFortessa instrument. BD CS&T beads were used immediately prior to every sample run to maintain instrument consistency throughout the entire study. Data were analysed using FlowJo version 10 (Ashland, OR, USA). [...] Statistical analysis was performed using SPSS statistics version 22 (IBM Corporation, Armonk, NY, USA) and R statistics package version 3.1 (R Foundation for Statistical Computing, Vienna, Austria). Biomarker performance was analysed by receiver operating characteristic (ROC) curve analysis, using the pROC package version 1.7.3 in R, and Youden’s index was used to select the optimal discriminatory threshold. A reduction in ECLAM score of 3 or more (∆ECLAM ≤ –3) was used to define “clinically meaningful” improvement in disease activity []. Delta parameters were calculated by subtracting the value on each individual’s first visit for each parameter. For analysis of ∆ECLAM, multiple linear regression was performed with stepwise selection based on Akaike information criteria (AIC), using a mixed-effects model to account for within-individual correlation because of repeated measures for each individual over time. The CD138+ plasma cell population size expressed as the percentage of B cells was analysed by multiple linear regression with stepwise selection and beta regression to account for the standard unit interval of this variable. Standardisation was applied to predictors in all models. […]

Pipeline specifications

Software tools FlowJo, SPSS
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Lupus Erythematosus, Systemic