How to Prepare Spectral Flow Cytometry Datasets for High Dimensional Data Analysis: A Practical Workflow.

Spectral flow cytometry is an upcoming technique that allows for extensive multicolor panels, enabling simultaneous investigation of a large number of cellular parameters in a single experiment. To fully explore the resulting high-dimensional single cell datasets, high-dimensional analysis is needed, as opposed to the common practice of manual gating in conventional flow cytometry. However, preparing spectral flow cytometry data for high-dimensional analysis can be challenging, because of several technical aspects. In this article, we will give insight into the pitfalls of handling spectral flow cytometry datasets. Moreover, we will describe a workflow to properly prepare spectral flow cytometry data for high dimensional analysis and tools for integrating new data at later time points. Using healthy control data as example, we will go through the concepts of quality control, data cleaning, transformation, correcting for batch effects, subsampling, clustering and data integration. This methods article provides an R-based pipeline based on previously published packages, that are readily available to use. Application of our workflow will aid spectral flow cytometry users to obtain valid and reproducible results.

IL-6 but Not TNFα Levels Are Associated With Time to Pregnancy in Female Rheumatoid Arthritis Patients With a Wish to Conceive.

Fertility issues are common amongst women with rheumatoid arthritis (RA). Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα), known key players in RA pathogenesis, have been associated with reproductive disorders. This study investigates the role of these cytokines in decreased fertility in women with active RA. Preconception cytokine measurements of 61 patients from the PARA-cohort, a prospective study on RA and pregnancy, were studied in relation to time to pregnancy as a measure for fertility. IL-6 levels were higher in patients with a time to pregnancy longer than 1 year (p = 0.016). Survival analysis of patients stratified by high or low serum IL-6 levels, shows a prolonged time to pregnancy in the high IL-6 group (p = 0.045). Univariate cox regression analysis of IL-6 in relation to time to pregnancy as well as multivariate cox regression analysis correcting for age, disease activity, nulliparity, NSAID use and prednisone use were performed, with hazards ratios for log transformed IL-6 of 0.68 (95% CI: 0.51-0.93, p = 0.015) and 0.66 (95% CI: 0.43-0.99, p = 0.044), respectively. For TNFα, no association with time to pregnancy was found. This study shows that high IL-6, but not TNFα, is associated with decreased fertility in women with RA. This finding provides a rationale to therapeutically target the IL-6 pathway in the time period before pregnancy. More research in the form of large cohort studies on drug safety and the effect of bDMARDS on fertility is needed for implementation of treatment strategies directed at fertility issues in women with RA.