Automated Sorting of Monocytes from Whole Blood for Reproducible Gene Expression Profiling.

Microarray-based gene expression profiling has been used to develop clinically relevant molecular classifiers for many different diseases. Furthermore, it has been shown for various chronic diseases that specific gene expression patterns are reflected at the level of blood cells. However, blood is a complex tissue comprising numerous cell types. Therefore, the contribution of rare cell types to a whole blood expression profile might not be detected and a substantial proportion of what is usually reported as “up-regulation” or “down-regulation” might actually be the result of a shift in cell populations and not of a true regulatory process. In order to circumvent these problems, several techniques have been established to analyze purified subpopulations rather than whole blood samples. Previously, it has been shown, for example, that reproducible gene expression profiles can be generated by positive selection of blood cell subsets from PBMCs¹. As the preparation of PBMCs by, for example, Ficoll is time-consuming, inconvenient, and not amenable to automation, we have set up a combined direct whole blood cell separation and gene expression profiling protocol. By using Whole Blood CD14 MicroBeads in combination with the autoMACS Pro™ Separator, the separation protocol generally allowed enrichment of monocytes from whole blood within 30 min with purities higher than 90%. In combination with the depletion of neutrophils, the major source of contaminating RNA, purities increased to over 95% for all tested blood donors. Monocytes included the CD14^bright/CD16⁻ as well as the CD14^dim/CD16⁺ populations. To assess the reproducibility of gene expression profiles and the influence of several experimental parameters, monocytes were sorted from 5 ml whole blood. RNA was extracted and hybridized to microarrays and the Pearson correlation coefficients of pairwise comparisons were calculated. Technical repeats of monocyte analysis from blood donated at different days showed a higher correlation coefficient than whole blood RNA. Blood storage at room temperature resulted in a strong deregulation of many genes, whereas blood stored at 4°C showed minimal changes, which is in agreement with previous studies. Skipping the centrifugation step, which is used to remove unbound MicroBeads did not alter the gene expression profiles. Incubation of sorted cells in PrepProtect™ Stabilization Buffer showed no alteration of gene expression thus enabling the shipping of cells without liquid nitrogen. Monocytes play a crucial role in diseases like atherosclerosis. Our rapid and simple protocol for combined direct cell sorting from whole blood and gene expression profiling of monocytes might help to ease the discovery of new biomarkers and to screen and monitor patients. ¹ Lyons et al., BMC Genomics (2007), 8:64.