The impact of pre-freezing storage time and temperature on gene expression of blood collected in EDTA tubes.

BACKGROUND: Blood is a common source of RNA for gene expression studies. However, it is known to be vulnerable to pre-analytical variables. Although RNA stabilization systems have been shown to reduce such influence, traditional EDTA tubes are still widely used since they are less expensive and enable to study specific leukocyte populations. This study aimed to assess the influence of storage time and temperature between blood sampling and handling on RNA from peripheral blood mononuclear cells (PBMCs). METHODS AND RESULTS: Nine blood samples were collected in EDTA tubes from 10 healthy donors. One tube from each donor was immediately processed for PBMC isolation, while the others were first incubated at either 4 degrees Celsius (°C) or room temperature for 2, 4, 6 and 24 h. RNA yield and quality and the expression level of fourt housekeeping (B2M, CASC3, GAPDH, HPRT1) and 8 target genes (CD14, CD19, CD20, IL10, MxA, TNF, TNFAIP3, NR4A2) were compared between samples. RNA yield, quality and integrity did not vary significantly with time and temperature. B2M was the most stable housekeeping gene, while the others were increasingly influenced by storing time, especially at 4 °C. Even when normalized to B2M, the expression level of some target genes, particularly TNFAIP3 and NR4A2, was highly affected by delays in blood processing at either temperature, already from 2 h. CONCLUSION: Pre-analytical processing has a great impact on transcript expression from blood collected in EDTA tubes, especially on genes related to inflammation. Standardized procedure of blood handling are needed to obtain reliable results.

Autologous Hematopoietic Stem Cell Transplantation (AHSCT): Standard of Care for Relapsing-Remitting Multiple Sclerosis Patients.

Autologous hematopoietic stem cell transplantation (AHSCT) has been used in the treatment of highly active multiple sclerosis (MS) for over two decades. It has been demonstrated to be highly efficacious in relapsing-remitting (RR) MS patients failing to respond to disease-modifying drugs (DMDs). AHSCT guarantees higher rates of no evidence of disease activity (NEDA) than those achieved with any other DMDs, but it is also associated with greater short-term risks which have limited its use. In the 2019 updated EBMT and ASBMT guidelines, which review the clinical evidence of AHSCT in MS, AHSCT indication for highly active RRMS has changed from "clinical option" to "standard of care". On this basis, AHSCT must be proposed on equal footing with second-line DMDs to patients with highly active RRMS, instead of being considered as a last resort after failure of all available treatments. The decision-making process requires a close collaboration between transplant hematologists and neurologists and a full discussion of risk-benefit of AHSCT and alternative treatments. In this context, we propose a standardized protocol for decision-making and informed consent process.