CD34+ immunomagnetic positive selection allows for CD34+ hematopoietic progenitors separation from CD3+ lymphocytes subsets, usually from an apheresis product collected from a previously mobilized donor. This T-cell depleted stem cell graft is primarily intended for rare cases (around 2% of allotransplanted patients in France) of severe, persistent, symptomatic bi- or tri-cytopenia post-allotransplantation, in order to allow for hematologic reconstitution without increasing the risk of GvHD occurrence. Although semi-manual and complex, the process is of sufficient robustness to consistently generate a cellular product with distinctive features and specifications, based on iterative in-process quality controls, that are discussed within these guidelines.

During the last decade, the development of nanomaterials to penetrate inside living cells has been the focus of a large number of studies, with applications for the biomedical field. However, the further dynamics of these nanomaterials inside the cells is dictated by the intracellular environment and in particular its mechanical properties. The mechanical characteristics of the cell interior can be probed with either active or passive microrheological approaches. However, active intracellular microrheology is still in its infancy, owing to the difficulty of inserting probes that can be manipulated by external forces. Here we review recent active microrheology studies using magnetic nanoprobes inserted into endosomes or phagosomes as useful approaches for measuring frequency-dependent viscoelasticity, for mapping the viscoelastic landscape, as well as for identifying the contribution of individual cytoskeleton components and the influence of cell motility. The results of such direct measurements challenge the validity of more typical passive approaches in which the spontaneous displacement of embedded nanoprobes is measured. Here we discuss that one must distinguish probes suitable for use in conditions of thermal equilibrium, whose movements reflect the mechanical environment from probes that interact actively with the cytoplasm and cytoskeleton, in a state of nonequilibrium for which fluctuation-dissipation theorem no longer holds. However, when data on these probes\' viscoelastic microenvironment is available, such passive probe movements can yield useful information on the forces responsible for intracellular activity.

BACKGROUND: Circulating endothelial cells (CECs) have been identified as markers of vascular damage in a variety of disorders, such as myocardial infarction, vasculitis, and transplantation. CD146-driven immunomagnetic isolation has gained widespread use, but the technique is hampered by the lack of a definition of CECs and the absence of a consensus for their enumeration.
OBJECTIVE: To evaluate several variables influencing immunomagnetic isolation of CECs, formulate a definition for CECs and propose a consensus protocol for their enumeration.
METHODS: We devised a protocol based on CD146-driven immunomagnetic isolation and a subsequent confirmatory step with Ulex-Europaeus-Lectin-1 staining. In a multi-center effort, we evaluated the preanalytical and analytical phases of this protocol. We evaluated the effects of storage, anticoagulation and density centrifugation, and compiled previous experience with this technique.
RESULTS: Our protocol permitted unequivocal identification of CECs with acceptable reproducibility. There was an effect of storage time in that median cell numbers declined to only 87.5% of their baseline values during 24 h of storage at 4 degrees C. Recovery was lower with citrate than with ethylene-diamine tetra-acetic acid after 4 h of storage; density centrifugation was also associated with lower recovery. We provide a comprehensive list of technical recommendations and potential pitfalls. Finally, based on our experience with this protocol and a recent consensus workshop, we formulated a working definition for CECs.
CONCLUSIONS: Our work represents an important step toward consensus regarding the CECs. Our recommendations represent the experience of three major centers and should now be scrutinized by others in the field.