In this issue

Optimizing Umbilical Cord T Cell Flow-based Assays

Flow cytometry is a powerful tool in evaluating T cells, but can be subject to high signal-to-noise ratio when there is experiment-based variability. Delay to cell processing is often unavoidable when using umbilical cord (UC) samples, which potentially introduces variation in results. There are no published studies that test delay prior to lymphocyte isolation as an independent variable affecting UC T cell viability, recovery, phenotype and function in flow-based assays. This study quantifies and compares variation in T cell parameters based on assay replicate, patient and delay to processing (0, 24 and 48 hours). In addition to detailed phenotypic and functional analysis of UC T cells, Scheible and coworkers provide methods for UC blood collection and processing that minimize variation at the replicate level, and describeobserved differences in T cell subpopulations and cytokine-positive frequencies in flow-based assays following a 24–48 hour delay to processing.

In this issue: page 937

Small is beautiful

Flow cytometry has been an approach of choice when analyzing individual cells or particles. However, one important limitation has been size, theoretically at the half micron boundary for most instruments. On the other hand, small entities such as viruses are typically probed collectively by looking at whole purified viral populations, infected cells or total infected lysates. However, these viral populations are highly heterogeneous. Unfortunately, it was not possible to push most analyses at the individual viral particle level. Loret, El Bilali, and Lippé have now solved this puzzle by not only analyzing but also sorting individual Herpes simplex viral particles by flow cytometry. In their landmark paper, they show they can sort one of many viral intermediates present in the cell, so-called nuclear C-capsids, with surprisingly high purity. Most interestingly, Herpes viral particles range from 125 to 250 nm in diameter depending on the stage of the infection. This de facto lowers the effective resolution of common FACS instruments and opens up new exciting avenues for flow cytometry and virology alike.

In this issue: page 950

Heterogeneity of breast cancer myoepithelial cells

Heterogeneity is a major obstacle to therapeutic success. The diagnosis of basal-like breast cancer has no molecular therapeutic implications; to date no targeted therapy is available for this subtype. “Basal-like” comprises a heterogeneous group that accounts for up to 15% of all breast cancers, displays distinctive patterns of relapse, and a poor prognosis. Breast cancer belonging to the basal-like subtype is thought to be stem cell derived, and myoepithelial cells are candidates for precursor cells. The Veneziani group (Leccia et al.), immunophenotyping breast cancer cells for 28 surface markers, analyzes inter- and intra-tumor heterogeneity of myoepithelial cells. Excluding markers sharing either high or low homogenous expression with established cell lines, they find a heterogeneous expression among the samples, representative of the inter-tumor diversity. Within each sample, the marker profile indicates remarkably restricted intra-tumor heterogeneity, suggesting enrichment for cellular phenotypes that have a hierarchical relationship. Their model---which addresses the contribution that myoepithelial cells can make to breast cancer at the cellular level and takes into account a large vocabulary of markers---can be a tool to identify molecular targets and test new drugs.

In this issue, page 960

Characterization of Circulating Tumor Cells

The majority of cancer-related deaths result from metastasis, which has been associated with the presence of circulating tumor cells (CTCs). The ability to characterize CTCs could be a powerful clinical tool, acting as a minimally invasive liquid biopsy that would inform clinical decision-making and help direct individualized therapy. Development of the FDA-cleared CellSearch® system (Veridex) has allowed for sensitive enumeration of CTCs. Although this platform also allows single-cell characterization of CTCs, the detailed process for developing user-defined protein marker assays using this platform is not well defined. Using the cancer stem cell marker CD44 and the apoptosis marker M-30 as examples, the work by Lowes and coworkers represents the first study in the literature to describe the detailed process of user-defined protein marker assay development and optimization using the CellSearch® system. This study provides an important resource for the future development of new marker assays by users of this platform.

In this issue, page 983