Sample preparation for flow cytometry benefits from some lateral thinking.

OVER the past 50 years, Flow Cytometry (FC) has established itself as one the most powerful analytical technologies for making controlled high throughput, multi-parameter measurements on large numbers of cells (1). As such FC has been pivotal in dissecting the phenotypes of key cellular populations from within complex, often highly heterogeneous samples both in the context of normal and disease states. The multi-parameter, high-throughput nature of FC very much relies on the fact that cells are first stained with fluorochrome labeled monoclonal antibodies specific for one of a panoply of key cellular antigens before being analyzed in liquid suspension by a flow cytometer system. The sample preparation process requires a significant degree of optimization, is laborintensive and can be affected by a host of potentially confounding variables. As such it is certainly fair to say that the “journey” from specimen to measurement is fraught with danger as fear of artifact generation lurks at every twist and turn. For those who analyze cells liberated from solid tissue by FC the challenge of sample preparation can be daunting. Even before the staining process begins the biopsy has to be digested and disaggregated to generate a suspension of viable cells while trying to avoid stripping off key surface receptors or changing the cellular composition in a way that no longer reflects the original tissue-resident populations (2). However there are also significant challenges to overcome when working with liquid biopsies such as blood. Blood is one of the most important analytical mediums because it is relatively non-invasive and cost effective to collect. Moreover the phenotypic status of various immune cells in the peripheral blood circulation may provide a set of highly informative biomarkers for diagnosing and monitoring various diseases. It is therefore imperative that the true in vivo cellular state is preserved as much as possible during the sample processing steps for FC analysis. The question any cytometrist must ask themselves when preparing samples for FC analysis is “what changes could I be eliciting in the cell population by treating the sample in this particular way”? There can be no debate whatsoever that the fewer manipulations carried out on a sample prior to analysis by FC the more likely it is to reflect the native cellular state. In this issue of Cytometry A, Civin and coworkers (page 1073) present a powerful microfluidics-based approach to aid in the analysis of blood samples that is based on a technology called Deterministic Lateral Displacement (DLD). The DLD-based microfluidics chip they describe is able to remove almost 99.99% of red blood cells (RBCs) and unbound monoclonal antibody (mAb) in under 20 min while still recovering 88% of the target lymphocyte population from within the sample. The authors have described the use of DLD for this purpose previously (3) but now in this issue of Cytometry A (page 1073; DOI: 10.1002/cyto.a.23019) they report the performance of a reformatted, high precision plastic version of the microfluidic chip that will invariably lead to improved accessibility and more widespread adoption across different cytometryreliant disciplines. Processing a blood sample for FC can take one of several directions but is always dictated by the fact that RBCs outnumber lymphocytes by a factor of 1000:1 in peripheral blood. Typically, fluorochrome-conjugated monoclonal antibodies against various key phenotypic surface receptors are added for a period of incubation before a 1–2 mL volume of RBC lysing agent is added to the sample. The sample is then