Rare cells, which exist alone or within a mixture of cells in relatively low numbers, are often indicative of health status. Cells present where they normally don’t exist, such as endothelial cells in circulating blood, suggesting serious pathology.
Flow cytometry has been instrumental in the discovery of rare cells and in understanding how they differ from more abundant cells. “In the past, microscopy was the way to go, but that technique requires a highly trained operator scanning visual fields for long periods of time,” says Sheree Friend, PhD, product manager for imaging flow cytometry at MilliporeSigma (Billerica, MA). Given the limitations of observation and human endurance, detecting numbers of cells sufficient for statistical validation of rare cells becomes difficult.
Microscopy still functions to characterize rare cells that have been isolated through flow methods. Cell-based morphological assessment, using event- and molecule-specific fluorescent tags, normally involves an additional step and instrumentation downstream of the cytometer. MilliporeSigma’s Amnis® imaging flow cytometer combines these critical analytical functions.
In one application, researchers from the University at Buffalo (NY) studied immunosuppression levels in organ transplant patients by quantifying the degree of translocation of NF-kB in peripheral blood T lymphocytes. Investigators concluded that data acquired through imaging flow cytometry could help guide appropriate dosing levels of immunosuppressive drugs in transplant patients. Although neither the cells nor protein is technically “rare,” this illustrates the detail with which cellular analysis can be performed.
In another experiment, researchers from Health Canada developed an assay to detect rare bi-nucleated cells to quantify micronuclei, a process currently performed by microscopy. Investigators concluded that imaging flow cytometry could deliver a more rapid and accurate method to estimate unknown radiation doses from accidental radiation exposure.
Combining microscopy and cytometry raises an interesting point about rare cell events. Friend explains, “When observing them, investigators often worry about artifacts—particles stuck to cells, for example. Combining imaging with flow cytometry affords the added ability to verify that rare events are not spurious, particularly when you don’t have thousands of cells or events. It lets you identify cellular morphology and occurrence simultaneously.”
Speed and numbers matter
From the clinical perspective, analysis of rare cytometry events can sometimes detect “minimum residual disease” (MRD), which occurs when certain illnesses appear to be eradicated but at a very basic level are not. It’s common to discuss MRD in patients treated for leukemia or lymphoma, where blood counts often are normal and patients feel well, yet they eventually relapse and die from their disease.
Conventional diagnostics fall short of the ability to detect dangerous MRD levels that indicate eventual relapse.
Assessing MRD in solid tumors is difficult because cells are attached within heterogeneous tissues. Blood cancers are much more “diagnostics-friendly” because dangerous agents are detectable as single, low-abundance cells.
MRD-related cells occurring at levels below one in 100,000 are considered unlikely to cause relapse within a patient’s normal life span. “But you can’t tell if concentrations are relevant from a sample that small,” says Jeannine Holden, MD, director of scientific affairs at Beckman Coulter (Miami, FL). Diagnosticians must count enough cells to confirm that observations are statistically relevant, which may involve counting millions of cells.
Counting that many discrete entities, even at flow cytometry speeds, is costly and time-consuming.
A fair number of cells are lost during sample preparation, and enrichment of target cell populations is not always practical. “You’re looking into a mixture of ten to 20 cell types that are not very different from each other,” Holden tells Lab Manager. Picking out target cells requires observing as many parameters, or colors, as possible. Colors refer to fluorescence emissions from different labels. Myeloma blood samples contain both malignant and normal B cells, mature and immature.
Disease-associated cells may change over the course of treatment. Cells that predominate at diagnosis are often different from those that interest clinicians during treatment or after long remission periods. Moreover, biologic drugs can apply selection pressure on tumor cell populations. “If a subset of cells that did not express the CD20 surface antigen escaped decimation, they might emerge later on,” Holden says. “Plus, cells might undergo phenotypic drift.”
Color my world
Researchers use cell sorting or cell analyzers to characterize and isolate pure subpopulations of cells, which are often rare cells with a frequency of 0.01 percent and below. Adding to the challenge is the often-limited availability of starting samples, for example, primary cells and circulating tumor cells. Tumor cells circulating in the blood are the basis of liquid biopsies, which take cell-based diagnostics beyond simple morphologic characterization to the protein and gene levels. Experts believe that liquid biopsies may one day replace many solid-tissue biopsies. Circulating tumor cells are quite rare, and our understanding of their significance is limited, but without flow cytometry, liquid biopsies would be impossible.
“Highly parametric experiments are the key to deep analysis of rare cells,” comments Nicole Ovadia, senior product manager at Bio-Rad (Hercules, CA). Here the terms parameter and color—a usable fluorescence frequency—are interchangeable. Super-fast detection permits cytometers to pick up several colors, representing distinct events, simultaneously as individual cells zip by.
For years, flow cytometry was stuck in the range of ten or fewer colors. Bio-Rad has recently introduced the ZE5™ Cell Analyzer, which detects 28 colors and up to 100,000 events or cells per second without loss of data. “This means detecting many more parameters, and much more rapidly, than with conventional flow methods,” Ovadia tells Lab Manager. Rare cell analysis by flow cytometry is advancing through innovations in instrumentation and detection reagents, consisting mostly of fluorescently tagged antibodies to proteins expressed on cell surfaces. “Once a platform exists, the reagents and tools improve alongside it,” notes MilliporeSigma’s Friend. Simultaneous detection of RNA and protein co-localization, a relatively new capability of flow cytometry, offers insights into transcription and translation. Adaptations of conventional cellbased assays, such as FISH, augment more narrowly focused detection methods. With this type of “codevelopment,” the utility of flow cytometry for detecting rare cells and related events continues to improve even after instruments have achieved legacy status.