Flow Cytometry in Human Studies

Flow cytometry, like mass spectrometry 20 years ago, has a reputation for inaccessibility and user-unfriendliness. The democratization of flow methods has added a new dimension—an extra level of specificity—to cell-based diagnostics, opening new paths for molecular techniques on individual cell types.

NeoGenomics Laboratories (Ft. Myers, FL), which specializes in cancer genetics testing and related information services, offers the usual array of current-generation diagnostics, but the company’s strength is the ability to combine any of those assays with flow cytometry.

“For clinical trials, we can adapt any of our tests on our clinical flow cytometry menu, but human studies often require customization; that is, adding or removing markers to a flow cytometry panel,” says Gina Wallar, PhD, VP of pharma services sales.

The NeoGenomics flow cytometry platform identifies activation state of cells, expression of biomarkers of interest, and quantification of specific cell populations. Assays may be directed toward diagnosis, response to drug treatment, or disease progression.

Once separated, cells may be tested further through any of 160+ additional assays for specific mutations, including by next-generation sequencing or any of the various PCR varieties. Downstream testing can be done to confirm, validate, or add to results from the flow cytometer.

“Where pure genomic methods give you DNA or RNA levels from a bulk sample, flow cytometry quantifies specific phenotypes,” Wallar says. Next-gen sequencing is extremely valuable in research settings, but flow cytometry has a faster turnaround time and is often less expensive. “Flow cytometry provides a rapid measure of a patient’s response to drugs, including phenotypic responses that may not yet be apparent through DNA analysis. That’s one reason for using flow cytometry along with genomic testing in a clinical trial.”

Solid tumor capability

Last year, IncellDx (Menlo Park, CA), which calls itself a “single-cell diagnostics company,” released the OncoTect iO® Lung Kit, a flow cytometry-based assay that quantifies programmed death ligand 1 (PD-L1) on tumor cells and immune cell subtypes in non-small cell lung cancer (NSCLC) tissue samples. OncoTect iO Lung Kit also quantifies tumor infiltrating lymphocytes (TILs).

OncoTect iO represents a new family of multiparametric molecular analysis systems for immune-oncology suitable for clinical research, pharmaceutical discovery, and the study of malignancies expressing PD-L1, which include those of the bladder, head and neck, and prostate.

OncoTect incorporates a non-enzymatic single cell tissue homogenization process, incellPREP™, for unfixed tumor biopsy samples. Suspended cells are labeled with antibodies, stained with a cell cycle dye, and analyzed via flow cytometry to enumerate cells of interest, particularly PD-L1 and its expression at various stages in the tumor cell cycle.

Related Article: Flow Cytometry for Detecting Rare Cells

Quantifying PD-L1 in tumor and immune cells may allow prediction of patient responses to novel PD-1 immunotherapies.

“We heavily promoted small, fluorescence-based cytometers in diagnostics that didn’t require a biophysicist to run,” says Bruce Patterson, MD, CEO and founder. “Luminex did us a big favor because their instrument is basically a cytometer, a fact that assisted in adoption of user-friendly models for clinical assays.”

IncellDx goes several steps further to detect not just cells but proteins, DNA, and mRNA in cells. By freeing cells from their tissue environments without chemicals or enzymes that might compromise salient features, incellPREP extends the range of useful flow methods to cells from solid tumors, which is a huge achievement since flow cytometry is normally associated with blood-based analysis.

Together, the company’s detection and sample preparation platforms enable cell-by-cell analysis, which adds a new dimension to molecular cancer diagnosis.

Molecular techniques like PCR and NGS are fine for detecting or confirming the presence of specific abnormalities, for answering the question: Is this mutation or translocation present anywhere in my sample? Cell-by-cell analysis provides that information, plus the source or origin of the abnormality.

“With NGS you put all your cells into a blender but you’ve lost the denominator,” Patterson explains. Actually, the denominator exists, but it is usually a nondescript entity with no real information on how a patient is faring. One could report, for example, on the number of mutations per unit volume of sample, or as a percentage of total (mutated plus wild-type) genes, but such knowledge is limited by the power to discriminate one cell from another, which in solid tumor homogenates is zero.

Solid tumors are as diverse cellularly as any other tissue, so which cells are affected could theoretically guide therapy or provide a prognosis. “With flow cytometry the denominator is the cell type,” Patterson continues. “You can identify the cell independently of whatever analyte you’re looking for, and determine exactly which cells carry that feature.”

What about the data?

With all the talk of multicolor, multiparameter flow experiments, it becomes easy to lose sight of the inevitable data overload. Last year, FlowJo (Ashland, FL) partnered with Becton Dickinson to apply FlowJo’s cloud-based data platform, Envoy, to the BD FACSymphony™, a cell analyzer that measures up to 50 characteristics in a single cell. FlowJo provides cloudbased storage of copious flow cytometry data plus onboard calculations and sharing of single-cell analysis data.

FlowJo CEO Mike Stadnisky, PhD, explains the need. “Flow experiments often involve two or more people, knowledge of instrument configurations and quality controls, generation of several different file types, and multiple analysis iterations. Keeping this information together is critical to drawing the correct conclusions about an experiment.”

Stadnisky is reluctant to call his product a laboratory information management system (LIMS) or electronic lab notebook (ELN). “Envoy holds the same details as an ELN but also retains communications between lab personnel and revisions to data analysis, and enables the users to share files— which an ELN does not.” LIMSs do a good job of storing data but the result is a massive database, access to which involves remembering keywords or aspects of a particular project. “Envoy provides similar storage capacity but unhindered access to its directory via search functions for finding any file, workspace, result, or communication.”

For additonal resources on flow cytometry, including useful articles and a list of manufacturers, visit www.labmanager.com/flow-cytometry