The growing role of biopharmaceuticals presents new challenges for the pharmaceutical and biotech companies guiding their development, as well as for the central laboratories performing the subject testing during a clinical trial. Many biopharmaceuticals are antibody-based compounds, which often elicit a response from the immune system. Thus, rigorous and objective quantification of immune cells and assessment of immune function is required to ensure optimal subject safety.

In addition, since much of the biology underlying the new classes of therapeutics remains unknown, clever and innovative experimental designs are necessary in clinical trials to enhance our knowledge regarding these treatment options.

One of the most robust methods available for immune system monitoring is flow cytometry. Flow cytometry utilizes lasers and sensitive optical detectors to measure properties of cells at rates upwards of 10,000 cells per second. The design of flow cytometers is such that measurements are recorded on individual cells as they pass the laser beam. Thus, although a large number of heterogeneous cells may be present in the sample the cytometer can identify subpopulations, and by appropriate gating strategies, measurements can be confined to particular cell subsets.

Flow cytometry is a powerful technology capable of quickly and accurately making single cell measure- ments of properties of cells that have been tagged with fluorescent compounds. Modern flow cytometers typically incorporate multiple lasers to confer the capability of simultaneously measuring many different fluorescent dyes, record digital data which facilitates later analysis, and possess multiple detectors so they have the ability to collect eight or more fluorescent channels depending on which tags have been attached to the cells. Thus, flow cytometers are powerful cellular analysis instruments capable of performing highly complex analyses for quantifying and monitoring multiple immune system parameters.

Since a single cell suspension is the required sample type for analysis on the cytometer, blood is ideally suited for assays designed for monitoring subject immune systems during a clinical trial.

Applications in trials

Flow cytometers quantify fluorescence tags on cells that have been added prior to placement of the sample on the instrument. Depending on the biological question and the assay being applied to answer it, these fluorescent tags can take different forms. Most common is the use of fluorochrome-conjugated monoclonal antibodies for phenotyping assessment. These antibodies can be chosen so that they bind to plasma membrane proteins whose expression is restricted to specific cell populations. For example, green, orange, and red fluorescently conjugated monoclonal antibodies might be added to subject blood samples to enumerate the percentages of T, B, and NK lymphocyte subpopulations and identify any effect from therapy. The flow cytometer is ideally suited to perform such tasks, as its function is to measure particle-associated fluorescence.

Flow cytometry technology can also be used to identify and quantitate intracellular proteins. Once again, a fluorochrome-conjugated monoclonal antibody will be incubated with the cell sample, in this case following a permeabilization step, which opens holes in the cell's membrane allowing the fluorescent tag to enter the cytoplasm of the cell. For example, the apoptotic regulatory protein bcl-2 can be quantified by labeling permeabilized cells using specific fluorochrome-conjugated monoclonal antibody.

Alternatively, the fluorescent tag can be a dye that binds to a specific cellular component. The common dye propidium iodide is fluorescent (so it is applicable for use in flow cytometric assays) and specifically binds to nucleic acid. Propidium iodide is a red dye, which will bind to both DNA and RNA inside the cell—by additionally adding RNase enzyme to eliminate the RNA, measured fluorescence will represent DNA content of the cells. Since cells increase their DNA in preparation to divide, the rate of cell division can be determined for a particular population. Such measurements are important in cancer to determine how quickly the tumor is growing.

In addition, flow cytometric assays are available that are capable of measuring particular processes within the cell. For example, many oncology therapies induce death of cancer cells by apoptosis, a process of self-directed cell destruction that typically occurs after initiation by an appropriate stimulus. As part of the cascade of events that occurs in the cell, DNA is cleaved into fragments. By permeabilizing cells and then incubating them with an enzyme, which incorporates fluorescently conjugated nucleic acids into sites of breakage in the DNA, cells that are undergoing apoptosis can be fluorescently tagged and identified by flow cytometry.