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A look into how flow cytometrycan benefit global clinical trials and what an IT savvy central lab can contribute.
Medical treatment of human disease is in a period of tremendous change. In the past, small molecule drugs comprised the overwhelming majority of treatment choices for physicians. The continued emergence of biopharmaceuticals is providing new hope to millions of patients around the world. There are approximately 250 biologic therapy products currently in the market, with hundreds more in development pipelines.
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.
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.
Diligent subject monitoring is an essential component of clinical trials, and central laboratories are well positioned to perform testing on subject samples during the course of a trial. Since subjects are dosed and clinically assessed at multiple sites, samples from these visits can be sent to a central laboratory, where all testing can be performed under one roof.
The central laboratory provides an environment characterized by strict quality control for all testing, technical staff who are trained and certified, and a highly evolved database for data transmission to the sponsor. In addition, central laboratories incorporate client service departments that assist with issues arising during the trial, and project management departments for monitoring sites and subject visits, while providing sites with kits stocked with all the necessary items for sample acquisition and shipment. In short, central labs have matured as highly efficient testing sites that can receive subject samples during a trial and provide high-quality data to the pharmaceutical or biotech company developing the therapy.
Now let's consider for a moment the dilemma of a biotech or pharmaceutical company entering into the clinical trial phase with its new biopharmaceutical therapeutic compound. The needs of this company consist of routine safety testing and complex immune monitoring of the samples from the participants in the trial. The central laboratory with the expertise and experience to perform high-end flow cytometry, which also has in place the complete lab and database infrastructure to perform hematology and chemistry, is the solution.
Since subject visits and sample acquisitions are taking place at multiple sites, a major issue to consider is the stability of samples during transport to the central lab. For most cell surface and intracellular proteins of interest, cells from whole blood samples can be successfully assayed within 72 hours of collection, following shipment at ambient temperature conditions. For unique or labile proteins, or for more complex methods such as functional assessment, time course studies are necessary to determine the window of time following sample acquisition during which the particular measurement can be made.
New BCT phlebotomy tubes have lengthened blood sample stability times; and for particularly sensitive assays, PBMC can be isolated from the whole blood sample at the site, cryo-preserved, and shipped to the central laboratory.
Another major issue for consideration is harmonization of subject sample testing during a clinical trial. Increasing utilization of global site selection has necessitated standardization between laboratories on different continents.
Consider a small biotech company that has developed an experimental design for its new compound based on investigator expertise at four academic medical centers located in Washington, DC, Glasgow, Beijing, and Mumbai. Blood samples from participants in this trial need to undergo testing in hematology and chemistry as well as complex flow cytometric assays for receptor occupancy and apoptosis for the target cancer cell population. What is the solution to the challenge this company's team faces? ICON Central Laboratories [ICL] believes it has the solution in its harmonized testing facilities in New York, Dublin, Singapore, and Bangalore.
These facilities perform hematology, chemistry, and flow cytometry, where samples from each of the four sites could be sent, respectively, at ambient temperature and processed for testing the following day. Both safety and flow cytometric assays would be performed at each of ICL's facilities, thus circumventing the concern of sample stability issues.
In addition, reagent optimization and validation is performed at the New York ICL location—all reagents are purchased in New York and shipped to the other facilities. The template for the flow cytometer, which includes gating strategy and analysis for data acquisition, is created in New York, locked down, and provided to the other ICL locations. A standardized cell preparation protocol is written and shared among centers; cross-training takes place and sample concordance studies between sites are completed. All of these steps help to ensure that laboratory testing from subjects seen in Glasgow (performed in Dublin) is comparable to laboratory testing from subjects seen in Mumbai (performed in Bangalore).
To further assist in standardization and quality control of flow cytometric data generated in the central laboratory environment, ICL has created a virtual flow cytometry network.
In the earlier example, samples are shipped to, prepared, and analyzed on the flow cytometer at each of the four laboratory locations. Subsequently the data, via computer server, is transmitted to the central database at the New York lab, where the reviewer[s] can assess quality control, interpret the data, provide final approval for the sample, and, if necessary, reanalyze the data to ensure consistency for all the data in the trial.
ICL uses the aftermarket flow cytometry software data analysis program FCS Express for this purpose. Flow cytometric listmode data from different laboratory sites around the world can be loaded into FCS Express and analysis regions can be reviewed, thus minimizing operator variation.
As a further step, ICL has in place a system that allows availability of flow cytometric list mode data to clients for review by logging into a secure account from their own computer. All these steps can provide reassurance to the sponsor that laboratory testing results impacting the outcome of their clinical trial are being generated in a setting that utilizes innovative strategies and procedures, which in the final analysis will provide the best data quality possible for their hematology and blood chemistry results as well as for their flow cytometric assays for receptor occupancy and quantification of apoptotic cells.
ICL combined the power of flow cytometric analysis with information technology capable of connecting the world. The result: a global solution that assured the integrity of subject samples, uniform test results, flow cytometric data directly correlated for individual subjects, consistent data review and final approval by a single team of flow cytometry experts, and access to the raw flow cytometric data so that the sponsor's scientists and those from ICL could work collaboratively to ensure final data quality.
Thomas W. McCloskey , PhD, is associate director, cellular immunology, research and development, Joseph Schappert , MD, is medical director, and Carol Rosenthal * is director of research and development at ICON Central Laboratories, 123 Smith Street, Farmingdale, NY 11735, email: Carol.Rosenthal@iconplc.com
*To whom all correspondence should be addressed.