Quality Assurance Issues
Achieving reproducible and accurate flow cytometry data depends on several critical factors that, if ignored, lead to significant inter-laboratory variation. Maria C. Jaimes, M.D., senior staff scientist at BD Biosciences, a segment of BD, provided a perspective.
“There are a number of ongoing clinical trials being performed simultaneously at several sites to assess the effectiveness of prophylactic HIV vaccines using flow cytometry functional assays, among others. The accuracy of these assays is critical because the data is utilized to make product-advancement decisions. BD was approached by the DAIDS/NIAID/NIH to develop a quality-assurance program to compare the reproducibility of the flow cytometry data being generated across many of these labs.”
According to Dr. Jaimes, these assays require the use of peripheral blood mononuclear cells from vaccines, and are able to discriminate T-cell subsets that respond to a specific antigen by using a technique called intracellular cytokine staining (ICS). The monitoring of cytokines such as interferon-gamma, interleukin-2, and/or tumor necrosis factor-alpha provides a measure of antigen-specific immune responses.
“We performed seven rounds of testing in 16 laboratories worldwide and found that the co-efficient of inter-laboratory variation was 35% on average. We utilized the data to identify key factors that led to variability among laboratories. One source of variability, for example, is how the cells were treated during the procedure: cells left in contact with certain buffers for extended periods of time will lead to a suboptimal result.”
Dr. Jaimes also indicated that the number of collected events is also important, i.e., how many relevant cells are acquired in the flow cytometer. “When few cells are acquired, the accuracy of the data decreased. Another factor was variable gating strategies. Finally, instrument set up and performance differences also accounted for inter-laboratory assay differences.”
The up side of BD’s extensive analysis is to draw attention to sources of variation in flow cytometry cell-based assays, often ignored by the laboratories. “Much of the field lacks rigor and reproducibility. This can be particularly problematic for clinical laboratories that use flow cytometry. Our studies allowed us to determine pass and fail criteria for ICS assays. This could easily be extrapolated for a quality assurance program for other flow cytometry assays.”
Dr. Jaimes said things are changing slowly. “I think we are seeing an increasing sense of awareness of these issues, just as the field of qPCR took some time to begin evolving more stringent guidelines. It’s a step-by-step process but the harmonization and optimization is well worth the effort.”
Acoustic Focusing Cytometer
Traditional flow cytometers manipulate cells with hydrodynamic forces. That is, a suspension of fluorescently labeled cells is withdrawn from the test tube by the instrument, focused with the sheath fluid, and propelled across the path of a laser that excites the fluors to create readouts. This can be a time-consuming process, depending on the number of cells and the volume of sample. One of the new technologies described at the meeting is Life Technologies’ Attune® Acoustic Focusing Cytometer that utilizes sound waves to precisely direct cells into the center of the sample stream.
“This new instrumentation is not your father’s flow cytometer,” remarked Greg Kaduchak, Ph.D., director of engineering. “This technology utilizes acoustic focusing to drive a single coherent stream of cells ultrafast through the instrument. This allows an unprecedented volumetric sample throughput and enables a 10-fold increase in the acquisition speed while at the same time promoting very low variation, especially as compared to current hydrodynamic focusing technology.”
According to Wenlan Hu, senior marketing manager, the Attune has been out for about a year and now the company has released a new blue/red laser configuration that will allow users a wider flexibility for applications.
“If researchers already have a panel set up on other instruments, they won’t have to reconfigure now that the blue/red is available. Also, for some researchers, especially performing marine and bacterial sampling, the ability to very precisely sample large volumes in a small time frame is an important benefit. Overall, this allows a simpler, faster, and more accurate workflow since the instrumentation does not require samples to be lysed or even washed.”
Cytometry literally means measuring cells. Although many people may be familiar with traditional flow cytometry, an emerging area called chemical and metabolic cytometry examines the composition of single cells. “While flow and image cytometry analyze a few components, chemical and metabolic cytometry can characterize thousands of components such as nucleic acids, proteins, and even metabolites. Indeed, it is now possible to detect down to the yoctomole levels of analytes within cells,” said Norman Dovichi, Ph.D., professor of chemistry and biochemistry, University of Notre Dame.
Chemical and metabolic cytometry can provide new layers of cellular information. “These types of analyses are important because single cells can differ dramatically from their neighbors; classical analytical methods average the composition of cells, but this masks cell-to-cell differences. Traditional flow cytometry measures physical properties such as cell size and the forward and side light-scatter patterns. However, only a few components can be measured utilizing affinity probes against known targets. Thus, the unexpected is invisible to the analysis.”
Chemical cytometry lyses cells, labels proteins and biogenic amines, and employs capillary-based separation of components (e.g., capillary electrophoresis or microbore liquid chromatography). Signals are generated typically from laser-induced fluorescent readings to describe cellular composition.
Another arm of chemical cytometry is metabolic cytometry, which measures with exquisite sensitivity selected metabolic pathways in single cells. It differs from chemical cytometry in that cells are first treated with a fluorescent substrate that is taken up and enzymatically processed within the cell. Then labeled cells are aspirated into a capillary, lysed, and components electrophoretically separated. The metabolites are detected using laser-induced fluorescence.
Dr. Dovichi is applying the technology to better understand the glycolipid metabolism of neurons. “We see that there are dramatic differences among individual neurons that could be important for understanding their functional roles and how this may impact illnesses such as Tay-Sach’s disease.”
For the future, Dr. Dovichi is working on interfacing the power of mass spectrometry with chemical and metabolic cytometry methodologies. “This would be especially powerful because of the great detail afforded by mass spec.”
The workhorse technology of flow cytometry continues to evolve and grow. As technologies advance, generating increasingly complex datasets, quick and accurate analysis will remain a challenge.