3 Ways The ZE5 Cell Analyzer Accelerates Flow Cytometry Research Opportunities
As new instruments come on the market, vendors are quick to provide data proving the systems’ prowess including sensitivity, speed, and such. These are important characteristics of the instrument, and should be reviewed. However, the real questions that should be asked about any new instrument should look beyond these benchmarks. Specifically, the questions that often come to mind include:
- Will the new instrument improve current experimental workflows?
- Will the new instrument enable new and novel experimental questions?
- Will the new instrument help improve the reproducibility of experiments?
Evaluating the instrument in the context of these questions will help determine if acquiring the instrument will expand the capabilities for the local research community. In the case of the ZE5 Cell Analyzer, it is clear that with the advancements that have been made by the Propel and Bio-Rad teams, this instrument offers significant expansion of capacity, resulting in improved reproducibility of the data.
Several features of the ZE5 stand out as prime examples of why this new instrument is a must-have for the research lab.
- Improve Reproducibility — A “Flying Collar Wash Station’” on the ZE5 is designed to wash the sample probe between samples to reduce carryover. For years, researchers have had to manually wash the SIP between samples, to help reduce sample-to-sample carryover. Automating this feature is a huge benefit of the ZE5.By automating the process of cleaning the SIP, carryover is reduced. This in turn reduces one source of data variation. This is even more critical when considering rare event analysis, where sample carryover can potentially skew the data. The data below shows how efficient this system is.
Figure 1: Carryover between samples on ZE5: (A) Lysed whole blood was run on the ZE5 in high-throughput (HT) mode. After each sample, the system carried out an automatic wash cycle of 0.25 sec. outside and 1.75 sec. inside the SIP. A clean tube of water was run immediately after the wash to evaluate carryover. (B) The resulting carryover data showing an average carryover of 0.046% (+/- 0.023%).
- Five-laser, 27 fluorescent parameter — More lasers and detectors is an excellent feature and offers improvement for standard assays, enables new assays, and can be used to improve reproducibility of experiments. More detectors allow for a deeper characteristic of a given population. In the case of a hard-to-obtain sample, more detectors allows the researcher to have a larger breadth of characterization, so that the critical data can be obtained without having to split the sample, thus reducing the sensitivity of measurement.With a large number of detectors, the ZE5 can also enable improved labeling of cells by allowing the researcher to ‘“barcode” their samples. In fluorescent barcoding experiments, each sample is labeled with a combination of 2 or more fluorochromes at one of several concentrations. For example, if one uses 2 different fluorochromes, with 3 different intensities (low, medium, and high), it is possible to mix 6 different samples together. A 3 by 3 barcoding results in 9 samples.All the samples are mixed together before they are labeled with the antibody mix at the same time, under the exact same conditions. This improves the staining, and thus the reproducibility of your data, and with the added speed the ZE5 has for sample acquisition (see below), barcoded samples can be read in the same time as a single sample on a slower instrument.If you are interested, you can read about fluorescent barcoding in these papers by Krutzik and Nolan, and Krutzik et al.
- Superfast electronics — The fluidics of the ZE5 can deliver a stable flow rate up to 2.5 μl/second. However, without matching fast processing electronics, the speed (and sample) would be wasted with increased coincident events and a high abort rate. The ZE5 delivers in the speed category, with very fast electronic and a cell laser transit time that is 3x as fast as other systems on the market.
Figure 2: Stability of Signal at high acquisition rate: Beads were acquired at increasing events per second, and singlet beads were gated using pulse geometry gating. The %CV of two parameters (Side Scatter and FITC) were plotted over a range from approximately 4,000 eps to 129,000 eps. The mean and standard deviation of the CVs over this range are shown below.
As can be seen, the electronics are stable, with a tight CV shown through a wide speed range. So, in addition to the barcoding discussed above, the fast electronics and stable flow rates are enabling for rare event analysis. Imagine trying to measure a cell that is found at a frequency of 1 in 105 cells. With rare events like this, the statistics are governed by Poisson distributions, rather than the more familiar Gaussian distributions. In Poisson statistics, it is the number of positive events that is important, not the total number of events.
Figure 3: Time to collect 400 positive events. The time to collect 400 events of a rare population (1 in 105 cells) is plotted versus the speed of acquisition (in events per second).
As this figure shows, to collect 40 million events with a typical flow cytometer is going to take 150 minutes (2.5 hours) for a single sample. However, with the speed of the ZE5, these rare event experiments become possible, as even at a moderately fast rate of 60,000 events per second, collection time drops to less than 12 minutes. Thus, the ZE5 enables researchers to study and characterize rarer cell populations in a reasonable time.
Some technological advances are incremental, while others are significant game-changing tools that offer the researcher the ability to significantly improve current assays while allowing for new and novel avenues of research to be performed. With speed, sensitivity, and capacity to spare, the ZE5 fits into the game-changing category. Reduced carryover, increased speed of acquisition, and a large number of parameters all open up new and novel assays, while improving the quality and reproducibility of ongoing ones.
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ABOUT TIM BUSHNELL, PHD
Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.More Written by Tim Bushnell, PhD