The Importance Of Quality Control And Quality Assurance In Flow Cytometry (Part 4 Of 6)
Incorporating quality control as a part of the optimization process in your flow cytometry protocol is important. Take a step back and consider how to build quality control tracking into the experimental protocol. When researchers hear about quality control, they immediately shift their attention to those operating and maintaining the instrument, as if the whole weight of QC should fall on their shoulders.
It is true that core facilities work hard to provide high-quality instruments and monitor performance over time so that the researchers can enjoy uniformity in their experiments.
That, however, is just one level of QC. As the experimental results are critical for the researcher’s long-term goals therefore they should not be so laissez-faire with QC. Instead, they should jump in with gusto to not only understand what those managing the instruments are doing, but also to develop specific QC protocols for their experiment. This should ideally be done during the optimization phase.
1. Instrument Quality Control
Instrument quality control is designed to ensure that the instrument is performing consistently over time. It is extremely important to perform this each time prior to instrument use. The instrument vendor will have their own recommendations for the daily QC. These directions are useful as if there are issues you can communicate with the vendor as you are providing them with information they expect.
Generally this is run when the instrument is first turned on. In many cases, it is worth performing at least a quick clean before starting the QC protocols. If you are a user who has access outside the normal hours of the instrument, it might be worth learning the QC protocols for yourself so that you can perform this necessary step before starting with your samples. A little QC goes a long way to making sure the instrument is behaving properly.
Figure 1: Levey-Jennings plot of voltage over time
Another important thing to remember is that while performing QC, make sure to examine the data, not just the pass/fail for the day, but the trends over the past week or month. Looking at how the data changes over time provides valuable insight into the stability of the instrument.
One of the most common ways to do this is to use the Levey-Jennings plot. In this plot, the cumulative data is plotted over time, with the mean, and +/- 2 (sometimes 3) standard deviations are overlaid. The user can see if the data they generated falls inside or outside of these control points.
It is pivotal to establish rules as when to intervene based on the data. The most common rules are called the Westgard rules, which specify when a process is out of control. In these cases, the process must be stopped and the process corrected. In the case of an instrument, this could be as simple as cleaning the instrument, or as complex as requiring a service call due to a bad part.
2. Voltage Control
If you have gone through the trouble of finding the optimal voltage for your specific experiment, it is worth the time to ensure that you maintain that intensity throughout the experiment. There are a variety of ways to do this, but one of the easiest is to use a bead to track the fluorescent intensity over time. The advantage of a bead is its consistency and traceability. You can order a given lot and use it for a longer period of time.
Of course, this means you need to set up your own tracking sheets, so that when you sit down at the flow cytometer. This way when you start working with the instrument, you can pull this sheet up, and run your bead control. This will let you both monitor the voltages and adjust it to hit the target values. An example of such a sheet is shown below.
With this, you will also need to export the appropriate values. In this case since you’re focusing on the MFI of the bead, you would use tracking voltage change in your Levey-Jennings plot. Any time you see significant issues (a major change greater than some percentage you define), it’s worth checking with the operators of the system to see if anything may have changed – new laser, new flow cell or the like.
When you are coming to the end of the beadlot you are using, it’s time to order a new lot and perform an overlapping experiment. In this experiment, you run the old lot and the new lot at the same time, so that you can define the new target values, as shown below.
In this case, the old bead lot has a mean fluorescent intensity of ~30,000. The new beadlot has an MFI of ~49,000. Thus, in your QC data, you would make a note of this, as well as the date when you transitioned over to the new value.
3. Reference Control
The third control to evaluate during development is the reference control. The reference control is a known sample that will behave in a defined way in your panel. This could either be frozen PBMCs, a cell line, or a mixture of different cells – but something that you can standardize. Each time you run an experiment, you take this reference control out and take it through the same process as the experimental sample. In doing so, this provides you with a control for the process of staining. This assures the researcher that the process worked, and that nothing was missing. It also provides a good check point – again running this before you run your sample will show if there are issues, allowing you to pause and troubleshoot before putting your valuable sample in the instrument.
During the optimization process of panel development, determining the important quality control metrics that need to be tracked is essential. These values are going to help determine if the data you generate is consistent and reproducible, as well as alert you to trouble on the instrument before you begin running your precious samples. While QC may not be as exciting as the actual data generation process, it is exceedingly important to ensure the quality of your data.
To learn more about important control measures for your flow cytometry lab, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.
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