Automated Software for the daily monitoring

of the performance of a flow cytometer

Clinical or research use of flow cytometry instrumentation requires that the cytometer be demonstrably proven to provide reproducible results. The QC Tracker software is a program from Phoenix Flow Systems which turns any personal computer into a Quality Control(QC) monitoring system for a flow cytometer. This QC program is designed to aid an operator in detecting and monitoring drift in the performance of a flow cytometer (or multiple cytometers). Stability in the QC data indicates that the data produced by the instrument are not the result of instrument artifact. Conversely, instability indicates the necessity for intervention by either the service personnel or the primary operator.

The QC Tracker software reads the information stored in the Flow Cytometry Standard (FCS)¹ 1.0/2.0 data files and Coulter Profile™ data files generated by a flow cytometer. The information is stored in a QC database so that it can be retrieved and graphed for comparison with previous information from the same flow cytometer. The following information is stored in the QC Tracker Database:

In addition, QC Tracker will calculate the mean channel and standard deviation of all of the databased information for each parameter of the first 10-30 (user selectable) cytometer data files that are entered into the QC Tracker database. Once the first 10-30 data files have been read into the QC database, the Means and standard deviations are calculated and locked until a service event or the particle lot number is changed. (If either of these two events is logged into the QC Tracker software, the standard deviations are recalculated and locked again after 10-30 new QC data files.)

Figure 1-Passing Daily QC Report

Figure 2-Failed QC Report-Note on the Failed QC Report, the parameters which failed are high lighted.

The QC Tracker software can also display and calculate Levi-Jennings graphs for any of the above-mentioned databased information. The user can query the database to generate Levi-Jennings graphs for 15, 30, 60, 90, 180 or 360-day time spans. See figure 3.

Figure 3-Levi-Jennings Report

In addition, the program includes a database for tracking and printing operator comments on changes made in the daily QC monitoring of the flow cytometer, i.e., the lot number of particles used for calibration, service calls performed on the flow cytometer or any other operator comments pertaining to the performance of each individual cytometer. See figure 4.

Figure 4-Comment Report

User comments can include items such as:

• Weekly Cleaning.
• Operator comments about current performance of the cytometer.
• Errors made in using the program.
• Who ran the daily QC particles.
• Event Comments.
• New lot number for particles.
• Service Calls.
• Changing the starting file number of a lot number.

The QC Tracker software can monitor multiple cytometers or types of alignment particles at one time. This means the software can monitor the particles used for alignment, such that the PMT voltages are held constant and the change in the mean channel of the particles is tracked. Or, the software can also monitor the particles used for setting fluorescence intensities such that the mean channel of the particles is held constant, and the PMT voltages are adjusted to keep the particles in their target channels. And, the software can also monitor chick red blood cells or a DNA standard.

Any particles or standard can be monitored by the QC Tracker software provided they contain only one peak, the peak generated is gaussian and the peak falls in the middle 80% of the histogram. Double humped peaks are unacceptable. The cytometer should be repaired or a different particle used before using the QC Tracker software. Phoenix Flow Systems recommends the Rainbow Particles from SpheroTech, catalogue #RFP-30-SA. Phone: (847)680-8922 or available from Phoenix Flow. (A free sample is included with the QC Tracker software.) An adequate quantity should be purchased that will last at least 6 months.

Once the flow cytometer is turned on and warmed up, the QC particles should be run every day to access the performance of the instrument. A protocol to acquire each type of QC particle data must be set up on the flow cytometer for each type of QC particle used with the following criteria:

• The protocol should consist of one ungated histogram for each parameter of the flow cytometer.
• The gain of the flow cytometer should be adjusted so the peak of the particle data does not fall within the first or last 10% of the histogram. Example: For a 256-channel histogram, the peak must be between channels 25 and 231.
• The QC particle data for each cytometer or type of particle must be stored in a unique directory. Do not store all particle data from multiple particle types or multiple cytometers in the same directory.
• The data should be stored as an ungated list mode file.
• The raw QC data file should consist of 10,000 events provided there is 100 events in the peak channel of the histogram. The QC Tracker software requires 100 events in the peak channel for valid statistical calculations and will give an error message to this effect when the file is read in if this is a problem.

Normally, two different types of particles should be run every day to check the performance of the flow cytometer: Alignment Particles and Fluorescence Standardization Particles. Alignment particles check the basic performance of the flow cytometer, i.e. are the PMT's, laser, electronics and fluidics functioning correctly. Fluorescence standardization particles have fluorescence intensities similar to cells and are used to check the performance of the flow cytometer at the normal operating conditions when analyzing a real sample. They are in a sense, a second level of QC for monitoring the performance of the flow cytometer.

When running alignment particles, the QC Tracker software will monitor the drift in the peak of the QC particles. Operate the amplifiers on the flow cytometer in linear mode. Make sure the alignment particles peak falls above the first and below the last 10% of the histogram. Do not adjust the amplifier and PMT gains from day to day. Make sure the amplifier and PMT gains are saved in the cytometer protocol and are the same values each time the QC particles are run. Store this QC data everyday and read it into the QC Tracker software to generate a daily QC report on the performance of the flow cytometer.

Adjust the amplifiers (log or linear) and PMT’s to the appropriate gains used for running immunofluorescence samples. Run the appropriate particles on the cytometer. The particles must have only one primary peak. Make sure the peak falls above the first and below the last 10% of the histogram. Record the mean channel of the particles in each parameter and keep this for handy reference. Save the QC particle data to its unique directory. The next day, noting the mean channels recorded from the previous day, run the QC particles. Adjust the gain of the PMTS of the flow cytometer so the particle peak comes up in the target channel means from the previous day. Store this data each day to the same QC data file directory (which is different from the alignment particle directory mentioned before). and read it into QC Tracker software to generate a daily QC report on the performance of the flow cytometer.