Determing PMT Linearity in flow cytomers using Sphero™ PMT
Posted: Tue 12 Jul 2016 11:50DETERMINING PMT LINEARITY IN FLOW CYTOMETERS USING THE SPHERO™ PMT QUALITY CONTROL EXCEL TEMPLATE
SPHERO™ - Spherotech, Inc.
Introduction
The fluorescence linearity of flow cytometers is affected by optical alignment, laser power, electronical offsets, and amplifier calibration(1). In addition, it is important to monitor and validate flow cytometers’ performance due to the nature of the information obtained during diagnostic testing. As a result, it is recommended that the linearity of the flow cytometer is determined on a monthly basis, after instrument repair, and after instrument relocation.
The SPHERO™ Calibration Particles and SPHERO™ PMT Quality Control Excel Template (PMT QC Template) are designed for linearity calibration and long term performance tracking of flow cytometers. They will help flow cytometer users verify the operation of their instruments. The PMT QC Template is a valuable tool for determining the linearity of log amplifies.
The information acquired from this template should be implemented into flow cytometer calibration documentation.
The user can determine a schedule for routine maintenance procedures and tolerance limits of linearity based on instrument trends or malfunctions using this template.
The SPHEROTM Rainbow Calibration Particles (RCPs) and Ultra Rainbow Calibration Particles (URCPs) contain a mixture of similar size particles with different fluorescence intensities. These products contain a mixture of fluorochrome compatible spectrally but not identical with the common fluorochromes used in fl ow cytometry such as FTIC, PE, PE-CY5, ECD and ACP. Each particle is assigned a Molecules of Equivalent value in multiple channels such as Fluorescein (MEFL), PE (MEPE), PE-CY5 (MECY), ECD (MEPTR), and APC (MEAP). In addition, they have very small coeffi cients of variation both in size and fluorescence(2). As a result, the linearity for each channel of the fl ow cytometer can be determined using SPHERO™ Calibration Particles, the assigned Molecules of Equivalent values, and the PMT QC Template. Refer to STN#9 for information regarding Molecules of Equivalent Fluorescence.
Results and Discussion
In the following example the SPHERO™ Rainbow Calibration Particles Cat. No. RCP-30-5 and PMT QC Template are used to determine the logarithmic amplifier linearity of a Dako CyanTM ADP. The following protocol is used to collect the data for the RCPs:
1. Set a live gate for the siglet population on the FSC vs SSC histogram to exclude aggregates. Since these beads are
much smaller than blood cells, the FSC gain has to be increase to place the beads on scale in the light scatter plot.
NOTE: The Relative Channel Number of the initial dot display screen may look cluttered due to the number of the populations and the aggregates. However, after setting a live gate on the FCS vs SSC, the dot display screen is cleaned.
2. Set PMT voltages: Input the instrument settings normally used for specimens in your laboratory. In most instances,
the number of peaks will correspond to the histograms as shown in the package insert.
3. Turn off compensation.
4. Collect the plots for your panel, for example: FSC vs. SSC, FL1-log, FL2-log, FL3-log, FL4-log, FL5-log, FL6-log,
FL7-log, FL8-log, FL9-log
5. Count a minimun of 5000 events inside the gate.
6. Record the peak value and channels of separation between adjacent peaks.
The calibration plots for the fluorescence channels MEPE, MECY, MEFL, and MEAP using Spherotech Cat. No.RCP-30-5 are shown in Figure 1:
]
These graphs were made by comparing the linear Relative Channel Number of each different fluorescent coated bead population against the MEF, i.e., MEFL, MEPE or MECY, and MEAP value. Originally, this data was collected in the 4 decade log scale in both 256 channel arithmetic/linear (Relative Channel Number) and Geometric (Mean Channel Number, i.e. Relative Brightness). These are the preferred scales for obtaining data when using the SPHERO™ PMT QC Template. However, the SPHERO™ PMT QC Template also provides tables to convert data from other log amplifi ers used in fl ow cytometers. For example, Figure 2 shows the SPHERO™ PMT QC Template Table #2 for converting 1024 Mean Channel Number to 256 Relative Channel Numbers for Beckman Coulter instruments.
BD FACS operators should use the SPHERO™ PMT QC Template Table #1 shown in Figure 3 to convert 1024 Relative Channel Number to the 256 Relative Channel Numbers, or the SPHERO™ PMT QC Template Table #3 show in Figure 3 to convert 104 Geometric Mean Channel Numbers to the 256 Relative Channel Numbers.
However, many of the new instrument provide data in a 5 decade log scale. Figure 5 shows the SPHERO™ PMT QC Template Table #4 for converting 105 Geometric Mean Channel Numbers to the 256 Relative Channel Numbers.
After converting the data into the Relative Channel Numbers these values were entered into the CH# Column on the PMT QC Template. An example of the PMT QC Template table for entering the Relative Channel Number for the MEFL channel is shown in Figure 6.
Another benefi t of the SPHERO™ PMT QC Template is that can calculate the MEF values of unknowns. The Cross Calibration Table of the PMT QC Template is used to determine the number of related fl uorophores for an unknown sample or other particles. Figure 7 shows an example of the Cross Calibration Table for the MEFL channel.
The MEF calculation for samples is accurate if the calibration and the evaluated of the unknown samples or particles is performed using the same instrument settings. To perform this operation, first the Relative Channel Number of the unknown is converted into a 256 Relative Channel Number. Then enter the converted Relative Channel Number into the Cross Calibration Table. This table will use the regression equation to solve for the MPE value.
Conclusion
The SPHERO™ PMT QC Template is used to standardize and monitor the linearity of a flow cytometer. A linear Calibration Graph from the SPHERO™ PMT QC Template should be obtained in all channels as shown in Figure 1. This will help identify any problems with the fl ow cytometer. Important data to note from the Calibration Graphs are the Average Residual Percentage and the Regression Coeffi cient. The Average Residual Percentage, should be less than 5%. It determines the average percent difference between the data points and the regression line. The Regression Coeffi cient shows the linearity of the PMT. The Regression Coeffi cient should be collect over time and graphed on Levy Jennings plots to determine the acceptable instrument operation. Please see STN-8 for more details on performance tracking of flow cytometers.
Due to the long-term stability of calibration particles with embedded fl uorochromes, the Calibration Graph, once generated, should not change on the same instrument. Any drastic change in the Average Residual Percentage or the Regression Coeffi cient indicates an instrumental problem. Even though signifi cant instrument-to-instrument variations in the Relative Channel Numbers are expected, the slope of the Calibration Graph should remain the same on similar instruments. The slope of the Calibration Graph is useful when normalizing different instrument within the same laboratory. See STN-9 for more information regarding normalization of different instruments.
The Cross Calibration Table of the SPHERO™ PMT QC Template also enables the users to assign the MEAP,
MEFL, MEPE and MEPCY of the unknown sample easily by using the fl ow cytometer.
References
(1) BD Biosciences. FACService. Vol 8, Jan 2002
(2) Spherotech. Sphero TechNote. STN-9 Rev D, August 2007
SPHERO™ - Spherotech, Inc.
Introduction
The fluorescence linearity of flow cytometers is affected by optical alignment, laser power, electronical offsets, and amplifier calibration(1). In addition, it is important to monitor and validate flow cytometers’ performance due to the nature of the information obtained during diagnostic testing. As a result, it is recommended that the linearity of the flow cytometer is determined on a monthly basis, after instrument repair, and after instrument relocation.
The SPHERO™ Calibration Particles and SPHERO™ PMT Quality Control Excel Template (PMT QC Template) are designed for linearity calibration and long term performance tracking of flow cytometers. They will help flow cytometer users verify the operation of their instruments. The PMT QC Template is a valuable tool for determining the linearity of log amplifies.
The information acquired from this template should be implemented into flow cytometer calibration documentation.
The user can determine a schedule for routine maintenance procedures and tolerance limits of linearity based on instrument trends or malfunctions using this template.
The SPHEROTM Rainbow Calibration Particles (RCPs) and Ultra Rainbow Calibration Particles (URCPs) contain a mixture of similar size particles with different fluorescence intensities. These products contain a mixture of fluorochrome compatible spectrally but not identical with the common fluorochromes used in fl ow cytometry such as FTIC, PE, PE-CY5, ECD and ACP. Each particle is assigned a Molecules of Equivalent value in multiple channels such as Fluorescein (MEFL), PE (MEPE), PE-CY5 (MECY), ECD (MEPTR), and APC (MEAP). In addition, they have very small coeffi cients of variation both in size and fluorescence(2). As a result, the linearity for each channel of the fl ow cytometer can be determined using SPHERO™ Calibration Particles, the assigned Molecules of Equivalent values, and the PMT QC Template. Refer to STN#9 for information regarding Molecules of Equivalent Fluorescence.
Results and Discussion
In the following example the SPHERO™ Rainbow Calibration Particles Cat. No. RCP-30-5 and PMT QC Template are used to determine the logarithmic amplifier linearity of a Dako CyanTM ADP. The following protocol is used to collect the data for the RCPs:
1. Set a live gate for the siglet population on the FSC vs SSC histogram to exclude aggregates. Since these beads are
much smaller than blood cells, the FSC gain has to be increase to place the beads on scale in the light scatter plot.
NOTE: The Relative Channel Number of the initial dot display screen may look cluttered due to the number of the populations and the aggregates. However, after setting a live gate on the FCS vs SSC, the dot display screen is cleaned.
2. Set PMT voltages: Input the instrument settings normally used for specimens in your laboratory. In most instances,
the number of peaks will correspond to the histograms as shown in the package insert.
3. Turn off compensation.
4. Collect the plots for your panel, for example: FSC vs. SSC, FL1-log, FL2-log, FL3-log, FL4-log, FL5-log, FL6-log,
FL7-log, FL8-log, FL9-log
5. Count a minimun of 5000 events inside the gate.
6. Record the peak value and channels of separation between adjacent peaks.
The calibration plots for the fluorescence channels MEPE, MECY, MEFL, and MEAP using Spherotech Cat. No.RCP-30-5 are shown in Figure 1:
These graphs were made by comparing the linear Relative Channel Number of each different fluorescent coated bead population against the MEF, i.e., MEFL, MEPE or MECY, and MEAP value. Originally, this data was collected in the 4 decade log scale in both 256 channel arithmetic/linear (Relative Channel Number) and Geometric (Mean Channel Number, i.e. Relative Brightness). These are the preferred scales for obtaining data when using the SPHERO™ PMT QC Template. However, the SPHERO™ PMT QC Template also provides tables to convert data from other log amplifi ers used in fl ow cytometers. For example, Figure 2 shows the SPHERO™ PMT QC Template Table #2 for converting 1024 Mean Channel Number to 256 Relative Channel Numbers for Beckman Coulter instruments.
BD FACS operators should use the SPHERO™ PMT QC Template Table #1 shown in Figure 3 to convert 1024 Relative Channel Number to the 256 Relative Channel Numbers, or the SPHERO™ PMT QC Template Table #3 show in Figure 3 to convert 104 Geometric Mean Channel Numbers to the 256 Relative Channel Numbers.
However, many of the new instrument provide data in a 5 decade log scale. Figure 5 shows the SPHERO™ PMT QC Template Table #4 for converting 105 Geometric Mean Channel Numbers to the 256 Relative Channel Numbers.
After converting the data into the Relative Channel Numbers these values were entered into the CH# Column on the PMT QC Template. An example of the PMT QC Template table for entering the Relative Channel Number for the MEFL channel is shown in Figure 6.
Another benefi t of the SPHERO™ PMT QC Template is that can calculate the MEF values of unknowns. The Cross Calibration Table of the PMT QC Template is used to determine the number of related fl uorophores for an unknown sample or other particles. Figure 7 shows an example of the Cross Calibration Table for the MEFL channel.
The MEF calculation for samples is accurate if the calibration and the evaluated of the unknown samples or particles is performed using the same instrument settings. To perform this operation, first the Relative Channel Number of the unknown is converted into a 256 Relative Channel Number. Then enter the converted Relative Channel Number into the Cross Calibration Table. This table will use the regression equation to solve for the MPE value.
Conclusion
The SPHERO™ PMT QC Template is used to standardize and monitor the linearity of a flow cytometer. A linear Calibration Graph from the SPHERO™ PMT QC Template should be obtained in all channels as shown in Figure 1. This will help identify any problems with the fl ow cytometer. Important data to note from the Calibration Graphs are the Average Residual Percentage and the Regression Coeffi cient. The Average Residual Percentage, should be less than 5%. It determines the average percent difference between the data points and the regression line. The Regression Coeffi cient shows the linearity of the PMT. The Regression Coeffi cient should be collect over time and graphed on Levy Jennings plots to determine the acceptable instrument operation. Please see STN-8 for more details on performance tracking of flow cytometers.
Due to the long-term stability of calibration particles with embedded fl uorochromes, the Calibration Graph, once generated, should not change on the same instrument. Any drastic change in the Average Residual Percentage or the Regression Coeffi cient indicates an instrumental problem. Even though signifi cant instrument-to-instrument variations in the Relative Channel Numbers are expected, the slope of the Calibration Graph should remain the same on similar instruments. The slope of the Calibration Graph is useful when normalizing different instrument within the same laboratory. See STN-9 for more information regarding normalization of different instruments.
The Cross Calibration Table of the SPHERO™ PMT QC Template also enables the users to assign the MEAP,
MEFL, MEPE and MEPCY of the unknown sample easily by using the fl ow cytometer.
References
(1) BD Biosciences. FACService. Vol 8, Jan 2002
(2) Spherotech. Sphero TechNote. STN-9 Rev D, August 2007