What is QuickCal Analysis Software ?
Q. I've heard of QuickCal¨ but I don't really know what it is or what it does.
A. QuickCal is an analysis programs which analyzes the calibration microbead data. This program is designed to calibrate the fluorescence detectors and to monitor the performance of your flow cytometer, as well as determine the MESF or ABC values of unknown samples against the calibration plots.
Q. Why should I calibrate the fluorescence detectors of my flow cytometer ?
A. Fluorescence intensity (FI) is the most fundamental way of distinguishing "positive" cells from "negative" cells. QuickCal provides an objective means, through calibration, of monitoring the reproducibility and stability of the fluorescence intensity over time. FI calibration also allows you to monitor and compare reagents, preparations, and samples.
Q. How often should the fluorescence calibration be done?
A. Calibration should be performed at least once a day for routine quality assurance. When used in conjunction with QC Windows, calibration may be reduced to once a week. If the settings of the flow cytometer are changed, then calibration must be performed again at these new settings. Calibration may be performed several times throughout the day to monitor the stability of the instrument.
Q. What are the units of measure for fluorescence intensity?
A. QuickCal expresses FI in "Molecules of Equivalent Soluble Fluorochrome" (MESF) when provided with Quantum MESF kits. The MESF value indicates the equivalent number of free fluorochrome molecules in solution that have the same emission intensity as the microbead standard. The use of MESF units is independent of the instrument and allows you to obtain consistent values and to avoid fluorescence measurement problems such as those created by quenching. Also, by expressing results in MESF units, you may validly compare data from different instruments. QuickCal also expresses FI in Antibody Binding capacity (ABC) when provided with Quantum Simply Cellular.
Q. How were the calibration values obtained?
A. The MESF calibrated FI values of each microbead population are directly traceable to spectrofluorometric quantitation against solutions of laser-grade fluorochromes. The ABC calibrated FI values of the microbeads are determined spectrofluorometric quantitation by solution depletion of the conjugated antibodies.
Q. You mentioned a reference blank. What purpose does it serve?
A. The reference blank is used to determine the fluorescence threshold (i.e., the noise level) of the instrument. This value, measured in MESF or ABC units against the calibration curve, indicates the noise level below which a true fluorescence signal can not be discerned. It is important to determine the noise level to insure that it will not interfere with the assay.
Q. What happens if the reference blank is off scale at the instrument settings for my assay?
A. With the reference blank off scale, a quantitative determination of noise level will not be possible; however, if the unstained cells are on scale and have a higher level of FI than the reference blank, then you can assume that the noise level will not interfere with your assay.
Q. How long does all this analysis take?
A. The entire QuickCal analysis takes less than five minutes.
Q. Is QuickCal available for all flow cytometers?
A. Yes. QuickCal will perform with all the modern flow cytometers including: Becton Dickinson's FACScan, FACStar, FACSort, FACS Calibur and FACS Vantage; Coulter's Profile I & II, EPICS C EPICS Elite and EPICS XL; as well as the Ortho CytoronAbsolute.
Q. What computer systems are compatible with QuickCal¨?
A. The QuickCal that is available for the Becton Dickinson instruments is designed to operate on the Macintosh computer of the instrument. The QuickCal available for the Coulter and Ortho instruments is written for Windows and to work with their respective work stations. A Windows and Macintosch versions of QuickCal are provided with each Quantum Kit.
Q. Is the software easy to install?
A. Yes. Just insert the disk into the floppy drive and copy the serial numbered folder into your hard drive.
Q. Does QuickCal keep track of the instrument performance data?
A. Yes. The data in the performance report that is printed out with each run is automatically logged into a database (quantum.dbf). The database accumulates perfromance parqameters throughout the year. QuickCal creates Levy-Jennings plots for six different parameters. QuickCal makes quality control less complicated and helps to eliminate clerical errors since the data is stored automatically. The QuickCal reports and Levy-Jennings plots can be printed out at any time.
Q. Does QuickCal¨ tell me if my instrument is performing properly?
A. Yes. QuickCal¨ presents the three most important parameters to check for instrument performance: linearity (Average Residual %); noise level (detection threshold); dynamic range (number of decades). Acceptable values for each of these parameters is somewhat dependent on the make and model of your flow cytometer, but as a general guideline: linearity should have an Average Residual of <5% and the MESF or ABC value of the noise level should be below 1000, or the intensity of your unstained sample. The number of decades is specified for specific instruments.
Q. How do I get an overall picture of the performance of my instrument over time?
A. Reviewing the Levy-Jennings plots provides a convenient and comprehensive method of determining the instrument's performance over time. The best understanding of how the instrument is working across its dynamic range can be gained by monitoring combinations of parameters.
Q. How do I calculate the fluorescence intensity for an unknown sample with QuickCal¨?
A. The MESF or ABC value for an unknown can be calculated using the regression equation. The QuickCal program allows the operator to obtain the FI values by simply entering the peak channel number of the unknown into the Sampler input form, then QuickCal calculates the corresponding MESF or ABC value.
Q. How long does the QuickCal System last?
A. In the B size there are enough Quantum Microbeads to last for 100 runs, equivalent to daily use for three months. The QuickCal Program is designed to expire in six months by not accepting new calibration data.
Q. Will variations between lots of QuickCal¨ Microbeads affect the evaluation of my flow cytometer's performance?
A. No. Although the Microbeads in each new QuickCal¨ System come with new FI values, these values are pre-programmed into the software so that the calibration line for the instrument for a particular setting will be constant.
Q. Do I have to start a new database every time I get a new Quantum/QuickCal kit?
A. Yes. However, the database is in a dBaseIII format, which allows the operator to combine the quantum.dbf files in a program such as Excel then read them with the QuickCal program.
Q. Why does the QuickCal¨ Program expire?
A. The lot numbers and the FI values of QuickCal¨ Microbeads are built into the program and, therefore, the software must expire with the microbeads.
Q. Are there any advantages for me in having the QuickCal¨ Program expire?
A. Yes. This design eliminates the need to enter microbead FI values every time you run the program, saving time and avoiding clerical errors. In addition, since new software comes with each new QuickCal¨ System, you will automatically receive the latest version of the Program, at no additional cost.
Q. Ordering individual Systems every half year can be "inconvenient". Is there any way to order a year's supply?
A. Yes! You may order a Quatum Subscription! The subscription consists of a size C Quantum kit and QuickCal software. This insures that you are not left with an expired System without having a new one on hand. Also, ordering by subscription provides you with significant savings.
Q. What additional information will I receive with my QuickCal order?
A. Every QuickCal program comes with a product insert which provides more detailed information on the theory, installation, application, and basic trouble-shooting of the System.
Q. How do I get more information about QuickCal¨?
A. You may obtain more information, request a catalog and/or place an order by calling us at (800) 227-8143.
