What is Unified Instrument Setup ?
Q. Setting up a flow cytometer to get consistent results can be confusing and time consuming. Is there an easy and reliable method available?
A. Yes. FCSC's standards kit, QC Windows, provides a simple, quick and unifed method for performing daily set up of your flow cytometer.
Q. What makes QC Windows different from other methods?
A. QC Windows, is a combination of specially designed microbead standards that is used in conjunction with your stained control cells to set the PMT voltages and compensation circuits, as well as to determine the noise level of the instrument relative to your particular assay.
Q. Which is contained in the QC Windows Kit?
A. The QC Windows kit contains a Table of Initial Target Channels, a bottle of Certified Blank microbeads and a bottle of QC3 reference standards.
Q. What are Certified Blank microbeads and what purpose do they serve?
A. Certified Blank microbeads are unlabeled standards guaranteed to have a lower fluorescence signal than the autofluorescence of unstained whole blood lysed human peripheral lymphocytes. The Certified Blank is used to determine and qualitatively evaluate the noise level of your flow cytometer. The noise level is the sum of the optical and electronic noise of your instrument. If the Certified Blank falls in a lower channel than the unstained cells, you are assured that noise will not interfere with your immunophenotyping assay.
Q. Are all blank microbeads the same?
A. No. Some blank microbeads have autofluorescence higher than unstained cells. Therefore, blank microbeads that are used to determine noise levels have to be certified.
Q. Does the Certified Blank have any other purpose?
A. Yes. It also approximates the pivot point for the compensation circuits. The pivot point is where zero fluorescence occurs and is the point around which the line of compensation rotates. The compensation line should be parallel to the axis to properly set the compensation for the entire intensity range, i.e., the fluorescence- of single-labeled cells should have the same fluorescence intensity in their secondary fluorescence channels as the unstained cells.
Q. I have noticed that when I adjust my instrument's compensation for a medium intensity cell, the brighter cells are under-compensated and the dimmer cells are over-compensated. Why does this happen?
A. This type of uneven compensation is usually caused by not having the pivot point (i.e., noise level) on scale, thus causing the compensation line not to be parallel with the axis.
Q. How is fluorescence compensation set using the QC Windows, method?
A. The fluorescence compensation circuits are set by gating a population of control cells labeled with antibodies and adjusting the compensation circuits so that the compensation lines are parallel with the axes. For example, FITC-labeled cells have the same fluorescence intensity in the PE channel as unstained cells. It is recommended that the control cells be representative of the samples and antibodies you will use for your assay.
Q. Are there other advantages in using the Certified Blank ?
A. Yes. By having the Certified Blanks on scale, stained and unstained cells will be away from the axes. Then it will be easier to see if a cell population is over-compensated.
Q. Other than setting compensation, do control cells have another purpose in the QC Windows methodology?
A. Yes. Including the stained control cells in your instrument set-up provides verification of sample preparation and reagent stability as an integral part of your daily Quality Control Program. If the QC3 microbeads fall in their Instrument Specific Target Channels, but the cells do not appear in their usual positions, then this may indicate a problem with the reagent stability and/or preparative method.
Q. You mentioned QC3 microbeads. What are they and how are they used?
A. The QC3 microbeads are reference standards which are double- or triple-labeled with FITC and PE or FITC, PE and PE-Cy5. They have the same spectral properties as cells labeled with antibodies conjugated to the same fluorochromes. The QC3 microbeads are used to set the Initial Target Channels in the various fluorescence detectors (e.g., FLl, FL2 and FL3) to establish consistent Windows of Analysis.
Q. What is a Window of Analysis?
A. A Window of Analysis is the range which is covered by the 256 or 1024 histogram channels of the various parameters of the flow cytometer. When using a two-dimensional histogram (dot plot), the data seem to appear in a window. The position of the Window of Analysis is controlled by the PMT or gain settings. Changing these settings does not affect the actual scatter and fluorescent signals, but instead just re-positions the window relative to the fluorescent signals.
Q. Why is it necessary to set and use Target Channels?
A. Target Channels are fixed reference positions in the Windows of Analysis. Placing the QC3 reference standard in the same Target Channels will ensure comparability of the Window of Analysis from day to day. Target Channels also allow the operator to determine the consistency of instrument performance by monitoring the instrument setting necessary to place the reference material (i.e., QC3) in the Target Channels.
Q. How do I establish the Window of Analysis with the QC3 microbeads?
A. The Initial Target Channels for QC3 in all fluorescence detectors are listed in the Table of Initial Target Channels for all instruments. With the compensation off, adjust the PMT voltages to place the QC3 reference standards in Initial Target Channels. Then, using labeled control cells, adjust the compensatoion circuits. Subsequently, when the instrument is set using the initial PMT and compensation settings, the QC3 microbeads should fall in their respective Instrument Specific Target Channels, validating the same Window of Analysis. Small variations in the Instrument Specific Target Channels (<1.5% of the Histogram scale) of QC3 reference standards is acceptable for most applications. The control cells, when properly compensated, should appear in the same previous positions in the Window of Analysis.
Q. If I use the QC Windows method will my "normal" instrument settings change?
A. Most likely both your PMT and compensation settings will be different from your usual settings if you use the QC Windows method, because you will be bringing the noise level (Certified Blank) on scale so that you can obtain uniform compensation. Compensation is very dependent on the PMT settings.
Q. Can QC Windows, help with comparison of data from different instruments?
A. Yes. Since the QC3 reference standards have the same spectral properties as FITC/PE/PE-Cy5 labeled cells, placing the QC3 microbeads in the same Target Channels on different instruments which have the same amplifier range (e.g., 4 decade log amplifiers) will set the same Windows of Analysis for all the instruments. This, in turn, will position cell populations in the same areas of the Windows of Analysis for all the instruments.
Q. What advantages does the QC Windows method have over current setup methods?
A. Current methods usually place unstained cells in the corner of the Window of Analysis, which results in non-uniform compensation across the intensity range. Placing the Certified Blank on scale allows correction for this non uniformity. In addition, many methods do not require a reference material which matches the spectra of labeled cells. Without such a matching standard (QC3), Windows of Analysis will not be comparable among different instruments.
Q. How do I get more information about QC Windows?
A. You may obtain more information, request a catalog and/or place an order by calling us at (800) 227-8143.
Q. Are there any reference materials which you could recommend, relating to the set up a flow cytometer?
A. Yes, you may consult the following sources:
1. Proposed Guideline: Clinical Applications of Flow Cytometry. Quality Assurance and Immunophenotyping of Peripheral Blood Lymphocytes. National Committee for Clinical Laboratory Standards. Document H42-P, 9(13), 1989.
2. Shapiro HM, Practical Flow Cytometry, 3rd Edition, John Wiley & Sons, Inc. New York 1995.
3. McCoy JP, Carey JL and Krause JR, Quality control in flow cytometry for diagnostic pathology, I. Cell surface phenotyping and general laboratory procedures. Am. J. Clin. Pathology 93(4) Suppl. 1, 1990.
4. Horan P and Loken MR, A practical guide to the use of flow systems, chapter in: Flow Cytometry Instrumentation and Data Analysis. MA Van Dilla, PN Dean, OD Laerum and MR Melamed, Eds. Academic Press, New York 1985.
5. Keren DF, Flow Cytometry in Clinical Diagnosis, ASCP Press, Chicago 1989.
6. Giorgi JV, Cheng HL, Margolick JB, et.al., Quality control in flow cytometry measurements of T-lymphocyte subsets: The multicenter AIDS cohort study experience. Clin. Immunol. Immunopathol. 55:173, 1990.
7. Parker JW, Adelberg B, Azen SP, et.al., Leukocyte immunophenotyping by flow cytometry in a multisite study: Standardization, quality control and normal values in the transfusion safety study. Clin Immunol. Immunopathol. 55:187, 1990.
8. Schwartz A, Monograph: Fluorescent Microbead Standards, Flow Cytometry Standards Corporation, Research Triangle Park, NC, 1988.
