BD Multicolor CompBeads

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1 4/ IVD BD Multicolor CompBeads 100 compensation setups Catalog No BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company BD Becton, Dickinson and Company BD Biosciences 2350 Qume Drive San Jose, CA USA Benex Limited Pottery Road, Dun Laoghaire, Co. Dublin, Ireland Tel Fax BD Biosciences European Customer Support Tel Fax Becton Dickinson Pty Ltd, 4 Research Park Drive, Macquarie University Research Park, North Ryde NSW 2113, Australia Becton Dickinson Limited, 8 Pacific Rise, Mt. Wellington, Auckland, New Zealand bdbiosciences.com ClinicalApplications@bd.com 1. INTENDED USE BD Multicolor CompBeads are intended for in vitro diagnostic use when acquired with BD FACSDiva software on a BD FACSCanto II flow cytometer. CompBeads are used to optimize fluorescence compensation settings for multicolor flow cytometric analysis. 2. PRINCIPLES OF THE PROCEDURE BD Multicolor CompBeads (CompBeads) are composed of microparticles coated with anti-mouse Ig-Kappa (Ig κ) that bind kappa light chain-bearing immunoglobulin. Unstained CompBeads and CompBeads stained with the same fluorochrome-labeled mouse Ig κ antihuman antibodies used in the experiment are acquired using BD FACSDiva software to calculate a compensation matrix of spectral overlap values for each fluorochrome into each of its non-primary detectors. This compensation matrix is then used to correct spectral overlap for any combination of fluorochrome-labeled mouse Ig κ anti-human antibodies conjugated to the following dyes: FITC PE PerCP-Cy 5.5 * PE-Cy 7 APC * Cy is a trademark of GE Healthcare. This product is subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and is made and sold under license from GE Healthcare. This product is licensed for sale only for in vitro diagnostics. It is not licensed for any other use. If you require any additional license to use this product and do not have one, return this material, unopened, to BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, and any money paid for the material will be refunded. 1

2 APC-H7 tandem (APC-H7) BD Horizon V450 BD Horizon V500-C 3. REAGENTS Reagent provided CompBeads (Catalog No ) are polystyrene microparticles supplied in phosphate-buffered saline (PBS) containing bovine serum albumin (BSA) and 0.09% sodium azide. Precautions For In Vitro Diagnostic Use. Do not add antibody to the unstained control tube. Compensation might not be accurate when this product is used with reagents that have a median fluorescence intensity (MFI) >100,000. Storage and handling CompBeads are stable until the expiration date shown on the label when stored undiluted between 2 C and 8 C. Do not use after the expiration date. Do not freeze the beads or expose them to direct light during incubation with fluorochrome-conjugated antibody. Do not use the beads if you observe any change in appearance. Precipitation or discoloration indicates instability or deterioration. 4. INSTRUMENT CompBeads are for use on a BD FACSCanto II flow cytometer. The cytometer must be equipped with BD FACSDiva software to set compensation. 5. PROCEDURE Reagents or materials required but not provided Disposable 12 x 75-mm capped polystyrene test tubes Micropipettor with tips Vortex mixer Centrifuge Stain buffer consisting of PBS (for example, Dulbecco s modified, ph 7.2 ±0.2) with 0.2% BSA and 0.1% sodium azide, or equivalent Wash buffer consisting of PBS (for example, Dulbecco s modified, ph 7.2 ±0.2) with 0.1% sodium azide, or equivalent BD FACS 7-color setup beads (Catalog No ) BD FACSDiva CS&T IVD beads (Catalog No and ) BD FACSCanto II flow cytometer See the cytometer reference manual and instructions for use (IFU) for information. Preparation of stained CompBeads 1. Label a 12 x 75-mm capped polystyrene test tube for an unstained control and each fluorochromeconjugated mouse Ig κ antibody to be used. 2

3 2. Pipette 100 µl of stain buffer into each tube. 3. Vortex the vial of CompBeads thoroughly and then add 1 full drop (approximately 60 µl) of CompBeads to each tube. CAUTION Avoid dripping the beads down the side of the tube while adding them to each tube. This can lead to low bead concentration and could impact the results of the compensation matrix. CAUTION Do not add more than one drop of CompBeads to each tube because it could impact the results of the compensation matrix. 4. Vortex the tubes thoroughly. 5. Pipette the appropriate volume for one test (see the reagent vial label) of fluorochrome-conjugated mouse Ig κ antibody sufficient to stain 10 6 cells into the corresponding tube and vortex thoroughly. CAUTION Do not add antibody to the unstained control tube. 6. Repeat step 5 for the remaining antibodies. 7. Incubate for 15 to 30 minutes in the dark at room temperature. 8. Add 2 ml of wash buffer to each tube. 9. Centrifuge the tubes at 300g for 10 minutes. 10. Remove the supernatant. 11. Resuspend the bead pellet by adding 0.5 ml of wash buffer to each tube. 12. Vortex the tubes thoroughly. 13. Store the tubes at 2 C to 8 C in the dark until acquired. CAUTION If you are using PE-Cy7, V450, or V500-C to set compensation, acquire the samples immediately. For all other fluorochromes, we recommend acquiring the samples within 24 hours of staining. Cytometer setup 1. Open BD FACSDiva software and create an experiment. 2. Set the photomultiplier tube (PMT) voltages for the specimen type you will be running according to the fluorochrome you are using. See the reagent IFU for instructions. 3. Create compensation controls, including label-specific tubes, as needed. (Be sure to include an unstained control). See the BD FACSDiva Software Reference Manual for instructions. 4. Display the unstained control tube in the normal worksheet view. 5. Load the unstained control tube on the cytometer. 6. Adjust the FSC vs SSC voltages and the FSC threshold to minimize debris and place the singlet bead population on scale in the FSC-A vs SSC-A dot plot. 7. Adjust the P1 gate around the singlet bead population and ensure that the P1 gate contains only singlet beads. 3

4 SSC-A Record data for all of the tubes. 9. Select the normal worksheet for each recorded tube and adjust the P1 and P2 gates as needed. NOTE Examine the histogram to ensure that P2 tightly brackets the singlet bead peak. If the peak has a shoulder, do not include it inside the gate. Count Unstained Control P1 50 FSC-A FITC Stained Control 10 2 FITC-A Calculate compensation, then link and save the settings. CAUTION Compensation might not be accurate when this product is used with reagents that have an MFI >100,000. You are now ready to acquire stained samples. If you used BD FACSDiva v7.0 and BD FACSDiva CS&T IVD beads to set PMT voltages, see Acquisition on page 4. If you used another method to set PMT voltages, see the reagent IFU for instructions. P2 Acquisition 1. Create a new experiment to acquire samples. 2. Link to the compensation setup that was created in step 10 of Cytometer setup. 3. Right-click Cytometer Settings and select Unlink From (Name of Settings) 4. Right-click Cytometer Settings and apply lyse/wash (LW) application settings that were determined using BD FACSDiva CS&T IVD beads. When prompted, select Keep the compensation value. 5. Acquire stained samples. Flow cytometry Figure 1 displays representative data from analysis of normal peripheral blood. Data was acquired using a BD FACSCanto II flow cytometer and BD FACSDiva software. Instrument setup was performed using BD FACS 7-color setup beads and CompBeads. Laser excitation is at 488 nm and 635 nm. Figure 1 Stained cells after compensation correction using CompBeads CD7 PE CD8 APC-H CD3 FITC CD4 APC 10 5 Instrument quality control Tracking daily cytometer setup values provides useful indicators of cytometer reproducibility and performance. 4

5 6. PERFORMANCE CHARACTERISTICS Reproducibility Two operators performed two separate runs per day over a period of eight days on two BD FACSCanto II flow cytometers using two lots of CompBeads. For each run on every day, duplicate sets of control tubes for each lot of CompBeads were stained with each fluorochromeconjugated antibody by each operator, and were acquired manually. The compensation matrix was then calculated using BD FACSDiva software. Inter-assay precision was calculated for the spillover values as two separate components. The first component (run/ operator-to-run/operator, lot-to-lot, dayto-day reproducibility) is shown in Table 1. The second component (instrument-to-instrument) is shown in Table 2. Table 1 Reproducibility of spillover values (run/ operator-to-run/operator, lot-to-lot, day-to-day) Parameter DF a SD %CV PE %FITC PerCP-Cy5.5 %PE PE-Cy7 %PerCP-Cy PE %PE-Cy APC-H7 %PE-Cy APC-H7 %APC APC %APC-H V450 %V500-C V500-C %V FITC %V500-C a. DF = degrees of freedom Table 2 Reproducibility of spillover values (instrument-to-instrument) Parameter DF a SD %CV PE %FITC PerCP-Cy5.5 %PE PE-Cy7 %PerCP-Cy PE %PE-Cy APC-H7 %PE-Cy APC-H7 %APC APC %APC-H V450 %V500-C V500-C %V FITC %V500-C a. DF = degrees of freedom Repeatability Two operators performed two separate runs per day over a period of eight days on two BD FACSCanto II flow cytometers using two lots of CompBeads. For each run on every day, duplicate sets of control tubes for each lot of CompBeads were stained with each fluorochromeconjugated antibody by each operator, and were acquired manually. The compensation matrix was then calculated using BD FACSDiva software. The intraassay precision (tube-to-tube repeatability) was calculated for the spillover values. See Table 3. Table 3 Repeatability of spillover values Parameter DF a SD %CV PE %FITC PerCP-Cy5.5 %PE PE-Cy7 %PerCP-Cy PE %PE-Cy APC-H7 %PE-Cy

6 Table 3 Repeatability of spillover values Parameter DF a SD %CV APC-H7 %APC APC %APC-H V450 %V500-C V500-C %V FITC %V500-C a. DF = degrees of freedom Accuracy Whole blood samples from a total of 20 normal donors were tested. Two operators performed two separate runs per day on two BD FACSCanto II flow cytometers. In addition to the CompBeads samples, each operator also stained duplicate sets of control tubes from a separate normal donor whole blood sample with each fluorochrome conjugated antibody. The single-stained cells were manually acquired. The compensation matrix was then calculated using BD FACSDiva software. The spillover values obtained from every donor sample were paired with the spillover values obtained on the same cytometer from CompBeads stained by the same operator in the same run on the same day. The absolute mean bias for the spillover values between the stained CompBeads and cells was calculated. Each operator tested whole blood samples from two different normal donors per day for a total of ten donors over five days. See Table 4. Table 4 Accuracy of spillover values between stained BD Multicolor CompBeads and stained cells (absolute mean bias) Mean bias Parameter N a SD (%) PE %FITC PerCP-Cy5.5 %PE PE-Cy7 %PerCP-Cy PE %PE-Cy APC-H7 %PE-Cy APC-H7 %APC APC %APC-H V450 %V500-C V500-C %V FITC %V500-C a. N = number of samples 7. LIMITATIONS CompBeads are recommended for use with a BD FACSCanto II flow cytometer and BD FACSDiva software. The beads are not intended to optimize compensation settings for fluorochrome-labeled mouse Ig κ antihuman antibodies conjugated to dyes other than those listed in Principles of the Procedure on page 1. CompBeads do not perform as a fluorescence calibrator and should not be used for setting up a flow cytometer for quantitative fluorescence measurements. MFI values can vary between lots of CompBeads. 6

7 WARRANTY Unless otherwise indicated in any applicable BD general conditions of sale for non-us customers, the following warranty applies to the purchase of these products. THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND NONINFRINGEMENT. BD S SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT. TROUBLESHOOTING Problem Possible Cause Solution Compensation calculation is not successful Gates are not properly adjusted CompBeads or conjugated antibodies are expired Unstained control tube is contaminated with conjugated antibody Age of CompBeads in stained tubes is >24 hours Problems with the cytometer fluidics Volume of CompBeads added to stained tubes is incorrect Adjust the gates to include the appropriate populations. Recalculate the compensation. Prepare freshly stained tubes using beads and conjugated antibodies that have not expired. Re-run the compensation setup. Prepare a new unstained control tube. Re-run the Prepare freshly stained tubes and rerun the Check cytometer fluidics for bubbles or debris. Prepare freshly stained tubes and ensure that one drop of CompBeads is added to each tube. Re-run 7