facscount user manual

facscount user manual
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facscount user manual

Sign in Forgot Password. My Bench Close Sign In Not A Member. Sign Up Join MedWrench OK name type Receive Summary Emails. Today clinicians in programs throughout Africa, Asia, Eastern Europe, and Latin America continue to rely on the system for cost-effective, simple, and dependable CD4 testing. FORUMS View All (5) Ask a New Question 0 Replies -juliet meme 6 years ago 6 years ago annual maintenance of bd facs count Reply 0 Replies -BKONE 8 years ago 8 years ago Depannage I wish I had the safety data sheets on the reagents used in this equipment. The instrument operates from a standard electrical outlet, requiring no hardware adjustments or calibration. Only minimal training is needed to set up the instrument and to run samples. Simple prompts on the display guide operators through testing, from input of patient information to final results. The software automatically identifies the lymphocyte populations of interest and summarizes patients’ results in a printed sample report. Quality controls in the software ensure that reported results are accurate by detecting and flagging error conditions and suppressing results when control limits are exceeded. Results are objective, requiring no interpretation or subjective analysis by the operators. BD FACSCount fluorescent reference beads are included in the reagent tube, and ensure accurate enumeration of lymphocytes of interest with no operator intervention. The control run generates a detailed analysis in a printed report summarizing system performance. The result of the last control run is reported on subsequent sample printouts to help determine linearity and pipetting accuracy. Since absolute counts are calculated from directly measured cell counts and reference bead counts on the BD FACSCount system, no additional analysis on a hematology analyzer is necessary.Sample preparation follows an easy three-step process. First, blood is added to a tube that contains pre-measured reagents.

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Then the sample is vortexed and incubated. In the second step, a fixative is added to the tube, and then it is vortexed and incubated. In step three, the sample is vortexed and run on the instrument.Prepare controls and patient samples by adding blood, then fixative solution, to the reagent tubes. By continuing to browse the site you are agreeing to our use of cookies. Please review our Privacy Policy for more details. All Rights Reserved. Toll Free Tel Fax Canada Toll Free (888) Tel (905) Fax (888) Mexico Toll Free Tel (52) Fax (52) 2 2008, Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, in any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written permission from BD Biosciences. The information in this guide is subject to change without notice. BD Biosciences reserves the right to change its products and services at any time to incorporate the latest technological developments. Although this guide has been prepared with every precaution to ensure accuracy, BD Biosciences assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information. BD Biosciences welcomes customer input on corrections and suggestions for improvement. BD FACSCount software 2008, Becton, Dickinson and Company. This software is the property of Becton, Dickinson and Company. Each sale of a stored unit of this software grants the purchaser a nontransferable, nonexclusive, personal license. This software may not be duplicated, reproduced, or copied in any form or by any means whatsoever, except as otherwise permitted by law. BD, BD logo, BD FACS, BD FACSCount, BD FACSFlow, and BD Vacutainer are trademarks or registered trademarks of Becton, Dickinson and Company.

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Vortex-Genie is a registered trademark of Scientific Industries, Inc. Maxi-Mix is a registered trademark of Thermo Fisher Scientific, Inc. Proline is a registered trademark of Biohit Oyi. All other company and product names might be trademarks of the respective companies with which they are associated. Patents The BD FACSCount flow cytometer is covered by one or more of the following US patents and foreign equivalents: 4,989,977 and 5,395,588. Regulatory Information For In Vitro Diagnostic use. Class I (1) laser product FCC Information WARNING Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment. NOTICE This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to Part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his or her own expense. Shielded cables must be used with this unit to ensure compliance with the Class A FCC limits. This Class A digital apparatus meets all requirements of the Canadian Interference-Causing Equipment Regulations. Once you become familiar with routine operation and need only a quick reminder of the main steps, use the quick reference guide or poster provided with this user s guide. Conventions The following tables list conventions in this guide. Table 1 lists the symbols that are used in this user s guide or on safety labels to alert you to a potential hazard. Text and keypad conventions are shown in Table 2.

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Table 1 Hazard symbols a Symbol Meaning CAUTION hazard or unsafe practice that could result in material damage, data loss, minor or severe injury, or death electrical danger laser radiation biological risk a. Although these symbols appear in color on the instrument, they are in black and white throughout this user s guide; their meaning remains unchanged.See Chapter 9, Troubleshooting. If additional assistance is required, contact your local BD Biosciences technical support representative or supplier. When contacting BD Biosciences, have the following information available: Product name, part number, and serial number Any error messages Details of recent system performance x BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents 11 1 Introduction The following topics are covered in this chapter: System Overview on page 12 Equipment and Materials Required on page 13 System Components on page 17 11 12 System Overview The BD FACSCount system, for use with BD FACSCount CD4 reagents, is an automated instrument and reagent system designed specifically for enumerating the absolute cell counts of CD4 T lymphocytes and the percentage of lymphocytes that are CD4 T lymphocytes in unlysed whole blood (CD4 counts and CD4 percentages). Figure 1-1 BD FACSCount system workstation coring station electronic pipette BD FACSCount instrument Principle of Operation Simply follow the whole blood staining procedure in Chapter 5 to prepare samples. Then run the samples on the BD FACSCount instrument. A built-in screen displays instructions for operation. The procedure requires minimal sample handling. When whole blood is added to the reagents, fluorochrome-labeled antibodies in the reagents bind specifically to lymphocyte surface antigens, and a fluorescent nuclear dye binds to the nucleated blood cells. After a fixative solution is added to the reagent tubes, the sample is run on the instrument.

During sample acquisition, the cells come in contact with the laser light, which causes the fluorochrome-labeled cells and fluorescently dyed cells to fluoresce. This fluorescent light provides the information necessary for the instrument to identify and count the lymphocytes and CD4 lymphocytes. 12 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents 13 In addition, the reagent tubes also contain a known number of fluorescent reference beads. A precise volume of whole blood is stained directly in the reagent tube. Results include CD4 counts and CD4 percentages, and are printed immediately after samples are run. Intended Use BD FACSCount CD4 reagents are used to enumerate the absolute counts of CD4 T lymphocytes and the percentage of lymphocytes that are CD4 T lymphocytes in unlysed whole blood (CD4 counts and CD4 percentages). The reagents are intended for in vitro diagnostic use on a BD FACSCount instrument. Equipment and Materials Required The following materials are used with the BD FACSCount system. For service and ordering information, see Appendix A. Materials Provided These materials are necessary to prepare and run samples and come with the instrument (see Figure 1-1 on page 12). For operating instructions, see Chapter 4. Workstation: holds blood specimens and operating supplies during sample and control preparation See Workstation on page 21 for details. Chapter 1: Introduction 13 14 Supplies Provided for Initial Use The following materials (Figure 1-2) are initially provided. Additional supplies must be purchased separately as needed. BD FACSCount CD4 reagents: each kit provides enough CD4 reagent, caps, and fixative solution for 50 tests See page 20 for more information. BD FACSCount control kit: offers four bead concentrations (Zero, Low, Medium, and High) NOTE The kit contains a Zero control that is not required for the CD4 assay. Do not use it. The kit provides sufficient controls for 25 control runs.

See page 21 for more information. For recommended brands, see Appendix A. Vortex: mixes samples For recommended brands, see Appendix A. Chlorine bleach (or BD FACS Clean solution): cleans the instrument NOTE Bleach contains 5 sodium hypochlorite. Distilled water (or BD FACSRinse solution): rinses the instrument before shutdown Deionized water or water purified by reverse osmosis can be substituted. Biosafety materials: include gloves worn when handling blood or biohazardous waste materials, and appropriate biohazard containers used for biohazardous waste materials Sheath fluid (saline solution): flows through the fluidics system For recommended sheath fluid, see page 128. Barcode Reader An optional barcode reader can be purchased from BD. See page 126 for purchasing information. Use the barcode reader to scan barcodes for reading the following: Patient accession numbers Reagent lot codes Reference bead counts Control lot codes Control bead counts 16 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents 17 System Components Before attempting to operate the BD FACSCount instrument, take a few minutes to review the system and to familiarize yourself with the locations and functions of the various instrument components. Instrument The BD FACSCount instrument is a compact cell counter with a built-in computer. Reagent tubes are introduced to the instrument via the sample holder that lifts the tubes to the sample injection probe. The sheath tank and waste tank, which are equipped with liquid level detectors to indicate empty and full conditions, are easily accessible through a hinged door at the front of the instrument.

Figure 1-4 BD FACSCount instrument printer front panel optics door fluidics compartment door sample holder Printer: prints control results, sample results, and any error messages Optics door: opens to a second, smaller door that allows access to the flow cell Chapter 1: Introduction 17 18 Sample holder: contains the sample and lifts tube to the sample injection probe Keep hands clear of the sample holder during instrument operation, except when a screen message prompts you to change tubes. Fluidics compartment door: opens to allow easy access to the sheath tank, waste tank, and sheath filter Sample injection probe (not visible): introduces sample to the flow cell via a stainless steel probe A laser beam intersects the sample stream within a flow cell. This flow cell is accessible through either of two hinged doors located to the right of the fluidics compartment. The screen (Figure 1-5) displays control and sample results, prompts, and messages that assist you with operation or inform you of errors. Results print automatically on thermal paper after samples are run. Figure 1-5 also shows the location of the parts of the front panel. Floppy drive: contains the BD FACSCount software disk that starts and runs the instrument Floppy disk release button: ejects the BD FACSCount software disk from the drive Display screen: displays prompts and messages for operation, control and sample run results, instrument status, and error messages Function keys: select control functions displayed on the screen The names and functions of these keys change depending on the screen displayed. This key is used to clear air bubbles or contaminants from the flow cell. Chapter 1: Introduction 19 20 BD FACSCount CD4 Software The floppy disk contains the BD FACSCount software required to start up and run the instrument.

The disk also stores the last entered reagent lot ID and control bead lot ID information, control run results, the last values entered in the Setup screen, the number of tubes run since the last daily clean, the date of the last long clean run, and the Results file. During operation, the software monitors the sheath fluid supply, waste level, and laser power. NOTE Multiple software disks are provided. Be sure to use the disk that contains the most recent control data. Use the following five software screens to operate the instrument. Sample: to run patient samples. See page 70. Control: to run controls. See page 60. Setup: to enter laboratory information, set the time and date, and select report and results options. See page 38. Utility: to prime, clean, and shut down the instrument. See page 36 and page 79. Restart: to restart the software. See page 32. BD FACSCount CD4 Reagents The following are included with BD FACSCount reagents. CD4 reagents (blue top), sufficient for 50 tests, conveniently packaged in a resealable foil bag Each CD4 tube fits into the workstation and the instrument sample holder. A tab allows you to label the tube with the appropriate patient accession number or ID. The CD4 tube is used for enumerating CD4 counts and CD4 percentages for each patient sample. For reagent specifications, see page 133 in Appendix B. 20 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents 21 BD FACSCount caps: 65 reagent tube caps used to prevent spillage of patient samples and controls while vortexing, during incubation, and before and after running samples on the instrument Keep reagent tubes capped except when pipetting or running tubes on the instrument. Fixative solution: one 5-mL vial of 5 formaldehyde in phosphate-buffered saline (PBS) BD FACSCount Controls The BD FACSCount control kit includes four levels of control beads: Zero, Low, Medium, and High.

BD FACSCount CD4 software for use with BD FACSCount CD4 reagents requires only three bead concentrations (Low, Medium, and High) to set up the instrument and check linearity. The Zero control bead concentration is not required for the CD4 assay, so do not use it. Each tube in the control kit provides enough beads for five control runs, a total of 25 runs per control kit. The beads are packaged in tubes similar to the reagent tubes, but with color-coded tops. The four bead concentrations are contained in two tube pairs. Figure 1-6 on page 22 shows how the various parts of the workstation are used. The BD FACSCount pipette comes with a charging stand, power supply adaptor, and plug kit. For information on operating the pipette, see Chapter BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents 23 2 Installing the System The following topics are covered in this chapter: Installing the Hardware on page 24 Installing the Pipette Charger Stand on page 25 Installing the Software on page 27 23 24 Installing the Hardware Your BD representative will install your BD FACSCount instrument. However, you should familiarize yourself with the power cord connection. Before connecting the power cord, read the BD FACSCount System Safety and Limitations booklet. Connect the AC power cord to the back of the instrument (Figure 2-1). Plug the other end of the power cord into the AC wall outlet. Position the instrument at least 6 inches from the back wall to allow adequate ventilation. Make sure there is sufficient room on the side of the instrument for direct access to the power cord. If you purchased the optional barcode reader, connect the cord to the barcode port at the back of the instrument (Figure 2-1). Follow these steps to install the charger stand. NOTE Make sure that the plug is compatible with your electrical outlet. 1 Connect the power supply adaptor to the back of the stand.

2 Connect the plug that is compatible with the electric outlet to the power supply adaptor. 3 Plug into the electrical outlet. The green light on the front of the charger stand indicates that it is operating. Charging the Pipette The pipette must be turned on while it is being charged. The pipette must be in the upper-most position of the charging head to ensure proper magnetic locking and optimal charging. Chapter 2: Installing the System 25 26 3 Keep the pipette in the stand for at least 10 hours to fully charge the battery the first time. When not in use, keep the pipette in the stand, in the ON position. If the pipette loses all charge, the display appears blank. To reactivate the battery, place the pipette into the charger stand until an arrow and an E appear in the display. A minimum of 10 hours is required to fully charge a dead battery. 4 Press the Start button. The pipette moves the piston to its home position, ready for operation.See Figure 2-2 on page BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents 27 Figure 2-2 Removing the pipette For more information on operating the pipette, see page 45. Installing the Software The BD FACSCount CD4 software disk stores the necessary information to run BD FACSCount CD4 reagents on the BD FACSCount instrument. 1 Insert the BD FACSCount CD4 software disk. NOTE The software disk also stores both control run information and the last values entered in the Setup screen. Multiple software disks are provided. The arrow at the front of the disk should be on the bottom left when inserting the disk. Leave the disk in the drive, even when the instrument is turned off. NOTE The disk should slide easily into the drive. If you encounter resistance when inserting it, check the orientation to see if you are inserting it backward. Chapter 2: Installing the System 27 28 2 Turn on the power to the BD FACSCount instrument. After approximately 1 minute, the copyright message briefly appears.

BD FACSCount CD4 Software Version 1.0, Jan 2008 BD Biosciences, Immunocytometry Systems San Jose, CA Copyright 2007 NOTE The first time the instrument is run, a message on the screen notifies you that a control file cannot be found. This is a reminder that you must run controls (see Figure 2-3). Figure 2-3 Error screen, no control file found Warning: No previous record of control run found Would you like to run controls now. The Control Run screen is displayed. After a 15-minute countdown, the BD FACSCount screen is displayed. If, during instrument troubleshooting, you are instructed to remove the disk, or if you need to remove the disk to back up the Results file, press the release button to eject it.Data can be lost or damaged. Chapter 2: Installing the System 29 30 THIS PAGE INTENTIONALLY LEFT BLANK 31 3 Getting Started The following topics are covered in this chapter: Starting Up the Instrument on page 32 Preparing the Fluidics on page 33 Entering Laboratory Information on page 38 Changing the Date and Time on page 40 Selecting Reports on page 41 31 32 Starting Up the Instrument Be sure that the BD FACSCount software disk is in the floppy drive. Then, turn on the power to the BD FACSCount instrument and allow the system to start up. After approximately one minute, the copyright message briefly appears, followed by the warmup screen. After a 15-minute countdown, the BD FACSCount screen appears. BD FACSCount CD4 Software Version 1.0, Jan 2008 BD Biosciences, Immunocytometry Systems San Jose, CA Copyright 2007 Instrument warmup. Whenever the instrument is not in use, perform the shutdown procedure (see page 82). This keeps the sample injection probe moist and free from salt deposits until the next sample is run. Restarting the Software Restart the software when instructed to in troubleshooting or when recommended by a BD service representative. To restart the software, press Restart on the BD FACSCount screen. Keep the same software disk in the floppy drive.

NOTE Each time you restart, there is a 15-minute warmup period. 32 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents 33 Preparing the Fluidics Before running controls or samples, prepare the fluidics by first filling the sheath tank, then emptying the waste tank. Study Figure 3-1 to familiarize yourself with the locations of fluidics components, then follow the steps for each procedure. Figure 3-1 Sheath and waste tanks and connectors sheath filter input tubing connector plastic pinch clamp air vent tubing sheath sensor connector sheath tank waste sensor connector sheath tubing connector waste tubing connector waste tank sheath filter sheath filter output tubing connector Filling the Sheath Tank The sheath tank is above the waste tank inside the fluidics compartment. The tank should be filled before the start of the run but can also be filled during operation. 1 Open the fluidics compartment door to access the sheath and waste tanks. Push the top center of the door, then release to open. 2 Disconnect the sheath sensor connector (white wire) from the fluidics panel. Squeeze the white tabs at the sides of the connector and pull. Chapter 3: Getting Started 33 34 3 Disconnect the sheath tubing from the fluidics panel. Squeeze the metal clip on the white connector and pull the tubing from the fitting. 4 Remove the sheath tank from the instrument. 5 Fill the tank with sheath fluid. Fill to 1 inch (2.5 cm) from the top. 6 Recap the tank and replace it in the instrument. Make sure the cap is not wet. Replace if wet. See Consumables on page 127 for replacement caps. Make sure the cap is firmly tightened. Slide the tank in with the cap facing up and the tubing at the top. 7 Reconnect the sheath tubing and sheath detection probe connector. Snap the sheath tubing connector back into place by pushing firmly until you hear a click. Connect the sheath sensor connector by squeezing the tabs and pushing the connector into place.

8 Make sure the sheath filter is filled with sheath fluid. If air is present in the filter, unclamp the plastic pinch clamp located on the air vent tubing to allow the air to escape through the vent tubing. If necessary, tap the filter gently to dislodge any bubbles and force them into the air vent tubing. When all air is removed, secure the clamp. 34 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents 35 Emptying the Waste Tank The waste tank is below the sheath tank inside the fluidics compartment. Always empty the waste tank when filling the sheath tank. This prevents the waste from overflowing. Blood specimens can contain infectious agents that are hazardous to your health. Wear gloves when handling blood or any materials with which it comes in contact. Follow local, state, or country biohazard handling regulations when disposing of biohazardous waste material. 1 Disconnect the waste tubing from the fluidics panel. Squeeze the metal clip on the connector and pull. 2 Disconnect the waste sensor connector (red wire) from the fluidics panel. Squeeze the white tabs at the sides of the connector and pull. 3 Remove the waste tank from the instrument. Do not dispose of waste tank contents until at least 30 minutes after the completion of the last run. This helps inactivate any biohazardous materials before disposal. A spare waste tank is provided, allowing you to immediately continue with a run while waiting 30 minutes before emptying the full tank. 4 Empty the tank according to local, state, or country biohazard handling regulations. Carefully remove the cap from an overfilled and potentially pressurized tank. 5 Add 200 ml of undiluted bleach to the empty tank. Chapter 3: Getting Started 35 36 6 Recap the tank and replace it in the instrument. Slide the tank in with the cap facing up and the tubing at the top. Do not allow the sheath tubing to get pinched when the waste tank is installed.

Make sure the vent filter at the top of the tank is free of fluid. 7 Reconnect the waste tubing and waste sensor connectors. Snap the waste tubing connector back into place by pushing firmly until you hear a click. Connect the waste sensor connector by squeezing the tabs and pushing the connector into place. Priming the System Check the flow cell each day after you turn on the instrument, and prime the system to make sure there are no air bubbles. If you run the system while the sheath tank is empty, the sheath filter will run dry. Drain Put on tube of distilled water. Press Drain to start draining. NoDrain 3 Install a tube of distilled water (or BD FACSRinse solution). 4 Open both optics doors to view the fluid as it drains from the flow cell. The following message is displayed. Drain Press Stop to stop draining. The flow cell fills automatically. The following message is displayed, followed by the BD FACSCount screen. Drain Refilling flow cell. Please wait. Draining typically takes less than 1 minute to complete. Do not leave the instrument unattended while draining. The fluid pump will run dry. 7 Verify that the flow cell doesn t contain air bubbles. If bubbles are visible, repeat the drain and fill. 8 Close both optics doors. Entering Laboratory Information If this is your first time using the instrument, follow these steps to enter the operator and laboratory identification information. The current character is displayed in the Character to add field. Continue scrolling until you reach the next character to be added. Select up to three characters. Select up to 30 characters. You can also select the results you want to print with the Sample Results printout. To change the default settings, follow these steps. NOTE Control run results are not optional. NOTE The absolute CD4 field is Yes and cannot be changed. The CD4 absolute count always prints on the printout and is always displayed on the screen.

A Results file saves data from the latest 100 tube runs on the software disk. To create a Results file, change the default from No to Yes. See page 76 for more information. Answering Yes to the fields in the following table reports (or prints) the noted value. Preprogramming eliminates the need to make program adjustments using the keypad. Therefore, the keypad on the pipette is inoperable. The arrow on the pipette display indicates the direction of the fluid once you press the Start button. For example, when the arrow points up toward the top of the pipette, pressing the Start button fills the tip. When the arrow points down toward the tip, pressing the Start button dispenses liquid. Follow the instructions in the procedure that starts on page 45 when pipetting blood, control beads, and fixative solution into the reagent tubes. Follow the procedures outlined in Method to Ensure Pipetting Precision on page 46 and Method to Ensure Pipette Accuracy on page 48. If the BD FACSCount pipette is inoperable, you can use another pipette provided it is capable of operating in the reverse pipetting mode. Make sure that you: Use the reverse pipetting technique. Qualify your technique by following the procedure in Method to Ensure Pipetting Precision on page 46. Pipetting Procedure 1 Place a clean tip on the pipette. 2 Place the tip 2 to 4 mm into the liquid. 3 Press the pipette Start button. Quickly release the button after pressing, but keep the tip in the liquid, pausing 1 to 2 seconds to allow the correct volume of liquid to fill the tip. Touch the tip to the inside surface of the vessel to remove the excess liquid from the outside of the tip before dispensing into the reagent tube. Avoid leaving the pipette on its side with liquid in the tip, which might seep back into the mechanism. 4 Dispense the liquid by placing the tip against the wall of the reagent tube just above the liquid level and pressing the Start button.

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