Bd Facscount Manual

Flow cytometry is a powerful and flexible analytical tool used during all stages of drug development. While substantial effort is invested in development and validation of analytical methods, instrument validation is often neglected. Flow cytometers are evolving at a pace that surpasses the protracted timeline of drug discovery and development.

Therefore, it becomes fundamentally important to the success of the study to document the validated state of the flow cytometer and verify data integrity at the time of study conduct. It is important to bear in mind that validation strategies are critical components of the entire process involved in bringing new therapeutic options to the medical community; drugs which eventually manifest as successful new treatments for those individuals afflicted with disease. Formal industry guidance is provided through Good Laboratory Practices GLP, which require validation of all computerized systems and equipment used to support pre-clinical studies for regulatory submissions.

Key elements of instrument validation processes have been delineated through guidance documents published by regulatory agencies and industry working groups to support the rigorous compliance needs of GLP. However, most testing to support drug development is conducted in less strict regulatory environments. Such comprehensive validation efforts may not be appropriate for laboratories supporting early discovery or basic research, however, laboratories involved in regulated stages of development, such as pre-clinical and clinical phases, should consider these recommendations. This paper presents a consensus methodological approach that the authors have used successfully to ensure data integrity in flow cytometric studies conducted during drug development.

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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 Reagents13 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. The software automatically identifies lymphocyte populations and calculates the CD4 counts (cells/µl) by comparing cellular events to bead events. 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).

Coring station: opens the sealed reagent and control tubes to prepare them for use Electronic pipette: accurately delivers 50 µl of fluid This pipette is automated and preprogrammed. 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.

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. Figure 1-5 Front panel floppy disk release button floppy drive numeric keypad display screen function keys fluidics keys on/off switch On/off switch: powers the instrument on and off Numeric keypad: includes numeric, Enter, and Delete keys 18 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents19 - Numeric entry keys, labeled 0 to 9, are used to input values such as patient accession numbers and reagent and control lot codes and bead counts.

Enter confirms the information input from the keypad. Enter also moves the cursor from one field to the next. Delete removes the last character that was entered.

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. Fluidics keys: include Run, Stop, and Drain for selecting the fluidics modes These keys are active on each screen during specific times only. Run lifts the sample holder to the sample injection probe to introduce samples, controls, and system cleaning agents. Stop lowers the sample holder and stops all fluid flow. The instrument goes into a standby mode when this key is pressed or when the sample holder is down.

Drain drains all fluid from the flow cell. This key is used to clear air bubbles or contaminants from the flow cell. Chapter 1: Introduction 1920 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. Control: to run controls. Setup: to enter laboratory information, set the time and date, and select report and results options. Utility: to prime, clean, and shut down the instrument. See page 36 and page 79. Restart: to restart the software. 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 Reagents21 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. Pair one Zero (yellow top) 0 beads/µl Low (red top) approximately 50 beads/µl pair two Medium (blue top) approximately 250 beads/µl High (purple top) approximately 1,000 beads/µl Workstation The BD FACSCount workstation provides a place to hold blood specimens, reagent tubes, controls, fixative solution, caps, and cleaning tubes while you are preparing and running samples. Figure 1-6 on page 22 shows how the various parts of the workstation are used.

Chapter 1: Introduction 21. 22 Figure 1-6 BD FACSCount workstation cleaning tubes control tubes BD Vacutainer tubes cover caps fixative solution reagent tubes sample prep instructions Electronic Pipette The pipette used in sample preparation must be properly calibrated to ensure that it is dispensing precisely 50 µl of blood. The BD FACSCount pipette comes with a charging stand, power supply adaptor, and plug kit.

The pipette is preprogrammed to accurately deliver 50 µl of fluid in reverse pipetting mode. For information on operating the pipette, see Chapter BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents23 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 2324 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). Figure 2-1 Plugging the AC power cord into the instrument keyboard/barcode port 24 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents25 Installing the Pipette Charger Stand The BD FACSCount pipette kit contains the pipette, charging stand, power supply adaptor, and plug kit. 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. Make sure you use the power supply adaptor that came with the pipette. 1 Turn on the pipette by pushing the red on/off switch to the far left position. On/off switch 2 Place the pipette in the charging stand. 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 2526 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.

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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. Start button To remove the pipette for use, swing the lower part out and away from the charger stand. See Figure 2-2 on page BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents27 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. Be sure to use the disk that contains the most recent control data. 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 2728 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?

No Yes Press Yes to run controls. The Control Run screen is displayed. Press No to acknowledge this message and continue. After a 15-minute countdown, the BD FACSCount screen is displayed. BD FACSCount CD4 v1.0 MM/DD/YY HH:MM:SS Sample Control Setup Utility Restart 28 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents.

29 NOTE During normal operation, the disk should not be removed. 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. Release button Do not remove the disk from the drive while the drive light is on. Data can be lost or damaged.

Chapter 2: Installing the System 2930 THIS PAGE INTENTIONALLY LEFT BLANK31 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 3132 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. Remaining time: MM: SS BD FACSCount CD4 v1.0 MM/DD/YY HH:MM:SS Sample Control Setup Utility Restart You can leave the instrument on for the entire day. 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 Reagents33 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 3334 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 Reagents35 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 3536 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. 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. If the sheath filter runs dry or if you change the filter, repeat the priming procedure five times to ensure all air has been removed. 1 Press Utility on the BD FACSCount screen to display the Utility screen. Utility Clean Shutdown Status Main 36 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents37 2 Press Drain to access the following message. 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. Figure 3-2 Flow cell flow cell 5 Press Drain again. The following message is displayed.

Drain Press Stop to stop draining. Chapter 3: Getting Started 3738 6 When all fluid has drained from the flow cell, press Stop. The flow cell fills automatically. The following message is displayed, followed by the BD FACSCount screen. Drain Refilling flow cell. 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. Once this information has been entered, it remains until you change it or change the software disk.

1 Press Setup on the BD FACSCount screen to display the Setup screen. Setup Op ID: MM/DD/YY HH:MM:SS Lab ID: Set IDs Set Date Reports Main 2 Press Set IDs. 38 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents39 3 Use NxtChar and PrvChar to scroll through the following options. The alphabet (A through Z) A space Hyphen (-) Period (.) Slash (/) Underscore Numbers 0 through 9 You can also use the numeric keypad to enter numbers. The current character is displayed in the Character to add field.

Setup Op ID: MM/DD/YY HH:MM:SS Lab ID: Character to add 'A' PrvChar NxtChar Select Clear Confirm 4 When the character to be added appears, press Select. Continue scrolling until you reach the next character to be added. Select up to three characters.

If you make a mistake, press Delete to remove the last character entered. To remove old IDs or to remove all characters in the field, press Clear. 5 Press Enter to move to the Lab ID field.

Again, use NxtChar and PrvChar to scroll through the alphabet, pressing Select when the appropriate letter appears, or use the numeric keypad to enter numbers. Select up to 30 characters.

Press Confirm to save this information to the software disk and return to the Setup screen. Chapter 3: Getting Started 3940 Changing the Date and Time 1 Press SetDate on the Setup screen.

2 Enter the new date and the new time using the numeric keypad. Setup Op ID: PJT MM/DD/YY HH:MM:SS Lab ID: VA Medical Center Set date and time using keypad. Cancel Confirm Press Enter after inputting the value for each field, for example, month, day, year (MM/DD/YY). Press Enter after inputting the value for each field, for example, hour, minute, second (HH:MM:SS). To exit this screen without changing the date and time, press Cancel.

3 Press Confirm to save this information to the software disk and return to the Setup screen. 40 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents41 Selecting Reports The Reports screen allows you to select the number of copies of sample and control run reports you want to print. 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. All control results are reported and saved to the software disk.

1 Press Reports on the Setup screen to access the Reports screen. Reports Number of copies: 1 CD4 count: Yes CD4%: Yes Results file: No Chg Val NextPage Setup Main 2 Press Chg Val to select the number of report printouts (1 to 3) that you want to print for each patient. Press Enter to move to the next field. 3 Press Chg Val to toggle between Yes and No for each of the remaining fields. 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. Chapter 3: Getting Started 4142 4 Press NextPage for more options.

Reports Lab ID: Yes Signature line: No Op ID: Yes Comment line: No Chg Val PrevPage Setup Main 5 Press Chg Val to toggle between Yes and No for these fields. Answering Yes to the fields in the following table reports (or prints) the noted value. Field CD4 count CD4% Lab ID Op ID Signature line Comment line Value Reported Absolute number of CD4 + T lymphocytes (This field remains Yes and cannot be changed.) Percentage of lymphocytes that are CD4 + T lymphocytes Name of your laboratory Initials of the operator Two lines for a handwritten signature Four lines for handwritten comments regarding the patient or sample 6 Press Main to return to the BD FACSCount screen.

42 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents43 4 Reverse Pipetting The following topics are covered in this chapter: Pipetting Procedure on page 45 Method to Ensure Pipetting Precision on page 46 Method to Ensure Pipette Accuracy on page 48 4344 Reverse pipetting is necessary for accurate results. The BD FACSCount electronic pipette is preprogrammed to operate in the reverse pipetting mode and to consistently deliver 50 µl of fluid. 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. Figure 4-1 BD FACSCount electronic pipette tip eject lever start button The pipette used in sample preparation must be properly calibrated to ensure that it is dispensing precisely 50 µl of blood. 44 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents45 We recommend checking the performance of your electronic pipette regularly, for example every 3 months, or when the control run repeatedly fails.

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. Pause 1 to 2 seconds to allow the correct volume of liquid to enter the reagent tube. Some liquid will remain in the tip.

Chapter 4: Reverse Pipetting 4546 5 Remove the tip from the reagent tube. 6 Place the pipette and tip over an appropriate biohazard container and squeeze the tip eject lever. Method to Ensure Pipetting Precision Use the following procedure to check that you are consistently pipetting a precise volume. Pipette 20 replicates of blood and record the weight of each. Calculate the coefficient of variation (%CV).

The%CV should be. 54 4 Open the reagent tubes with the coring station. NOTE Make sure the coring station is clean before you use it (see page 89).

Slide the reagent tubes upright into the coring station. When the tubes are securely positioned, pull the lever down to core the tubes. Allow the lever to return to its original position. 5 Transfer the tubes from the coring station to the workstation, keeping the tubes upright. 6 Close the workstation cover to protect the reagents from light.

54 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents55 Adding Blood to Controls 1 Invert the BD Vacutainer EDTA blood tube (or equivalent) 5 to 10 times to adequately mix the normal whole blood. Use proper precaution and wear suitable protective clothing, eyewear, and gloves. 2 Pipette 50 µl of normal whole blood into the first control tube. Reverse pipetting is critical.

For information on pipetting, see page Discard the used tip in an appropriate biohazard container. 4 Cap the tube and vortex upright for 6 seconds. 5 Repeat steps 2 through 4 for the 2 remaining control tubes. Chapter 5: Preparing Controls and Samples 5556 Adding Blood to Samples 1 Invert the BD Vacutainer EDTA blood tube (or equivalent) 5 to 10 times to adequately mix the whole blood. Use proper precaution and wear suitable protective clothing, eyewear, and gloves. 2 Pipette 50 µl of specimen whole blood into the first sample tube. Reverse pipetting is critical.

For information on pipetting, see page Change tips between tubes. Discard the used tip in an appropriate biohazard container.

4 Cap the tube and vortex upright for 6 seconds. 5 Repeat steps 1 through 4 to prepare sample tubes for the remaining patient specimens. 6 Inspect the pipette. If you observe blood, clean the pipette with a soft cloth dipped in a mild detergent solution.

Incubating Tubes 1 Place the reagent tubes in the workstation and close the cover to protect the reagents from light. 2 Incubate the tubes for 30 minutes at room temperature (20 to 25 C, 68 to 77 F). NOTE Correct incubation time is critical and should not exceed 40 minutes or be less than 30 minutes for each reagent tube. For this reason, BD recommends preparing no more than 15 control and sample reagent tubes at one time. 56 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents. 57 Adding Fixative 1 Uncap the first control or patient sample tube and pipette 50 µl of fixative solution into the tube. 2 Recap the reagent tube and vortex upright for 6 seconds.

3 Change to a new tip. Discard the used tip in an appropriate biohazard container. 4 Repeat steps 1 through 3 for each remaining control and patient sample tube.

Store reagent tubes in the workstation until you are ready to add control beads to the control tubes. Stained controls can be held up to 24 hours before adding control beads.

Adding Control Beads 1 Uncap the control tubes. 2 Remove one pair of Zero/Low control beads and one pair of Medium/High control beads from the control kit and place them in the control area of the workstation. Control area of workstations NOTE The kit contains a Zero control that is not required for the CD4 assay. Do not use it. 3 Vortex the Zero/Low control bead pair.

Before opening the Zero/Low control bead pair with the coring station, vortex the pair upside down for 6 seconds, then upright for 6 seconds. Cap the controls after use and store upright. For subsequent uses of the control bead pair, vortex upright for 6 seconds.

Chapter 5: Preparing Controls and Samples 5758 4 Pipette 50 µl of the Low control beads (red top) into the reagent tube labeled Low. Discard the tip.

5 Vortex the Medium/High control bead pair. Before opening the Medium/High control bead pair with the coring station, vortex the pair upside down for 6 seconds, then upright for 6 seconds. Cap the controls after use and store upright.

For subsequent uses of the control bead pair, vortex upright for 6 seconds. 6 Pipette 50 µl of the Medium control beads (blue top) into the reagent tube labeled Medium.

Discard the tip. 7 Pipette 50 µl of the High control beads (purple top) into the reagent tube labeled High. Discard the tip. 8 Recap the reagent tubes. You should have three reagent tubes containing the control beads.

NOTE Run the tubes on the BD FACSCount instrument within 2 hours of adding control beads to reagent tubes. Store samples at room temperature in the workstation, protected from light, until they are run on the instrument. 58 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents59 6 Running Controls The following topics are covered in this chapter: Entering Control Run Information on page 60 Running Controls on page 62 Aborting the Control Run on page 65 Control Results Printout on page 65 5960 Control runs check system accuracy and linearity. Run controls each day before you run patient samples or whenever you open a new reagent lot. Data for the last control run is stored on the software disk until the next time controls are run.

NOTE Multiple BD FACSCount CD4 software disks are provided. Be sure to use the disk that contains the most recent control data. Controls are reported as Passed or Failed. If the control run fails, patient samples are flagged with the following message: Last Control Run: Failed.

This message appears on Sample Results printouts until controls are rerun and pass. Entering Control Run Information BD FACSCount reagents and control beads are each assigned specific lot codes and specific bead counts. Carefully enter the lot codes and bead counts before running controls or samples. This information is stored and does not need to be changed between runs unless a new lot of controls or a new lot of reagent is used.

Do not mix different reagent lots when running controls or samples. 1 Press Control on the BD FACSCount screen (see page 32) to access the control beads. Control Beads Control lot code: Counts Low: 50 Med: 248 High: 999 Confirm Main The lot code and bead count values that appear on the screen are those entered at the last run. If this is your first run, the lot code and counts are default values. 60 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents61 2 Enter the eight-digit control bead lot code.

This information is found on the control bead box. Alternatively, use a barcode reader to enter the control bead lot code.

Press Enter to move to the next field. 3 Enter the bead counts for the Low, Medium, and High controls. Always make sure the lot code and bead count numbers on the screen match the number on the packaging. If a number does not match, scan the barcode again.

If the scan is still unsuccessful, contact BD Biosciences. This information is found inside the control bead box.

Alternatively, use a barcode reader to enter the bead counts. 4 Press Confirm to save this information to the software disk. The Reagent screen is displayed. CD4 Reagent Lot Code: xxxxxxxx Counts CD4: xxxx Confirm Main 5 Enter the eight-digit reagent lot code. This information is found on the foil bag label and on the reagent tube tab. Alternatively, use a barcode reader to enter the reagent lot code. Press Enter to move to the next field.

6 Enter the CD4 reference bead counts for the reagent lot. This information is found on the foil bag label. Alternatively, use a barcode reader to enter reference bead counts. It is critical to enter these counts correctly.

Chapter 6: Running Controls 6162 7 Press Confirm to save this information to the software disk. A message appears informing you the system is setting up controls, followed by the Control Low screen for entering the normal ID number. Figure 6-1 Control Low screen Running Controls Control NormID: Type in the lab NormID number and press the Enter key. Clear ID Main Run the controls in the following order: Low, Medium, and then high. 1 Enter the control normal ID (up to 15 numbers) and press Enter. The normal ID is the laboratory number that identifies the tube of blood.

If the normal ID from the previous run exists, press Clear ID to remove it. The instrument prompts you for the CD4 Low control with the following screen. Control Low NormID: 5035 Move the CD4 Low tube into position and press the Run key. Main Be sure you have added control beads to the reagent tubes (see page 57). 2 Vortex the CD4-Low reagent tube upright for 6 seconds. Set the vortex speed to a setting that causes the liquid to rise to the top of the reagent tube.

Inadequate suspension of all white blood cell populations might cause inaccurate results. 62 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents. 63 3 Uncap the CD4-Low tube and set the cap aside.

4 Place the tube in the sample holder. 5 Press Run.

The event rate (events/second), total events, and acquisition time (mm:ss) are displayed, followed by messages informing you of the acquisition and analysis status. (Figure 6-2 is an example). Figure 6-2 Acquisition and analysis status message Control Low NormID: Rate: 310 Total: 2000 Time: 1:23 Acquiring/analyzing data. To abort run, press Stop.

When analysis of the Control-Low tube is complete, the sample holder lowers and the following message appears. NormID: Move the CD4 Medium tube into position and press the Run key. To abort run, press Stop. 6 Remove the CD4-Low tube and recap it. 7 Repeat steps 2 through 5 for both the CD4-Medium and CD4-High controls. The instrument prompts you for each control.

Chapter 6: Running Controls 6364 At the end of the CD4-High control run, control results are displayed and then printed. If Results file is selected in the Setup menu, control results are saved in the Results file. See page 76 for details on information stored in the Results file.

Do not turn off the instrument while the printer is printing. Results will be lost. Do not remove the disk from the drive while the drive light is on.

Data can be lost or damaged. 8 Choose one of the following when you see the following message.

Control NormID: 5035 Control run: Passed Control results are on printout. Sample Control Service Report Main Press Sample to run samples (see Running Samples on page 69). Press Control to rerun controls. Press Service when instructed to do so by BD Biosciences technical support. Press Report to print another report.

Press Main to return to the BD FACSCount screen. If you encounter any errors while running the controls, you will see the following message.

Control xxxx NormID: Tube failure. Test stopped. See printout for error code(s). Continue 64 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents65 Press Continue to display the control results. See page 108 for explanations of printed error codes. 9 If you are not running any sample tubes following the control run, perform the daily cleaning procedure (see page 80) and then the shutdown procedure (see page 82).

Aborting the Control Run Press Stop on the keypad during the control run to abort the run. Pressing Stop displays the following message. Control xxxx NormID: Rate: xxxx Total: xxxxx Time: MM: SS Do you really want to stop the test? No Yes Press No to continue the run.

Press Yes to abort. The following message is displayed, the control run is aborted, and the sample tube is lowered. Control NormID: 5035 User abort Sample Control Main Control Results Printout Control results are displayed on the screen (Figure 6-3 on page 67), automatically printed, and are saved in the Results file if that option is enabled. Controls are reported as Passed or Failed. If the control run fails, patient samples are flagged with the message: Last Control Run: Failed.

This message appears on Sample Results printouts until controls are rerun and pass. Chapter 6: Running Controls 6566 The control printout contains the following information. Control information, including control lot code, control bead counts, reagent lot code, and the reference bead count entered for the control run Date (mm/dd/yy) and time (hh:mm) the controls were run Control Run results: Passed or Failed (see Figure 6-3 on page 67) Measured counts for control beads Statistical results for linearity check Reagent reference bead locations Measured count and percentage for CD4 T lymphocytes (statistical mean and CV of the CD4 count across the three tubes) NOTE Do not place tape over the printout. Adhesive or transparent tape erases thermal printing over time. Avoid exposing printouts to the sun or other source of heat. Under normal storage conditions printouts should last 5 to 7 years. If permanent records are required, photocopy the printout.

Although the control printout contains various statistical values, the actual control run result reports the success or failure of the control run. This result is reported as Passed or Failed. If the control run fails, the results on the report are suppressed.

Check the printout for printed error codes. See Error Codes on page 108 for possible causes. Do not report patient results if the control run fails. A successful control run is necessary to ensure reliable patient results. 71 4 Press Enter. The instrument prompts you for the CD4 tube (see the following section, Running Patient Samples).

Running Patient Samples After you enter sample information, the instrument prompts you for the CD4 tube. #: 1234 Move the CD4 tube into position and press the Run key.

Main 1 Vortex the reagent tube upright for 6 seconds. Set the vortex speed to a setting that causes the liquid to rise to the top of the reagent tube. Inadequate suspension of all white blood cell populations can result in inaccurate results.

2 Uncap the CD4 tube and set the cap aside. 3 Place the reagent tube in the sample holder and press Run. The event rate (events/second), total events, and acquisition time (mm:ss) are displayed, followed by messages informing you of the acquisition and analysis status (see Figure 7-1 on page 72). Chapter 7: Running Samples 7172 Figure 7-1 Event rate, total events, and acquisition and analysis status Sample CD4 Acc.#: 1234 Rate: 230 Total: 4600 Time: 0:20 Collecting initial data.

To abort run, press Stop. Sample CD4 Acc.#: 1234 Rate: 220 Total: Time: 1:10 Acquiring/analyzing data. To abort run, press Stop. When analysis of the CD4 tube is complete, the sample holder lowers. 4 Remove the CD4 tube and recap it. Discard the CD4 tube as biohazardous waste in accordance with local regulations.

Patient results are displayed and printed. If Results file is selected in the Setup menu, patient results are saved in the Results file. Do not turn off the instrument while the printer is printing. Results will be lost. Do not remove the disk from the drive while the drive light is on.

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Data can be lost or damaged. 5 Choose one of the following when you see this message on the screen. #: 1234 CD4: 642 cells/l CD4%: 46.5 Sample Control Service Report Main 72 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents73 Press Sample to run the next sample; enter the patient accession number, and follow steps 1 through 4 in this section. Press Control to rerun controls.

Press Service when instructed to do so by BD Biosciences technical support. Press Report to print another copy of the Sample Report. Press Main to return to the BD FACSCount screen. If you encountered any errors while running the sample, the following message is displayed. Sample CD4 Acc. #: 1234 Tube failure.

Test stopped. See printout for error code(s). Continue Press Continue. See page 108 for information on error codes. Some error codes can suppress results. See Suppressed Numeric Results on page 76. NOTE Re-use caps during sample preparation; throw them away after acquisition.

6 After running the last sample, perform the daily cleaning procedure on page 80 and the shutdown procedure on page 82. For performances characteristics, refer to the BD FACSCount CD4 Reagents package insert. Chapter 7: Running Samples 7374 Aborting a Sample Run Press Stop during the sample run to abort the run. Pressing Stop displays the following message. Sample CD4 Acc. #: 1234 Rate: 260 Total: Time: 1:10 Do you really want to stop the test?

No Yes Press No to continue the run. Press Yes to abort. The following message (Figure 7-2) is displayed and the sample run is aborted. Figure 7-2 Sample abort message Sample CD4 Acc. #: 1234 User abort Sample Control Main 74 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents75 Sample Results Printout Patient results are displayed on the screen and are printed automatically (see example on right). The sample printout contains the following information. Reagent information: reagent lot code and reference bead count entered for the sample run This number should match the reagent lot code for the control run.

85 3 Remove the sheath tank from the instrument and discard the sheath fluid (Figure 8-1). Figure 8-1 sheath filter, sheath tank, and waste tank connections 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 4 Rinse the tank with DI water (or BD FACSRinse solution), and fill it with 2 liters of a 1:10 bleach solution (or BD FACS Clean solution). 5 Fill one cleaning tube with a 1:10 bleach solution (or BD FACS Clean solution). 6 Place the sheath tank back on the instrument, bypassing the sheath filter.

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See Figure 8-2 on page 86. Bypass the sheath filter to prevent damage from bleach (or BD FACS Clean solution) and distilled water (or BD FACSRinse solution). Chapter 8: Cleaning and Maintenance 85.

86 Figure 8-2 Sheath filter configured for Long Clean sheath filter bypassed Disconnect the sheath filter output tubing connector and set it aside. Connect the sheath tank output tubing connector to where the sheath filter output tubing connector was connected. 7 Press Confirm on the Long Clean Bleach screen. Long Clean Bleach Bypass the sheath filter and press Confirm. Cancel Confirm 8 Press Confirm on the next screen. Long Clean Bleach Connect the sheath tank filled with 1:10 bleach solution and press Confirm.

Confirm 9 Empty the waste tank, and press Confirm. Long Clean Bleach Make sure the waste tank is empty and press Confirm.

Confirm 86 BD FACSCount System User s Guide for Use with BD FACSCount CD4 Reagents.