BD Trucount Tubes

1 1. INTENDED USE. BD Trucount Tubes are used for BD Trucount Tubes determining absolute counts of leucocytes in blood. For determining absolute counts of leucocytes in blood BD Trucount Tubes are designed for use with in vitro diagnostic products such as 25 Tubes Catalog No. 340334 BD Tritest reagents, and a suitably equipped flow cytometer. BD Trucount Tubes can be used with the BD FACS . Loader. 2. PRINCIPLES OF THE PROCEDURE. Procedures described in this instructions for use (IFU) apply to immunophenotyping applications. For 11/2016 23-3483-08 other applications, refer to the appropriate product-specific IFU. IVD. Add the appropriate monoclonal antibody 2016 BD. BD, the BD Logo and all other trademarks reagent and whole blood directly to the are property of Becton, Dickinson and Company. BD Trucount tube. The lyophilized pellet in the tube dissolves, releasing a known number of fluorescent beads. During Becton, Dickinson and Company analysis, the absolute number (cells/ L) of BD Biosciences positive cells in the sample can be 2350 Qume Drive, San Jose, CA 95131 USA determined by comparing cellular events Benex Limited to bead events.

2 If the appropriate Pottery Road, Dun Laoghaire, software, such as BD Multiset , is used, Co. Dublin, Ireland absolute counts will be determined by the Tel + Fax + software. If you are manually performing BD Biosciences data analysis using software such as European Customer Support BD CellQuest Pro , simply divide the Tel + number of positive cellular events by the Fax + number of bead events, then multiply by the BD Trucount bead concentration. Becton Dickinson Pty Ltd, 4 Research Park Drive, Macquarie University Research Park, 3. REAGENT. North Ryde NSW 2113, Australia Becton Dickinson Limited, Each pouch contains 25 BD Trucount 8 Pacific Rise, Mt. Wellington, Tubes , sufficient for 25 tests. Auckland, New Zealand Precautions For In Vitro Diagnostic Use. 1. BD Trucount Tubes are designed for use turned from blue to lavender, discard with a specific lyse/no-wash procedure. the remaining Tubes . Use Tubes within For absolute counting, prepare and 1 hour after removal from the foil analyze samples within BD Trucount pouch and do not use beyond the Tubes .

3 Do not transfer beads to another expiration date indicated on the tube. The presence of proteins, such as packaging. serum proteins contained in whole WARNING All biological specimens and blood, is necessary for proper materials coming in contact with them are performance of BD Trucount beads in considered biohazards. Handle as if absolute Follow specific capable of transmitting infection2,3 and assay IFUs when diluting samples. Do dispose of with proper precautions in not attempt to threshold on forward accordance with federal, state, and local scatter (FSC) for data collection. Do not regulations. Never pipette by mouth. remove the metal retainer in the Wear suitable protective clothing, BD Trucount tube. eyewear, and gloves. It is the responsibility of the user to BD FACS lysing solution is required validate any other method or use. and contains diethylene glycol and The addition of a precise volume of formaldehyde. Refer to the BD FACS.

4 Blood is critical to achieving the result. Lysing Solution instructions for use for Pipettes must be calibrated to deliver warnings. exactly 50 L of sample. If this or a similar type of pipette is not used, 4. INSTRUMENT. perform the reverse pipetting technique BD Trucount applications are designed for (see Reverse Pipetting in Section 6 for a flow cytometers equipped with brief description). Refer to the pipette appropriate computer hardware and manufacturer's instructions for more software. The flow cytometer must be information. equipped to detect three-color Always be sure to use the bead count fluorescence, forward scatter (FSC), and from the current lot of BD Trucount side scatter (SSC). We recommend the Tubes when entering this value in the BD FACSC alibur flow cytometer;. software or when manually calculating however, results can be achieved using an absolute count. The correct bead other platforms. Refer to the appropriate count is critical for determining a cell reagent IFU for specific instrument count.

5 Do not mix multiple lots of Tubes limitations. The BD FACS Loader can also in the same assay. be used with this product. BD has developed BD Multiset software, for use Store BD Trucount Tubes in their with specific reagents and BD Trucount original foil pouch at 2 C 25 C. To Tubes , which automatically calculates avoid potential condensation, open the absolute counts. However, you can also pouch only after it has reached room use software such as BD CellQuest Pro for temperature and carefully reseal the data acquisition and analysis and pouch immediately after removing a manually calculate absolute counts. tube. Examine the desiccant each time you open the pouch. If the desiccant has 2. 5. SPECIMEN COLLECTION AND Staining the Cells PREPARATION Stain whole blood samples following Collect blood aseptically by specific instructions in the appropriate venipuncture4,5 into a sterile EDTA reagent IFU. Lyse red blood cells after (lavender top) BD Vacutainer blood staining using diluted (1X) BD FACS.

6 Collection tube. Follow the collection tube lysing solution. Use care to protect the manufacturer's guidelines for the Tubes from direct light. Perform the minimum volume of blood to be collected. procedure at room temperature (20 C . Store anticoagulated blood at room 25 C). temperature (20 C 25 C) until ready for Reverse Pipetting staining. A precise volume of whole blood is critical. If a pipette that delivers a precise 6. PROCEDURE. volume of blood is not used, perform Reagent Provided reverse pipetting. This technique takes BD Trucount Tubes (Catalog No. advantage of two stops in a pipette. 340334), containing a freeze-dried pellet For normal pipetting, typically, the of fluorescent beads in a single-use tube. button is depressed to the first stop;. Reagents and Materials Required but Not sample is drawn up by releasing the Provided button, then expelled by pressing to the first stop again. BD FACSComp beads. Refer to your product catalog for information on the For reverse pipetting, the button is specific BD FACSComp product for depressed to the second stop.

7 When the your application. button is released, excess sample is drawn up into the tip. A precise volume BD FACS lysing solution (10X), 100 mL of sample is expelled by pressing the (Catalog No. 349202). Refer to the button to the first stop, leaving excess BD FACS Lysing Solution IFU for sample in the tip. dilution instructions and warnings. Staining Reagent-grade (distilled or deionized). water. Refer to the appropriate reagent IFU for detailed sample preparation instructions. EDTA BD Vacutainer blood collection 1. For each patient sample, label a Tubes (Catalog No. 356457), or BD Trucount tube with the reagent equivalent. and sample identification number. Vortex mixer. NOTE Before use, verify that the Micropipettor with tips. BD Trucount bead pellet is intact and Bulk dispenser or pipettor (450 L) for within the metal retainer at the dispensing BD FACS lysing solution. bottom of the tube. If this is not the case, discard the BD Trucount tube BD FACSFlow sheath fluid (Catalog and replace it with another.)

8 No. 342003) or equivalent. 2. Pipette 20 L of the appropriate BD Trucount Controls (Catalog No. reagent just above the stainless steel 340335). retainer. Do not touch the pellet. 3. 3. Pipette 50 L of well-mixed, Before acquiring samples, adjust the anticoagulated whole blood onto the threshold to minimize debris and ensure side of the tube just above the retainer. populations of interest are included. NOTE Avoid smearing blood down Figures 1, 2, and 3 show an example of the side of the tube. If whole blood BD Trucount Tubes used with remains on the side of the tube, it will BD Tritest CD3/CD4/CD45 reagent. not be stained with the reagent. If you are not using a BD software program that automatically calculates Accuracy is critical. Use a BD. absolute cell counts, you can obtain the electronic pipette or use the reverse absolute count of the cell population (A), pipetting technique to pipette sample by dividing the number of positive cell onto the side of the tube just above the events (X) by the number of bead events retainer.

9 (Y), and then multiplying by the 4. Cap the tube and vortex gently to mix. BD Trucount bead concentration (N/V, Incubate for 15 minutes in the dark at where N = number of beads per test* and room temperature (20 C 25 C). V = test volume). A = X/Y N/V. 5. Add 450 L 1X BD FACS lysing Gate the lymphocyte population (2) from solution to the tube. an FL3 vs SSC dot plot. Then, obtain the 6. Cap the tube and vortex gently to mix. number of events in the quadrant or Incubate for 15 minutes in the dark at region containing the cell population from room temperature. The sample is now a gated FL1 vs FL2 dot plot (see Figure 2). ready to be analyzed on the flow Obtain the number of events in the cytometer. absolute count bead (1) region from an ungated FL1 vs FL2 dot plot (see Flow Cytometry Figure 3). Refer to the appropriate reagent IFU for Figure 1 FL3 (CD45) vs SSC dot plot specific instructions. Vortex the samples thoroughly (at low speed) to resuspend beads and reduce cell aggregation before running them on the flow If using the BD FACS Loader for acquisition, vortex Tubes immediately before placing them into the Loader racks.

10 Acquire and analyze list-mode data using the appropriate software. We recommend using BD FACSComp beads and the appropriate software such as BD FACSComp , version or later, for setting the photomultiplier tube (PMT). voltages, setting the fluorescence compensation, and checking instrument sensitivity prior to use. * This value is found on the BD Trucount tube foil pouch label and might vary from lot to lot. 4. Figure 2 Gated FL1 vs FL2 dot plot 7. PERFORMANCE CHARACTERISTICS. Performance was established by comparison with the BD FACSC ount . system. These results with BD Tritest CD3/CD4/CD45 are representative of results obtained with other IVD. phenotyping reagents. Refer to specific reagent IFUs for more details. Accuracy Whole blood was stained with BD Tritest CD3/CD4/CD45 using BD Trucount Tubes and acquired and analyzed using BD CellQuest Pro software. Two samples of each specimen were stained and Figure 3 Ungated FL1 vs FL2 dot plot analyzed in parallel using the BD.