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BD Trucount™ Tubes: For Determining Absolute Counts of Leucocytes in Blood
BD Trucount™ Tubes: For Determining Absolute Counts of Leucocytes in Blood
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.
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. 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 counting.1 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
blood is critical to achieving the result. Lysing Solution instructions for use for
Pipettes must be calibrated to deliver warnings.
exactly 50 µL 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 FACSCalibur™ 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. 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 2ºC–25ºC. 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
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. 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 µL) 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.
No. 342003) or equivalent.
2. Pipette 20 µL 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 µL 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.
(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 µL 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 cytometer.6 If
using the BD FACS Loader for
acquisition, vortex tubes immediately
before placing them into the Loader racks.
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 2.0 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 FACSCount™
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
FACSCount system. Data was analyzed to
determine the average differences between
the results obtained using BD Tritest/
BD Trucount versus results with BD
FACSCount. The results appear in
Figure 4 and Figure 5, and Table 1 and
Table 2.
Figure 4 BD FACSCount results versus BD Tritest/
BD Trucount results
Quality Control
+
BD Tritest Absolute CD3
5
Figure 5 BD FACSCount results versus BD Tritest/ to assess reproducibility. The results
BD Trucount results appear in Table 3.
Table 3 Reproducibility: BD Trucount with
BD Tritest CD3/CD4/CD45
+
BD Tritest Absolute CD3 CD4
+
a. SD = standard deviation
6
b. CV = coefficient of variation WARRANTY
Unless otherwise indicated in any applicable BD
Table 4b: Site 2
general conditions of sale for non-US customers,
Mean Mean the following warranty applies to the purchase
CD4 SDa CV%b CD3 SDa CV%b
of these products.
Low 86 7.6 8.9 768 29.5 3.8
THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO
266 24.6 9.2 1,047 53.5 5.1 CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL
422 23.3 5.5 3,533 247.6 7.0 OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE
CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES,
Med 517 12.3 2.4 1,083 7.0 0.6 EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF
MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND
689 27.7 4.0 1,623 126.5 7.8 NONINFRINGEMENT. BD’S SOLE LIABILITY IS LIMITED TO EITHER
903 45.4 5.0 1,589 67.6 4.3 REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE
PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY
High 1,197 93.4 7.8 2,254 149.8 6.6 INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL
INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT.
1,201 37.4 3.1 1,758 40.5 2.3
1,363 66.7 4.9 2,330 115.5 5.0
REFERENCES
a. SD = standard deviation
1. Brando B, Göhde W Jr Scarpati B, D'Avanzo G. The
b. CV = coefficient of variation
“vanishing counting bead” phenomenon: effect on
absolute CD34+ cell counting in phosphate-buffered
Table 4c: Site 3 saline–diluted leukapheresis samples. Cytometry.
2001;43:154-160.
Mean Mean
CD4 SDa CV%b CD3 SDa CV%b 2. Protection of Laboratory Workers from
Occupationally Acquired Infections; Acquired
Low 10 1.0 10.4 267 10.5 4.0 Guidelines–Third Edition. Wayne, PA: Clinical and
49 3.2 6.4 1,385 80.2 5.8 Laboratory Standards Institute; 2005. CLSI
document M29-A3.
236 11.7 5.0 1,748 46.6 2.7
3. Centers for Disease Control. Perspectives in disease
Med 715 16.8 2.3 2,214 64.2 2.9 prevention and health promotion update: universal
878 40.3 4.6 1,328 49.9 3.8 precautions for prevention of transmission of human
immunodeficiency virus, hepatitis B virus, and other
947 61.1 6.4 1,590 120.1 7.6
bloodborne pathogens in health-care settings.
High 1,056 43.5 4.1 2,904 164.5 5.7 MMWR. 1988;37:377-388.
1,299 79.0 6.1 1,970 121.8 6.2 4. Procedures for the Collection of Diagnostic Blood
Specimens by Venipuncture; Approved Standard–
1,502 65.3 4.3 2,303 63.6 2.8
Sixth Edition; Wayne, PA: Clinical and Laboratory
Standards Institute; 2007. CLSI document GP41-A6.
a. SD = standard deviation
b. CV = coefficient of variation 5. Enumeration of Immunologically Defined Cell
Populations by Flow Cytometry; Approved
Guideline–Second Edition. Wayne, PA: Clinical and
At these clinical sites, the range of CVs for Laboratory Standards Institute; 2007. CLSI
CD3+ cells, observed for all samples, was document H42-A2.
<1% (count of 1,083 cells/µL) to 13% 6. Jackson AL, Warner NL. Preparation, staining, and
(count of 187 cells/µL). The range of CVs analysis by flow cytometry of peripheral blood
leukocytes. In: Rose NR, Friedman H, Fahey JL, eds.
for CD3+CD4+ cells, observed for all Manual of Clinical Laboratory Immunology. 3rd ed.
samples, was <1% (count of 271 cells/µL) Washington, DC: American Society for
to 80% (count of 24 cells/µL). Microbiology; 1986:226-235.