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1. INTENDED USE
BD Simultest™ Leucogate™ (CD45/
CD14) is a two-color direct
immunofluorescence reagent for
establishing an optimal lymphocyte gate
for immunophenotyping of erythrocytelysed whole blood (LWB). BD Simultest
Leucogate is for in vitro diagnostic use
with BD in vitro diagnostic reagents. See
the appropriate BD in vitro diagnostic
package insert for additional information.
BD Simultest™
Leucogate™ (CD45/CD14)
For lymphocyte gating and quality control in
immunophenotyping
50 Tests—Catalog No. 342408
2. SUMMARY AND EXPLANATION
3/2014
Immunophenotyping of human
lymphocytes by flow cytometry can
employ Leucogate, a negative control, and
one or more monoclonal antibody
reagents reactive with lymphocyte cellsurface antigens. BD Simultest Leucogate
reagent is used to create a light-scatter
analysis gate around the lymphocytes.
Gating is necessary because monoclonal
antibody immunophenotyping reagents
can react not only with lymphocytes but
also with nonlymphocytes in LWB
preparations. BD Simultest Leucogate
permits the gated cells to be characterized
and enumerated, thereby permitting
quality control evaluation of the data.
Nonlymphocyte components of LWB that
can contaminate the lymphocyte analysis
gate include monocytes, granulocytes
(neutrophils, eosinophils, basophils), and
debris (residual erythrocytes, erythrocyte
ghosts, and platelets).1
23-5297-02
IVD
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North Ryde NSW 2113, Australia
NOTE
For additional information and
an overview of human lymphocyte
immunophenotyping, see the appropriate
BD in vitro diagnostic immunophenotyping reagent instructions for use (IFU).
Becton Dickinson Limited,
8 Pacific Rise, Mt. Wellington,
Auckland, New Zealand
bdbiosciences.com
[email protected]
1
gating efficiency by determining the
number of lymphocytes within the gate
compared to total lymphocytes in the
sample.
3. PRINCIPLES OF THE PROCEDURE
When monoclonal antibody reagents are
added to human whole blood, the
fluorochrome-labeled antibodies bind
specifically to antigens on the surface of
leucocytes. Monoclonal antibodies can be
used to identify lymphocyte subpopulations.
The percentage of each identifiable
nonlymphocyte contaminant included in
the lymphocyte gate can also be
determined for quality control purposes.
The maximum allowable percentages of
nonlymphocytes that can be included in
the gate are 3% monocytes, 6%
granulocytes, and 10% debris events.
Values greater than these limits will be
flagged by the software.
An aliquot of the stained patient sample is
introduced into the flow cytometer and
passed in a narrow stream through the
path of a laser beam. The stained cells
fluoresce when excited by the laser beam
and the emitted light is collected and
processed by the flow cytometer.
We also recommend using BD Simultest™
Control γ1/γ2a (IgG1 FITC/IgG2a PE) and
BD Simulset software to set
fluorescence 1 (FL1) and fluorescence 2
(FL2) markers around the negative
lymphocyte population and to assess the
amount of nonantigen-specific binding
(nonspecific staining) present, particularly
that caused by Fc receptors.
We recommend using BD Simultest
Leucogate with BD Simulset™ software to
establish a lymphocyte analysis gate that
includes greater than or equal to 98% of
the normal mature (nonblast)
lymphocytes in the sample. However, if
the gate contains greater than 3%
monocytes, the software automatically
reduces or tightens the light-scatter gate to
collect greater than or equal to 95% of the
lymphocytes contained in the sample.
4. REAGENTS
Reagents Provided, Sufficient for 50 Tests
The BD Simultest Leucogate reagent,
sufficient for 50 tests, is provided in 1 mL
of buffered saline with gelatin and 0.1%
sodium azide. It contains FITC-labeled
CD45 (Anti–HLe-1), clone 2D1,2-4 for
identification of leucocytes, and PElabeled CD14, clone MϕP9,5-7 for
identification of monocytes. The
fluorescein-to-protein ratio (F:P) for
BD IgG monoclonal antibody reagents is
2 to 5. The F:P ratio for CD45 (Anti–
HLe-1) FITC has been optimized for its
intended use.
The process by which the BD Simultest
Leucogate tube data is analyzed is a
multistep procedure (see Figure 1). The
procedure is performed automatically
with BD Simulset software, but can also
be performed manually with
BD CONSORT™ 30 or BD LYSYS™ II
software (see the captions of Figure 1 to
set gates manually).
Quality control criteria for the
lymphocyte analysis gate require that
greater than or equal to 95% of all the
lymphocytes in the sample be included in
the analysis gate. The software evaluates
The CD45 (Anti–HLe-1)4 antibody is
composed of mouse IgG1 heavy chains
and kappa light chains.
2
The CD45 antigen is present on all human
leucocytes, including lymphocytes,
monocytes, granulocytes, eosinophils, and
basophils in peripheral blood and has a
role in signal transduction, modifying
signals from other surface molecules.8
The CD45 antibody recognizes human
leucocyte antigens, 180 to 220 kilodaltons
(kDa), that are members of the T200
family.8
Figure 1 Steps used to establish the optimal
lymphocyte light-scatter gate
The CD14 antibody is composed of
mouse IgG2b heavy chains and kappa light
chains. CD14 recognizes a human
monocyte antigen of 53 kDa.9 The CD14
antigen is present on the majority of
normal peripheral blood monocytes.10
Precautions
No.
Description
1
Set a gate around the lymphocyte
population that expresses low side
scatter (SSC) and is negative for CD14
PE. Record the x- and y-coordinates
for SSC.
2
Set a gate around the leucocytes to
exclude most of the debris events.
Record the x- and y-coordinates for
forward scatter (FSC).
3
Use the coordinates for FSC and SSC
from steps 1 and 2 to establish the
optimal lymphocyte gate.
3
•
For in vitro diagnostic use.
•
When stored at 2°C–8°C, antibody
reagents are stable until the expiration
date shown on the label. Do not use
after the expiration date.
•
The antibody reagent should not be
frozen or exposed to direct light
during storage or during incubation
with cells. Keep the reagent vial dry.
•
Alteration in the appearance of the
reagent, such as precipitation or
discoloration, indicates instability or
deterioration. In such cases, the
reagent should not be used.
•
The antibody reagents contain sodium
azide as a preservative. However, care
should be taken to avoid microbial
contamination, which can cause
erroneous results.
A white blood cell (WBC) count and a
differential white cell count should be
obtained from the same sample of whole
blood before staining. BD Simultest
Leucogate can be used on samples with
WBCs in the usable range of any BD in
vitro diagnostic reagent. Check the
appropriate BD in vitro diagnostic IFU for
the usable range of the
immunophenotyping reagent. For samples
with counts lower than the lower limit,
more blood might be needed and a
separation procedure might be required to
concentrate the cells. Samples with counts
higher than the upper limit of the range
might need to be diluted with 1X
phosphate-buffered saline (PBS)
containing 0.1% sodium azide.
WARNING All biological specimens and
materials coming into contact with them
are considered biohazards. Handle as if
capable of transmitting infection11,12 and
dispose of with proper precautions in
accordance with federal, state, and local
regulations. Never pipette by mouth.
Wear suitable protective clothing,
eyewear, and gloves.
5. INSTRUMENT
BD Simultest Leucogate reagent is
designed for use on a BD FACS™ brand
flow cytometer equipped with appropriate
computer hardware, software, and gating
electronics. The flow cytometer must be
equipped to detect two-color fluorescence,
FSC, and SSC. We recommend using
BD Simulset software, version 2.5 or later,
for data acquisition and analysis.
Interfering Conditions
Previously fixed and stored patient
specimens should not be used. Whole
blood samples refrigerated prior to
staining can give aberrant results. For
optimal results, blood samples should be
stained within 6 hours of venipuncture.
Samples obtained from patients taking
immunosuppressive drugs can yield poor
resolution.15 The presence of blast cells or
unlysed or nucleated red blood cells
(RBCs) can interfere with test results.
Hemolyzed samples should be rejected.
Follow the collection tube manufacturer’s
guidelines for the minimum volume of
blood to be collected.
All performance characteristics were
obtained using a BD FACScan™ flow
cytometer. Other systems can have
different characteristics and should be
verified by the user.
6. SPECIMEN AND COLLECTION
PREPARATION
Collect blood aseptically by
venipuncture13,14 into a sterile (lavender
top) BD Vacutainer® EDTA blood
collection tube or equivalent. A minimum
of 1 mL of whole blood is required for
this procedure. Blood should be stained
within 6 hours of drawing for optimal
results. Anticoagulated blood can be
stored at room temperature (20°C–25°C)
for up to 6 hours until ready for staining.
Blood samples refrigerated prior to
staining can give aberrant results.
CAUTION Use standard precautions when
obtaining, handling, and disposing of all
human blood samples and potentially
carcinogenic reagents.
7. PROCEDURE
Reagents Provided
See Reagents Provided and Precautions in
Section 4, Reagents.
4
Reagents and Materials Required But Not
Provided
•
Reagent-grade (both distilled and
deionized) water.
•
BD Simultest Control.
•
Appropriate immunophenotyping
reagent.
•
BD FACS™ lysing solution (10X)
(Catalog No. 349202). For dilution
instructions and warnings, see the
IFU.
•
BD Vacutainer EDTA blood collection
tubes or equivalent.
•
BD Calibrite™ beads (Catalog No.
349502). For detailed information on
use, see the IFU.
Staining and Fixing the Cells
Whole blood samples are first stained
with BD Simultest Leucogate (tube A),
BD Simultest Control (tube B), and the
appropriate BD lymphocyte
immunophenotyping reagent (tube C).
Diluted (1X) BD FACS lysing solution is
then used to lyse RBCs following staining.
Use care to protect the tubes from direct
light. Perform the procedure at room
temperature (20°C–25°C) using room
temperature reagents. See Precautions in
Section 4, Reagents.
•
Falcon®* disposable 12 x 75-mm
polystyrene test tubes or equivalent.
•
Vortex mixer.
•
Low-speed centrifuge (200g) with
swinging bucket rotor and
12 x 75-mm tube carriers.
•
Vacuum aspirator with trap.
•
Micropipettor with tips
•
BD CellWASH™ (Catalog No.
349524) or a wash buffer of PBS with
0.1% sodium azide.
•
BD CellFIX™ (Catalog No. 340181)
or 1% paraformaldehyde solution in
PBS with 0.1% sodium azide. Store at
2°C–8°C in amber glass for up to
1 week.
•
BD FACSFlow™ sheath fluid (Catalog
No. 342003) or equivalent.
1. For each patient sample, label three
12 x 75-mm tubes A, B, and C. If
there are other reagents in the panel,
label additional tubes as required.
Also label each tube with the sample
identification number.
2. Place 20 µL of BD Simultest
Leucogate reagent into tube A, 20 µL
of BD Simultest Control into tube B,
20 µL of the appropriate
immunophenotyping reagent into tube
C, and 20 µL of each additional
immunophenotyping reagent in the
panel into separate tubes as required.
3. For each patient sample tube, use a
fresh micropipettor tip and carefully
add 100 µL of the correct
concentration of well-mixed, anticoagulated, whole blood patient
sample into the bottom of each of the
labeled tubes. Use care to prevent
blood from running down the side of
CAUTION Use only BD FACSFlow
sheath fluid diluent to dilute
BD Calibrite beads.
* Falcon is a registered trademark of Corning
Incorporated.
5
9. Vortex thoroughly at low speed to
resuspend the cell pellet in the residual
fluid, and then add 0.5 mL of
BD CellFIX solution or 1%
paraformaldehyde to each tube.
Vortex thoroughly at low speed for
3 seconds. Make sure that the cells are
well mixed with the fixing solution.
the tube. Vortex thoroughly at low
speed for 3 seconds and incubate for
15 to 30 minutes at room
temperature.
NOTE
Protect samples from direct
light during this incubation procedure
and use care to prevent blood from
running down the side of the tube. If
whole blood remains on the side of the
tube, it may not be stained with the
reagent.
10. The cells are now ready to be analyzed
on the flow cytometer. Cap or cover
the prepared tubes and store at 2°C–
8°C in the dark until flow cytometric
analysis can be performed. Analyze
the fixed cells within 24 hours after
staining. Vortex the cells thoroughly
(at low speed) before putting them
through the flow cytometer to help
reduce cell aggregation.16
4. Add 2 mL of room temperature 1X
BD FACS lysing solution to each tube.
Immediately vortex thoroughly at low
speed for 3 seconds and incubate for
10 to 12 minutes at room temperature
in the dark. Do not exceed
12 minutes.
Flow Cytometry
Follow the BD instructions for two-color
flow cytometric analysis.
NOTE
Avoid prolonged exposure of
the cells to lytic reagents, which can
cause white cell destruction.
For information on tubes B and C, see the
appropriate reagent IFU.
5. Immediately after incubation,
centrifuge tubes at 300g for 5 minutes
at room temperature.
Quality Control
For optimal results, we recommend using
BD Calibrite beads and BD FACSComp™
software for setting the photomultiplier
tube (PMT) voltages, setting the
fluorescence compensation, and checking
instrument sensitivity prior to use of
BD Simultest reagents on a BD FACScan
flow cytometer.
6. Aspirate the supernatant, leaving
approximately 50 µL of residual fluid
in the tube to avoid disturbing the cell
pellet.
7. Vortex thoroughly at low speed to
resuspend the cell pellet in the residual
fluid, and then add 2 mL of
BD CellWASH solution or PBS with
0.1% sodium azide to each tube.
Vortex thoroughly at low speed for
3 seconds. Centrifuge at 200g for
5 minutes at room temperature.
We recommend that a control sample
from a normal adult subject be run daily
to optimize instrument settings and as a
quality control check of the system.
Correct results for a hematologically
normal patient are illustrated in Figure 1.
8. Aspirate the supernatant, leaving
approximately 50 µL of residual fluid
in the tube to avoid disturbing the
pellet.
6
BD Simulset software will automatically
inspect the data and alert the operator
with a number of possible error messages.
See the BD Simulset Software User’s
Guide for a list of possible messages. The
software uses the following criteria for
inspection of the dot plots obtained for
each sample to evaluate the quality of the
data obtained.
•
•
•
8. RESULTS
Percent Lymphocyte Conversion
When the Percent Lymphocyte
Conversion computation is performed, the
lymphocyte subset for the in vitro
diagnostic immunophenotyping reagent is
reported as a percentage of lymphocytes
in the lymphocyte analysis gate. If the
computation is not performed, results will
be reported as a percentage of the gated
events.
The operator should reject the results
if any one of the following error
messages is received for the normal
control: no separation between
cellular populations; too few
lymphocytes (less than 500); excessive
RBC or nucleated RBC contamination
and debris (greater than 10%); or
excessive monocyte (greater than 3%)
or granulocyte (greater than 6%)
contamination of the lymphocyte gate.
9. LIMITATIONS
Three-Part Differential
For lysed whole blood, it is possible to
estimate monocytes, lymphocytes, and
granulocytes as a percentage of leucocytes
using BD Simultest Leucogate reagent
(tube A). BD Simulset software
automatically calculates a three-part
differential. See the BD Simulset Software
User’s Guide for representative data
printouts.
If there is no obvious reason for the
normal control to fail, a sample from
another normal control should be
restained and rerun and the entire
staining procedure repeated on all
subsequent samples.
NOTE
Do not use the differential from
the BD Simulset software report to
compute absolute counts. The differential
provided by BD Simulset software is
printed only for comparison with results
from an independent laboratory
differential white cell count for quality
control purposes and should not be used
in place of an independent laboratory
differential white cell count in patient
charts or entered into BD Simulset
software to obtain absolute counts.
Samples with nucleated RBCs can
contain too much debris because of
incomplete lysis of the nucleated
erythrocytes with BD FACS lysing
solution. Too much debris can also
occur when assaying blood samples
from patients with certain
hematological disorders where red
cells are difficult to lyse (for example,
myelofibrosis and spherocytosis).
Nucleated erythrocytes will be
counted as debris and, if debris
exceeds 10%, the software will flag
the sample as “too many nonlymphs
in the gate” and the sample results
should be rejected.
•
7
Variation in either automatic or
manual lymphocyte acquisition gate
settings will change the relative
amounts of subsets assayed.
BD Simulset software uses the
BD Leucogate tube to include at least
95% of the total lymphocytes in the
sample to set the lymphocyte analysis
gate and requires a visual inspection
of the gate setting for validation.
•
•
Linearity-Recovery
Linearity of response over a wide range of
WBCs is determined for each BD in vitro
diagnostic immunophenotyping reagent
using Leucogate in the assay procedure.
See the appropriate BD reagent IFU for
the usable WBC range.
BD Simultest Leucogate reagent is not
intended for screening samples for the
presence of leukemic cells or for use in
phenotyping samples from leukemia
patients. The presence of blast cells
might not allow the Leucogate reagent
to set an adequate lymphocyte
analysis gate. The software will flag
the sample and results will not be
printed.
WARRANTY
Unless otherwise indicated in any applicable BD
general conditions of sale for non-US customers,
the following warranty applies to the purchase
of these products.
THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO
CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL
The ability of the flow cytometer to
select lymphocytes and to eliminate
platelets, red blood cells, debris,
granulocytes, and monocytes from the
lymphocyte gate depends on the
existence of a clear demarcation
between these formed elements and
lymphocytes on a display of FSC
versus SSC. For some patients, this
demarcation is not clear and
lymphocyte gating will be less
effective.
OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE
CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES,
EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF
MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND
NONINFRINGEMENT. BD’S SOLE LIABILITY IS LIMITED TO EITHER
REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE
PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY
INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL
INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT.
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LFA-1 family, and leucocyte common antigens: new
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1984:82-108.
10. PERFORMANCE CHARACTERISTICS
For lymphocyte immunophenotyping
performance data using BD Simultest
Leucogate and other BD
immunophenotyping reagents, see the
appropriate reagent IFU.
Cross-Reactivity
CD14 reacts weakly with granulocytes as
well as monocytes/macrophages.17 The
CD45 antibody has been reported to
weakly react with mature circulating
erythrocytes and platelets.8,18
8
6. Dimitriu-Bona A, Burmester G, Waters S,
Winchester R. Human mononuclear phagocyte
differentiation antigens. I. Patterns of antigenic
expression on the surface of human monocytes and
macrophages defined by monoclonal antibodies. J
Immunol. 1983;130:145-152.
7. Herrmann F, Komischke B, Odenwald E, Ludwig W.
Use of monoclonal antibodies as a diagnostic tool in
human leukemia. I. Acute myeloid leukemia and
acute phase of chronic myeloid leukemia. Blut.
1983;47:157-163.
8. Schwinzer R. Cluster report: CD45/CD45R. In:
Knapp W, Dörken B, Gilks WR, eds. Leucocyte
Typing IV: White Cell Differentiation Antigens. New
York, NY: Oxford University Press; 1989:628-634.
9. Goyert S, Tesio L, Ashman L, et al. Report on the
CD14 Cluster Workshop. In: Knapp W, Dörken B,
Gilks WR, eds. Leucocyte Typing IV: White Cell
Differentiation Antigens. New York, NY: Oxford
University Press; 1989:789-794.
10. Bernstein I, Self S. Joint report of the myeloid section
of the Second International Workshop on Human
Leukocyte Differentiation Antigens. In: Reinherz E,
Haynes B, Nadler L, Bernstein I, eds. Leukocyte
Typing II: Human Myeloid and Hematopoietic
Cells. New York, NY: Springer-Verlag; 1984:1-25.
16. Jackson A, Warner N. Preparation, staining, and
analysis by flow cytometry of peripheral blood
leukocytes. In: Rose N, Friedman H, Fahey J, eds.
Manual of Clinical Laboratory Immunology. 3rd ed.
Washington, DC: American Society for
Microbiology; 1986:226-235.
17. Jayaram Y, Hogg N. Surface expression of CD14
molecules on human neutrophils. In: Knapp W,
Dörken B, Gilks W, et al, eds. Leucocyte Typing IV:
White Cell Differentiation Antigens. New York, NY:
Oxford University Press; 1989:796-797.
18. Jackson A. Basic phenotyping of lymphocytes:
selection and testing of reagents and interpretation
of data. Clin Immunol Newslett. 1990;10:49-55.
11. Protection of Laboratory Workers from
Occupationally Acquired Infections; Approved
Guideline—Third Edition. Wayne, PA: Clinical and
Laboratory Standards Institute; 2005. CLSI
document M29-A3.
12. Centers for Disease Control. Perspectives in disease
prevention and health promotion update: universal
precautions for prevention of transmission of human
immunodeficiency virus, hepatitis B virus, and other
bloodborne pathogens in health-care settings.
MMWR. 1988;37:377-388.
13. Procedures for the Collection of Diagnostic Blood
Specimens by Venipuncture; Approved Standard—
Six Edition. Wayne, PA: Clinical and Laboratory
Standards Institute; 2007. CLSI document H3-A6.
14. Enumeration of Immunologically Defined Cell
Populations by Flow Cytometry; Approved
Guideline—Second Edition. Wayne, PA: Clinical
and Laboratory Standards Institute; 2007. CLSI
document H42-A2.
15. Giorgi JV. Lymphocyte subset measurements:
significance in clinical medicine. In: Rose NR,
Friedman H, Fahey JL, eds. Manual of Clinical
Laboratory Immunology. 3rd ed. Washington, DC:
American Society for Microbiology; 1986:236-246.
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