1. INTENDED USE
Anti–TCR-α/β is intended for in vitro
diagnostic use in the identification of cells
expressing TCR-α/β antigen, using a
BD FACS™ brand flow cytometer.
Anti–TCR-α/β (WT31)
Monoclonal mouse anti-human reagent for
identification of cells expressing TCR-α/β antigen
Form
Catalog No.
FITC
333140
11/2013
The flow cytometer must be equipped to
detect light scatter and the appropriate
fluorescence, and be equipped with
appropriate analysis software (such as BD
CellQuest™ or BD LYSYS™ II software)
for data acquisition and analysis. Refer to
your instrument user’s guide for
instructions.
Applications
Expression of TCR-α/β antigen in the
characterization of hematologic
neoplasia1,2
23-7550-02
IVD
2. COMPOSITION
BD, BD Logo and all other trademarks are property of
Becton, Dickinson and Company. © 2013 BD
Anti–TCR-α/β, clone WT31, is derived
from hybridization of mouse Sp2/0-Ag14
myeloma cells with spleen cells from
BALB/c mice immunized with human
thymocytes.3 Anti–TCR-α/β is composed
of mouse IgG1 heavy chains and kappa
light chains.3
Becton, Dickinson and Company
BD Biosciences
2350 Qume Drive
San Jose, CA 95131 USA
Benex Limited
Pottery Road, Dun Laoghaire,
Co. Dublin, Ireland
Tel +353.1.202.5222
Fax +353.1.202.5388
Each reagent is supplied in phosphatebuffered saline (PBS) containing gelatin
and 0.1% sodium azide. Concentrations
are listed in Table 1.
BD Biosciences
European Customer Support
Tel +32.2.400.98.95
Fax +32.2.401.70.94
help.biosciences@europe.bd.com
Table 1 Bottling concentrations
Becton Dickinson Pty Ltd,
4 Research Park Drive,
Macquarie University Research Park,
North Ryde NSW 2113, Australia
Form
Amount provided
Conca (µg/mL)
FITC
100 µg in 2.0 mL of PBS
50
a. Conc = concentration
Becton Dickinson Limited,
8 Pacific Rise, Mt. Wellington,
Auckland, New Zealand
Antibody purity is as follows.
•
bdbiosciences.com
ClinicalApplications@bd.com
1
FITC: ≤5% free fluorophore at bottling,
as measured by size-exclusion
chromatography (SEC)
3. STORAGE AND HANDLING
5. SPECIMEN(S)
The antibody reagent is stable until the
expiration date shown on the label when
stored at 2°C–8°C. Do not use after the
expiration date. Do not freeze the reagent
or expose it to direct light during storage
or incubation with cells. Keep the outside
of the reagent vial dry.
Reagents can be used for
immunophenotyping by flow cytometry
with a variety of specimen types, including
peripheral blood, bone marrow aspirates
or biopsies, and other body fluids or
tissues. Each type of specimen can have
different storage conditions and
limitations that should be considered
prior to collection and analysis.4,5
Do not use the reagent if you observe any
change in appearance. Precipitation or
discoloration indicates instability or
deterioration.
Samples with large numbers of nonviable
cells can give erroneous results due to
selective loss of populations and to
increased nonspecific binding of
antibodies to nonviable cells. Viability of
samples should be assessed and a cut-off
value established. A cut-off value of at
least 80% viable cells has been suggested.4
4. REAGENTS OR MATERIALS
REQUIRED BUT NOT PROVIDED
•
Falcon®* disposable 12 x 75-mm
polystyrene test tubes or equivalent
•
Micropipettor with tips
•
Vortex mixer
•
CD3 PE (Catalog No. 347347)
•
Centrifuge
•
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.
•
WARNING All biological specimens and
materials coming in contact with them are
considered biohazards. Handle as if
capable of transmitting infection6,7 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.
6. PROCEDURE
1. In a 12 x 75-mm tube, add 20 µL of
reagent to 106 peripheral blood
mononuclear cells (PBMCs) in 50 µL
of medium containing 0.1% sodium
azide.
BD FACS brand flow cytometer. Refer
to the appropriate instrument user’s
guide for information.
2. Mix thoroughly and incubate for
15 minutes in the dark at 2°C–8°C.
3. Add 20 µL of CD3 PE.
4. Mix thoroughly and incubate again
for 15 minutes at 2°C–8°C.
5. Wash with BD CellWASH solution (or
wash buffer).
* Falcon is a registered trademark of Corning
Incorporated.
2
6. Add 0.5 mL of BD CellFIX solution
(or 1% paraformaldehyde solution)
and mix thoroughly. Store at 2°C–8°C
until analyzed. We recommend
analyzing within 24 hours of staining.
gated on lymphocytes. Laser excitation is
at 488 nm.
Figure 1 Representative data analyzed with a
BD FACS brand flow cytometer
Number of Cells
CAUTION Anti–TCR-α/β is partially
inhibited by the binding of antibodies
against the CD3 antigen.8 Anti–TCR-α/β
FITC and Anti-CD3, clone SK7, PE at the
supplied concentrations have been tested
and are recommended for two-color
immunofluorescence studies. Problems
can result from the use of other antibodies
directed against the CD3 antigen when
used in conjunction with Anti–TCR-α/β.
The reciprocal combination, Anti-CD3
FITC and Anti–TCR-α/β Biotin with a
second-step reagent, is likewise not
recommended due to steric hindrance
considerations. Since the TCR-1 antigen is
labile in tissue section preparations, the
performance of Anti–TCR-α/β in
immunohistologic applications is
suboptimal.
Mouse IgG1 FITC
69%
1
Anti-TCR-α/β FITC
2
3
Internal Quality Control
We recommend using BD Calibrite™
beads and BD FACSComp™ software to
set photomultiplier tube (PMT) voltages,
fluorescence compensation, and to check
instrument sensitivity prior to use. Refer
to the BD Calibrite Beads instructions for
use (IFU) and the BD FACSComp
Software User’s Guide.
Analytical Results
Abnormal numbers of cells expressing this
antigen or aberrant expression levels of
the antigen can be expected in some
disease states. It is important to
understand the normal expression pattern
for this antigen and its relationship to
expression of other relevant antigens in
order to perform appropriate analysis.
We recommend running a control sample
daily from a normal adult subject or a
commercially available whole blood
control to optimize instrument settings
and as a quality control check of the
system.10
7. PERFORMANCE CHARACTERISTICS
Flow Cytometry
Vortex the cells thoroughly at low speed
to reduce aggregation before running
them on the flow cytometer.9 Acquire and
analyze list-mode data using appropriate
software. Before acquiring samples, adjust
the threshold to minimize debris and
ensure populations of interest are
included. Figure 1 displays representative
data performed on normal blood and
Specificity
Anti–TCR-α/β recognizes a
conformational epitope formed by the
T-cell receptor (TCR) for antigen and the
CD3 epsilon chain.8,11 The α/β TCR is a
disulfide-linked 80-kilodalton (kDa)
heterodimer consisting of a 44-kDa α
chain and a 37-kDa β chain.8,12
3
WT31 (Anti–TCR-α/β) identifies
approximately 97% of normal peripheral
blood T lymphocytes that express the
CD3 antigen.13 The TCR-α/β antigen is
also expressed on 50% to 70% of
thymocytes.13 A small subset of normal
peripheral blood T lymphocytes (1% to
9%) and thymocytes (less than 2%) lacks
reactivity or reacts weakly with Anti–
TCR-α/β.3,13,14 This CD3+ TCR-α/β–
subset predominantly expresses the γ/δ
chains of the TCR complex.3,13,14
concentrations. Each concentration of
reagent was tested on PBMCs. The
separation of TCR-α/β+ from TCR-α/β–
was determined for each sample and
averaged within each concentration. The
bottled antibody concentration for each
reagent provided optimum sensitivity in
resolving the TCR-α/β+ cells from the
negative. See Table 1.
Repeatability
To determine the repeatability of staining
with each reagent, samples were stained
with multiple lots of reagents. The
different samples used in the evaluation
provided an average mean fluorescence
intensity (MFI) value as shown in Table 2.
For each sample, two different lots of
reagents generated a pair of results.
Individual standard deviations (SDs) were
determined from the paired results for
each sample. Individual SDs were
combined to derive a pooled SD for each
reagent that provides an estimate of
within-sample repeatability.
NOTE
The weak reactivity described is
caused by WT31 recognizing a
conformational epitope that exists when γ
or δ subunits are associated with the
CD3ε chain.11 Figure 2 (plots A and B)
show the effects of the absence or
presence of CD3 on WT31 and TCR-γ/δ
staining patterns. In cases where a CD3ε–
transfectant (or other system) is
employed, the WT31 antibody might fail
to detect the presence of α/β subunits.
Anti–TCR-α/β is mitogenic for resting
peripheral blood T lymphocytes from
responding individuals.15-17
Table 2 Repeatability of MFI of Anti–TCR-α/β+
lymphocytes across different lots and across
multiple donors (N)
Figure 2 Two-parameter analysis of lysed whole
blood stained with WT31 (Anti–TCR-α/β-1) FITC and
TCR-γ/δ PE
Plot B
FITC
WT31 FITC
Average
MFI
Pooled
SD
Pooled
%CVb
7
97.06
6.34
6.53
a. N = number of samples
b. CV = coefficient of variation
TCR-γ/δ PE
TCR-γ/δ PE
Plot A
Na
8. LIMITATIONS
Conjugates with brighter fluorochromes
(PE, APC) will give greater separation
than those with other dyes (FITC, PerCP).
When populations overlap, calculation of
the percentage of cells positive for the
marker can be affected by the choice of
fluorochrome.
WT31 FITC
Sensitivity
Sensitivity is defined as resolution of the
TCR-α/β+ population from the TCR-α/β–
population. Sensitivity was measured by
evaluating a range of antibody
4
Use of monoclonal antibodies in patient
treatment can interfere with recognition
of target antigens by this reagent. This
should be considered when analyzing
samples from patients treated in this
fashion. BD Biosciences has not
characterized the effect of the presence of
therapeutic antibodies on the performance
of this reagent.
TROUBLESHOOTING
Single reagents can provide only limited
information in the analysis of leukemias
and lymphomas. Using combinations of
reagents can provide more information
than using the reagents individually.
Multicolor analysis using relevant
combinations of reagents is highly
recommended.5
Problem
Possible Cause
Poor
resolution
between debris
and
lymphocytes
Cell interaction Prepare and stain
with other cells another sample.
and platelets
Staining dim
or fading
Solution
Rough handling Check cell viability;
of cell
centrifuge cells at
preparation
lower speed.
Inappropriate
instrument
settings
Follow proper
instrument setup
procedures; optimize
instrument settings
as required.
Cell
concentration
too high at
staining step
Check and adjust cell
concentration or
sample volume; stain
with fresh sample.
Insufficient
reagent
Repeat staining with
increased amount of
antibody.
Cells not
Repeat staining with
analyzed within fresh sample; analyze
24 hours of
promptly.
staining
As reagents can be used in different
combinations, laboratories need to
become familiar with the properties of
each antibody in conjunction with other
markers in normal and abnormal samples.
Reagent performance data was collected
typically with EDTA-treated blood.
Reagent performance can be affected by
the use of other anticoagulants.
Improper
medium
preparation
(sodium azide
omitted)
Use sodium azide in
staining medium and
washing steps.
Few or no cells Cell
concentration
too low
Resuspend fresh
sample at a higher
concentration; repeat
staining and
analysis.
Cytometer
malfunctioning
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.
Troubleshoot
instrument.
REFERENCES
1. Spits H, Paliard X, Vandekerckhove Y, van
Vlasselaer P, de Vries JE. Functional and phenotypic
differences between CD4+ and CD4– T cell receptorgamma delta clones from peripheral blood. J
Immunol. 1991;147:1180-1188.
2. Knowles DM. Immunophenotypic markers useful in
the diagnosis and classification of hematopoietic
neoplasms. In: Knowles DM, Thompson DD, eds.
Neoplastic Hematopathology. 2nd ed. Philadelphia:
Lippincott Williams & Wilkins; 2001:93-226.
3. Weiss A, Newton M, Crommie D. Expression of T3
in association with a molecule distinct from the
T cell antigen receptor heterodimer. Proc Natl Acad
Sci USA. 1986;83:6998-7002.
THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO
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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.
5
4. Rothe G, Schmitz G. Consensus protocol for the
flow cytometric immunophenotyping of
hematopoietic malignancies. Leukemia.
1996;10:877-895.
5. Stelzer GT, Marti G, Hurley A, McCoy P Jr,
Lovett EJ, Schwartz A. US-Canadian Consensus
recommendations on the immunophenotypic
analysis of hematologic neoplasia by flow
cytometry: standardization and validation of
laboratory procedures. Cytometry. 1997;30:214230.
6. Protection of Laboratory Workers from
Occupationally Acquired Infections; Approved
Guideline—Third Edition. Wayne, PA: Clinical and
Laboratory Standards Institute; 2005. CLSI
document M29-A3.
7. 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.
8. Spits H, Borst J, Tax W, Capel P, Terhorst C, de Vries
J. Characteristics of a monoclonal antibody (WT-31)
that recognizes a common epitope on the human T
cell receptor for antigen. J Immunol.
1985;135:1922-1928.
9. Jackson AL, Warner NL. Preparation, staining, and
analysis by flow cytometry of peripheral blood
leukocytes. In: Rose NR, Friedman H, Fahey JL, eds.
Manual of Clinical Laboratory Immunology. 3rd ed.
Washington, DC: American Society for
Microbiology; 1986:226-235.
10. 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.
11. Salmerón A, Sanchez-Madrid F, Ursa M, Fresno M,
Alarcón B. A conformational eptitope expressed
upon association of CD3ε with either CD3-δ or
CD3-γ is the main target for recognition by
Anti-CD3 monoclonal antibodies. J Immunol.
1991;147:3047-3052.
12. Oettgen H, Kappler J, Tax W, Terhorst C.
Characterization of the two heavy chains of the T3
complex on the surface of human T lymphocytes.
J Biol Chem. 1984;259:12039-12048.
13. Lanier LL, Weiss A. Presence of Ti (WT31) negative
T lymphocytes in peripheral blood and thymus.
Nature. 1986;324:268-270.
14. Brenner MB, McClean J, Dialynas DP, et al.
Identification of a putative second T cell receptor.
Nature. 1986;322:145-149.
15. Gupta S, Shimizu M, Ohira K, Vayuvegula B. T-cell
activation via the T-cell receptor: a comparison
between WT31 (defining alpha/beta TCR)-induced
and anti-CD3-induced activation of human
T lymphocytes. Cell Immunol. 1991;132:26-44.
16. Tax W, Leeuwenberg H, Willems H, Capel P,
Koene R. Monoclonal antibodies reactive with
OKT3 antigen or OKT8 antigen. In: Bernard A,
Boumsell L, Dausset J, Milstein C, Schlossman S,
eds. Leucocyte Typing. New York, NY: SpringerVerlag; 1984:721-722.
17. Tax WJ, Willems HW, Reekers PP, Capel PJ, Koene
RA. Polymorphism in mitogenic effect of IgG1
monoclonal antibodies against T3 antigen on human
T cells. Nature. 1983;304:445-447.
6