1.

INTENDED USE
CD33 is intended for in vitro diagnostic
CD33 (P67.6) use in the identification of cells expressing
CD33 antigen, using a BD FACS™ brand
Monoclonal mouse anti-human reagent for
identification of cells expressing CD33 antigen flow cytometer.
The flow cytometer must be equipped to
Form Catalog No. detect light scatter and the appropriate
FITC 345798 fluorescence, and be equipped with
PE 345799 appropriate analysis software (such as BD
PerCP-Cy5.5 333146 CellQuest™ Pro or BD LYSYS™ II
PE-Cy7 333952 software) for data acquisition and
APC 345800 analysis. Refer to your instrument user’s
guide for instructions.
Applications
Expression of CD33 in the
8/2013 23-5079-03
characterization of hematologic
neoplasia1,2
IVD
BD, BD Logo and all other trademarks are property of 2. COMPOSITION
Becton, Dickinson and Company. © 2013 BD
CD33, clone P67.6, is derived from
hybridization of mouse Sp2/0 myeloma
cells with spleen cells from BALB/c mice
Becton, Dickinson and Company immunized with FMY9S5 cells containing
BD Biosciences
2350 Qume Drive the CD33 gene. CD33 is composed of
San Jose, CA 95131 USA mouse IgG1 heavy chains and kappa light
Benex Limited chains.
Pottery Road, Dun Laoghaire,
Co. Dublin, Ireland Each reagent is supplied in phosphate-
Tel +353.1.202.5222 buffered saline (PBS) containing gelatin
Fax +353.1.202.5388
and 0.1% sodium azide. Concentrations
BD Biosciences are listed in Table 1.
European Customer Support
Tel +32.2.400.98.95 Table 1 Bottling Concentrations
Fax +32.2.401.70.94
help.biosciences@europe.bd.com
Conca
Becton Dickinson Pty Ltd, Form Amount provided (µg/mL)
4 Research Park Drive,
Macquarie University Research Park, FITC 3 µg in 1.0 mL of PBS 3
North Ryde NSW 2113, Australia
PE 24 µg in 2.0 mL of PBS 12
Becton Dickinson Limited,
8 Pacific Rise, Mt. Wellington, PerCP-Cy™5.5b 6.3 µg in 1.0 mL of PBS 6.3
Auckland, New Zealand
PE-Cy™7 12.5 µg in 0.5 mL of PBS 25

APC 6.2 µg in 0.5 mL of PBS 12.4

bdbiosciences.com a. Conc = concentration

ClinicalApplications@bd.com

1
b. Cy™ is a trademark of GE Healthcare. This product is subject to • Centrifuge
proprietary rights of GE Healthcare and Carnegie Mellon
University, and is made and sold under license from GE • BD CellWASH™ solution (Catalog No.
Healthcare. This product is licensed for sale only for in vitro
diagnostics. It is not licensed for any other use. If you require any 349524) or PBS buffer with 0.1%
additional license to use this product and do not have one, return sodium azide
this material, unopened, to BD Biosciences, 2350 Qume Drive, San
Jose, CA 95131, and any money paid for the material will be • BD CellFIX™ solution (Catalog No.
refunded. 340181) or 1% paraformaldehyde
solution in PBS with 0.1% sodium
Antibody purity is as follows. azide. Store at 2°C–8°C in amber glass
• FITC: ≤5% free fluorophore at bottling, for up to 1 week.
as measured by size-exclusion • BD FACS brand flow cytometer. Refer
chromatography (SEC) to the appropriate instrument user’s
• PE, PerCP-Cy5.5, PE-Cy7, APC: ≤20% guide for information.
free fluorophore at bottling, as
measured by SEC 5. SPECIMEN(S)
Reagents can be used for
3. STORAGE AND HANDLING immunophenotyping by flow cytometry
The antibody reagent is stable until the with a variety of specimen types, including
expiration date shown on the label when peripheral blood, bone marrow aspirates
stored at 2°C–8°C. Do not use after the or biopsies, and other body fluids or
expiration date. Do not freeze the reagent tissues. Each type of specimen can have
or expose it to direct light during storage different storage conditions and
or incubation with cells. Keep the outside limitations that should be considered
of the reagent vial dry. prior to collection and analysis.3,4
Do not use the reagent if you observe any Samples with large numbers of nonviable
change in appearance. Precipitation or cells can give erroneous results due to
discoloration indicates instability or selective loss of populations and to
deterioration. increased nonspecific binding of
antibodies to nonviable cells. Viability of
4. REAGENTS OR MATERIALS samples should be assessed and a cut-off
REQUIRED BUT NOT PROVIDED value established. A cut-off value of at
least 80% viable cells has been suggested.3
• Falcon®* disposable 12 x 75-mm
polystyrene test tubes or equivalent WARNING All biological specimens and
materials coming in contact with them are
• Micropipettor with tips considered biohazards. Handle as if
• Vortex mixer capable of transmitting infection5,6 and
dispose of with proper precautions in
• BD FACS™ lysing solution (10X)
accordance with federal, state, and local
(Catalog No. 349202). For dilution
regulations. Never pipette by mouth.
instructions and warnings, refer to the
Wear suitable protective clothing,
instructions for use (IFU).
eyewear, and gloves.
* Falcon is a registered trademark of Corning
Incorporated.

2
6. PROCEDURE
1. Add the appropriate volume of CD33
fluorochrome-conjugated monoclonal
antibody to 100 µL of whole blood in
a 12 x 75-mm tube. Refer to the
appropriate vial label for volume.
2. Vortex gently and incubate for 15 to
30 minutes in the dark at room
temperature (20°C–25°C).
3. Add 2 mL of 1X BD FACS lysing
solution.
4. Vortex gently and incubate for 10
minutes in the dark at room
temperature.
5. Centrifuge at 300g for 5 minutes.
Remove the supernatant.
6. Add 2 to 3 mL of BD CellWASH
solution (or wash buffer) and
centrifuge at 200g for 5 minutes.
Remove the supernatant.
7. 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.
CAUTION Some PE-Cy7 conjugates
show changes in their emission spectra
with prolonged exposure to
paraformaldehyde. For overnight
storage of stained cells, wash and
resuspend in buffer without
paraformaldehyde after 1 hour of
fixation.
3
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.
Flow Cytometry
Vortex the cells thoroughly at low speed
to reduce aggregation before running
them on the flow cytometer.7 Before
acquiring samples, adjust the threshold to
minimize debris and to ensure that
populations of interest are included.
Acquire and analyze list-mode data using
appropriate software. Figure 1 displays
representative data performed on whole
blood and gated on monocytes and
lymphocytes. Laser excitation is at
488 nm and 635 nm.
Figure 1 Representative data analyzed with a BD FACS
brand flow cytometer
APC
PE-Cy7
PerCP-Cy5.5
PE
FITC
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 IFU and the BD
FACSComp Software User’s Guide.
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.8
7. PERFORMANCE CHARACTERISTICS
Specificity
CD33 recognizes a human
myelomonocytic antigen with a molecular
weight of 67 kilodaltons (kDa).9
The CD33 antigen is present on
monocytes (bright) and granulocytes
(dim).10 Granulocytes can be further
subdivided into neutrophil, eosinophil,
and basophil populations based on CD33
staining in combination with other
cell-surface antigens.10 The CD33 antigen
is also found on CFU-Mix, CFU-GM,
CFU-Meg, a portion of BFU-E,
myeloblasts, promyelocytes, myelocytes,
and metamyelocytes, but not on earlier
precursors.1,11-13 The CD33 antigen is
expressed on blast cells in greater than
85% of acute myeloid leukemias (AMLs),
and it can be aberrantly expressed in acute
lymphoblastic leukemias (ALLs).2 Normal
lymphocytes, platelets, and erythrocytes
do not express the CD33 antigen.11
Sensitivity
Sensitivity is defined as resolution of the
CD33+ population from the CD33–
population. Sensitivity was measured by
4
evaluating a range of antibody
concentrations. Each concentration of
reagent was tested on whole blood. The
separation of CD33+ from CD33– was
determined for each sample and averaged
within each concentration. The bottled
antibody concentration for each reagent
provided optimum sensitivity in resolving
the CD33+ cells from the negative. See
Table 1.
Reproducibility
CD33 was submitted to the Fourth
International Workshop and Conference
on Human Leucocyte Differentiation
Antigens. Participating laboratories
evaluated clone P67.6 as part of a blind
panel of antibodies and reported
consistent results.9
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.
Table 2 Repeatability of MFI of monocytes across
different lots and across multiple donors (N)
Average Pooled Pooled
Na MFI SD %CVb
FITC 3 70.23 0.51 0.73
PE 26 492.5 51.48 10.45
PerCP-Cy5.5 4 81.0 21.9 27.04
Table 2 Repeatability of MFI of monocytes across Reagent performance can be affected by
different lots and across multiple donors (N) the use of other anticoagulants.
Average Pooled Pooled WARRANTY
Na MFI SD %CVb
Unless otherwise indicated in any applicable BD
PE-Cy7 3 626.83 69.55 11.10 general conditions of sale for non-US customers,
APC 3 271.2 18.05 6.66 the following warranty applies to the purchase
of these products.
a. N = number of samples
b. CV = coefficient of variation THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO
CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL
OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE
8. LIMITATIONS CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES,
EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF

Conjugates with brighter fluorochromes MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND
NONINFRINGEMENT. BD’S SOLE LIABILITY IS LIMITED TO EITHER
(PE, APC) will give greater separation REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE

than those with other dyes (FITC, PerCP). PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY
INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL
When populations overlap, calculation of INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT.

the percentage of cells positive for the

marker can be affected by the choice of TROUBLESHOOTING
fluorochrome. Problem Possible Cause Solution
Use of monoclonal antibodies in patient Poor Cell interaction Prepare and stain
treatment can interfere with recognition resolution with other cells another sample.
between debris and platelets
of target antigens by this reagent. This and
lymphocytes Rough handling Check cell viability;
should be considered when analyzing of cell centrifuge cells at
samples from patients treated in this preparation lower speed.
fashion. BD Biosciences has not Inappropriate Follow proper
instrument instrument setup
characterized the effect of the presence of settings procedures; optimize
therapeutic antibodies on the performance instrument settings
as required.
of this reagent.
Staining dim Cell Check and adjust cell
Single reagents can provide only limited or fading concentration concentration or
too high at sample volume; stain
information in the analysis of leukemias staining step with fresh sample.
and lymphomas. Using combinations of Insufficient Repeat staining with
reagents can provide more information reagent increased amount of
than using the reagents individually. antibody.
Multicolor analysis using relevant Cells not Repeat staining with
analyzed within fresh sample; analyze
combinations of reagents is highly 24 hours of promptly.
recommended.4 staining
Improper Use sodium azide in
As reagents can be used in different medium staining medium and
combinations, laboratories need to preparation washing steps.
(sodium azide
become familiar with the properties of omitted)
each antibody in conjunction with other
markers in normal and abnormal samples.
Reagent performance data was collected
typically with EDTA-treated blood.

5
Problem Possible Cause Solution 10. Terstappen LW, Hollander Z, Meiners H, Loken
MR. Quantitative comparison of myeloid antigens
Few or no cells Cell Resuspend fresh on five lineages of mature peripheral blood cells. J
concentration sample at a higher Leuk Biol. 1990;48:138-148.
too low concentration; repeat
staining and 11. Bernstein ID, Singer JW, Andrews RG, et al.
analysis. Treatment of acute myeloid leukemia cells in vitro
Cytometer Troubleshoot with a monoclonal antibody recognizing a myeloid
malfunctioning instrument. differentiation antigen allows normal progenitor
cells to be expressed. J Clin Invest. 1987;79:1153-
1159.
REFERENCES 12. Andrews RG, Torok-Storb B, Bernstein ID. Myeloid-
1. Dinndorf PA, Andrews RG, Benjamin D, associated differentiation antigens on stem cells and
Ridgway D, Wolff L, Bernstein ID. Expression of their progeny identified by monoclonal antibodies.
normal myeloid-associated antigens by acute Blood. 1983;62:124-132.
leukemia cells. Blood. 1986;67:1048-1053. 13. Andrews RG, Takahashi M, Segal GM, Powell JS,
2. Foon KA, Todd RF III. Immunologic classification of Bernstein ID, Singer JW. The L4F3 antigen is
leukemia and lymphoma. Blood. 1986;68:1-31. expressed by unipotent and multipotent colony-
forming cells but not by their precursors. Blood.
3. Rothe G, Schmitz G. Consensus protocol for the
1986;68:1030-1035.
flow cytometric immunophenotyping of
hematopoietic malignancies. Leukemia.
1996;10:877-895.
4. 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;30:214-
230.
5. Protection of Laboratory Workers from
Occupationally Acquired Infections; Approved
Guideline—Third Edition. Wayne, PA: Clinical and
Laboratory Standards Institute; 2005. CLSI
document M29-A3.
6. 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.
7. 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.
8. 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.
9. Köller U, Peschel CH. Cluster report: CD33. In:
Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte
Typing IV: White Cell Differentiation Antigens. New
York, NY: Oxford University Press; 1989:812-813.