1. INTENDED USE
BD CellFIX™ is intended for the fixation
of human peripheral blood cell
suspensions after immunofluorescence
staining with monoclonal antibodies and
prior to flow cytometric analysis.
BD CellFIX™
10X concentrate—Catalog No. 340181
2. SUMMARY AND EXPLANATION
BD CellFIX is a premixed fixing
concentrate, especially formulated for the
treatment of samples prior to analysis
with (automated) flow cytometers such as
the BD FACS™ brand instruments.
3. PRINCIPLES OF THE PROCEDURE
03/2015
The interaction of BD CellFIX with
human peripheral blood cells results in
cross-linking of membrane-bound
proteins. BD CellFIX effectively preserves
stained human lymphocytes for at least 24
hours without significant alteration of
lymphocyte subset enumeration values
and fluorescence intensity results. The
fixative may be used in conjunction with
monoclonal antibodies conjugated to
FITC, PE, PerCP, PerCP-Cy™5.5*, PECy™7, APC and APC-Cy7†. Prior to flow
cytometric analysis, whole blood is
stained with monoclonal antibodies,
treated with BD FACS™ lysing solution to
lyse erythrocytes, then washed and fixed
with BD CellFIX.
23-12416-02
IVD
BD, BD Logo and all other trademarks are property of
Becton, Dickinson and Company. © 2015 BD
Becton Dickinson Benelux N.V.
Becton, Dickinson and Company
Erembodegem-Dorp 86
B-9320 Erembodegem
Belgium
Tel 32.2.400.98.95
Fax 32.2.401.70.94
Benex Limited
Pottery Road, Dun Laoghaire,
Co. Dublin, Ireland
Tel +353.1.202.5222
Fax +353.1.202.5388
BD Biosciences
European Customer Support
Tel +32.2.400.98.95
Fax +32.2.401.70.94
help.biosciences@europe.bd.com
* Cy™ is a trademark of GE Healthcare. This
product is subject to proprietary rights of GE
Healthcare and Carnegie Mellon University, and is
made and sold under license from GE Healthcare.
This product is licensed for sale only for in vitro
diagnostics. It is not licensed for any other use. If
you require any additional license to use this
product and do not have one, return this
material, unopened, to BD Biosciences, 2350
Qume Drive, San Jose, CA 95131, and any money
paid for the material will be refunded.
† APC-Cy7: US patent 5,714,386
bdbiosciences.com
ClinicalApplications@bd.com
1
capable of transmitting infection1,2 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.
4. REAGENT
Reagent Provided
BD CellFIX 10X concentrate, 50 mL
(sufficient for 1000 fixations) of a
proprietary buffered solution.
Precautions
Dilution Instructions
For use, dilute BD CellFIX 1:10 with
reagent-grade (distilled and deionized)
water. Store in a glass container at room
temperature (20°C–25°C) for up to one
month.
Solution contains 10.0% formaldehyde,
CAS number 50-00-0, 3.55% methanol,
CAS number 67-56-1, and 0.93% sodium
azide, CAS number 26628-22-8.
Danger
Storage and Handling
When stored at 20°C–25°C, the reagent
concentrate is stable until the expiration
date shown on the label. Do not use after
the expiration date.
H311 Toxic in contact with skin.
H331 Toxic if inhaled.
H341 Suspected of causing genetic
defects.
H350 May cause cancer. Route of
exposure: Inhalative.
H371-H335 May cause damage to
organs. May cause respiratory irritation.
H373 May cause damage to the kidneys
through prolonged or repeated exposure.
Route of exposure: Oral.
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.
H318 Causes serious eye damage.
5. SPECIMEN COLLECTION AND
PREPARATION
Collect blood aseptically by venipuncture
into a sterile BD Vacutainer® EDTA
blood collection tube or equivalent. Store
anticoagulated blood at room temperature
(20°C–25°C) for up to 6 hours until ready
for staining.
H302 Harmful if swallowed.
H315 Causes skin irritation.
H317 May cause an allergic skin
reaction.
H413 May cause long lasting harmful
effects to aquatic life.
Wear protective gloves / eye protection.
Wear protective clothing. Avoid
breathing mist/vapours/spray. IF IN
EYES: Rinse cautiously with water for
several minutes. Remove contact lenses,
if present and easy to do. Continue
rinsing. IF INHALED: Remove victim to
fresh air and keep at rest in a position
comfortable for breathing. IF
SWALLOWED: Call a POISON
CENTER/doctor if you feel unwell.
CAUTION Use standard precautions when
obtaining, handling, and disposing of all
human blood samples and/or potentially
carcinogenic reagents.
6. PROCEDURE
Reagent provided
BD CellFIX, 10X concentrate (Catalog
No. 340181)
WARNING All biological specimens and
materials coming into contact with them
are considered biohazards. Handle as if
2
use (IFU). Use care to protect tubes
from direct light.
Reagents and Materials Required but Not
Provided
•
BD Vacutainer EDTA blood collection
tubes or equivalent.
•
Falcon®‡ disposable 12 x 75-mm
polystyrene test tubes or equivalent.
•
Micropipettor with tips.
•
5- or 10-mL serological pipet.
•
BD monoclonal antibodies to human
leucocyte antigens.
•
Vortex mixer.
•
Low-speed centrifuge (minimum speed
200g) with swinging bucket rotor with
12 x 75-mm tube carriers.
•
Vacuum aspirator with trap.
•
Reagent-grade (both distilled and
deionized) water.
•
BD FACS lysing solution (10X)
(Catalog No. 349202)
•
BD CellWASH™ (Catalog No. 349524)
or a wash buffer of phosphate buffered
saline (PBS) with 0.1% sodium azide.
•
2. In the case of PBMCs, proceed to
step 3. In the case of whole blood
samples, lyse red blood cells following
staining, using diluted BD FACS lysing
solution (1X). Use care to protect
tubes from direct light. Perform the
procedure (incubation and
centrifugation) as directed in the
specific reagent data sheet or IFU or
the BD FACS lysing solution IFU.
Discard or aspirate the supernatant,
leaving approximately 50 µL of
residual fluid in the tube to avoid
disturbing the pellet.
3. Vortex thoroughly at low speed to
resuspend the cell pellet. Add 2 mL of
BD CellWASH solution or 1X PBS
with 0.1% sodium azide to the cell
suspensions. Vortex thoroughly at low
speed for 3 seconds. Centrifuge at
200g for 5 minutes at room
temperature (20°C–25°C).
4. Discard or aspirate the supernatant,
leaving approximately 50 µL of
residual fluid in the tube to avoid
disturbing the pellet.
1X PBS (Dulbecco’s modified PBS, pH
7.2 ±0.2, 0.01 mol/L PO4 and 0.15 mol/
L NaCl)3. This reagent does not contain
calcium, magnesium, phenol red, or
sodium azide. Filter PBS through a 0.2µm filter before use. Store at 2°C–8°C.
5. Vortex thoroughly at low speed to
resuspend the cell pellet in the residual
fluid.
6. Dilute room-temperature (20°C–
25°C) 10X BD CellFIX to 1X,
following the instructions in Reagent,
Section 4. Add 0.5 mL of 1X
BD CellFIX solution to each tube
except if the monoclonal antibody
instructions for use specify other
volumes. Vortex thoroughly at low
speed for 3 seconds. Make sure that
the cell suspension is well mixed with
BD CellFIX.
Preparing Samples
1. Stain whole blood or peripheral blood
mononuclear cells (PBMCs) with
monoclonal antibodies using the
amounts, incubation times, and
temperatures specified in the
appropriate reagent instructions for
‡ Falcon is a registered trademark of Corning
Incorporated.
3
7. Tubes 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. Analyze the fixed cells within
24 hours. Vortex the cells thoroughly
at low speed to help reduce
aggregation before running them on
the flow cytometer.
8. PERFORMANCE CHARACTERISTICS
Performance
Results obtained for cell preparations
fixed with BD CellFIX were compared to
non-fixed cell preparations (PBS) and to
cell preparations fixed with a freshly
prepared fixative solution (1X PBS, 1%
paraformaldehyde, 0.1% sodium azide).
Blood samples of one donor were
prepared for analysis with BD Multitest
reagents CD3/CD8/CD45/CD4 and CD3/
CD16+CD56/CD45/CD19. Lymphocyte
subsets (in %) were determined in nonfixed samples, in samples treated with the
reference fixative solution, and in samples
treated with 6 different BD CellFIX lots
identified as a through f. Cells were
analyzed immediately after preparation
(T=0 h) and after 24 hours (T=24 h).
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.
7. EXPECTED RESULTS
Figure 1 % T lymphocytes in non-fixed blood cell
preparations (--), in cell preparations fixed with 1%
paraformaldehyde solution/0.1% sodium azide
(reference fixative), and in blood cell preparations fixed
with BD CellFIX lot a, b, c, d, e or f. % NK lymphocytes
in non-fixed blood cell preparations (--),in cell
preparations fixed with 1% paraformaldehyde
solution/0.1% sodium azide (reference fixative), and in
blood cell preparations fixed with BD CellFIX lot a, b, c,
d, e, or f. % B lymphocytes in non-fixed blood cell
preparations (--), in cell preparations fixed with 1%
paraformaldehyde solution/0.1% sodium azide
Aliquots of blood specimens from normal
and abnormal donors were stained with
BD Multitest™ reagents CD3/CD8/CD45/
CD4 and CD3/CD16+CD56/CD45/
CD19, then lysed with BD FACS lysing
solution, washed, and fixed with
BD CellFIX (see Section 6, Preparing
Samples). Samples were analyzed on a
BD FACSCalibur™ flow cytometer with
BD Multiset™ software, and lymphocyte
subset populations (in %) were
determined. The reference ranges for the
parameters studied are presented in the
following table. Refer to Section 9 for
limitations about reference ranges.
Lymphocyte
subset
BD Multiset
software (n=29)
Reichert et al4
(n=271)
Mean
Reference
range
Mean
Reference
range
%CD3+
68
55–84
73 (6)a
61–85
%CD16+CD56+
15
5–27
14 (6)a
6–29
%CD19+
14
6–25
14 (4)a
7–3
a. Values given in parentheses as standard deviations (SD).
4
with BD CellFIX, and lymphocyte subsets
were determined. Samples were run on the
BD FACSCalibur instrument immediately
after treatment (T = 0 h) and after 24
hours (T = 24 h).
(reference fixative), and in blood cell preparations fixed
with BD CellFIX lot a, b, c, d, e, or f.
T=0h
T=24h
% T Lymphs, no fixation
CellFIX lot
f
CellFIX lot
e
CellFIX lot
d
CellFIX lot
c
CellFIX lot
b
Mean
SD
%CVa
30
71
0.79
1.11
%CD16+CD56+
30
12
0.63
5.30
%CD19+
30
14
0.77
5.35
n
Mean
SD
%CVa
T=0h
%CD3+
30
72
0.81
1.12
T=24h
%CD16+CD56+
30
11
0.96
8.79
% NK Lymphs, no fixation
%CD19+
30
14
0.97
7.12
40
30
30
20
20
10
10
a. CV = coefficient of variation
Accuracy
To determine accuracy of BD CellFIX,
flow analysis of BD CellFIX treated and
non-BD CellFIX treated 4-color stained
cell preparations was performed, and
resulting lymphocyte subsets were
compared. Aliquots of blood specimens
from 29 normal or abnormal donors were
stained, lysed, washed, and fixed with
BD CellFIX and immediately run on a
BD FACSCalibur flow cytometer. Aliquots
of same blood specimens were prepared in
a similar way for 4-color analysis but
were not fixed with BD CellFIX.
CellFIX lot
f
CellFIX lot
e
CellFIX lot
d
CellFIX lot
c
0
CellFIX lot
b
0
CellFIX lot
a
n
%CD3+
a. CV = coefficient of variation
T=0h
T=24h
% B Lymphs, no fixation
40
40
% B Lymphocytes
Lymphocyte
subset at T=0h
Lymphocyte
subset at T=24h
40
Reference
Fixative
% NK Lymphocytes
CellFIX lot
a
100
90
80
70
60
50
40
30
20
10
0
Reference
Fixative
% T Lymphocytes
100
90
80
70
60
50
40
30
20
10
0
30
30
20
20
10
10
0
Figure 2 % T lymphocytes in non-fixed blood cell
preparations vs % T lymphocytes in cell preparations
fixed with BD CellFIX; % NK lymphocytes in non-fixed
blood cell preparations vs % NK lymphocytes in cell
preparations fixed with BD CellFIX; % B lymphocytes in
CellFIX lot
f
CellFIX lot
e
CellFIX lot
d
CellFIX lot
c
CellFIX lot
b
CellFIX lot
a
Reference
Fixative
0
Precision
Repeatability was assessed using the
BD Multitest reagents CD3/CD8/CD45/
CD4 and CD3/CD16+CD56/CD45/
CD19. Thirty aliquots of the same blood
sample were stained, lysed, and treated
5
non-fixed blood cell preparations vs % B lymphocytes
in cell preparations fixed with BD CellFIX.
% T Lymphocytes
% T Lymphs in blood cell
preparations fixed
with CellFIX
Expected values
100
90
80
70
60
50
40
30
20
10
0
EDTA is the anticoagulant of choice.
BD Biosciences has limited information
concerning use of other anticoagulants.
•
Samples should be processed within 6
hours of collection. Store blood samples
in BD Vacutainer blood collection tubes
or equivalent at room temperature prior
to staining, lysing, and fixing.
•
Incubation times or temperatures other
than those specified may be a source of
error.
•
The volumes of reagent recommended
are based on studies of normal human
blood.
y = 0,9774x + 1,1417
R 2 = 0,9894
0
10 20 30 40 50 60 70 80 90 100
% T Lymphs in non-fixed blood cell preparations
% NK Lymphocytes
WARRANTY
Expected values
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.
40
% NK Lymphs in blood cell
preparations fixed with
CellFIX
•
y = 1,022x - 0,1176
30
R 2 = 0,9818
20
THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO
CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL
10
OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE
CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES,
0
0
10
20
30
EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF
40
MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND
% NK Lymphs in non-fixed blood cell preparations
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
% B Lymphocytes
INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT.
Expected values
REFERENCES
50
40
% B Lymphs in blood cell
preparations fixed
with CellFIX
1. Protection of Laboratory Workers from
Occupationally Acquired Infections; Approved
Guideline—Third Edition. Wayne, PA: Clinical and
Laboratory Standards Institute; 2005. M29-A3.
2. 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.
3. 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.
y = 0,9774x + 0,3194
2
R = 0,9884
30
20
10
0
0
10
20
30
40
50
% B Lymphs in non-fixed blood cell preparations
9. LIMITATIONS
•
Each laboratory should establish
normal ranges for leucocyte subsets
using its own test conditions.
6
4. Reichert TA, DeBruyere M, Deneys V, et al.
Lymphocyte subset reference ranges in adult
Caucasians. Clin Immunol Immunopathol. 1991;
60:190-208.
7