BD Simultest™
IMK-Lymphocyte
Catalog No. 340182
IVD
BD, BD Logo and all other trademarks are property of
Becton, Dickinson and Company. © 2015 BD
02/2015
23-2568-06
Becton, Dickinson and Company
BD Biosciences
2350 Qume Drive
San Jose, CA 95131 USA
Becton Dickinson Pty Ltd,
4 Research Park Drive,
Macquarie University Research Park,
North Ryde NSW 2113, Australia
Benex Limited
Pottery Road, Dun Laoghaire,
Co. Dublin, Ireland
Tel +353.1.202.5222
Fax +353.1.202.5388
Becton Dickinson Limited,
8 Pacific Rise, Mt. Wellington,
Auckland, New Zealand
BD Biosciences
European Customer Support
Tel +32.2.400.98.95
Fax +32.2.401.70.94
[email protected]
bdbiosciences.com
[email protected]
CONTENTS
1. INTENDED USE ............................................................................ 5
2. SUMMARY AND EXPLANATION ................................................ 5
Clinical Applications .................................................................... 7
3. PRINCIPLES OF THE PROCEDURE................................................ 8
4. REAGENTS ................................................................................. 11
Reagents Provided, Sufficient for 50 Tests .................................. 11
Precautions ................................................................................. 17
5. INSTRUMENT............................................................................. 18
6. SPECIMEN COLLECTION AND PREPARATION .......................... 19
Interfering Conditions ................................................................ 20
7. PROCEDURE .............................................................................. 20
Reagents Provided ...................................................................... 20
Reagents and Materials Required But Not Provided................... 20
Staining and Fixing the Cells ...................................................... 21
Flow Cytometry.......................................................................... 23
Quality Control .......................................................................... 28
8. RESULTS..................................................................................... 30
Percent Lymphocyte Conversion................................................. 30
Three-Part Differential................................................................ 33
Absolute Counts ......................................................................... 34
9. LIMITATIONS ............................................................................. 35
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10. EXPECTED VALUES ................................................................... 39
Leucocyte Subsets ....................................................................... 39
Absolute Counts ......................................................................... 41
11. PERFORMANCE CHARACTERISTICS.......................................... 43
Within-Sample Reproducibility................................................... 43
Between-Instrument Reproducibility........................................... 44
Between-Laboratory Reproducibility .......................................... 46
Simultest IMK-Lymphocyte vs Comparative Method ................. 46
Stability of Stained Cell Preparations.......................................... 47
Cross-Reactivity ......................................................................... 48
Linearity-Recovery ..................................................................... 48
12. TROUBLESHOOTING ................................................................. 49
WARRANTY..................................................................................... 52
REFERENCES .................................................................................... 52
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1. INTENDED USE
BD Simultest™ IMK-Lymphocyte is a two-color direct
immunofluorescence reagent kit for enumerating percentages of the
following mature human leucocyte subsets in erythrocyte-lysed whole
blood (LWB): T (CD3+) lymphocytes, B (CD19+) lymphocytes, helper/
inducer T (CD3+CD4+) lymphocytes, suppressor/cytotoxic
T (CD3+CD8+) lymphocytes, and natural killer (NK) (CD3–CD16+
and/or CD56+) lymphocytes. The helper/suppressor T-lymphocyte
ratio (CD3+CD4+/ CD3+CD8+) can also be determined.
2. SUMMARY AND EXPLANATION
Human lymphocytes can be divided into three major populations based
on their biologic function and cell-surface antigen expression:
T lymphocytes, B lymphocytes, and NK lymphocytes. T lymphocytes
participate in antigen-specific cell-mediated immunity and regulate the
secretion of immunoglobulin by B lymphocytes. T lymphocytes can
also be classified based on their functional properties as helper/inducer
and suppressor/cytotoxic. NK lymphocytes mediate cytotoxicity
against certain tumors and virus-infected target cells, without requiring
the presence of class I or II major histocompatibility complex (MHC)
molecules on the target.1
Historical methods for assaying lymphocyte subsets were problematic
because they were technique-dependent, time-consuming, or not highly
cell lineage–specific. Classic methods included identifying
T lymphocytes by rosetting them with sheep red blood cells (SRBCs)2-4
and identifying B lymphocytes with fluorochrome-labeled polyclonal
antibodies to surface immunoglobulin.5-7
Antibody reagents have been used to distinguish cell-surface antigens
by fluorescence microscopy, electron microscopy, and radioactive label
assay.8 Microscopy methods are not highly sensitive because of the
limited number of cells that can be analyzed, while methods employing
the use of radioactive isotopes require additional safety and waste
5
disposal considerations. Cytolytic lymphocyte assays included the
hemolytic plaque assay9 and a cytotoxicity assay to detect the release
of radioactive chromium.10 The development of monoclonal
antibodies to human leucocyte antigens provided reagents of singledefined specificity, a major improvement over heterologous antisera.11
Flow cytometry improved the sensitivity of antigen detection and the
reproducibility of leucocyte subset identification compared to
microscopy.12 Flow cytometry using indirect immunofluorescence,
while providing assay sensitivity,13 requires an additional step to add
the fluorochrome-labeled secondary reagent.
Lymphocytes from human whole blood traditionally were prepared for
flow cytometric analysis using density-gradient separation methods.
Studies have shown that these methods were time-consuming, involved
multiple blood-handling steps, and could result in the loss of
lymphocyte subsets.12,14-16 The LWB method used with BD Simultest
IMK-Lymphocyte does not require density-gradient separation and
therefore allows for shorter sample preparation time and less handling
of whole blood12 (refer to the BD Monoclonal Antibodies Source
Book, Section 2.2). Studies have also shown that the LWB method is
less likely to show loss of lymphocyte subsets and can help improve
assay reproducibility when compared to earlier methods.12,15,16
The development of monoclonal antibodies and flow cytometry
technology has made possible new approaches to leucocyte subset
identification and immune monitoring. Fluorochrome-labeled
monoclonal antibodies and multicolor flow cytometry permit
simultaneous quantification of two or more leucocyte subpopulations.
Immune monitoring is facilitated by flow cytometers, such as the
BD FACScan™ and BD FACStrak™, which characterize cells by means
of four-parameter analysis. The BD FACScan and BD FACStrak, when
used with BD Simultest™ IMK-Lymphocyte software, characterize up
to 50,000 cells in a single sample by simultaneous analysis of forward
scatter (FSC), side scatter (SSC), and multicolor fluorescence.
6
Clinical Applications*
Total T- and B-lymphocyte percentages can be used to characterize
some forms of immunodeficiency17-19 and autoimmune diseases.20,21
Determining percentages of CD4+ and CD8+ lymphocytes can be
useful in monitoring the immune status of patients with immune
deficiency diseases, autoimmune diseases, or immune reactions. The
relative percentage of the CD4+ subset has been reported to be
depressed and the relative percentage of the CD8+ subset to be elevated
in many patients with congenital or acquired immune deficiencies such
as severe combined immunodeficiency (SCID)17 and acquired immune
deficiency syndrome (AIDS).22
The percentage of suppressor/cytotoxic T lymphocytes can lie outside
the normal reference range in some autoimmune diseases23 and in
certain immune reactions such as acute graft-versus-host disease
(GVHD)24 and transplant rejection.25 The relative percentage of the
CD8+ lymphocyte population can often be decreased in active systemic
lupus erythematosus (SLE) but can also be increased in SLE patients
undergoing steroid therapy.21
Helper/inducer T lymphocytes are a subset of T (CD3+) lymphocytes
that also express the CD4 antigen. Suppressor/cytotoxic T lymphocytes
express the CD8 antigen and are principally a subset of T (CD3+)
lymphocytes, although a subset of NK lymphocytes is also CD8–
positive.26
The CD4+/CD8+ lymphocyte ratio can be quantified as the ratio of
CD4 FITC–positive lymphocytes to CD8 PE–positive lymphocytes. The
most common method that has been used to determine the CD4/CD8
ratio has been a single two-color reagent containing CD4 and CD8
antibodies. The CD4+/CD8+ ratio has been used to evaluate the
immune status of patients with autoimmune disorders or those
suspected of developing immune disorders21,22 or known to have
* Not all the studies in this section employed BD reagents.
7
immunodeficiencies.4,22 In addition, the ratio has been used to monitor
bone marrow transplant patients for onset of acute GVHD.24
The conventional helper/suppressor ratio (CD4/CD8) does not
distinguish between T- and NK-lymphocyte CD8 antigen expression,
or between helper/inducer T lymphocytes and monocytes that express
the CD4 antigen in low copy numbers.22,27 The reagent combinations
provided in the kit allow the CD3+CD4+ helper/inducer
T lymphocytes and CD3+CD8+ suppressor/cytotoxic T lymphocytes to
be identified and enumerated separately from contaminating CD3–
CD4+ monocytes and CD3–CD8+ NK lymphocytes.22 Instead of using
a single reagent containing CD4 and CD8, the BD Simultest IMKLymphocyte kit uses two monoclonal antibody reagents (CD3/CD4
and CD3/CD8) to determine the true helper/suppressor T-lymphocyte
ratio (CD3+CD4+/CD3+CD8+). The true helper/suppressor
T-lymphocyte ratio does not include CD4-positive monocytes and
CD8-positive NK lymphocytes.
NK lymphocytes, identified as being CD3–, CD16+ and/or CD56+
have been shown to mediate cytotoxicity against certain tumors and
virus-infected target cells.
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. The stained samples are treated with BD FACS™
lysing solution (reagent G) to lyse erythrocytes. Samples are then
washed and fixed prior to flow cytometric analysis.
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. The
use of two fluorochromes permits simultaneous two-color analysis
because each fluorochrome emits light at a different wavelength when
8
excited at 488 nm by an argon-ion laser. The FITC-stained
lymphocytes emit yellow-green light (emission maximum
approximately 515 nm) while the PE-stained lymphocytes emit redorange light (emission maximum approximately 580 nm).
The cells also interact with the laser beam by scattering the light. The
FSC light provides a measure that correlates well with cell size, while
the SSC light is an indicator of cellular granularity. The BD FACScan
and BD FACStrak flow cytometers used with BD Simultest
IMK-Lymphocyte software count a sufficient number of cells to ensure
that a minimum of 2,000 lymphocytes are included in the analysis gate.
The data files should be saved in list mode and given logical names to
aid in retrieval for subsequent analysis by the BD Simultest
IMK-Lymphocyte software.
The BD Simultest IMK-Lymphocyte software uses the BD Leucogate™
sample, Reagent A, to automatically establish a lymphocyte analysis
gate that includes greater than or equal to 98% of the normal mature
lymphocytes in the sample. However, if the gate contains greater than
or equal to 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.
The software identifies and calculates the percentages of contaminating
monocytes, granulocytes, and debris that are included within the
BD Leucogate lymphocyte analysis gate on the basis of FSC, SSC, and
fluorescence properties. Refer to the BD Simultest IMK-Lymphocyte
Software User’s Guide for details on how the software sets the gate and
automatically adjusts subset percentages by using the percent purity of
the gated lymphocyte population.
The presence of blast cells can interfere with the gating procedure and
result in a processing failure. Samples containing blast cells can
therefore require testing by other methods. If the software is unable to
set a gate, a message will appear on the printout. Refer to the
9
BD Simultest IMK-Lymphocyte Software User’s Guide for a complete
listing of software error messages.
The software uses the negative control, Reagent B, to set fluorescence-1
(FL1) and fluorescence-2 (FL2) markers around the negative
lymphocyte population and to assess the amount of nonantigen-specific
antibody binding (nonspecific staining) present, particularly that
caused by Fc receptors. When greater than 5% of the negative control
events are above the FL1 or FL2 negative control markers, an error
message of “too much nonspecific staining” will appear on the
computer display screen and on the laboratory printout for the
negative control tube. When the negative control tube is being
processed by the software, the operator should check the screen for
error messages that would indicate nonspecific staining.
The negative control is a mixture of conjugated monoclonal antibodies
with the same fluorochromes (FITC and PE) as the test reagents. The
negative control antibodies are specific for antigens not present on
human leucocytes. The Control should be used to stain a separate
aliquot of each patient sample.
For each patient sample, the lymphocyte gate set with BD Leucogate
and the fluorescence markers determined using the Control are used to
analyze the remaining tubes C through F. When the Percent
Lymphocyte Conversion option has been selected from the Main Menu
of the BD Simultest IMK-Lymphocyte software, the results are
expressed as percentages of lymphocytes in the analysis gate. If this
software option is not selected, results will be expressed as percentages
of the total gated events. BD Simultest IMK-Lymphocyte software
provides a report quantifying immunologically significant lymphocyte
subsets as percentages of total circulating human lymphocytes and the
helper/suppressor T-lymphocyte ratio (CD3+CD4+/ CD3+CD8+).
For the purposes of quality control, a three-part differential with
percentages of monocytes, granulocytes, and lymphocytes is
automatically determined by the software and printed only for
10
comparison with results from a laboratory differential. To obtain the
three-part differential, the software uses an algorithm to distinguish the
three cell populations based on FSC, SSC, and fluorescence. Monocytes
are CD14-positive and have an SSC signal intermediate between that of
lymphocytes and granulocytes. Lymphocytes exhibit a low and
granulocytes a high SSC signal.
NOTE The three-part differential provided by BD Simultest IMKLymphocyte software should be used only for comparison with an
independent 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 Simultest IMK-Lymphocyte software to obtain absolute counts.
4. REAGENTS
Reagents Provided, Sufficient for 50 Tests
Reagents A through F contain two murine monoclonal antibodies, one
conjugated with FITC, and one conjugated with PE. Each antibody
reagent, sufficient for 50 tests, is provided in 1 mL of buffered saline
with gelatin and 0.1% sodium azide. The monoclonal antibodies are
produced by cell lines derived from the fusion of mouse myeloma cells
with spleen cells of BALB/c mice. The fluorescein-to-protein ratio (F:P)
for BD IgG monoclonal antibody reagents is 2 to 5. The F:P ratio for
each reagent has been optimized for its intended use.
Reagent A, BD Leucogate (CD45/CD14), 1 mL
BD Leucogate is used to define and evaluate the light-scatter gate that
distinguishes lymphocytes from granulocytes, monocytes, debris, and
unlysed or nucleated red blood cells. This reagent contains FITClabeled CD45, clone 2D1,28-30 for identification of leucocytes, and PElabeled CD14, clone MφP9,31-33 for identification of monocytes.
11
The CD45 antibody was derived from hybridization of mouse NS-1
myeloma cells with spleen cells of BALB/c mice immunized with human
peripheral blood mononuclear cells (PBMCs). The antibody is
composed of mouse IgG1 heavy chains and kappa light chains. The
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.34 The CD45 antibody has been reported to
react weakly with mature circulating erythrocytes and platelets.12,34
The CD45 antibody recognizes human leucocyte antigens, 180 to 220
kilodaltons (kDa), that are members of the T200 family.34
The CD14 antibody was derived from the hybridization of mouse Sp2/
0 myeloma cells with spleen cells of BALB/c mice immunized with
peripheral blood monocytes from a rheumatoid arthritis patient. The
antibody is composed of mouse IgG2b heavy chains and kappa light
chains. The CD14 antibody recognizes a human monocyte antigen of
53 kDa.35 The CD14 antigen is present on the majority of normal
peripheral blood monocytes.36 The CD14 antibody reacts weakly with
granulocytes.37
Reagent B, Control, 1 mL
The Control reagent is used to set FL1 and FL2 quadrant markers
around the reference unstained (negative) lymphocyte population and
to estimate the amount of nonantigen specific antibody binding
(nonspecific staining), present, in particular that caused by Fc
receptors. It contains FITC-labeled IgG1, clone X40, and PE-labeled
IgG2a, clone X39.
12
Both X40 and X39 react specifically with keyhole limpet hemocyanin
(KLH), an antigen not expressed on human cells or human cell lines.
Clones X40 and X39 are both derived from hybridization of mouse
Sp2/0-Ag14 myeloma cells with spleen cells from BALB/c mice
immunized with KLH. Clone X40 is composed of mouse IgG1 heavy
chains and kappa light chains. Clone X39 is composed of mouse IgG2a
heavy chains and kappa light chains.
Reagent C, CD3/CD19, 1 mL
CD3/CD19 is used to identify T and B lymphocytes. It contains
FITC-labeled CD3, clone SK7,38-41 for identification of
T lymphocytes, and PE-labeled CD19, clone 4G7,42 for identification
of B lymphocytes.
The CD3 antibody was derived from the hybridization of mouse NS-1
myeloma cells with spleen cells from BALB/c mice immunized with
human thymocytes. The antibody is composed of mouse IgG1 heavy
chains and kappa light chains. The CD3 antibody reacts with the
epsilon chain of the CD3/T-cell antigen receptor (TCR) complex.43
This complex is composed of at least six proteins that range in
molecular weight from 20 to 30 kDa.44 The antigen recognized by
CD3 antibodies is noncovalently associated with either α/β or γ/δ TCR
(70 to 90 kDa).45
The CD19 antibody was derived from hybridization of mouse
P3-X63-Ag8.653 myeloma cells with spleen cells of BALB/c mice
immunized with human chronic lymphocytic leukemia cells. The
antibody is composed of mouse IgG1 heavy chains and kappa light
chains. The antigen recognized by CD19 antibodies is present on
human B lymphocytes at all stages of maturation, but is lost on plasma
cells. The CD19 antigen can be involved in activation and proliferation
of B lymphocytes. The molecular weight of the antigen recognized by
this antibody is 90 kDa.46
13
Reagent D, CD3/CD4, 1 mL
CD3/CD4 is used to identify helper/inducer T lymphocytes. This
reagent combination permits CD3–CD4+ monocytes in quadrant 1 (see
Figure 1 on page 24 for quadrant definition) to be separated from the
CD3+CD4+ helper/inducer T lymphocytes in quadrant 2 (See Figure 2
on page 25). It contains FITC-labeled CD3, clone SK7, for the
identification of T lymphocytes, and PE-labeled CD4, clone SK3,31,47
for the identification of helper/inducer T lymphocytes.
The CD4 antibody was derived from the hybridization of mouse NS-1
myeloma cells with spleen cells from BALB/c mice immunized with
human peripheral blood T lymphocytes. It is composed of mouse IgG1
heavy chains and kappa light chains. The CD4 antibody recognizes the
CD4 antigen, which interacts with class II MHC molecules and is the
primary receptor for the human immunodeficiency virus (HIV).48,49
The cytoplasmic portion of the antigen is associated with the protein
tyrosine kinase p56lck. The CD4 antigen can regulate the function of
the CD3 antigen/TCR complex.50 The CD4 antibody reacts with
monocytes/macrophages, which have a CD4 antigen density lower than
that on helper/inducer T lymphocytes.51
Reagent E, CD3/CD8, 1 mL
CD3/CD8 is used to identify suppressor/cytotoxic T lymphocytes. This
reagent combination permits CD3–CD8+ NK lymphocytes in quadrant
1 to be separated from the CD3+CD8+ suppressor/cytotoxic
T lymphocytes in quadrant 2 (see Figure 2 on page 25). It contains
FITC-labeled CD3, clone SK7, for identifying T lymphocytes, and PElabeled CD8, clone SK1,31 for identifying suppressor/cytotoxic
T lymphocytes.
The CD8 antibody is derived from the hybridization of mouse NS-1
myeloma cells with spleen cells from BALB/c mice immunized with
human peripheral blood T lymphocytes. It is composed of mouse IgG1
heavy chains and kappa light chains. The CD8 antigen is present on the
14
human suppressor/cytotoxic T-lymphocyte subset39,47 as well as on a
subset of NK lymphocytes.26 The CD8 antigenic determinant interacts
with class I MHC molecules, resulting in increased adhesion between
the CD8+ T lymphocytes and the target cells.52-54 Binding of the CD8
antigen to class I MHC molecules enhances the activation of resting
T lymphocytes.52-55 The CD8 antigen is expressed as a disulfide-linked
bimolecular complex with a 32-kDa α subunit.56,57 The cytoplasmic
domain of the α subunit of the CD8 antigen is associated with the
protein tyrosine kinase p56lck.54,55
Reagent F, CD3/CD16+CD56, 1 mL
The CD3/CD16+CD56 reagent is used to identify T and NK
lymphocytes. It contains PE-labeled CD16, clone B73.1,58-61, and PElabeled CD56, clone MY31,62,63, to ensure identification of the NK
lymphocyte population. Since some T lymphocytes also express the
CD16 and/or CD56 antigens, the reagent also contains FITC-labeled
CD3 to identify T lymphocytes and the T-lymphocyte subset that
expresses the CD16 and/or CD56 antigens.64
The CD16 antibody was derived from the hybridization of mouse
P3-X63-Ag8.653 myeloma cells with spleen cells of BALB/c mice
immunized with NK lymphocytes. The antibody is composed of mouse
IgG1 heavy chains and kappa light chains.58-60 The antigen recognized
by CD16 antibodies is a 50 to 65-kDa protein that is the IgG Fc III
receptor present on NK lymphocytes and neutrophils.61
The CD56 antibody was derived from the hybridization of mouse Sp2/
0 myeloma cells with spleen cells of (B6xBALB/c)F1 mice immunized
with the KG-1a cell line. The antibody is composed of mouse IgG1
heavy chains and kappa light chains. The CD56 antigen is present on
NK lymphocytes.64,65 The antibody recognizes the 140-kDa isoform of
the neural cell adhesion molecule (NCAM).61,63 The 140-kDa core
protein is extensively glycosylated to give a mature antigen.62,64 The
CD56 antigen is present on essentially all resting and activated CD16+
NK lymphocytes and approximately 5% of CD3+ peripheral blood
15
lymphocytes.65 CD56 antigen density increases when NK lymphocytes
are activated.66 CD3+CD56+ T lymphocytes compose a unique subset
of cytotoxic T lymphocytes that mediates non-MHC–restricted
cytotoxicity.65 The CD56 antigen is involved in neuronal homotypic
cell adhesion and cell differentiation during embryogenesis.62
Reagent G, 10X BD FACS Lysing Solution, 60 mL
Reagent G contains 10X buffered BD FACS lysing solution, with less
than 50% diethylene glycol and less than 15% formaldehyde. When
stored at 2°C–25°C, the 10X concentrate is stable until the expiration
date on the label. For use, dilute 1:10 with room temperature (20°C–
25°C) reagent-grade water. The prepared solution is stable for 1 month
when stored in a glass container at room temperature.
Reagent
Component
A
CD45
25
CD14
12.5
IgG1
12.5
IgG2a
12.5
B
C
D
E
F
Concentration (µg/mL)
CD3
25
CD19
3.1
CD3
25
CD4
1.5
CD3
25
CD8
6.25
CD3
25
CD16
6.25
CD56
12.5
16
Precautions
•
•
•
•
•
•
•
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 reagents should not be frozen or exposed to direct
light during storage or during incubation with cells. Keep the
reagent vial dry.
Incubation or centrifugation times or temperatures other than
those specified can be a source of error.
For optimal results, stain blood samples within 6 hours of
venipuncture.
Alteration in the appearance of any of the reagents, 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.
Reagent G contains 30.0% diethylene glycol, CAS number 111-46-6,
9.99% formaldehyde, CAS number 50-00-0, and 3.51% methanol,
CAS number 67-56-1.
Danger
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.
17
H318 Causes serious eye damage.
H302 Harmful if swallowed.
H315 Causes skin irritation.
H317 May cause an allergic skin reaction.
Wear protective clothing / eye protection. Wear protective gloves.
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: Immediately call a doctor.
WARNING All biological specimens and materials coming in
contact with them are considered biohazards. Handle as if capable
of transmitting infection67,68 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 IMK-Lymphocyte is designed for use on a BD 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.
All performance characteristics were obtained using the BD FACScan
and verified on the BD FACStrak. Other systems can have different
characteristics and should be verified by the user.
The following instrument system is recommended:
•
BD FACScan or BD FACStrak flow cytometer system equipped for
two-color fluorescence detection and two-parameter light-scatter
18
•
•
•
•
detection. For detailed information on use, refer to the
BD FACScan User’s Guide or the BD FACStrak User’s Guide.
BD CONSORT™ 30 or CONSORT 32 computer system and
peripherals (included in the BD FACScan system) or CONSORT
31 computer system and peripherals (included in the BD FACStrak
system). For detailed information on use, refer to the CONSORT
30 Software User’s Guide, the CONSORT 32 System User’s
Guide, or the CONSORT 31 System User’s Guide.
BD Simultest IMK-Lymphocyte software. For detailed information
on use, refer to the BD Simultest IMK-Lymphocyte User’s Guide.
BD Calibrite™ beads (Catalog No. 349502). These beads are used
for setting the photomultiplier tube (PMT) voltages, setting the
fluorescence compensation, checking instrument sensitivity, and
monitoring the performance of the flow cytometer. For detailed
information on use, refer to the BD Calibrite Beads instructions
for use (IFU).
BD FACSComp™ software. For detailed information on use, refer
to the BD FACSComp Software User’s Guide.
6. SPECIMEN COLLECTION AND PREPARATION
Collect blood aseptically by venipuncture69,70 into a sterile EDTA
(lavender top) BD Vacutainer® blood collection tube. A minimum of 1
mL of whole blood is required for this procedure. Blood should be
stained within 6 hours of venipuncture 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.
A white blood cell (WBC) count and a differential white count should
be obtained from the same sample of whole blood before staining. An
acceptable WBC concentration range is from 3.5 x 103 to 9.4 x 103
WBC/µL. Samples with counts greater than 9.4 x 103 WBC/µL might
need to be diluted with 1X phosphate buffered saline (PBS) containing
19
0.1% sodium azide, or BD CellWASH™ solution; check with your
local BD Biosciences representative for availability. For samples with
counts less than 3.5 x 103 WBC/µL, more blood might be needed and a
separation procedure can be required to concentrate the cells.
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.71 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.
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.
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.
Vortex mixer.
Low-speed centrifuge (minimum speed 200g) with swinging bucket
rotor and 12 x 75-mm tube carriers.
Vacuum aspirator with trap.
* Falcon is a registered trademark of Corning Incorporated.
20
•
•
•
•
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.
CAUTION Use only BD FACSFlow sheath fluid diluent to dilute
BD Calibrite beads.
•
Reagent-grade (both distilled and deionized) water.
Staining and Fixing the Cells
Whole blood samples are first stained with Reagents A through F.
Diluted Reagent G is used to lyse RBCs following staining. See
Interfering Conditions under Section 6, Specimen Collection and
Preparation, and Section 9, Limitations. Use care to protect the tubes
from direct light. Perform the procedure at room temperature (20°C–
25°C) using room temperature reagents. Refer to Precautions in
Section 4, Reagents.
1. For each patient sample, label six 12 x 75-mm tubes with A, B, C,
D, E, and F. Also label each tube with the sample identification
number.
2. Place 20 µL of Reagent A into tube A, 20 µL of Reagent B into
tube B, 20 µL of Reagent C into tube C, 20 µL of Reagent D into
tube D, 20 µL of Reagent E into tube E, and 20 µL of Reagent F
into tube F.
3. For each sample tube, use a fresh micropipettor tip and carefully
add 100 µL of well-mixed, anticoagulated whole blood into the
bottom of each of the labeled tubes. The recommended WBC
concentration is 3.5 x 103 to 9.4 x 103 WBC/µL. Vortex
21
thoroughly at low speed for 3 seconds and incubate for 15 to 30
minutes at room temperature (20°C–25°C).
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
will not be stained with the reagent.
4. Dilute 10X lysing solution to 1X following the instructions in
Section 4, Reagent G. Add 2 mL of room temperature (20°C–
25°C) 1X lysing solution to each tube. Immediately vortex
thoroughly at low speed for 3 seconds and incubate for 10 to 12
minutes at room temperature (20°C–25°C) in the dark. Do not
exceed 12 minutes.
NOTE Avoid prolonged exposure of the cells to lytic reagents,
which can cause white cell destruction. See Section 9, Limitations
on page 35.
5. Immediately after incubation, centrifuge tubes at 300g for 5
minutes at room temperature (20°C–25°C).
6. Aspirate the supernatant, leaving approximately 50 µL of residual
fluid in the tube to avoid disturbing the 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 (20°C–25°C).
8. Aspirate the supernatant, leaving approximately 50 µL of residual
fluid in the tube to avoid disturbing the pellet.
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
22
for 3 seconds. Make sure that the cells are well mixed with the
fixing solution.
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. Analyze the fixed cells within 24 hours
after staining. Vortex the cells thoroughly (at low speed) to reduce
aggregation before running them through the flow cytometer.13
Flow Cytometry
Follow the BD instructions for two-color flow cytometric analysis and
see Section 5, Instrument. The following general approach is
recommended. The BD FACScan or BD FACStrak flow cytometer is
first prepared for sample analysis using BD Calibrite beads and
BD FACSComp software.
The stained samples are then run on the flow cytometer and analyzed
with BD Simultest IMK-Lymphocyte software. Refer to the
BD FACScan User’s Guide or BD FACStrak User’s Guide, the
BD Calibrite Beads IFU, the BD FACSComp Software User’s Guide,
and the BD Simultest IMK-Lymphocyte Software User’s Guide for
detailed instructions on use. We recommend that patient data be stored
in list mode to allow subsequent analysis of data files.
Set up and adjust compensation of the BD FACScan or BD FACStrak
flow cytometer. Using BD Calibrite beads and BD FACSComp
software, set PMT voltages, adjust fluorescence compensation, and
check detector sensitivity. For details, refer to the BD Calibrite Beads
IFU, the BD FACSComp Software User’s Guide, and either the
BD FACScan User’s Guide or the BD FACStrak User’s Guide.
Run all samples through the flow cytometer and acquire data in list
mode files using BD Simultest IMK-Lymphocyte software. Refer to the
BD Simultest IMK-Lymphocyte Software User’s Guide for detailed
instructions for use of the software. The software automatically
collects a sufficient number of events to obtain a minimum of 2,000
23
lymphocytes within the lymphocyte gate. The software counts the
number of events in each quadrant and then computes a percentage of
positive lymphocyte events for quadrant 1 (low yellow-green/high redorange), quadrant 2 (high yellow-green/high red-orange, or dualfluorescence), quadrant 3 (low yellow-green/low red-orange), and
quadrant 4 (high yellow-green/low red-orange) (see Figure 1).
Figure 1 The fluorescence-display quadrants (indicated as Q1 through Q4) and the
corresponding colors
Q1
Q2
Low yellowgreen/high
red-orange
High yellow-green/high redorange (dual fluorescence)
Q3
Q4
Low yellowgreen/low
red-orange
High yellow-green/low red-orange
24
The BD Leucogate tube (A) is used to gate on lymphocytes. The
BD FACScan or BD FACStrak with BD Simultest IMK-Lymphocyte
software automatically sets a lymphocyte analysis gate to eliminate
most debris, monocytes, and granulocytes (see Figure 2). Refer to the
BD Simultest IMK-Lymphocyte Software User’s Guide for information
on how the gate is set. If there is inadequate separation between
populations, the sample will be flagged to alert the operator and a
message will appear. Poor sample preparation or incorrect instrument
setup can contribute to errors in sample gating.
Figure 2 BD FACScan LWB sample, from a hematologically normal patient, stained with
BD Simultest IMK-Lymphocyte reagents. BD Leucogate was used to reduce debris,
monocytes, and granulocytes in the gate shown under tube A. Dot plot displays of FL1
(x-axis) vs FL2 (y-axis) are shown for tube B through F.
25
Tube B
SSC
IgG2a
Tube A
IgG1
Tube D
CD4
CD19
FSC
Tube C
CD3
CD3
Tube F
CD8
CD16+CD56
Tube E
CD3
CD3
26
The Control tube (B) is used to set fluorescence intensity markers and
gives an indication of nonspecific staining. BD Simultest IMKLymphocyte software automatically uses the lymphocyte analysis gate
set with BD Leucogate and then establishes FL1 and FL2 marker
placement. These markers define the boundaries between negative
events and positively stained events in the lymphocyte gate.
Fluorescence markers should be set around the negative population
that appears as the cluster of events that are low in both yellow-green
and red-orange fluorescence.
Data for tubes C, D, E, and F is acquired and analyzed by BD Simultest
IMK-Lymphocyte software using the gate and markers established with
tubes A and B. The lymphocyte populations present in each quadrant
for each tube are shown in Table 1. See the BD Simultest
IMK-Lymphocyte Software User’s Guide for detailed instructions on
use of the software. Analyze these results using the criteria provided
under Quality Control and according to the instructions in Section 8,
Results.
Table 1. Mononuclear cell populations separated by quadrant for tubes C through F
Quadrant
Tube
Q2
Q3
C:CD3/CD19 CD3–CD19+
B lymphocytes
CD3+CD19+
Nonspecific
staininga
CD3–CD19–
CD3+CD19–
lymphocytes,
T lymphocytes
monocytes,
granulocytes, and
debris
D: CD3/CD4 CD3–CD4+
monocytes
CD3+CD4+
helper/inducer
T lymphocytes
CD3–CD4–
CD3+CD4–
lymphocytes,
T lymphocytes
granulocytes, and
debris
CD3+CD8+
suppressor/
cytotoxic
T lymphocytes
CD3–CD8–
CD3+CD8–
lymphocytes,
T lymphocytes
monocytes,
granulocytes, and
debris
E: CD3/CD8
Q1
CD3–CD8+
NK lymphocyte
subset
27
Q4
Table 1. Mononuclear cell populations separated by quadrant for tubes C through F
Quadrant
Tube
Q1
Q2
Q3
Q4
F: CD3/
CD16+
CD56
CD3–/CD16+ and/
or CD56+
NK lymphocytes,
granulocytes
CD3+/CD16+
and/or CD56+
T-lymphocyte
subset
CD3–/CD16–
CD56–
lymphocytes,
monocytes, and
debris
CD3+/CD16– and
CD56–
T lymphocytes
a. See section 9, Limitations.
Quality Control
For optimal results, we recommend using BD Calibrite beads and
BD Autocomp™ software for setting the PMT voltages, setting the
fluorescence compensation, and checking instrument sensitivity prior
to use of BD Simultest IMK-Lymphocyte on the BD FACScan or
BD FACStrak flow cytometer.
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 2.
Negative controls are run with each patient sample to set FL1 and FL2
markers between negative and positively stained lymphocyte clusters
and to detect the presence of nonspecific staining that would indicate
erroneous patient results. Examine the computer screen display and the
Laboratory Report for Control tube B. If more than 5% of the events
occur outside of Q3 for the Control tube, a message of “too much
nonspecific staining” will be reported for tube B. In this case, the
results for tubes C through F should be considered suspect. See
Troubleshooting at the end of this IFU. Visual inspection of the dot
plots obtained for tubes C through F is necessary to ensure that
fluorescence markers are correctly set and that there is minimal
nonspecific staining.
28
Visually inspect the dot plot for Control tube B. If the negative cluster
is diffuse and smeared over the FL2 intensity range, the marker might
not be set correctly and results can be suspect.
If poor separation between positive and negative clusters is seen on
visual inspection of the dot plots for the normal control, especially for
CD3 and CD4, nonspecific staining or incomplete staining can be
inferred and the run should be rejected. Nonspecific staining might be
seen because of poor condition of the cells. Consult Section 12,
Troubleshooting, if nonspecific or suboptimal staining is
observed.BD Simultest IMK-Lymphocyte software automatically
checks to see that there is agreement in T-lymphocyte percentages
between tubes containing CD3 (tubes C, D, E, and F). The run will be
rejected if the total T-lymphocyte value in any two tubes differs by
more than 8%.
Internal assay consistency can be indicated when the total percentages
of T, B, and NK lymphocytes total 100% ±5% when using the Percent
Lymphocyte Conversion option with BD Simultest IMK-Lymphocyte
software. Refer to the BD Simultest IMK-Lymphocyte User's Guide for
instructions when selecting this option.
BD Simultest IMK-Lymphocyte software will automatically inspect the
data and alert the operator with a number of possible error messages.
Refer to the BD Simultest IMK-Lymphocyte Software User’s Guide for
a list of possible messages. Use the following criteria for inspection of
the dot plots obtained for each sample to evaluate the quality of the
data obtained.
1. The operator should reject the results if one or more 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.
29
2. If there is no obvious reason for the normal control to fail, a
sample from another normal control should be stained and run,
and the entire staining procedure repeated on all subsequent
samples.
3. Samples with nucleated RBCs can contain too much debris because
of incomplete lysis of nucleated erythrocytes with BD FACS lysing
solution (reagent G). Too much debris can also occur when
assaying blood samples from patients with certain hematologic
disorders for which red cells are difficult to lyse, as in 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 gate” and the sample results should be
rejected.
4. The tube-to-tube difference in gated events must be less than or
equal to 500 events for all tubes in the panel except the
BD Leucogate tube (A). (However, if the Control tube is
problematic, then the panel is suspect.) See also Troubleshooting at
the end of this IFU.
8. RESULTS
Percent Lymphocyte Conversion
When the Percent Lymphocyte Conversion software option is selected,
BD Simultest IMK-Lymphocyte software automatically calculates each
reported lymphocyte subset as a percentage of lymphocytes in the
lymphocyte analysis gate based on the lymphocyte percentage
determined with the BD Leucogate tube A. The software also subtracts
nonlymphocytes from Q3. Refer to the BD Monoclonal Antibodies
Source Book, Section 8.3.
When all quality control criteria listed in Section 7, Procedure, have
been met, the Percent Lymphocyte Conversion software option
provides appropriate estimations of the true subset values. However, if
the quality control criteria are not met (for example, excessive
30
nonlymphocyte contamination of the gate), results can be suspect. In
this case, the data should be analyzed manually to determine the effect
of cross-reacting nonlymphocytes on results. If the Percent Lymphocyte
Conversion option is turned off, results will be reported as a percentage
of the total gated events. (Refer to the BD Simultest IMK-Lymphocyte
Software User’s Guide or the BD Monoclonal Antibodies Source Book,
Section 8.3).
The principle of the computation follows (Q values refer to counts in
quadrants):
N = total number of events in the lymphocyte gate defined by
BD Leucogate
= L + M + G + D where:
L = number of lymphocytes in the gate
M = number of monocytes in the gate
G = number of granulocytes in the gate
D = number of debris events in the gate
Equation 1. Percent gated lymphocytes (%L) = L/N x 100
Equation 1 computes the percentage of lymphocyte events within the
BD Leucogate lymphocyte analysis gate. This value is given as “%L” in
tube A in Table 2 and reflects the purity of lymphocytes within the gate
drawn using the BD Leucogate tube (A).
Equation 2a. Percent gated converted lymphocytes (Conv %L) in Q1,
Q2, or Q4 =
Number of gated events in Q1, Q2, or Q4
--------------------------------------------------------------------------------------------------- × 100
N x (%L/100)
The software uses Equation 2a to calculate the converted percentage of
lymphocytes for Q1, Q2, and Q4. It assumes that all nonlymphocytes
are unstained and appear in Q3.
31
NOTE The Percent Lymphocyte Conversion option does not correct
for nonlymphocyte events that can occur in Q1, Q2, and Q4.
Therefore, CD16+ granulocyte events are not corrected in Q1 by the
software.
Equation 2b. Percent gated converted lymphocytes (Conv %L) in Q3 =
(Number of gated events in Q3 - Number of gated nonlymphs)
------------------------------------------------------------------------------------------------------------------------------------------------------ × 100
N x (L/100)
where:
Number of gated nonlymphocytes = number of gated debris events +
number of gated granulocytes + number of gated monocytes. See
Table 2, tube A.
The software uses Equation 2b to calculate the converted percentage of
lymphocytes for Q3. Since it assumes that all nonlymphocytes are in
this quadrant, it subtracts them from the number of gated events in Q3
and expresses the remainder as a percentage of the lymphocytes in the
gate.
Table 2 summarizes the data in Figure 2.
Table 2. Summary of the representative data from Figure 2 and identification of
quadrants used to compute subsets
Tube
A
%La
%Ma
%Ga
%Da
95
1
3
1
% of Total Gated Lymphsb
99
a. %L, M, G, and D are lymphocytes, monocytes, granulocytes, and debris in the lymphocyte gate defined by
BD Leucogate, expressed as percentages of gated events. These values reflect the quality and purity of the gate.
Data was collected on a BD FACScan flow cytometer.
b. % of total gated lymphocytes are the lymphocytes in the gate defined by BD Leucogate, expressed as a
percentage of all lymphocytes in the ungated sample. This value measures the proportion of all lymphocytes
included in the gate.
32
Quadrant(s)
% of Lymphocytes
(Converted %L)a Ratiob
Tube
Cell Type
C:
CD3/CD19
Total T lymphocytes
Q4
Total B lymphocytes
Q1
7
D:
CD3/CD4
Total T lymphocytes
Q2 and Q4
75
Q2
52
E:
CD3/CD8
Total T lymphocytes
Q2 and Q4
72
Suppressor/cytotoxic
T lymphocytes
Q2
24
D,E:
CD3/CD4;
CD3/CD8
Helper/inducer;
suppressor/cytotoxic
T lymphocytes
Q2
—
F:
CD3/CD16 + CD56
Total T lymphocytes
Q2 and Q4
74
Q1
16
Helper/inducer
T lymphocytes
NK lymphocytes
74
2.2§
a. See Section 8, Results, for an explanation of Percent Lymphocyte Conversion.
b. Helper/suppressor T-lymphocyte ratio = (CD3+CD4+)/(CD3+CD8+).
Table 3 gives representative data for the three-part differential obtained
on a healthy adult donor using the BD FACScan with the BD Simultest
IMK-Lymphocyte software.
Three-Part Differential
For lysed whole blood, it is possible to estimate monocytes,
lymphocytes, and granulocytes as a percentage of leucocytes using the
BD Leucogate tube (A) (see Table 2). BD Simultest IMK-Lymphocyte
software automatically calculates a three-part differential (Table 3).
Refer to the BD Simultest IMK-Lymphocyte Software User’s Guide for
representative data printouts.
33
NOTE The differential provided by BD Simultest IMK-Lymphocyte
software should be used only for comparison with an independent
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 Simultest IMK-Lymphocyte
software to obtain absolute counts.
Table 3. Representative data for the three part differential obtained on a healthy adult
donor using the BD FACScan with BD Simultest IMK-Lymphocyte software
Three-Part Differentiala
% Leucocytes
Lymphocytes
21
Monocytes
5
Granulocytes
74
a. The three-part differential is obtained by expressing lymphocytes, monocytes, and granulocytes in the entire
ungated sample as a percentage of the sum of lymphocytes, monocytes, and granulocytes in the entire sample.
Absolute Counts
An absolute cell count can be computed if a WBC count and the
lymphocyte percentage from an independent differential white cell
count are obtained using standard laboratory procedures. BD Simultest
IMK-Lymphocyte software automatically calculates an absolute count
for each subset if the operator enters a WBC count and the lymphocyte
percentage from a differential white cell count for each patient sample
of whole blood. Refer to the BD Simultest IMK-Lymphocyte Software
User’s Guide. The principle of the computation follows:
1. Enter the WBC count in WBC/µL obtained on the same blood
sample.
2. Enter the percentage of lymphocytes from an independent
laboratory differential white cell count obtained for the same
34
sample. Do not use the BD Simultest IMK-Lymphocyte three-part
differential values.
3. The absolute counts for each subset are then computed as in the
following example:
Absolute count of CD3+CD4+ cells/µL =
+
+
%CD3 CD4 events- × -------%L- × (WBC/μL )
-------------------------------------------------100
100
If the data is presented in absolute counts, the value and precision of
the absolute count reference range will be a function of (a) the
laboratory normal reference range and precision for WBC count, (b)
percent lymphocytes, and (c) the normal reference range and precision
for percent lymphocytes positive for the specific marker.
9. LIMITATIONS
•
•
•
•
Use freshly drawn blood and stain within 6 hours of venipuncture.
Prior to staining, store blood at room temperature (20°C–25°C).
Cells that have been refrigerated before staining can give aberrant
results. Previously fixed cells are not recommended for use.
Stained and fixed cells should be assayed within 24 hours of
staining.
Whole blood samples with counts less than 3.5 x 103 WBC/µL and
greater than 9.4 x 103 WBC/µL can require special handling to
obtain correct results. Samples with counts greater than 9.4 x 103
WBC/µL might need to be diluted with PBS containing 0.1%
sodium azide, or BD CellWASH. Samples with counts less than
3.5 x 103 WBC/µL can require more blood and a separation
procedure to concentrate the cells.
Confounding variables such as medications that affect properties
of blood cells can yield inaccurate results.71 For example, poor
resolution between positive and negative cells has been observed
35
•
•
•
•
with transplant patients receiving immunosuppressive drugs. If
there is poor separation between negative and positive clusters, the
run should be rejected (see Troubleshooting, at the end of this
IFU).
If the difference in the total T-lymphocyte percentage for any tube
differs from the average total T-lymphocyte percentage by greater
than 8%, the software will print a notice on the Physician Report.
In this case, the run should be rejected.
Laboratories must establish their own normal reference ranges for
each of the BD Simultest IMK-Lymphocyte parameters that can be
affected by sex of patient, age of patient, and preparative
technique. Race of patient can also have an effect, although
sufficient data is not available to establish this. Age, sex, clinical
status, and race of subjects should be known when a reference
range is determined.
If the results are to be expressed in absolute counts, an independent
differential white cell count and a WBC count must also be run on
the same sample of blood. Do not use the differential from the
BD Simultest IMK-Lymphocyte report to compute absolute counts.
The differential provided by BD Simultest IMK-Lymphocyte
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 Simultest IMK-Lymphocyte software to
obtain absolute counts.
Variation in either automatic or manual lymphocyte analysis gate
settings will change the relative amounts of subsets assayed.
BD Simultest IMK-Lymphocyte software uses the BD Leucogate
tube (A) 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.
36
•
•
•
•
•
Abnormal states of health are not always represented by abnormal
percentages of lymphocyte subsets. That is, an individual who
might be in an abnormal state of health can exhibit the same T-, B-,
or NK-lymphocyte percentages as a healthy individual. Results
from BD Simultest IMK-Lymphocyte must be used in conjunction
with other information available from the clinical evaluation and
additional independent diagnostic procedures.
A normal subject has been reported to have no reaction with
CD4.72 However, this lack of reactivity has not been observed in
studies of over 300 subjects.71
The information obtained from the use of this reagent must be
combined with other information and interpreted by a medically
qualified diagnostician to establish presence or absence of specific
disease states.
The BD Simultest IMK-Lymphocyte Percent Lymphocyte
Conversion software option assumes that all nonlymphocytes are
unstained and appear in quadrant 3. Therefore CD16+
granulocytes that appear in quadrant 1 will be included as CD16+
lymphocytes. The sample is flagged if the granulocyte
contamination is greater than 6%. Potentially interfering
granulocytes should be minimized by eliminating as many as
possible from the lymphocyte gate using their light-scatter
properties. Refer to the BD Simultest IMK-Lymphocyte Software
User’s Guide and the BD Monoclonal Antibodies Source Book,
Section 8.3, for more information on Percent Lymphocyte
Conversion.
Samples with nucleated RBCs can show incomplete lysis because
BD FACS lysing solution (reagent G) might not lyse nucleated
erythrocytes. This can also occur when assaying blood samples
from patients with certain hematologic disorders in which red cells
are difficult to lyse, as in myelofibrosis and spherocytosis.
Nucleated erythrocytes will be counted as debris and, if debris is
greater than 10%, the software will flag the sample as “too many
37
•
•
•
•
nonlymphs in gate.” We recommend that such sample results be
rejected. When the Percent Lymphocyte Conversion software
option has been selected, nonlymphocytes such as nucleated or
unlysed red cells identified using BD Leucogate are assumed to be
unstained and are subtracted from Q3 by the software.
BD Simultest IMK-Lymphocyte software checks for nonspecific
staining in Control tube B only. The marker settings from tube B
should be verified using the negative population from tube C. If the
markers appear to be set inappropriately, reset them using the
negative population from tube C. The operator should examine the
printout for any evidence of nonspecific antibody staining in
subsequent tubes (C through F). Nonspecific staining can be
indicated when there is poor separation between positive and
negative clusters on the fluorescence dot plot for tubes C through
F. The presence of such nonspecific staining can reflect a change in
the reagent or an error in the preparation of the samples. Such
samples should be restained.
BD Simultest IMK-Lymphocyte 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 BD Leucogate to set an adequate lymphocyte analysis
gate. The software will flag the sample and results will not be
printed.
Samples containing leukemic cells with volume and scatter
characteristics similar to mature cells will be counted as such.
Examples of these are chronic lymphocytic leukemia (CLL), FABL1 type acute lymphoblastic leukemia (ALL), and adult T-cell
leukemia/lymphoma (ATL).
For whole blood samples, exposure of cells to BD FACS lysing
solution (reagent G) beyond 12 minutes can cause white cell
destruction due to osmotic shock when the wash solution is added
(see item 4 under Staining and Fixing the Cells in Section 7,
Procedure).
38
•
•
•
•
•
The ability of the flow cytometer to select lymphocytes and to
eliminate platelets, red cells, debris, granulocytes, and monocytes
from the lymphocyte gate depends on the existence of a clear
separation between these formed elements and lymphocytes on a
display of FSC vs SSC. For some patients, this separation is not
clear and lymphocyte gating will be less effective.
Performance characteristics have been determined with a
BD FACScan using BD Simultest IMK-Lymphocyte software, and
verified on the BD FACStrak. Performance characteristics have not
been established with any other instrument.
Absolute count values of lymphocyte subsets might not be
comparable across laboratories due to variations in methods for
determining white blood cell counts and/or white cell differential
values.
Note that some BD Simultest IMK-Lymphocyte antibodies react
with other nonlymphocyte-formed elements in blood (See CrossReactivity under Performance Characteristics, Section 11).
Note that normal reference ranges established by BD for the
(CD3+CD4+)/(CD3+CD8+) lymphocyte ratio extend below a ratio
of 1.0 (Table 4 and Table 5).
10. EXPECTED VALUES
Leucocyte Subsets
Data for four different quadrants is collected and displayed for each
tube on the BD FACScan and BD FACStrak display (Figure 2).
The cell populations present in each quadrant for each tube are shown
in Table 1 and in the BD Simultest IMK-Lymphocyte Software User’s
Guide. The clinically significant results are found in the Physician
Report.
BD has investigated the normal reference ranges for BD Simultest
IMK-Lymphocyte parameters in 160 normal male and female subjects
using the BD FACScan flow cytometer at three sites (two US clinical
39
sites and BD in San Jose, California). The expected normal reference
ranges for LWB are shown in Table 4.72 Ranges are presented as
percentages of lymphocytes in the analysis gate using the BD Leucogate
tube and the Percent Lymphocyte Conversion option in BD Simultest
IMK-Lymphocyte software.
Table 4. Representative reference rangesa for BD Simultest IMK-Lymphocyte
parameters in hematologically normal adults as percentages of total gated lymphocytes
(converted) (data pooled from three clinical sites)
Parameter
Sex
Age
n
Mean (%)
95%
Rangeb
Total T lymphocytes
Both
18–70
160
72
59–85
Total B lymphocytes
Both
18–70
160
13
6.4–23
Helper/inducer T lymphocytes
Male
18–70
84
43
29–57
Female
18–70
75
46
31–61
Suppressor/cytotoxic T lymphocytes
Both
18–70
159
25
11–38
Helper/suppressor T-lymphocyte
ratio
Both
18–70
159
1.9
0.9–3.6
NK lymphocytes
Both
18–70
160
14
5.6–31
a. This data was collected on a BD FACScan flow cytometer using BD Simultest IMK-Lymphocyte reagents and
software. Software employed was BD Simultest IMK-Lymphocyte software, one of the BD Simulset™ family of
software.
b. The 95% range is obtained by fitting an appropriate distribution to the data and then estimating the central 95%
area of the fitted distribution.
The ranges obtained were tested for differences by clinical site, by sex
of subject, and by age of subject. For each cell population,
nonparametric comparisons were made between values obtained at
each study site, between values obtained for males and females, and
between values obtained for subjects ages 18 to 70. When the
comparison indicated a significant difference, the data from the groups
was not pooled, and separate reference ranges are given. The normal
reference range for percentages of positive cells is calculated from a
40
fitted distribution.73 The ability to pool reference ranges for the
BD Simultest IMK-Lymphocyte parameters (Table 4) is an indication of
between-laboratory reproducibility.
There are small but significant differences between the sexes for helper/
inducer T lymphocytes. More than one upper and lower limit for
helper/inducer T-lymphocyte ranges are presented (Table 4 and
Table 5) because of sex differences that have been observed.
Adult reference ranges should not be used with pediatric blood
samples.
Race can be a variable in the establishment of normal reference
ranges,74 although insufficient data was collected by BD to determine
this.
NOTE Expected normal values can vary depending upon age, sex, or
race of patient. Because of these differences, each laboratory should
establish its own normal reference range for each parameter.
Absolute Counts
The BD FACScan/CONSORT 30 or 32 system and BD FACStrak/
CONSORT 31 system with BD Simultest IMK-Lymphocyte software
automatically calculate the absolute count if a WBC count and
percentage of lymphocytes or an absolute lymphocyte count are
obtained and entered into the software.
Table 5. Representative reference rangesa (absolute counts from one clinical site) for
BD Simultest IMK-Lymphocyte parameters in hematologically normal adults (ages 18–70)
Sex
n
Mean
95% Rangeb
WBCc
Both
63
5.66
3.50–9.40
Lymphocyte percentagec
Both
62
29.8
19.0–43.0
Total T lymphocytes
Both
62
1.20
0.72–2.33
41
Table 5. Representative reference rangesa (absolute counts from one clinical site) for
BD Simultest IMK-Lymphocyte parameters in hematologically normal adults (ages 18–70)
95% Rangeb
Sex
n
Mean
Total B lymphocytes
Both
62
0.23
0.10–0.43
Helper/inducer T lymphocytes
Male
42
0.67
0.43–1.01
Female
20
0.90
0.50–1.76
Suppressor/cytotoxic T lymphocytes
Both
61
0.44
0.17–1.05
Helper/suppressor T-lymphocyte ratio
Both
62
1.89
0.87–3.40
NK lymphocytes
Both
62
0.29
0.09–0.43
a. This data was collected on a BD FACScan flow cytometer using BD Simultest IMK-Lymphocyte reagents and
software. Software employed was BD Simultest IMK-Lymphocyte software, one of the BD Simulset family of
software. Absolute counts are expressed as 103 WBC/µL.
b. The 95% range is estimated directly from the data using the 2.5 and 97.5 percentiles.
c. Hematology data were obtained from a Sysmex K1000 hematology analyzer (Sysmex Corporation of Japan)
NOTE Do not use the BD Simultest IMK-Lymphocyte three-part
differential values for absolute count determinations. The differential
provided by BD Simultest IMK-Lymphocyte software should be used
only for comparison with an independent 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 Simultest IMK-Lymphocyte software to obtain
absolute counts.
Absolute counts will exhibit significant interlaboratory variation
depending upon the procedure employed for obtaining the WBC count
and differential white count.
Representative absolute counts are shown in Table 5. The table is
generated from data acquired at one US clinical site. BD Simultest
IMK-Lymphocyte parameters are expressed as a percentage of
lymphocytes in the lymphocyte analysis gate. Refer to the BD
Monoclonal Antibodies Source Book, Section 8.3.
42
11. PERFORMANCE CHARACTERISTICS
Performance of the BD Simultest IMK-Lymphocyte reagent kit was
established by testing at two US clinical sites and at BD in San Jose,
California.
Within-Sample Reproducibility
Blood samples from each of six normal and four abnormal subjects
were obtained, aliquoted (two times for both normals and abnormals),
stained with the BD Simultest IMK-Lymphocyte reagents, lysed,
washed, and fixed within 6 hours of sample collection. Flow
cytometric analysis was performed within 24 hours on a BD FACScan
flow cytometer in the same laboratory. Table 6 and Table 7 show the
average within-sample reproducibility obtained for both normal and
abnormal subjects.
Table 6. Within-sample reproducibilitya for BD Simultest IMK-Lymphocyte parameters
(6 normal subjects) as percentages of gated lymphocytes (converted)
dfd
SD as an
Estimate of
Between-Stain
Reproducibility
dfe
12
0.858
15.6
0.816
12
0.816
18.0
1.080
12
1.486
10.4
27.2
0.817
12
1.061
11.1
Helper/suppressor
T-lymphocyte ratio
1.72
0.063
12
0.071
15.0
NK lymphocytes
13.3
1.384
12
1.384
17.8
Meanb as %
Lymphocytes
SD as an Estimate
of Within-Stain
Reproducibilityc
Total T lymphocytes
72.7
0.845
Total B lymphocytes
13.5
Helper/inducer
T lymphocytes
45.8
Suppressor/cytotoxic
T lymphocytes
Parameter
a. This data was collected on a BD FACScan flow cytometer using BD Simultest IMK-Lymphocyte reagents and
software. Software employed was BD Simultest IMK-Lymphocyte software, one of the BD Simulset family of
software.
43
b. Mean is the pooled mean, Y = the mean of the individual means. Means are expressed as percentages of
lymphocytes (except for helper/suppressor ratio).
c. SD = standard deviation, the pooled standard deviation.
d. df (normal) = degrees of freedom, the number of observations (4) minus the number of means (2), multiplied by
the number of subjects (6) = 12.
e. df = degrees of freedom using Satterthwaite’s method.74
Table 7. Within-sample reproducibilitya for BD Simultest IMK-Lymphocyte parameters
(4 abnormal subjects) as percentages of gated lymphocytes (converted)
Meanbas %
Lymphocytes
SD as an Estimate
of Within-Stain
Reproducibilityc
dfd
SD as an
Estimate of
Between-Stain
Reproducibility
Total T lymphocytes
82.9
0.766
8
0.927
8.1
Total B lymphocytes
8.81
0.559
8
0.662
8.4
Helper/inducer
T lymphocytes
31.6
1.061
8
1.118
9.9
Suppressor/cytotoxic
T lymphocytes
51.4
1.369
8
1.561
8.8
Helper/suppressor
T-lymphocyte ratio
0.69
0.031
8
0.031
11.8
NK lymphocytes
8.00
0.707
8
0.707
11.5
Parameter
dfe
a. This data was collected on a BD FACScan flow cytometer using BD Simultest IMK-Lymphocyte reagents and
software. Software employed was BD Simultest IMK-Lymphocyte software, one of the BD Simulset family of
software.
b. Mean is the pooled mean, Y= the mean of the individual means. Means are expressed as percentages of
lymphocytes (except for helper/suppressor ratio).
c. SD = standard deviation, the pooled standard deviation.
d. df (abnormal) = degrees of freedom, the number of observations (4) minus the number of means (2), multiplied by
the number of subjects (4) = 8.
e. df = degrees of freedom using Satterthwaite’s method.74
Between-Instrument Reproducibility
Using a protocol similar to that above, samples of whole blood from
five normal subjects were divided into two aliquots, stained with the
44
BD Simultest IMK-Lymphocyte reagents, lysed, washed, and fixed.
Samples were each run twice on two different BD FACScan flow
cytometers. Results are shown in Table 8.
Table 8. Between-instrument reproducibilitya for BD Simultest IMK-Lymphocyte
parameters* (5 normal subjects and 2 instruments) as percentages of gated lymphocytes
(converted)
IMK-Lymphocyte Parameter
Total T lymphocytes
Total B lymphocytes
Instrument
Mean (%)b
SDc
CVd
1
68.2
0.77
1.13
2
68.7
0.71
1.04
1
17.2
0.80
4.66
2
17.8
0.88
4.97
1
38.6
1.04
2.68
2
38.1
0.88
2.32
Suppressor/cytotoxic
T lymphocytes
1
28.9
1.04
3.58
2
27.6
1.00
3.62
Helper/suppressor
T-lymphocyte ratio
1
1.51
0.06
3.74
2
1.54
0.07
4.47
NK lymphocytes
1
12.9
0.46
3.58
2
13.4
0.41
3.06
Helper/inducer T lymphocytes
a. This data was collected on a BD FACScan flow cytometer using BD Simultest IMK-Lymphocyte reagents and
software. Software employed was BD Simultest IMK-Lymphocyte software, one of the BD Simulset family of
software.
b. Mean is the pooled mean, Y= the mean of the individual means. Means are expressed as percentages of
lymphocytes (except for helper/suppressor ratio).
45
c. SD = standard deviation, the pooled standard deviation.
SD =
2
2
2
( n – 1 )s 1 + ( n – 1 )s 2 + … + ( n – 1 )s k
1
2
k
----------------------------------------------------------------------------------------------------------------n +n +…+n –k
1
2
k
si2 = variance of the ith sample for 1≤i≤k.
k = number of samples.
n = number of observations in a sample.
d. CV = average coefficient of variation (= SD/Y x 100).
Between-Laboratory Reproducibility
Between-laboratory reproducibility is indicated by the ability to pool
the normal reference ranges for BD Simultest IMK-Lymphocyte
parameters as shown in Table 4.
Simultest IMK-Lymphocyte vs Comparative Method
Aliquots from the same blood samples from both the normal and
abnormal subjects used to determine normal reference ranges were
analyzed with the BD Simultest IMK-Lymphocyte reagents (using
BD Simultest IMK-Lymphocyte software) and the BD Simultest IMK
Plus reagents (using BD Simultest™ IMK Plus software.)
Samples were stained, lysed, washed, and fixed and flow cytometric
analysis performed using the BD FACScan. Results were compared
using linear regression. A summary of the results is presented in
Table 9.
Table 9. BD Simultest IMK-Lymphocyte vs comparative methoda (BD Simultest IMKLymphocyte reagent panel and BD Simultest IMK Plus reagent panel)b
Range of Data (%)
Slope
Intercept
r
n
IMK Plus
IMKLymphocyte
Total T lymphocytes
0.94
3.66
0.96
85
50–97
47–96
Total B lymphocytes
0.98
0.62
0.97
85
1–25
1–29
Parameter
46
Table 9. BD Simultest IMK-Lymphocyte vs comparative methoda (BD Simultest IMKLymphocyte reagent panel and BD Simultest IMK Plus reagent panel)b
Range of Data (%)
Slope
Intercept
r
n
IMK Plus
IMKLymphocyte
Helper/inducer
T lymphocytes
0.95
–0.27
0.96
84
8–68c
8–65
Suppressor/cytotoxic
T lymphocytes
1.01
–6.03
0.93
84
19–74d
12–67
Helper/suppressor
T-lymphocyte ratio
1.25
–0.08
0.90
84
0.17–3.0e
0.21–3.17
NK lymphocytes
0.96
0.37
0.96
85
2–32
1–33
Parameter
a. This data was collected on a BD FACScan flow cytometer using BD Simultest IMK-Lymphocyte reagents and
software. Software employed was BD Simultest IMK-Lymphocyte software, one of the BD Simulset family of
software.
b. The two methods differ in that the BD Simultest IMK Plus kit uses CD4/CD8 to identify helper/inducer and
suppressor/cytotoxic T lymphocytes, whereas the BD Simultest IMK-Lymphocyte uses CD3/CD4 and CD3/CD8 to
distinguish true helper/inducer and true suppressor/cytotoxic T-lymphocyte subsets.
c. Includes monocytes.
d. Includes NK lymphocytes.
e. Includes monocytes and NK lymphocytes.
Stability of Stained Cell Preparations
Cell preparations from six normal and four abnormal subjects were
acquired and analyzed on the BD FACScan. The preparations were
stored at 2°C–8°C in the dark for 24 hours and flow cytometric
analysis repeated. Results demonstrate small but statistically significant
differences for helper/inducer T-lymphocyte values and for helper/
suppressor T-lymphocyte ratio values between day 0 and day 1. All
other differences were not significant. We recommend analyzing
samples within 24 hours of staining.
47
Cross-Reactivity
The specificity of these monoclonal antibodies has been established by
blind testing at a number of laboratories by the International Leucocyte
Workshop Group.29,31,38,42,61,62
The CD45 antibody has been reported to react weakly with mature
circulating erythrocytes and platelets.12,34 The CD14 antibody reacts
with granulocytes,37 as well as monocytes/macrophages. The CD4
antibody reacts with monocytes as well as helper/inducer
T lymphocytes.51 The CD8 antigen is present on a subset of NK
lymphocytes.26 The CD16 antibody reacts weakly with neutrophils.61
Potentially interfering granulocytes should be eliminated from the
lymphocyte gate using their light-scatter properties.
Although the Control reagent (Reagent B) is designed to detect
nonspecific staining, nonspecific staining due to poor condition of cells
can be observed with any antibody reagent. Nonspecific staining can
be inferred if no clear separation between negative and positive cells is
seen on the fluorescence plot or if more than 5% of events for the
Control tube appear outside Q3. Consult the Troubleshooting section
at the end of this IFU if nonspecific staining is seen.
Linearity-Recovery
All lots of BD Simultest IMK-Lymphocyte reagents are tested on cell
suspensions to ensure optimal performance with 100 µL of whole
blood. For this study, the normal range was 3.5 x 103 to 9.4 x 103
WBC/µL. Results are expected to be linear from 3.5 x 103 to 9.4 x 103
WBC/µL.
48
12. TROUBLESHOOTING
Problem
Possible Causes
Insufficient separation between negative 1) Decreased fluorescence due to incorrect PMT
and positive populations.
voltage or amplifier gain.
2) Insufficient antibody reagent.
No separation found between debris
1) Presence of excess platelets or nucleated red
and lymphocyte clusters on light-scatter blood cells or unlysed red blood cells.
dot plot.
2) FSC amplifier gain improperly set.
3) Sample not stained within 6 hours of collection.
4) FSC threshold set too high or too low.
Suboptimal antibody-staining
performance.
1) Sample not stained within 6 hours of collection.
2) Analysis gate selected by software included too
many nonlymphocyte events.
3) Fluorescence markers set incorrectly.
Solution
1) Run BD Autocomp software with BD Calibrite beads. Must meet specifications for
intensity in fluorescence channels.
2) Check pipet calibration, reagent volume added, as well as volume and concentration of
cells added. Check reagent storage conditions.
1) Restain a fresh sample. Wash cells at lower speed, for example, 100g for 15 minutes at
room temperature (20°C–25°C).
2) Adjust FSC amplifier gain such that the left edge of the lymphocyte cluster begins at
approximately channel 50 (256-channel scale). This is especially important when going from
setup/compensation with BD Autocomp software to whole blood determinations. Refer to
the appropriate user’s guide or instrument manual.
3) Collect a fresh sample to stain.
4) Adjust FSC threshold so that there are approximately 10 channels of debris (256-channel
scale).
49
Solution
1) Collect a fresh sample to stain.
2) Reprocess data using CONSORT 30, BD Lysys™ II, or BD Simulset software and set a
manual gate. NOTE: A tight lymphocyte gate might exclude large lymphocytes, for example,
NK lymphocytes, lymphoblasts. If possible, reacquire samples after optimization of scatter,
adjust SSC PMT, FSC amplifier gain, and FSC threshold to separate lymphocyte clusters
from debris. Refer to the appropriate software user’s guide.
3) Reprocess data using CONSORT 30, BD Lysys II, or BD Simulset software and set
manual fluorescence markers.
Problem
Possible Causes
Software unable to set an acceptable
lymphocyte gate.
1) Sample is lymphopenic.
2) FSC threshold set too high or too low.
3) FSC and/or SSC amplifier gain improperly
set.
4) Sample not stained within 6 hours of
collection.
5) Software marker setting not appropriate.
Unstained cells in the stained sample move
over marker set with Control tube (B) into
Q4 or Q1.
1) Nonspecific staining due to sample
containing dead or damaged cells.
Staining is dim or inconsistent.
1) Cell concentration too high at staining
step.
2) Nonspecific staining due to Fc receptors.
2) Insufficient reagent.
3) Cells not analyzed within 24 hours of
staining.
4) Improper medium preparation (sodium
azide omitted).
5) Buffer or fixative at improper pH.
6) Possible contamination of PBS, or
fixative.
50
Solution
1) Concentrate cells using a density-gradient method or reacquire a larger number of events
using CONSORT 30 or BD Lysys II.
2) Adjust FSC threshold so that there are approximately 10 channels of debris (256-channel
scale).
3) Adjust FSC amplifier gain such that the left edge of the lymphocyte cluster begins at
approximately channel 50 (256-channel scale). Adjust SSC amplifier gain in order to
optimize cell populations. This is especially important when going from setup/
compensation with BD Autocomp software to whole blood determinations. Refer to the
appropriate user’s guide.
4) Collect a fresh sample to stain.
5) Reacquire data using CONSORT 30 or BD Lysys II.
1) Collect a fresh sample to stain.
2) Check for excessive monocyte, granulocyte, or debris contamination in the lymphocyte
analysis gate.
1) Check and adjust cell concentration; repeat staining with fresh sample.
2) Repeat staining with proper amount of reagent.
3) Repeat staining with fresh sample; analyze promptly.
4) Use sodium azide in staining medium and washing steps.
5) Prepare freshly diluted PBS with reagent-grade water and fresh fixative with PBS with
sodium azide, or prepare fresh BD CellWASH. Adjust to the proper pH. Filter through a
0.2-µm filter.
6) See 5 above.
Problem
Few or no cells.
Possible Causes
1) Cell concentration too low.
2) Centrifuge speed too low.
3) Flow cytometer malfunctioning.
51
Problem
Increased autofluorescence.
Possible Causes
1) Possible contamination of PBS, or fixative.
2) Poor sample preparation.
Solution
1) Resuspend fresh sample at a higher concentration. Repeat staining and analysis.
2) Check centrifuge calibration. Repeat prep using correct centrifuge speed.
3) Troubleshoot instrument.
1) Prepare freshly diluted PBS with reagent-grade water and fresh fixative; filter PBS and
fixative through a 0.2-µm filter.
2) Collect a fresh sample and stain.
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 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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