BD Simultest™ IMK Plus
Catalog No. 349217
IVD
BD, BD Logo and all other trademarks are property of
Becton, Dickinson and Company. © 2015 BD
02/2015
23-3178-04
Becton, Dickinson and Company
BD Biosciences
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San Jose, CA 95131 USA
Becton Dickinson Pty Ltd,
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Tel +353.1.202.5222
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Becton Dickinson Limited,
8 Pacific Rise, Mt. Wellington,
Auckland, New Zealand
BD Biosciences
European Customer Support
Tel +32.2.400.98.95
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[email protected]
bdbiosciences.com
[email protected]
CONTENTS
1. INTENDED USE ............................................................................ 5
2. SUMMARY AND EXPLANATION ................................................ 5
Clinical Applications .................................................................... 6
3. PRINCIPLES OF THE PROCEDURE................................................ 8
4. REAGENTS ................................................................................. 11
Reagents Provided, Sufficient for 50 Tests .................................. 11
Precautions ................................................................................. 17
5. INSTRUMENT............................................................................. 18
6. SPECIMEN AND COLLECTION 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 .......................................................................... 27
8. RESULTS..................................................................................... 29
Calculation of Corrected Counts ................................................ 29
Calculation of Absolute Counts.................................................. 33
Three-Part Differential................................................................ 34
9. LIMITATIONS ............................................................................. 35
iii
10. EXPECTED VALUES ................................................................... 38
Leucocyte Subsets ....................................................................... 38
Absolute Counts ......................................................................... 41
11. PERFORMANCE CHARACTERISTICS.......................................... 43
Within-Sample Reproducibility................................................... 43
Between-Instrument Reproducibility........................................... 44
Between-Laboratory Reproducibility .......................................... 44
BD Simultest IMK Plus vs Comparative Methods....................... 44
Stability of Stained Cell Preparations.......................................... 46
Cross-Reactivity ......................................................................... 47
Linearity-Recovery ..................................................................... 48
12. TROUBLESHOOTING ................................................................. 49
REFERENCES .................................................................................... 52
WARRANTY..................................................................................... 58
iv
1. INTENDED USE
The BD Simultest™ IMK Plus reagent kit is a two-color direct
immunofluorescence method for enumerating percentages of the
following mature (nonblast) human lymphocyte subsets in erythrocytelysed whole blood (LWB): T (CD3+) and activated T (CD3+HLA-DR+)
lymphocytes, B (CD19+) lymphocytes, helper/inducer (CD4+) and
suppressor/cytotoxic (CD8+) lymphocytes, and natural killer (NK)
(CD16+ or CD56+ or both) lymphocytes. The helper/suppressor ratio
(CD4+/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 that
correlates with function: T lymphocytes, B lymphocytes, and NK
lymphocytes. T lymphocytes participate in antigen-specific cellmediated immunity and regulate the secretion of immunoglobulin by
B lymphocytes. T lymphocytes can also be classified based on their
functional properties as helper/inducer, suppressor/cytotoxic, or
activated T lymphocytes.
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)1
and identifying B lymphocytes using SRBCs coated with fluorochromelabeled polyclonal antibodies to surface immunoglobulin.1 Cytolytic
lymphocyte assays included the hemolytic plaque assay2 and a
cytotoxicity assay to detect the release of radioactive chromium.3
Antibody reagents have been used to distinguish cell-surface antigens
by fluorescence microscopy, electron microscopy, and radioactive label
assay.4 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. Flow cytometry using indirect
immunofluorescence, while providing assay sensitivity,5 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.6–8 The LWB method used with this kit does not
require density-gradient separation and therefore allows for shorter
sample preparation time and less handling of whole blood6 (see the BD
Monoclonal Antibodies Source Book, Section 2.2).
The development of monoclonal antibody 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™, which accurately characterize cells by means of fourparameter analysis. The BD FACScan, when used with BD Simultest™
IMK Plus software, characterizes up to 50,000 cells in a single sample
by simultaneous analysis of forward scatter (FSC), side scatter (SSC),
and multicolor fluorescence.
Clinical Applications*
Total T- and B-lymphocyte percentages are used to characterize some
forms of immunodeficiency diseases9,10 and some types of autoimmune
disease.11,12
* Not all studies cited in this section employed BD reagents.
6
Activated T lymphocytes (HLA-DR+ T lymphocytes) can be elevated in
states of immune activation,13 which can be caused by infection,14 or
impending rejection in the case of transplant monitoring.15 The actual
cause of immune activation must be verified by additional clinical and
laboratory tests.
Helper/inducer lymphocytes are a subset of T (CD3+) lymphocytes,
which also express the CD4 antigen. Suppressor/cytotoxic lymphocytes
express the CD8 antigen and are principally a subset of T (CD3+)
lymphocytes, although a subset of NK lymphocytes is also CD8+.16
Determining the 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 is depressed and the relative
percentage of the CD8+ subset is elevated in many patients with
congenital or acquired immunodeficiencies9 such as severe combined
immunodeficiency (SCID)9 and acquired immune deficiency syndrome
(AIDS).17
The percentage of suppressor/cytotoxic cells can be outside the normal
reference range in some autoimmune diseases11 and in certain immune
reactions such as acute graft-versus-host disease (GVHD)18 and
transplant rejection.15 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.1
The CD4+/CD8+ (helper/suppressor) lymphocyte ratio, quantified as
the ratio of CD4 FITC-positive lymphocytes to CD8 PE-positive
lymphocytes, has been used to evaluate the immune status of patients
with, or suspected of developing, autoimmune disorders11,12 or
immune deficiencies.1,17 In many cases, the relative percentages of
helper lymphocytes decline and suppressor lymphocytes increase in
immune deficiency states. These states can also be marked by T-cell
lymphopenia.10,19 In addition, the ratio has been used to monitor bone
7
marrow transplant patients for onset of acute GVHD.18 While a useful
indicator, use of the CD4+/CD8+ (helper/suppressor) lymphocyte ratio
has specific limitations discussed in items Section 9, Limitations, of this
instructions for use (IFU).
NK lymphocytes, identified as being CD3-negative and CD16- or
CD56-positive or both,20 have been shown to mediate cytotoxicity
against certain tumors and virus-infected target cells. NK-mediated
cytotoxicity does not require class I or class II major histocompatibility
complex (MHC) molecules to be present on the target cell.21 NKlymphocyte activity is depressed in AIDS patients and, in some
instances, in AIDS-related complex and lymphadenopathy syndrome.22
3. PRINCIPLES OF THE PROCEDURE
The kit contains six pairs of BD Simultest murine monoclonal antibody
reagents conjugated with FITC and PE that include BD Leucogate™
(CD45/CD14) (Reagent A) for establishing a lymphocyte acquisition
gate and BD Simultest™ Control IgG1 FITC/IgG2a PE (Reagent B) for
setting fluorescence markers around the negative population and
detecting nonantigen-specific antibody binding. The kit also contains
BD FACS™ lysing solution (Reagent G) for preparing cells by the LWB
method. We recommend the BD FACScan for flow cytometry and
BD Simultest IMK Plus software for evaluating the data.
A fresh peripheral blood sample is collected by venipuncture and
stained within 6 hours with each of the six antibody reagents from the
BD Simultest IMK Plus kit. When the 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 then treated with BD FACS lysing solution to lyse
erythrocytes, and washed prior to flow cytometric analysis.
8
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
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
forward-scattered light provides a measure that correlates well with
cell size, while the side-scattered light is an indicator of cellular
granularity. The BD FACScan flow cytometer used with BD Simultest
IMK Plus software counts a sufficient number of cells to ensure that a
minimum of 2,000 lymphocytes are included in the acquisition gate.
The data files should be saved in list mode and given logical names to
aid in retrieval for subsequent analysis by BD Simultest IMK Plus
software.
BD Simultest IMK Plus software uses BD Leucogate, Reagent A, to
establish a lymphocyte acquisition gate that includes greater than or
equal to 98% of the normal mature (nonblast) lymphocytes in the
sample. However, if the gate contains greater than or equal to 3%
monocytes, the software automatically reduces or tightens the lightscatter 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 acquisition gate on the basis of SSC, FSC,
and fluorescence properties. Refer to the BD Simultest IMK Plus User’s
Guide for details on how the software sets the gates and automatically
9
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. See the BD Simultest
IMK Plus 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 present, particularly that caused
by Fc receptors. When greater than 5% of the 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 the laboratory printout for the Control Tube B. When the negative
Control tube is being processed by the software, the operator should
check for error messages that would indicate nonantigen-specific
antibody binding.
The negative Control is a mixture of conjugated monoclonal antibodies
with the same fluorochromes (FITC and PE) as the test reagents. The
Control antibodies are specific to 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 acquisition gate set with
BD Leucogate (Tube A) and the fluorescence markers determined using
the Control (Tube B) are used to analyze the subsequent tubes (C
through F). When the Quadrant Correction software option has been
selected from the Main Menu of BD Simultest IMK Plus software, the
lymphocyte subpopulations in Tubes C through F are enumerated and
then expressed as percentages of lymphocytes in the acquisition gate.
BD Simultest IMK Plus software provides a report quantifying these
10
immunologically significant lymphocyte subsets in LWB as percentages
of total circulating (nonblast) human lymphocytes and reports a CD4+/
CD8+ lymphocyte ratio. If this software option is not selected, results
will be expressed as percentages of the total gated events.
For quality control purposes, a three-part differential for percentages
of monocytes, granulocytes, and lymphocytes is determined
automatically by the software and is printed only for comparison with
results from a standard laboratory differential white count.23 To obtain
the three-part differential, the software uses an algorithm to identify
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 and
granulocytes can also be distinguished on the basis of their SSC signal.
Lymphocytes exhibit a low SSC signal and granulocytes a high SSC
signal.
NOTE The differential count provided by BD Simultest IMK Plus
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 nor entered into BD Simultest IMK Plus software to
obtain absolute counts.
4. REAGENTS
Reagents Provided, Sufficient for 50 Tests
All monoclonal antibody reagents contain murine immunoglobulins
conjugated to either FITC or PE in 1.0 mL of buffered saline with
gelatin and 0.1% sodium azide. The monoclonal antibodies are derived
from fusion of mouse myeloma cells with spleen cells or lymph node
cells of BALB/c mice. The fluorescein-to-protein ratio (F:P) for BD
monoclonal antibody reagents is within the range of 2 to 10. The F:P
ratio for each reagent has been optimized for its intended use.
11
Reagent A, BD Leucogate (CD45/CD14), 1.0 mL
BD Leucogate is used to define and evaluate the light-scatter gate that
distinguishes lymphocytes from granulocytes, monocytes, unlysed or
nucleated red blood cells (RBCs), and debris. This reagent contains
FITC-labeled CD45, clone 2D1,24–26 for identification of leucocytes,
and PE-labeled CD14, clone MφP9,27–29 for identification of
monocytes.
The CD4530 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 IgG1 heavy chains and kappa light chains. The CD45
antigen is present on all human leucocytes and has a role in signal
transduction, modifying signals from other surface molecules.30 The
molecular weight of the antigen recognized by this antibody is 170 to
220 kilodaltons (kDa).30
The CD1427 antibody was derived from 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 molecular weight of the CD14 antigen recognized by this
antibody is 53 kDa.31 The antigen is a myeloid differentiation
antigen31 and is present on 75% to 90% of human monocytes.32 The
CD14 antibody reacts weakly with granulocytes.33
Reagent B, Control, 1.0 mL
The isotype control reagent is used to set the FL1 and FL2 quadrant
markers around the unstained (negative) lymphocyte population to
establish a boundary between negative and positive cell populations
and estimates nonantigen-specific antibody binding, in particular that
caused by Fc receptors. It contains FITC-labeled IgG1, clone X40, and
PE-labeled IgG2a, clone X39, murine monoclonal antibodies that react
12
specifically with keyhole limpet hemocyanin (KLH), an antigen not
present on human leucocytes.
Reagent C, CD3/CD19, 1.0 mL
CD3/CD19 is used to enumerate T and B lymphocytes. It contains
FITC-labeled CD3, clone SK7,34–37 for the identification of
T lymphocytes, and PE-labeled CD19, clone 4G7,38 for the
identification of B lymphocytes.
The CD3 antibody36 is derived from hybridization of mouse NS-1
myeloma cells with spleen cells from BALB/c mice immunized with
human thymocytes. This antibody is composed of mouse IgG1 heavy
chains and kappa light chains. CD3 reacts with the epsilon chain of the
CD3 antigen/T-cell antigen receptor (TCR) complex. This complex is
composed of at least four proteins that range in molecular weight from
20 to 30 kDa.39 The antigen recognized by CD3 antibodies is non
covalently associated with either α/β or γ/δ TCR (70 to 90 kDa).40
The CD19 antibody38 is derived from hybridization of mouse
P3-X63-Ag8.653 myeloma cells with spleen cells of BALB/c mice
immunized with human chronic lymphocytic leukemia (CLL) cells. The
antibody is composed of IgG1 heavy chains and kappa light chains.
The CD19 antigen is present on human B lymphocytes at all stages of
maturation but is lost on plasma cells. The antigen can be involved in
activation and proliferation of B lymphocytes.41 The molecular weight
of the antigen recognized by this antibody is 90 kDa.41
Reagent D, CD4/CD8, 1.0 mL
CD4/CD8 is used to simultaneously characterize helper/inducer and
suppressor/cytotoxic lymphocytes. It contains FITC-labeled CD4, clone
SK3,42,43 for the identification of helper/inducer lymphocytes, and PElabeled CD8, clone SK1,42,43 for the identification of suppressor/
cytotoxic lymphocytes.
13
The CD4 antibody42 is derived from 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).44,45
The antigen has a molecular weight of 59 kDa. 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.46 CD4 reacts with monocytes/macrophages and helper/
inducer T lymphocytes.47
The CD8 antibody42 is derived from 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
human suppressor/cytotoxic T-lymphocyte subset35,43 as well as on a
subset of NK lymphocytes.16 The CD8 antigenic determinant interacts
with class I MHC molecules, resulting in increased adhesion between
the CD8+ T lymphocytes and the target cells.48-50 Binding of the CD8
antigen to class I MHC enhances the activation of resting
T lymphocytes.48–51 The CD8 antigen is expressed as a disulfide-linked
bimolecular complex with a 32-kDa α subunit.52,53 The CD8 antigen
is coupled to p56lck. The CD8:p56lck complex can play a role in Tlymphocyte activation through mediation of the interactions between
the CD8 antigen and the CD3 antigen/TCR complex.50,51
Reagent E, CD3/Anti–HLA-DR, 1.0 mL
CD3/Anti–HLA-DR is used to enumerate T lymphocytes, DR+ nonT lymphocytes (primarily B lymphocytes), and activated
T lymphocytes. It contains FITC-labeled CD3, clone SK7,34–37 for the
identification of T lymphocytes, and PE-labeled Anti–HLA-DR, clone
L243,54 for the identification of DR+ non-T lymphocytes and activated
T lymphocytes.
14
The Anti–HLA-DR antibody is derived from hybridization of mouse
NS1/1-AG4 myeloma cells with spleen cells from BALB/c mice
immunized with the human lymphoblastoid B-cell line RPMI 8866. It
is composed of mouse IgG2a heavy chains and kappa light chains. The
HLA-DR antigen (human leucocyte antigen, D-related) is a human
class II MHC molecule. The antigen is a transmembrane glycoprotein
composed of α- and β subunits that have molecular weights of 36 and
27 kDa, respectively.54–55 The antibody reacts with a nonpolymorphic
HLA-DR epitope.54–56 The antigen is expressed on B lymphocytes,
monocytes, macrophages, activated T lymphocytes, activated NK
lymphocytes, and human progenitor cells.14,19,57–60
Reagent F, CD3/CD16+CD56, 1.0 mL
CD3/CD16+CD56 is used to identify T and NK lymphocytes. It
contains FITC-labeled CD3, clone SK7,34–37 to identify T lymphocytes.
It also contains PE-labeled CD16, clone B73.1,61–64 and PE-labeled
CD56, clone MY31,63,65 to identify NK-lymphocyte populations as
well as T-lymphocyte subsets.
The CD16 antibody64 was derived from hybridization of mouse
P3X-63-Ag8.653 myeloma cells with spleen cells of BALB/c mice
immunized with NK lymphocytes. The antibody is composed of IgG1
heavy chains and kappa light chains.61–63 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.20
The CD56 antibody65 was derived from hybridization of mouse Sp2/0
myeloma cells with cells of (B6xBALB/c)F1 mice immunized with the
KG1a cell line. The antibody is composed of IgG1 heavy chains and
kappa light chains. The molecular weight of the glycosylated antigen
recognized by this antibody ranges from 175 to 220 kDa20,66 (or 137
kDa when deglycosylated67) and is present on NK lymphocytes. The
CD56 antigen is involved in neuronal homotypic cell adhesion and cell
differentiation during embryogenesis.65
15
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 shown on the label. For use, dilute 1:10 with room temperature
(20°C–25°C) reagent-grade water. Store in a glass container at room
temperature. The prepared solution is stable for 1 month at room
temperature.
Concentration values are listed in the following table:
Reagent
Component
A: BD Leucogate
CD45 FITC
Concentration (µg/mL)
25
CD14 PE
B: Control
12.5
IgG1 FITC
25
IgG2a PE
C: CD3/CD19
D: CD4/CD8
E: CD3/HLA-DR
F: CD3/CD16+CD56
12.5
CD3 FITC
25
CD19 PE
6
CD4 FITC
1.5
CD8 PE
25
CD3 FITC
25
Anti-HLA-DR PE
3.1
CD3 FITC
25
CD16 PE
12.5
CD56 PE
25
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.
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 the reagents, such as precipitation
or discoloration, indicates instability or deterioration. In such
cases, the reagents 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,
10% 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 into
contact with them are considered biohazards. Handle as if capable
of transmitting infection68,69 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
The BD Simultest IMK Plus kit 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.
18
The following instrument system is recommended:
•
•
•
•
•
BD FACScan flow cytometer system equipped for three-color
fluorescence detection and two-parameter light-scatter detection.
For detailed information on use, refer to the BD FACScan User’s
Guide.
BD CONSORT™ 30 or CONSORT 32 computer system and
peripherals (included in the BD FACScan system). For detailed
information on use, refer to the BD CONSORT 30 Software
User’s Guide or the BD CONSORT 32 System User’s Guide.
BD Simultest IMK Plus software. For detailed information on use,
refer to the BD Simultest IMK Plus User’s Guide.
BD Calibrite™ beads (Catalog No. 349502). These beads can be
used for setting the photomultiplier tube (PMT) voltages, setting
the fluorescence compensation, and checking instrument sensitivity
on the BD FACScan flow cytometer. For detailed information on
use, refer to the BD Calibrite Beads IFU.
BD Autocomp™ software. For detailed information on use, refer
to the BD Autocomp Software User’s Guide.
All performance characteristics were obtained using the above
instrument system. Other systems can have different characteristics.
6. SPECIMEN AND COLLECTION PREPARATION
Collect blood aseptically by venipuncture5,70 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 drawing for optimal results. Anticoagulated
blood can be stored at room temperature (20°C–25°C) for up to
6 hours until ready for staining. Blood samples refrigerated prior to
staining can give aberrant results.
A white blood count (WBC) and a differential white count should be
obtained from the same sample of whole blood before staining. An
19
acceptable WBC concentration range is from 3.5 x 103 to 9.8 x 103
WBC/µL. Samples with counts greater than 9.8 x 103 WBC/µL must be
diluted with 1X phosphate-buffered saline (PBS) containing 0.1%
sodium azide (see Section 9, Limitations). 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 cells 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. The presence of
abnormal (blast) cells or unlysed or nucleated RBCs can interfere with
test results. Hemolyzed samples or samples with less than 1 mL of
whole blood in the collection tube should be rejected.
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, Sufficient for 50 Tests, 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.
* Falcon is a registered trademark of Corning Incorporated.
20
•
•
•
•
•
•
Low-speed centrifuge (200g) with swinging bucket rotor and
12 x 75-mm tube carriers.
Vacuum aspirator with trap.
Micropipettor with tips.
BD CellWASH™ (Catalog No. 349524) or a wash buffer of PBS
with 0.1% sodium azide.
BD CellFIX™ (Catalog No. 340181) or 1% paraformaldehyde
solution in PBS with 0.1% sodium azide. Store at 2°C–8°C in
amber glass for up to 1 week.
BD FACSFlow™ sheath fluid (Catalog No. 342003).
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 stained with Reagents A through F. Diluted
Reagent G, 1X BD FACS lysing solution, is then used to lyse RBCs
following staining. If the WBC count is sufficient, cell separation is not
required prior to staining. See Section 6, Specimen and Collection
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 A through 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 patient sample, use a fresh micropipettor tip and carefully
add 100 µL of the correct concentration of well-mixed,
21
anticoagulated whole blood patient sample into the bottom of each
of the six labeled tubes. The required WBC concentration is 3.5 x
103 to 9.8 x 103 WBC/µL. Vortex 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
might not be stained with the reagent.
4. Dilute 10X BD FACS lysing solution to 1X following the
instructions for Reagent G under Reagents Provided, Sufficient for
50 Tests, in Section 4, Reagents. Add 2 mL of room temperature
(20°C–25°C) 1X BD FACS lysing solution to each tube.
Immediately vortex thoroughly at low speed for 3 seconds and
incubate for 10 to 12 minutes at room temperature (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.
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 each 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.
22
9. Vortex thoroughly at low speed to resuspend the cell pellet in the
residual fluid and then add 0.5 mL of BD CellFIX solution or 1%
paraformaldehyde to each tube. Vortex thoroughly at low speed
for 3 seconds. Make sure that the cells are well mixed with the
fixing solution.
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) before
putting them through the flow cytometer to help reduce
aggregation.
Flow Cytometry
Follow the BD instructions for two-color flow cytometric analysis and
refer to Section 5, Instrument. The following general approach is
recommended. The BD FACScan flow cytometer is first prepared for
sample analysis using BD Calibrite beads and BD Autocomp software.
The stained sample tubes are then run on the flow cytometer and
analyzed with the BD Simultest IMK Plus software. Refer to the
BD FACScan User’s Guide, the BD Calibrite Beads IFU, the
BD Autocomp Software User’s Guide, and the BD Simultest IMK Plus
User’s Guide for detailed instructions for use. We recommend that
patient data be stored to allow subsequent analysis of data files.
Set up and adjust compensation of the BD FACScan flow cytometer.
Using BD Calibrite beads and BD Autocomp software, set
photomultiplier tube (PMT) voltages, adjust fluorescence
compensation, and check detector sensitivity. Refer to the BD Calibrite
Beads IFU, the BD Autocomp Software User’s Guide, and the
BD FACScan User’s Guide for details.
Run all sample tubes through the flow cytometer and acquire data in
list mode files using BD Simultest IMK Plus software. Refer to the
BD Simultest IMK Plus User’s Guide for detailed instructions for use
of the software. The software automatically collects a sufficient
23
number of events to obtain a minimum of 2,000 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 (Q1) (low yellow-green/high red-orange), Q2
(high yellow-green/high red-orange, or dual-fluorescence), Q3 (low
yellow-green/low red-orange), and Q4 (high yellow-green/low redorange) (see Figure 1).
Figure 1 The fluorescence display quadrants (indicated as Q1–Q4) and the corresponding
colors.
Q1
Low yellowgreen/high
red-orange
Q2
High yellow-green/high
red-orange
(dual fluorescence)
Q3
Low yellowgreen/low
red-orange
Q4
High yellow-green/low
red-orange
The BD Leucogate tube A is used to gate on lymphocytes. The
BD FACScan with BD Simultest IMK Plus software automatically sets
a lymphocyte acquisition gate to eliminate most debris, monocytes, and
granulocytes (see Figure 2 and Figure 3). Refer to the BD Simultest
IMK Plus 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. Errors in
sample gating can be caused by the sample preparation or instrument
setup.
The Control tube B is used to set fluorescence intensity markers.
BD Simultest IMK Plus software automatically uses the lymphocyte
24
acquisition gate set with BD Leucogate and then establishes FL1 and
FL2 markers. These markers define the boundaries between positively
stained and unstained 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.
Figure 2 BD FACScan LWB sample from a hematologically normal male patient stained
with BD Simultest IMK Plus 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) versus FL2
(y-axis) are shown for tubes B through F.
Tube B
Tub e C
CD19
S SC
Ig G 2 a
Tub e A
Ig G 1
CD3
Tube D
Tube E
Tube F
CD4
C D 1 6+ C D 5 6
C D8
An ti – H L A- D R
FSC
CD3
CD3
After the markers are set, if more than 5% of the total counts for tube
B remain in Q1 (low yellow-green/high red-orange), Q2 (high yellowgreen/high red-orange), and Q4 (high yellow-green/low red-orange),
nonantigen-specific antibody binding is suspected. The software
notifies the operator with a message “too much nonspecific staining.”
25
If this error message appears, a new sample should be obtained from
the original aliquot of anticoagulated whole blood and the entire
staining procedure should be repeated.
Figure 3 Mononuclear cell populations in Q1 through Q4 for tubes C through F. The
populations represent the primary reagent reactivity. (For a complete list of reactivity, see the
reagent descriptions under Reagents Provided, Sufficient for 50 Tests, in Section 4,
Reagents.)
Tube C (CD3/CD19)
Tube D(CD4/CD8)
Q1
Q2
Q1
Q2
CD3–CD19+
B lymphocytes
Nonantigen-specific
antibody bindinga
CD4–CD8+
suppressor/
cytotoxic and NK
lymphocytes
CD4+CD8+
lymphocytes
Q3
Q4
Q3
Q4
Unstained
lymphocytes;
contaminating
monocytes,
granulocytes, and
debris
CD3+CD19–
T lymphocytes
Unstained
lymphocytes;
granulocytes and
debris
CD4+CD8–
helper/inducer
lymphocytes;
monocytes
a. See Section 9, Limitations.
Tube E (CD3/Anti–HLA-DR)
Tube F (CD3/CD16+CD56)
Q1
Q2
Q1
Q2
CD3-Anti–HLA-DR+
B lymphocytes,
activated NK
lymphocytes;
monocytes/
macrophages
CD3+Anti–HLA-DR+
activated
T lymphocytes
CD3–CD16+CD56+
NK lymphocytes
CD3+CD16+CD56+
T lymphocyte
subset
Q3
Q4
Q3
Q4
Unstained
lymphocytes;
contaminating
monocytes,
granulocytes, and
debris
CD3+Anti–HLA-DR–
T lymphocytes
Unstained
lymphocytes;
contaminating
monocytes,
granulocytes, and
debris
CD3+CD16–CD56–
T lymphocytes
26
Data for tubes C, D, E, and F is acquired and analyzed by BD Simultest
IMK Plus software using the gates and markers established with tubes
A and B. Analyze these results using the criteria provided under
Quality Control and according to the instructions in Section 8, Results.
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 Plus reagents on the BD FACScan flow
cytometer. Refer to Appendix A in the BD Simultest IMK Plus User’s
Guide for information on optimizing the flow cytometer prior to
analyzing patient samples.
The negative Control provided in the BD Simultest IMK Plus reagent
kit is run with each patient sample to set FL1 and FL2 markers
between negatively and positively stained lymphocyte clusters and to
detect the presence of nonantigen-specific antibody binding 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 quadrant 3 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, Section 12, 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 nonantigen-specific antibody binding.
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
sample are shown in Figure 2.
27
If there is poor separation between negative and positive clusters as
seen on visual inspection of the dot plots or contour displays,
nonantigen-specific antibody binding can be inferred and the run
should be rejected. Nonantigen-specific antibody binding might be seen
because of poor condition of the cells. Consult 12.Troubleshooting,
Section 12, if nonantigen-specific antibody binding is observed.
BD Simultest IMK Plus software will automatically inspect the data
and alert the operator with a number of possible error messages. Refer
to Appendix D of the BD Simultest IMK Plus User’s Guide for a list of
possible messages. The software uses the following criteria for
inspection of the dot plots obtained for each sample to evaluate the
quality of the data obtained.
1. The operator should reject any results if any one of the following
error messages is received for the normal control: no separation
between cellular populations; too few lymphocytes (less than 500);
excessive RBC or nucleated RBC contamination and debris
(greater than 20%); or excessive monocyte (greater than 4%) or
granulocyte (greater than 4%) contamination of the lymphocyte
gate.
2. If there is no obvious reason for the normal control to fail, a
sample from another normal control should be restained and rerun
and the entire staining procedure repeated on all subsequent
samples.
3. Samples with nucleated RBCs can contain too much debris because
of incomplete lysis of nucleated erythrocytes with Reagent G,
BD FACS lysing solution. Too much debris can also occur when
assaying blood samples from patients with certain hematologic
disorders where red cells are difficult to lyse, for example,
myelofibrosis, spherocytosis. Nucleated erythrocytes will be
counted as debris and, if debris exceeds 20%, the software will flag
28
the sample as “too many nonlymphs in gate” and the sample
results should be rejected.
4. The operator should check that the BD Simultest IMK Plus
lymphocyte differential count is within ±10% of the independent
differential white count. If the BD Simultest IMK Plus count is
outside the range, a discrepancy exists and the run should be
questioned. Either the BD Simultest IMK Plus count is in error or
the independent laboratory differential white count is incorrect.
See Section 9, Limitations.
5. The operator should check that there is agreement in T-lymphocyte
percentages between tubes C, E, and F, which contain CD3+. The
run should be rejected if the total T-lymphocyte values in any two
of the three tubes differ by more than 8%.
6. Helper/inducer and suppressor/cytotoxic lymphocytes will not add
to total T lymphocytes because the CD8 antigen is also expressed
on NK lymphocytes. Internal assay consistency can be indicated
when the sum of the percentages of T, B, and NK lymphocytes
totals 100% ±5% when the Quadrant Correction option of
BD Simultest IMK Plus software is selected from the Main Menu.
Refer to the BD Simultest IMK Plus User’s Guide for instructions
on selecting this option.
See also Troubleshooting, Section 12, at the end of this IFU.
8. RESULTS
Calculation of Corrected Counts
When the Quadrant Correction software option is selected, the
BD FACScan system with BD Simultest IMK Plus software
automatically calculates each reported lymphocyte subset as a
percentage of total lymphocytes in the lymphocyte acquisition gate.
The software first subtracts nonlymphocytes from quadrant 3 (Q3) and
29
then reports the results as percentages of lymphocytes in the
lymphocyte acquisition gate set using the BD Leucogate tube (A).
When all quality control criteria listed under Quality Control in
Section 7, Procedure, have been met, the Quadrant Correction
software option provides appropriate estimations of the true subset
values. However, if the quality control criteria are not met (for
example, because of excessive 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 Quadrant Correction option is turned off, results will be
reported as a percentage of the total gated events. See the BD Simultest
IMK Plus User’s Guide.
The principle of the computation follows (Q values refer to counts in
quadrants):
Equation 1 computes the percentage of lymphocyte events within the
BD Leucogate lymphocyte acquisition gate. This value is given as
“%L” in tube A in Table 1 and reflects the purity of lymphocytes
within the gate drawn using the BD Leucogate tube (A).
30
Table 1 Summary of the representative data from Figure 2 and identification of
quadrants used to compute subsets
Tube
A (BD Leucogate)
Tube
C (CD3/CD19)
D (CD4/CD8)
%L*
%M*
%G*
%D*
% of Total Gated Lymphs†
95
1
3
1
99
Cell Type
Quadrant (s)
% of
Helper/
Lymphocytes( Suppressor
Corrected)
Ratio‡
Total T lymphocytes
Q4
Total B Lymphocytes
Q1
82
9
Helper/Inducer
lymphocytes;
monocytes
Q2, Q4
50
Suppressor/cytotoxic
and NK lymphocytes
Q1, Q2
41
E (CD3/Anti–
HLA-DR)
Total T lymphocytes
Q4
81
Activated T
lymphocytes
Q2
18
F (CD3/
CD16+CD56)
Total T lymphocytes
Q4
81
NK lymphocytes
Q1
7
1.2
* %L, %M, %G, and %D are lymphocytes, monocytes, granulocytes, and debris in the gate defined by BD Leucogate
expressed as percentages of all the events in that gate. These values reflect the quality and purity of the gate.
† Percent 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.
‡ Helper/suppressor ratio = %CD4+ lymphocytes/%CD8+ lymphocytes
31
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 2a. Percent gated, corrected lymphocytes in Q1, Q2, or Q4 =
Number of gated events in Q1, Q2, or Q4- × 100
--------------------------------------------------------------------------------------------N × (%L/100)
In Equation 2a, percentages of positive cells in each of the three
positive quadrants (Q1, Q2, and Q4) are expressed as corrected
percentages of lymphocytes, assuming all the non-lymphocytes in the
BD Leucogate-defined gate are unstained cells. To calculate the
correction factor in these quadrants, the software uses Equation 2a for
Q1, Q2, and Q4.
NOTE The Quadrant Correction option does not correct for
nonlymphocyte events that can occur in Q1, Q2, and Q4. Therefore,
CD4-positive monocyte events are not corrected in Q4 by the software.
On the Laboratory Report sheet, values for unstained cells (Q3) are
reported under “Corr %L” as percent lymphocytes corrected in Q3.
Q3 represents unstained cells and, presumably, all the nonlymphocyte
events in the gate as well as unstained lymphocytes. Therefore, in
addition to converting the results from the percentage of total events to
the percentage of lymphocytes in the gate, Equation 2b also subtracts
the gated nonlymphocyte events from the Q3-gated events.
32
Equation 2b. Percent gated, corrected, unstained lymphocytes in Q3
(Corr %L) =
(Number gated events in Q3 – Number gated nonlymphocytes) × 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 1, tube A.
Calculation of Absolute Counts
An absolute cell count can be computed if a WBC count and the
lymphocyte percentage from a differential white count are obtained
using standard laboratory procedures. The BD FACScan with
BD Simultest IMK Plus software automatically calculates an absolute
count for each BD Simultest IMK Plus parameter 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 Plus 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 a laboratory differential
white cell count obtained for the same sample. Do not use the
BD Simultest IMK Plus software three-part differential values.
3. The absolute counts for each subset are then computed as follows:
Absolute count of CD4+ cells/µL =
+
%CD4
---------- × (WBCs/µL)
----------------------- × %L
100
100
33
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 the WBC count,
(b) percent lymphocytes, and (c) the normal reference range and
precision for percent lymphocytes positive for the specific marker.
Three-Part Differential
For lysed whole blood, it is possible to determine monocytes,
lymphocytes, and granulocytes as a percentage of leucocytes using the
BD Leucogate tube A (see Table 2). The BD FACScan with
BD Simultest IMK Plus software automatically calculates a three-part
differential (Table 2). Refer to the BD Simultest IMK Plus User’s Guide
for representative data printouts.
Table 2. Three-part differential data from Figure 2
Three-Part Differentiala
% Leucocytes
Lymphocytes
25
Monocytes
5
Granulocytes
70
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.
NOTE The differential count provided by BD Simultest IMK Plus
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 nor entered into BD Simultest IMK Plus
software to obtain absolute counts.
34
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)
because 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 of less than 3.5 x 103 WBC/µL
and greater than 9.8 x 103 WBC/µL will require special handling to
obtain correct results. Samples with counts of greater than
9.8 x 103 WBC/µL will need to be diluted with PBS containing
0.1% sodium azide. Samples with counts of 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. For example, poor
resolution between positive and negative cells has been observed
with transplant patients receiving immunosuppressive drugs. If
there is poor separation between negative and positive clusters, the
run should be rejected (see Troubleshooting, Section 12, at the end
of this IFU). If the difference in the total T-cell percentage between
any two of the three tubes containing CD3 is greater than 8%, the
run should be rejected.
Laboratories must establish their own normal reference ranges for
each of the BD Simultest IMK Plus parameters, which can be
affected by sex, 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 count from
35
•
•
•
•
the BD Simultest IMK Plus software report to compute absolute
counts. The differential count provided by BD Simultest IMK Plus
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 nor entered into BD Simultest IMK Plus software to obtain
absolute counts.
If the BD Simultest IMK Plus lymphocyte differential count differs
from the independent differential white count by more than ±10%,
there is a significant discrepancy and care should be taken in
interpreting absolute count results. Either the BD Simultest IMK
Plus differential count or the laboratory differential white count is
in error. See Quality Control under Section 7, Procedure.
Variation in either automatic or manual lymphocyte acquisition
gate settings will change the relative amounts of subsets assayed.
BD Simultest IMK Plus software uses the BD Leucogate tube (A) to
include at least 95% of the total lymphocytes in the sample to set
the lymphocyte acquisition gate and requires a visual inspection of
the gate setting for validation.
Abnormal states of health are not always represented by abnormal
percentages of T or B lymphocytes or their subsets. That is, an
individual who can be in an abnormal state of health can exhibit
the same T- or B-lymphocyte percentages as a healthy individual.
Results from BD Simultest IMK Plus must be used in conjunction
with other information available from the clinical evaluation and
additional independent diagnostic procedures.
The information obtained from this kit must be combined with
other information and interpreted by a medically qualified
diagnostician to establish presence or absence of specific disease
states.
36
•
•
•
•
•
Note that normal reference ranges established by BD for the CD4+/
CD8+ (helper/suppressor) lymphocyte ratio extend below a ratio of
1.0 (see Table 3 under Leucocyte Subsets in Section 10, Expected
Values).
The BD Simultest IMK Plus software Quadrant Correction option
assumes that all nonlymphocytes are unstained. Therefore, CD4positive monocytes that appear in Q4 will be included as CD4positive lymphocytes. The limit of monocyte contamination
allowed is 4% before the sample is flagged. See the BD Simultest
IMK Plus User’s Guide for more information on quadrant
correction.
Note that some BD Simultest IMK Plus antibodies react with other
non-lymphocyte-formed elements in blood. See Cross-Reactivity in
Section 11, Performance Characteristics.
The helper/suppressor T-lymphocyte ratio as determined by
BD CONSORT 30 or BD CONSORT 32 with BD Simultest IMK
Plus software can vary because of changes in various subsets of
CD8+ lymphocytes that are also positive for CD16, CD56, or both.
Dual-fluorescence studies employing each of these antibodies with
CD8 can determine which populations are responsible for
variations.
Samples with nucleated RBCs can show incomplete lysis because
Reagent G, BD FACS lysing solution, 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, for example, myelofibrosis, spherocytosis.
Nucleated erythrocytes will be counted as debris and, if debris is
greater than 20%, the software will flag the sample as “too many
nonlymphs in gate.” We recommend that such sample results be
rejected. When the Quadrant Correction software option has been
selected, nonlymphocytes such as nucleated or unlysed red cells
determined using BD Leucogate are assumed to be unstained and
are subtracted from Q3 by the software.
37
•
•
•
•
•
BD Simultest IMK Plus software checks for nonantigen-specific
antibody binding in Control tube B only. The operator should
examine the printout for any evidence of nonantigen-specific
antibody binding in subsequent tubes (C through F). For example,
on the dot plot display of tube C, if excessive events occur in Q2,
nonantigen-specific antibody binding should be suspected. The
presence of such nonantigen-specific antibody binding can reflect a
change in the reagents or an error in the preparation of the
samples. Such samples should be repeated.
The presence of abnormal (blast) cells might not allow
BD Leucogate to set an adequate lymphocyte acquisition gate. The
software will flag the sample and results will not be printed.
For whole blood samples, prolonged exposure of cells to Reagent
G, BD FACS lysing solution, beyond 12 minutes can cause white
cell destruction (see item 4 under Staining and Fixing the Cells in
Section 7, Procedure).
The ability of the flow cytometer to select lymphocytes and to
eliminate platelets, red cells, debris, granulocytes, and monocytes
from the lymphocyte count depends on the existence of a clear
demarcation between these formed elements and lymphocytes on a
display of FSC vs SSC. For some patients, this demarcation is not
clear and lymphocyte gating will be less effective.
Performance characteristics have been determined with
BD FACScan flow cytometers. Performance characteristics with
any other instruments have not been established.
10. EXPECTED VALUES
Leucocyte Subsets
Four sets of counts are collected for each tube, including the Control
tube B. Each count is represented in a different quadrant on the
BD FACScan display (Figure 2).
38
The cell populations present in each quadrant, by tube, are shown in
Figure 3 in this IFU and in the BD Simultest IMK Plus User’s Guide.
The clinically significant results are found in the Physician’s Report.71
BD has reported a mean helper/suppressor lymphocyte ratio of 1.4 and
a 95% range of the ratio between 0.6 to 2.8 in the peripheral blood of
304 healthy male and female subjects whose ages ranged from 20 to
70 years.71
BD has investigated the normal reference ranges for BD Simultest IMK
Plus parameters in normal male and female subjects using the
BD FACScan flow cytometer at six sites (five European clinical centers
and one US site). The expected normal reference ranges for LWB are
shown in Table 3.71 Ranges are presented as corrected percentages of
lymphocytes.
The ranges obtained were tested for differences by clinical site, by sex,
and by age of subject. For each BD Simultest IMK Plus parameter,
nonparametric comparisons were made between values obtained at
each study center, between values obtained for males and females, and
between values obtained for subjects age 18 to 40 and age 41 to 70. If
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 fitted distribution.71 The ability to pool reference ranges for the
BD Simultest IMK Plus parameters (Table 3) is an indication of
between-laboratory reproducibility.
There are small but significant differences between sexes for ranges of
total T lymphocytes determined by Reagent F and differences between
sexes and subjects 40 or below versus subjects over 40 years old for Thelper/inducer lymphocytes, the CD4+/CD8+ lymphocyte ratio,
activated T lymphocytes, and NK lymphocytes.
39
Table 3. Representative reference rangesa for BD Simultest IMK Plus parameters in
hematologically normal adults as percentages of total lymphocytes (corrected) (data
pooled from six clinical centers)
Parameter
Sex
Age
n
Mean (%)
95% Rangeb
Total T lymphocytes
Male
Female
18–70
18–70
161
143
71.3
73.9
58.2–84.3
62.8–85.0
Total B lymphocytes
Both
18–70
319
14.0
7.1–23.3
Helper/inducer lymphocytes
Male
Male
Female
Female
≤40
>40
≤40
>40
77
84
85
58
39.9
43.9
44.0
48.9
27.3–52.5
28.5–59.2
31.4–56.7
34.0–63.8
Suppressor/cytotoxic lymphocytes Both
18–70
304
33.4
18.9–47.9
Helper/suppressor ratio
Male
Male
Female
Female
≤40
>40
≤40
>40
77
84
85
58
1.2
1.5
1.3
1.7
0.6–2.2
0.6–3.0
0.7–2.3
0.8–3.3
Activated T lymphocytes
Male
Male
Female
Female
≤40
>40
≤40
>40
58
67
64
44
9.4
11.0
7.8
9.1
3.5–20.6
3.6–25.9
2.8–17.3
4.2–17.2
NK lymphocytes
Male
Male
Female
Female
≤40
>40
≤40
>40
80
88
91
60
13.2
16.3
12.3
13.6
5.4–27.1
6.7–33.5
4.8–26.1
6.0–26.7
a. This data was collected using BD Simultest IMK Plus reagents, BD Simultest IMK Plus software, and the BD FACScan
flow cytometer.
b. The 95% range is obtained from fitting an appropriate distribution to the data and then estimating the central 95%
area of the fitted distribution.
40
Adult reference ranges should not be used with pediatric blood samples
(neonate to 13 years of age). Preliminary results on 50 pediatric
samples (infants and children) suggest that percentage values relative to
adult values can be slightly higher for total B lymphocytes. Children
less than one year old tended to have significantly lower total Tlymphocyte values.
Race can also be a variable in the establishment of normal reference
ranges,72 although insufficient data was collected by BD to determine
this. BD presents more than one upper and lower limit for the ranges
for most BD Simultest IMK Plus parameters (Table 3 on page 40)
because of the age and sex differences that have been observed.
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 system with BD Simultest IMK Plus software
automatically calculates the absolute count if a WBC and a differential
percentage of lymphocytes or an absolute lymphocyte count is
obtained and entered into the software.
NOTE Do not use the BD Simultest IMK Plus three-part differential
values for absolute count determinations. The differential count
provided by BD Simultest IMK Plus 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
nor entered into BD Simultest IMK Plus software to obtain absolute
counts.
Absolute counts will exhibit significant inter-laboratory variation
depending upon the procedure employed for obtaining the WBC and
differential white counts.
41
Representative absolute counts are shown in Table 4. The table is
generated from data acquired at Laboratoire d’Hématologie, Ecole de
Santé Publique, Université Catholique de Louvain, Belgium, the site
with the largest single population of subjects between the ages of 20
and 70. These subjects represent a selected population between the ages
of 20 to 70 to which the criterion T + B + NK = 100 ±5% was applied.
BD Simultest IMK Plus parameters were expressed as a percentage of
lymphocytes in the lymphocyte acquisition gate after subtracting
nonlymphocytes (granulocytes, monocytes, and debris) from
quadrant 3.
Table 4. Representative reference rangesa for BD Simultest IMK Plus parameters in
hematologically normal adults (absolute count datab from one clinical site)
95% Rangec
Parameter
Sex
Age
n
Mean
WBC
Both
20–70
98
6.08d
3.45–9.76
% Lymphs
Both
20–70
98
30.96
20.7–44.6
Total T lymphocytes
Male
Female
20–70
20–70
40
55
1.31
1.39
0.594–1.992
0.772–2.201
Total B lymphocytes
Both
20–70
98
0.27
0.109–0.532
Helper/inducer lymphocytes
Male
Male
Female
Female
≤40
>40
≤40
>40
11
29
27
28
0.77
0.82
0.86
0.86
0.439–1.112
0.356–1.351
0.309–1.571
0.496–1.354
Suppressor/cytotoxic
lymphocytes
Both
20–70
95
0.58
0.282–0.999
Helper/suppressor ratio
Male
Male
Female
Female
≤40
>40
≤40
>40
11
29
27
28
1.28
1.57
1.37
1.84
0.865–1.885
0.784–3.100
0.775–2.375
0.846–3.500
42
Table 4. Representative reference rangesa for BD Simultest IMK Plus parameters in
hematologically normal adults (absolute count datab from one clinical site) (continued)
Parameter
Sex
Age
n
Mean
95% Rangec
Activated T lymphocytes
Male
Male
Female
Female
≤40
>40
≤40
>40
11
30
29
28
0.14
0.17
0.12
0.15
0.050–0.268
0.042–0.451
0.034–0.321
0.054–0.496
NK lymphocytes
Male
Male
Female
Female
≤40
>40
≤40
>40
11
30
29
28
0.19
0.26
0.21
0.23
0.078–0.345
0.102–0.511
0.072–0.543
0.106–0.605
a. This data was collected using BD Simultest IMK Plus reagents, BD Simultest IMK Plus software, and the
BD FACScan flow cytometer.
b. Absolute counts are expressed as 1 x 103 cells/µL
c. The 95% range is estimated directly from the data using the 2.5 and 97.5 percentiles.
d. Data was obtained from a Technicon™ H6000 automated cell counter. (Technicon is a trademark of Miles
Instruments.)
11. PERFORMANCE CHARACTERISTICS
Performance of BD Simultest IMK Plus was established by testing at
either one US clinical center or at BD laboratories in San Jose,
California, or both.
Within-Sample Reproducibility
Blood samples from each of five normal and five abnormal donors
were obtained, aliquoted (four times for normals, three for abnormals),
stained with the BD Simultest IMK Plus reagents, lysed, and fixed
within 2 hours of staining. Flow cytometric analysis was performed
within 24 hours on a BD FACScan flow cytometer in the same
laboratory. Table 5 shows the average within-sample reproducibility
obtained for both normal and abnormal subjects.
43
Between-Instrument Reproducibility
Using a protocol similar to that above, samples of whole blood from
ten normal subjects were aliquoted, stained with the BD Simultest IMK
Plus reagents, and lysed. Single samples from each donor were then
analyzed on three different BD FACScan flow cytometers. Table 6
shows these results.
Between-Laboratory Reproducibility
Between-laboratory reproducibility is indicated by the ability to pool
normal reference ranges for BD Simultest IMK Plus parameters
(Table 3 on page 40).
BD Simultest IMK Plus vs Comparative Methods
Results of aliquots from the same blood samples from normal and
abnormal subjects were analyzed with the BD Simultest IMK Plus
reagents, the BD Simultest™ Immune Monitoring Kit, CD3 FITC, and
CD19 PE, using LWB and the BD FACScan. Results were compared
using linear regression. A summary of the results is presented in
Table 7.
Table 5. Within-sample reproducibility for BD Simultest IMK Plus parameters (five
normal subjects and five abnormal subjects) as percentages of lymphocytes (corrected)
Parameter
Subjects
n
Meana
(%)
SDb
CVc
dfd
Total T lymphocytes
Normal
Abnormal
5
5
68.4
80.4
1.6
1.9
2.2
2.0
14e
15e
Total B lymphocytes
Normal
Abnormal
5
5
9.5
9.3
2.4
1.0
21.5
9.0
14
15
Helper/inducer lymphocytes
Normal
Abnormal
5
5
46.3
18.8
1.5
2.1
3.2
12.2
15
15
Suppressor/cytotoxic lymphocytes
Normal
Abnormal
5
5
34.8
65.5
1.3
2.6
3.6
3.7
15
15
44
Table 5. Within-sample reproducibility for BD Simultest IMK Plus parameters (five
normal subjects and five abnormal subjects) as percentages of lymphocytes (corrected)
Parameter
Subjects
n
Meana
(%)
SDb
CVc
dfd
Helper/suppressor ratio
Normal
Abnormal
5
5
1.4
0.3
0.1
0.04
5.2
13.4
15
15
Activated T lymphocytes
Normal
Abnormal
5
5
6.4
38.9
1.5
1.6
24.5
4.3
15
15
NK lymphocytes
Normal
Abnormal
5
5
24.7
12.6
2.0
0.9
7.0
6.7
14e
15
a. Mean is the pooled mean, Y = the mean of the individual means.
b. SD = standard deviation, the pooled standard deviation.
SD =
2
2
2
( n – 1 ) × s + ( n – 1 ) × s + ... + ( n – 1 ) × s
k
k
2
2
1
1
-----------------------------------------------------------------------------------------------------------------------------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.
c. CV = coefficient of variation
d. df = degree of freedom = (number of replicates – number of donor means) x number of subjects; 4 aliquots – 1
mean = 3 degrees of freedom. Three degrees of freedom for 5 subjects = 15 degrees of freedom, or 1 aliquot x 3
instruments – 1 mean = 2 degrees of freedom. Two degrees of freedom for 10 subjects = 20 degrees of freedom.
e. One of the replicates was discarded by the clinician.
Table 6. Between-instrument reproducibility for BD Simultest IMK Plus parameters (ten
normal subjects) as percentages of lymphocytes (corrected)
Meana (%)
SDb
CVc
Total T lymphocytes
72.1
2.13
2.29
20
Total B lymphocytes
12.7
1.26
10.59
20
Helper/inducer lymphocytes
45.8
1.26
2.47
20
Suppressor/cytotoxic lymphocytes
39.0
1.37
3.28
20
Parameter
45
dfd
Table 6. Between-instrument reproducibility for BD Simultest IMK Plus parameters (ten
normal subjects) as percentages of lymphocytes (corrected)
Meana (%)
SDb
CVc
Helper/suppressor ratio
1.2
0.06
4.80
20
Activated T lymphocytes
11.8
1.56
14.09
20
NK lymphocytes
16.8
1.28
6.93
20
Parameter
dfd
a. Mean is the pooled mean, Y = the mean of the individual means.
b. SD = standard deviation, the pooled standard deviation.
SD =
2
2
2
( n – 1 ) × s + ( n – 1 ) × s + ... + ( n – 1 ) × s
1
1
2
2
k
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.
c. CV = coefficient of variation =
SD
x 100
Y
d. df = degree of freedom = (number of replicates – number of donor means) x number of subjects; 4 aliquots – 1
mean = 3 degrees of freedom. Three degrees of freedom for 5 subjects = 15 degrees of freedom, or 1 aliquot x 3
instruments – 1 mean = 2 degrees of freedom. Two degrees of freedom for 10 subjects = 20 degrees of freedom.
Stability of Stained Cell Preparations
Cell preparations from five normal 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.
Studies indicated no significant difference between day 0 and day 1
46
readings in any parameter. We recommend analyzing stained samples
within 24 hours.
Table 7. BD Simultest IMK Plus vs comparative method using LWB and the BD FACScan
Slope
Intercept
r
na
Range of
Data (%)
Total T lymphocytesb
0.98
4.06
0.98
58
10–90
Total B lymphocytesc
0.97
1.47
0.99
65
0–80
Helper/inducer lymphocytes
1.16
–1.81
0.96
31
10–60
Suppressor/cytotoxic lymphocytesd
0.96
7.05
0.93
31
25–80
Helper/suppressor ratiod
1.02
–0.02
0.98
31
0.125–2.0
Activated T lymphocytes
1.06
0.34
0.92
27
5–50
Parameter
a.
b.
c.
d.
Composed of normal and abnormal samples.
Defined by CD3 FITC.
Defined by CD19 PE.
As compared to the BD Simultest Immune Monitoring Kit.
Cross-Reactivity
The specificity of monoclonal antibodies with a CD number has been
established by blind testing at a number of laboratories by the
International Leucocyte Workshop group.25,27,34,38,42,64,65
The CD4 antibody reacts with monocytes as well as lymphocytes.47
The Anti–HLA-DR antibody reacts with monocytes/macrophages as
well as B lymphocytes.14,19,57–60 The CD14 antibody reacts with
granulocytes as well as monocytes/macrophages.33 The CD16 antibody
reacts weakly with neutrophils.20 Potentially interfering granulocytes
should be eliminated from the lymphocyte count by their light-scatter
properties.
Although Reagent B is designed to detect nonantigen-specific antibody
binding, nonspecific binding due to poor condition of cells can be
observed with any antibody reagent. Nonantigen-specific antibody
47
binding can be inferred if no clear demarcation between negative and
positive cells is seen on the fluorescence plot or if there are more than
5% of counts from the negative Control outside quadrant 3. Consult
Troubleshooting, Section 12, at the end of this IFU if nonantigenspecific antibody binding is seen.
Linearity-Recovery
All lots of BD Simultest IMK Plus reagents are titered against actual
cell suspensions to provide optimal performance at 1.0 x 106 WBC/
sample. For an LWB sample, the normal range is 3.5 x 103 to
9.8 x 103 WBC/µL. Results are expected to be linear from 3.5 x 103 to
9.8 x 103 WBC/µL.
48
12. TROUBLESHOOTING
Problem
Possible Cause
Solution
• Decreased fluorescence due to 1. Run BD Autocomp software
Insufficient
incorrect PMT voltage or
separation between
with BD Calibrite beads. Must
amplifier gain.
positive and negative
meet specifications for
•
Insufficient antibody reagent.
fluorescent
intensity in fluorescence
populations.
channels.
2. Check pipet calibration,
reagent volume added, as well
as volume and concentration
of cells added. Check reagent
storage conditions.
No demarcation
found between
debris and
lymphocyte clusters
on light-scatter dot
plot.
• Presence of excess platelets or 1. Restain a fresh sample. Wash
nucleated red blood cells or
cells at lower speed, for
unlysed red blood cells.
example 100g for 15 minutes
• Amplifier gain improperly set.
at room temperature (20°C–
• Sample not stained within 6
25°C).
hours of collection.
• FSC threshold set too high or 2. Adjust amplifier gain such that
too low.
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 to whole blood
determinations. See 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 (256channel scale).
49
Problem
Possible Cause
Solution
Suboptimal
antibody-staining
performance.
• Sample not stained within 6
hours of collection.
• Analysis gate selected by
software included too many
nonlymphocyte events.
• Fluorescence markers set
incorrectly.
1. Collect a fresh sample to stain.
2. Reprocess data using
BD 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. See the
appropriate software user's
guide.
3. Reprocess data using
BD CONSORT 30, BD LYSYS
II, or BD Simulset software and
set manual fluorescence
markers.
50
Problem
Possible Cause
Software unable to
set an acceptable
lymphocyte gate.
• Sample is lymphopenic.
1. Concentrate cells using a
• FSC threshold set too high or
density-gradient method or
too low.
reacquire a larger number of
• Amplifier gain improperly set.
events using
• Sample not stained within 6
BD CONSORT 30 or
hours of collection.
BD LYSYS II.
• Software marker setting not
appropriate.
2. Adjust FSC threshold so that
there are approximately 10
channels of debris (256channel scale).
Solution
3. Adjust 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 to whole blood
determinations. See the
appropriate user's guide.
4. Collect a fresh sample to stain.
5. Reacquire data using
BD CONSORT 30 or
BD LYSYS II.
Unstained cells in the • Nonantigen-specific antibody
binding due to sample
stained sample move
containing dead or damaged
over marker set with
cells.
Control tube B into
• Nonantigen-specific antibody
Q4 or Q1.
binding due to Fc receptors.
Staining is dim or
inconsistent.
1. Collect a fresh sample to stain.
2. Check for excessive monocyte,
granulocyte, or debris
contamination in the
lymphocyte acquisition gate.
• Cell concentration too high at 1. Check and adjust cell
staining step.
concentration; repeat staining
• Insufficient reagent.
with fresh sample.
• Cells not analyzed within 24
2. Repeat staining with proper
hours of staining.
• Improper medium
amount of reagent.
preparation (sodium azide
3. Repeat staining with fresh
omitted).
sample; analyze promptly.
4. Use sodium azide in staining
medium and washing steps.
51
Problem
Possible Cause
Solution
Few or no cells.
• Cell concentration too low.
• Flow cytometer
malfunctioning.
1. Resuspend fresh sample at a
higher concentration. Repeat
staining and analysis.
2. Troubleshoot instrument.
Increased
autofluorescence.
• Possible bacterial
contamination of PBS or
fixative.
• Poor sample preparation.
1. Prepare fresh 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.
Weak positive
fluorescence.
Possible bacterial contamination
of PBS or fixative. Buffer or
fixative at improper pH.
Prepare fresh PBS with reagentgrade water and fresh fixative
with PBS with sodium azide.
Adjust to the proper pH. Filter
through 0.2-µm filter.
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