2
Analyzing Samples for CD34 Enumeration
Using the BD™ Stem Cell Enumeration Kit
Presented by
Ellen Meinelt, MS MLS(ASCP)CM, Technical Applications Specialist and
Calin Yuan, Product Course Developer, BD Biosciences
February 29, 2012
3
Learning Objectives
• Describe the purpose of stem cell enumeration.
• Describe the purpose and uses of stem cell controls.
• List critical aspects of staining stem cell specimens.
• List the main steps in stem cell acquisition and
analysis on FACSCalibur or FACSCanto II.
4
BD Stem Cell Enumeration Kit Highlights
ISHAGE
PROCOUNT VS SCE
5
BD Stem Cell Enumeration Kit Highlights
6
Hematopoietic Stem Cells
Neutrophil
 Hematopoietic stem cells can generate all lymphoid and myeloid lineages.
7
Sources & Phenotype of Hematopoietic Stem Cells
bone marrow
cord blood
mobilized
peripheral blood
 Hematopoietic stem cells are CD34+ / CD45dim / SSClow / FSClow to intermediate.
8
Why Perform Stem Cell Enumeration?
 To determine when to perform bone
marrow stem cell transplants.
 To determine whether to continue
apheresis.
 To assess bone marrow and cord blood
viability.
 The viable CD34+ absolute count is a critical parameter
for rapid and sustained engraftment.
9
Let’s Review. . .
Why are stem cells enumerated?
10
BD Stem Cell Enumeration Kit Highlights
ISHAGE
History of CD34 Enumeration Protocols
1985 Siena, et al.
Milan Protocol
 Dual platform
 FSC / SSC
 50K or 50 CD34+ events
 Class III CD34 clone (1991)
History of CD34 Enumeration Protocols
1985 Siena, et al.
1994 Bender
Milan Protocol
 CD45
 Dual platform
 FSC / SSC
 50K or 50 CD34+ events
 Class III CD34 clone (1991)
History of CD34 Enumeration Protocols
1985 Siena, et al.
1994 Bender
Milan Protocol
 CD45
 Dual platform
1995 Owens and Loken
 FSC / SSC
 7-AAD viability dye
 50K or 50 CD34+ events
 Class III CD34 clone (1991)
History of CD34 Enumeration Protocols
1985 Siena, et al.
1994 Bender
Milan Protocol
 CD45
 Dual platform
1995 Owens and Loken
 FSC / SSC
 7-AAD viability dye
 50K or 50 CD34+ events
 Class III CD34 clone (1991)
1994, 1996 Sutherland, et al.
Original ISHAGE Protocol
 Dual platform
 4 Parameter, 2 Colors:
 FSC / SSC
 CD34 PE / CD45 FITC
 Requires isotype control
History of CD34 Enumeration Protocols
1985 Siena, et al.
1994 Bender
Milan Protocol
 CD45
 Dual platform
1995 Owens and Loken
 FSC / SSC
 7-AAD viability dye
 50K or 50 CD34+ events
 Class III CD34 clone (1991)
1994, 1996 Sutherland, et al.
Original ISHAGE Protocol
 Dual platform
 4 Parameter, 2 Colors:
 FSC / SSC
 CD34 PE / CD45 FITC
 Requires isotype control
1998 BD Procount Kit
Single platform with BD™ Trucount beads
 Nucleic acid staining
 Isotype control included
 Lyse/no wash methodology
 Leukopheresis and Peripheral Blood
History of CD34 Enumeration Protocols
1985 Siena, et al.
1994 Bender
Milan Protocol
 CD45
 Dual platform
1995 Owens and Loken
 FSC / SSC
 7-AAD viability dye
 50K or 50 CD34+ events
 Class III CD34 clone (1991)
1994, 1996 Sutherland, et al.
Original ISHAGE Protocol
 Dual platform
 4 Parameter, 2 Colors:
 FSC / SSC
 CD34 PE / CD45 FITC
1998 Keeney, et al.
 Requires isotype control
Modified ISHAGE Protocol
 Single platform method with
1998 BD Procount Kit
counting beads
Single platform with BD™ Trucount beads
 75K or 100 CD34
 Nucleic acid staining
 7-AAD viability dye
 Isotype control included
 4 Parameter, 2 Colors:
 Lyse/no wash methodology
 FSC / SSC
 Leukopheresis and Peripheral Blood
 CD34 PE/ CD45 FITC
 Sequential gating eliminates use of
isotype control.
17
BD Stem Cell Enumeration Kit Highlights
18
Features of the BD Stem Cell Enumeration Kit
 FDA Cleared IVD test
 Meets ISHAGE guidelines
 Single tube assay
 Compatible with various
sample types and
anticoagulants
Kit contents (50 tests/kit)
• CD34 PE/ CD45 FITC
• 7-AAD Viability Dye
• NH4Cl Lysing Solution
• Trucount Tubes
 Single platform with
BD Trucount Tube
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BD SCE Kit Sample Preparation*
* Dilute samples with
> 40,000 WBC/uL in
PBS with 0.5% BSA.
A FEW SIMPLE STEPS IN A SINGLE TUBE
BD
TrucountTM
tube
Add 20 µL of CD45 FITC/CD34 PE reagent,
20 µL of 7-AAD, and
100 µL of blood (by reverse pipetting) to a Trucount
tube.
Vortex and incubate in the dark at RT for 20 min.
Add 2 mL of 1X ammonium chloride lysing solution.
Vortex and incubate in the dark at RT for 10 min.
Put samples into an ice bath!
Acquire samples within 1 hour after lyse.
20
BD Stem Cell Control Kit
 BD Stem Cell Control Kit (Catalog # 340991)
 CD34 High (approximately 35 cells/L)
 CD34 Low (approximately 10 cells/L)
 Functions as
 Instrument setup and performance
 Process control for:
 antibody staining for CD34+
 7-AAD staining of non-viable cells
 red blood cell lysis
 Always run process controls prior to staining specimens.
21
Let’s Review. . .
How are stem cell controls used in the BD SCE Assay?
22
Let’s Review. . .
What are the critical aspects of
sample handling and preparation?
23
BD Stem Cell Enumeration Kit Highlights
PROCOUNT VS SCE
24
Comparison of BD SCE Kit and BD Procount Kit
BD SCE Kit
BD Procount Kit
1
Meet ISHAGE Guidelines
Yes
No
2
No. of Tubes per Sample
1
2 (requires isotype)
3
Viability Dye
7-AAD
None
4
Incubation Time
30 minutes (20 + 10)
45 minutes (15 + 30)
5
Reagent Stability
20 months
5 months
Peripheral Blood
Leukapheresis
Bone Marrow
Cord Blood
Peripheral Blood (PB)
Leukapheresis
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Specimen Type
7
Specimen Stability
8
9
Anticoagulant
Cytometer
24 hours for all samples
EDTA
Heparin
ACD-A
CPD
BD FACSCalibur
(OS 9 and OS X)
BD FACSCanto II
24 hours for PB
6 hours for Leukapheresis
EDTA
BD FACSCalibur OS 9 only
The Winner: BD SCE Kit
BD SCE KIT
26
27
BD Stem Cell Enumeration Kit Highlights
28
Analyzing samples for CD34
enumeration using BD FACSCalibur
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Workflow overview using BD FACSCalibur
Start Up System
Perform QC
Optimize Settings
Start up the system.
Stain the stem cell controls for instrument
optimization and process control.
Perform cytometer QC.
Perform instrument optimization with specific
application setup (using a stem cell control).
Acquire Data
Acquire process controls and verify results.
Stain and acquire samples.
Analyze Data
Analyze data and adjust gates as needed.
Shut Down System
Perform daily clean and shut down system.
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Instrument Start Up and Perform QC
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
•
Set up the cytometer using BD
Calibrite™ beads and BD
FACSComp™ software with
3- or 4-color lyse/no-wash assay
(Calib.LNW settings).
•
Verify that all parameters pass.
Analyze
Data
Shut Down
System
Optimize Instrument Settings
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
Plot 1
Use high stem cell control
(w/o 7-AAD)
• Adjust Threshold.
Plot 6
• Adjust FSC gain.
Optimize Instrument Settings
Start Up
System
Perform
QC
Optimize
Settings
Use high stem cell control
(w/o 7-AAD)
Acquire
Data
Analyze
Data
Shut Down
System
Plot 8
• Adjust compensation.
Plot 7
33
Acquire Data
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Verify settings for
acquisition and
storage.
•
Add custom keywords:
Dilution Factor,
Trucount, and
Sample Volume.
• Gently vortex sample and acquire. Do not use the BD FACS
•
Check that 100 viable CD34 events have been acquired.
TM
Loader.
34
Analyze the Acquired Data
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
BD CellQuestTM Pro Software
BD CellQuestTM Software
•
•
Enter the sample volume,
dilution factor, and BD
Trucount bead value in the
expression editors to
obtain results.
Calculate SCE results using
the statistics and equations
provided.
35
Analyze the Acquired Data
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
•
Check each plot and verify that the gates are
properly placed.
•
How do we identify the stem cells?
1. Identify viable cells.
2. Identify the leukocytes and lymphocytes.
3. Identify the CD34+ cluster among the viable cells.
4. Gate the Trucount Beads.
Shut Down
System
SCE Template in BD CellQuest Pro Software
1
Plots and Gates
For every sample, gates must be
checked and adjusted.
1
2
2 Expression Editors*
Enter values prior to acquisition.
- Trucount
- Dilution factor
- Sample volume
3
3 Gates & Statistics
Statistics are provided for
additional optional analysis.
4
SCE Results*
CQP automatically
provides SCE results.
4
• Expression editors and automatic
results are not available in BD
CellQuest software.
SCE Template in BD CellQuest Software
1
Plots and Gates
For every sample, gates must
be checked and adjusted.
2 Gates & Statistics
Statistics are provided for
additional optional analysis.
1
2
3 CD34+ SCE Equation
The equation enables
calculation of CD34+ cells/L .
3
• Expression editors and automatic
results are not available in BD
CellQuest software.
SCE Equations for BD CellQuest Software
Statistics provided in the
BD SCE CellQuest Template
Equations provided in the BD Stem Cell Enumeration
Application Guide for BD FACSCalibur Flow Cytometers
39
Gate List for Acquisition Template
40
SCE Analysis
• The BD SCE Kit acquisition and
analysis template is shown.
• Plot numbers (1–8) in orange
are shown for reference and do
not appear in the actual
template.
41
Verifying Viability Gate in Plot 8
a. Adjust R8:
42
Verifying Gates in Plots 1 and 6
b. Adjust R1:
c. Adjust R5 to include most of the lymphocytes.
d. Adjust R4 to include viable lymphocytes.
43
Verifying Gate in Plot 2
e. Adjust R2 to include all CD34+ events.
44
Verifying Gate in Plot 3
f.
Adjust R3
45
Verifying Gates in Plots 4 and 6
g. Adjust R4
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Verifying Gate in Plot 5
h. Adjust R6:
Optional: Adjust the quadrant marker to establish the lower
limit of CD45 expression by the CD34+ events, as in Plot 1.
CD34+
events
Red events
are 7-AAD
positive
(dead cells)
SCE Report in BD CellQuest Pro Software
SCE Report in BD CellQuest Software
49
Let’s Review. . .
What are the main steps in stem cell enumeration
on the BD FACSCalibur?
50
51
BD Stem Cell Enumeration Kit Highlights
52
Analyzing samples for CD34
Enumeration using BD FACSCanto II
Workflow Overview using BD FACSCanto II
Start Up
System
Start up the system.
Set up the software for SCE acquisition.
Perform
QC
Stain the stem cell controls for instrument optimization
and process control.
Perform cytometer QC.
Optimize
Settings
Perform instrument optimization with specific application
setup (using a stem cell control).
Acquire
Data
Acquire process controls and verify results.
Stain and acquire samples.
Analyze
Data
Analyze data and adjust gates as needed.
Shut Down
System
Perform daily clean and shut down system.
54
Workflow
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Perform fluidics startup in BD FACS Canto clinical software.
• Enter or confirm reagent lot IDs and acquisition targets.
55
Workflow
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
Perform QC with BD FACS 7-Color Setup Beads
• Track settings over time to
measure the cytometer’s
performance and consistency.
• Establish instrument settings that
are used as a starting point before
optimizing settings.
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Workflow
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Optimize Settings for BD Stem Cell Panel
• Optimize with either high or low
BD Stem Cell process control
tube sample (with 7-AAD).
• Confirm that spillover of 7-AAD
into PE is determined
successfully.
Analyze
Data
Shut Down
System
57
Workflow
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
Acquire process controls and confirm that they pass.
Tip: Confirm that the high and low process control results meet the
expected values for absolute CD34+ and %CD34+.
58
Workflow
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Acquire specimens.
Tip: Vortex each tube just prior to acquiring manually.
Shut Down
System
59
Workflow
Start Up
System
•
•
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
Check each plot and verify that the gates are properly placed.
How do we identify the stem cells?
1. Eliminate debris.
2. Identify viable cells.
3.
Identify the leukocytes and lymphocytes.
4.
Identify the CD34+ cluster among the viable cells.
5.
Gate the Trucount Beads.
60
Review Gates
• Plot numbers (1 – 8) in orange are shown for reference and do not appear in the
actual SCE module.
•
Letters (a – h) indicate the order in which gates should be verified.
2
1
3
e
d
6
c
a
f
8
7
5
4
b
h
g
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1. Identify debris in plot 6.
Identify debris in the lower left corner of the plot.
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2. Identify viable events
Identify viable events in plot 8.
Use plot 7 to confirm the placement of the viable gate.
63
3. Identify lymphocytes in plot 1.
Identify lymphocytes among CD45+ events.
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4. Identify stem cells.
Use plots 2 and 3 to identify stem cells.
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4. Confirm the Viable CD34 Gate
Plot 4 displays the viable CD34 population.
Viable lymphocytes are shown in
light blue and viable CD34+ cells
are shown in red.
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5. Identify beads
Confirm placement of the Trucount beads gate.
SCE Report in Canto Clinical Software
Header
Provides information on sample
ID, acquisition and analysis
times, and status.
Dot Plots
Display populations and gates
NOTE:
For process control lab reports,
the two viability data plots
(outlined here) are omitted.
Results
QC Messages
and Comments
68
Let’s Review. . .
What are the main steps in stem cell enumeration
on the BD FACSCanto II?
69
Let’s Review. . .
What are key advantages of the BD SCE Kit?
70
Let’s Review. . .
What are key advantages of the BD SCE Kit?
ISHAGE
PROCOUNT VS SCE
71
References
•
Siena S, Castro-Malapina H, Gulati, SC, et. al. Effects of in-vitro purging with 4-hydroxycyclophosphamide on the
hematopoietic and micro –environmental elements of human bone marrow. Blood;1985:655-662.
•
Siena S, Bregni M, Brando B, et. al. Flow Cytometry for clinical estimation of circulating hematopoietic progenitors for
autologous transplantation in cancer patients. Blood. 1991;77:400-409.
•
Bender JG, Unverzagt K, Walker DE et. Al. Phenotypic analysis and characterization of CD34+ cells from normal human
bone marrow, cord blood, peripheral blood, and mobilized peripheral blood from patients undergoing autologous stem cell
transplantation. Clin Immunol Immunopathol. 1994 Jan;70(1):10-8.
•
Sutherland DR, Keating A, Nayar R, et. al. Sensitive detection and enumeration of CD34+ cells in peripheral and cord blood
by flow cytometry. Ex. Hematol. 1994;22:1003-1010.
•
Loken MP. Peripheral blood stem cell quantitation. In: Owens MA, Loken MP (eds) Flow Cytometry Principles for Clinical
Laboratory Practice Wiley-Liss: New York, 1995, pp 111-127.
•
Sutherland DR, Anderson L, Keeney M, et. al. The ISHAGE guidelines for CD34+ cell determination by flow cytometry.
J Hematotherapy. 1996;5:213-226.
•
Keeney M, Chin-Yee I, Weir J, et. al. Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE
guidelines. Cytometry. 1998;34:61-70.
•
Dauber K., et al. Enumeration of viable CD34+ cells by flow cytometry in blood, bone marrow and cord blood: results of a
study of the novel BD ™ stem cell enumeration kit. Cytotherapy (2011) 13:449-458.
•
Lemarie C., et al. A new single-platform method for the enumeration of CD34+ cells Cytotherapy (2009) 11(6):804–80
For In Vitro Diagnostic Use. CE marked to the European In Vitro Diagnostic Medical Devices Directive 98/79/EC.
Class I (1) Laser Products.
BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2012 BD
23-14029-00
72
Q & A from the BD™ Stem Cell
Enumeration (SCE) Webinar
March 7, 2012
73
Q & A by Topic
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
BD Procount™ Kit vs BD SCE Kit
BD SCE Kit US Clinical Trial and EU Performance
BD SCE Kit Contents
Sample Types and Processing
Controls for the SCE Assay
Instruments Cleared for the SCE Kit
BD SCE Software
BD SCE Assay
Instrument Setup
Gating
Result Reports
1. BD Procount kit vs BD SCE kit
Q. Does the BD Procount kit use a different viability
reagent than the BD SCE kit?
Answer:
Yes, the BD Procount kit uses a nucleic acid dye to stain nucleated cells in
addition to the CD45 for the leucocytes. Since the BD Procount kit does not
contain the viability dye, it is not based on the ISHAGE guidelines. See the table
for additional differences.
74
2.BD SCE kit US clinical trial and EU performance
Q. Describe the results of the BD SCE kit US clinical trial
and EU performance evaluation.
Answer:
a. A total of 1,032 samples was tested across 8 sample types (fresh and
normal peripheral blood, frozen and thawed apheresis, bone marrow,
and cord blood) in the EU performance evaluation and the US clinical
trials.
b. The BD SCE reagent kit, templates, and application module results
were demonstrated to be substantially equivalent to the predicate
device.
c. The kit has been in use in Europe for over a year, and papers have
been published.*
*Dauber K, Odendahl M, Seifried E, Bonig H, Tonn T. Enumeration of viable CD34(+) cells by flow cytometry in blood,
bone marrow and cord blood: results of a study of the novel BD™ stem cell enumeration kit. Cytotherapy. 2011;4:449458.
75
3. BD SCE kit contents
Q. How many tests will the BD SCE kit run?
Answer:
The BD SCE kit contains components sufficient for 50
tests.
The use of process controls will reduce the total number of
tests within a kit; thus, the number of tests per kit will vary
based on the daily workload of the lab.
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3. BD SCE kit contents
77
Q. Can I swap out antibodies in the kit for research use?
Answer:
The CD45 and CD34 antibodies are combined in the vial. It
is not possible to remove one without the other. BD does
not promote off-label use (use of a device or software not
covered in the manufacturer’s Instructions for Use).
4. Sample types and processing
Q. Have you validated different cord blood processing
methods such as HESPAN® solution and the
Prepacyte® processing system?
Answer:
BD does not have experience in methodologies for stem
cell collection and processing. BD worked with labs that
have developed and validated methods for cord blood
processing. The BD SCE kit was used with cord blood
specimens processed with different methods.
78
4. Sample types and processing
Q. Please reiterate what the 24-hour rule applies to.
Answer:
Freshly collected samples can be stored at 2° to 8°C for
up to 24 hours before staining.
79
4. Sample types and processing
Q. Can we detect the number of viable stem cells per
volume after thawing and washing frozen stem cell
samples?
Answer:
Yes, the kit is designed and used for this process.
80
4. Sample types and processing
Q. What is the longest acceptable elapsed time between
collection and running of a sample? We sometimes
receive samples that were collected more than 24
hours before.
Answer:
The BD SCE kit does not support the use of samples older
than 24 hours.
81
4. Sample types and processing
Q. What is the best way to process frozen cord blood
samples for stem cell enumeration?
Answer:
There are several protocols available for thawing frozen
stem cell specimens. See the Clinical and Laboratory
Standards Institute (CLSI) H42-A2 guidelines.*
*Enumeration of Immunologically Defined Cell Populations by Flow Cytometry;
Approved Guideline—Second Edition (H42-A2); Volume 18, No. 21.
82
4. Sample types and processing
Q. Have you done any studies of samples that have an
obvious hematogone* population?
Answer:
These cells are CD34+/CD19+. Hematagones are
generally not taken into account with the ISHAGE
protocol. Since hematagones are CD34+ and CD45dim,
they would be included in the CD34 enumeration because
there is no other marker or characteristic that would
exclude them from the gating.
*Hematogones are benign, immature B cells that commonly populate the bone marrow of children.
Their presence has been noted to interfere with the flow-cytometric analysis of cases of suspected
acute lymphoblastic leukemia (ALL) because their immunophenotype (positive for CD19, CD10,
CD34, and terminal deoxynucleotidyl transferase) is similar to that of pre-B cell lymphoblasts.
83
4. Sample types and processing
Q. Does the 24-hour rule apply to thawed samples?
Answer:
The 24-hour rule does not apply to thawed samples. They
must be stained as soon as possible and not longer than 1
hour after thawing.
84
4. Sample types and processing
Q. What are the critical aspects of sample handling and
preparation?
Answer:
a. Perform dilution of samples containing greater than 40,000
WBCs/µL, record the correctly calculated dilution factor, and
enter the dilution factor into the software.
b. Stain samples within 24 hours of collection.
c. Reverse pipette to ensure that an accurate volume of blood is
dispensed.
d. Maintain sample viability; this is important.
e. Use wet ice to store stained samples.
f.
Acquire within 1 hour of staining (to limit toxicity of the
7-AAD viability dye and ammonium chloride lysing solution).
85
5. Controls for the BD SCE assay
86
Q. Are the two levels of controls sold with the BD SCE kit?
Answer:
No. The BD™ Stem Cell Control kit must be ordered
separately.
We recommend using the BD Stem Cell Control kit
(Catalog No. 340991) when using the BD SCE kit.
5. Controls for the BD SCE assay
87
Q. Must I run the two levels of controls daily? CD34 is
very expensive, and reimbursement from Centers for
Medicare & Medicaid Services (CMS) is low. This will be
hard to justify with our administration.
Answer:
a. College of American Pathologists (CAP) requires that two
levels of controls be run daily.
b. It is also good laboratory practice.
c. The Stem Cell Controls are used to optimize the
instrument settings.
5. Controls for the BD SCE assay
Q. Can I track the control values in a QC method?
Answer:
Yes, control values are automatically tracked in BD
FACSCanto™ clinical software using the Levey-Jennings
plots. To enable that feature, “Control” should be entered
in the Sample Name field for both the High and Low
controls.
88
6. Instruments cleared for the BD SCE kit
Q. Can the BD SCE kit be used with the BD LSRFortessa™,
BD™ LSR II, and BD FACSAria™ III flow cytometer?
Answer:
The BD SCE kit is IVD cleared for use with the
BD FACSCalibur™ and BD FACSCanto™ II flow
cytometers, both of which are also cleared for IVD use.
BD does not promote off-label use.
89
6. Instruments cleared for the BD SCE kit
Q. We use a BD FACScan™ system with BD CellQuest™
Pro software. Can the BD FACScan be used for this
assay?
Answer:
The BD SCE kit is IVD cleared for use with BD CellQuest™
or BD CellQuest Pro software on the BD FACSCalibur flow
cytometer.
BD does not promote off-label use.
90
7. BD SCE software
Q. Is there software for the BD SCE kit?
Answer:
Yes.
For BD FACSCanto II users, the BD™ SCE software module
for BD FACSCanto clinical software v2.4 is needed.
For BD FACSCalibur users, BD CellQuest or BD CellQuest
Pro templates are needed.
Please contact BD customer service or your local sales
representative about how to obtain CDs.
91
7. BD SCE software
Q. Can I use the BD SCE module on a BD FACSCanto™
flow cytometer?
Answer:
The BD SCE kit is FDA cleared for use on the BD FACSCanto II and
BD FACSCalibur systems. Running the BD SCE kit on a
BD FACSCanto system is considered off-label use.
When the BD SCE module is installed on a BD FACSCanto system, a
warning message is displayed during installation and the lab reports
always indicate “RUO – Research Use Only.” The individual lab must
validate its results.
92
8. BD SCE assay
Q. We currently use 1 mL of lysing solution. Why do we
need 2 mL?
Answer:
The 2 mL of lysing solution is specified in the ISHAGE
protocol created by Sutherland et al,* and this is how BD
validated the use of the kit.
*Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE Guidelines
for CD34+ Cell determination by flow cytometry. J Hematother. 1996;5:213-226.
93
9. Instrument setup
Q. Can the BD FACS™ Loader be used?
Answer:
No. Due to temperature requirements of the assay, samples
must be acquired manually. 7-AAD and ammonium chloride
are toxic to cells. Therefore, cell viability of stained SCE
samples decreases over time. Store stained samples in a
wet ice bath to maintain viability and acquire samples within
1 hour of staining.
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9. Instrument setup
Q. Can BD™ CS&T beads be used for setting up the
BD FACSCanto II for the BD SCE assay?
Answer:
No. The assay is designed such that BD FACS™ 7-Color
Setup beads must be run, since compensation is
generated with these beads. BD™ Cytometer Setup and
Tracking (CS&T) beads do not generate any compensation
values and are not compatible with BD FACSCanto clinical
software. Immediately after running your 7-Color Setup
beads, the optimization for the BD SCE assay needs to
be performed.
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9. Instrument setup
Q. Can we optimize the BD FACSCalibur for the BD SCE assay
and then save the settings as instrument settings? We could
then move to the Leukemia/Lymphoma panels and then come
back to the BD SCE assay at the end of the day.
Answer:
Yes, the sequence of running both the lyse/no-wash (LNW)
and lyse/wash settings using BD FACSComp™ software
can be done. The BD SCE assay settings could then be
optimized in BD CellQuest software, and those instrument
settings saved. Finally, the Leukemia/Lymphoma settings
could be optimized and saved.
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9. Instrument setup
Q. My lab runs different panels in BD FACSCanto clinical
software. How do I optimize for all the panels?
Answer:
Perform setup using 7-Color Setup beads. Ensure that it
passes. Perform stem cell optimization. Then proceed with
optimizing for the remaining panels. Always perform stem
cell optimization immediately after setup with 7-Color
Setup beads.
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9. Instrument setup
Q. Do you need to do multiple runs of the compensation setup?
The scenario:
1. Start up the BD FACSCanto II cytometer and BD FACSCanto
clinical software and run 7-color setup and stem cell optimization.
2. Acquire stem cell samples.
3. Exit BD FACSCanto clinical software and start
BD FACSDiva™ software.
4. Restart BD FACSCanto clinical software.
5. Acquire stem cell samples? Or re-run 7-Color Setup beads and
compensation?
Answer:
You can run samples later after exiting and restarting as long as the
7-color setup and the panel-specific optimization are completed within
24 hours. If 7-color setup is run again later on, then the panel-specific
optimization has to be run again.
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9. Instrument setup
Q. What is the acceptable range for PE-%7-AAD spectral
overlap?
Answer:
a. In BD FACSCanto clinical software, a value between -0.5%
and 10.5% indicates a successful setup optimization.
b. For the BD FACSCalibur, add 4 to the FL3-%FL2 value
obtained from running a 3- or 4-color LNW setup.
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9. Instrument setup
Q. Are all acquisition settings in the BD SCE acquisition
template applied simultaneously?
Answer:
No. Use the Debris gate (R7) for the Acquisition Rejection
Gate. Use 75,000 Viable CD45 for the collection criterion.
Once the sample is acquired, check that 1,000 BD
Trucount™ beads and 100 viable CD34 cells have been
acquired. If these collection criteria are not satisfied, reacquire the sample with the collection criterion set to 100
viable CD34. There is a maximum acquisition time of 900
seconds (15 minutes).
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9. Instrument setup
Q. Are all acquisition settings in the BD SCE module for
the BD FACSCanto II applied simultaneously?
Answer:
Yes. Before acquisition, check that the acquisition settings
are defined as: 75,000 viable CD45, 125 viable CD34, and
1,000 BD Trucount beads. The maximum acquisition time
is 900 seconds (15 minutes).
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9. Instrument setup
Q. What is the BD FACSCanto II threshold? Can I change
it?
Answer:
The threshold in the BD FACSCanto II SCE module is set
to FITC 400 and cannot be changed.
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10. Gating
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Q. We currently use a plot to gate on singlet beads plotted
over time. Is there any accommodation for this?
Answer:
Please do not make any changes to the template. Plotting
for singlet beads is not recommended for BD Trucount
beads because all the beads are included in the count at
the factory. Results will be inaccurate if you eliminate the
doublets.
10. Gating
Q. Please walk me through the sequential gating that is
used for the BD SCE assay.
Answer:
When using the BD FACSCalibur, a series of regions
and gates is utilized to enumerate the CD34 cells. First
the cells must be viable as defined in R8 in plot 8.
Then we look at the CD45 staining (plot 1) on the
leucocytes in the sample. Some debris may be
excluded, yet too much cannot, to avoid removing the
stem cells. Then we look at the lymphocyte gate, R5
(plot 1). From there we move to the Forward Scatter vs
Side Scatter as displayed in plot 6. This R4 region is
adjusted to capture the cluster of lymphocytes. We then
move to the CD34 vs Side Scatter, plot 2, to capture
the CD34+/CD45+ cells. This sequential gating now
specifies that to be a viable CD34+ stem cell, the event
must be viable (R8), CD45+ (R1), Forward Scatter as
gated (R4), CD34+ (R2), and CD45 vs Side Scatter
(R3).
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10. Gating
Q. What is the population that is 7-AAD positive in plot 7?
Answer:
Plot 7 displays only CD34 events, so the 7-AAD positive
events are CD34 dead events. The ratio between the
negatives and positives on this plot gives the viability
value.
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10. Gating
Q. There are too many events to set the viability gate in
plot 8. What should I do?
Answer:
Display fewer % of events.
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10. Gating
Q. Why is the R7 debris gate not included in any of the
other gate definitions?
Answer:
R7 is an exclusion gate; events in R7 are excluded from
the data file. R7 should not exceed FSC and SSC 200.
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10. Gating
Q. Why doesn't the Total CD34 gate include R4?
Answer:
The total CD34 does not include R4 because some of the
dead stem cells are outside this region. R4 identifies the
healthy lymphocytes or healthy stem cells.
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11. Result reports
Q. We need to know the total viability of the sample. How
is that done?
Answer:
The BD SCE templates (BD CellQuest or BD CellQuest
Pro) and the SCE acquisition and analysis module (BD
FACSCanto II) do not provide this information.
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11. Result reports
Q.
There is no plot showing total CD34 events to adjust the gate.
We find that the total CD34 event count is higher than the
viable CD34 events. Since the ISHAGE protocol reports out only
the viable CD34 count, a total CD34 count is not displayed or
reported.
Answer:
Total CD34 events are displayed on plot 7 with the
live/dead discriminating gate. The total CD34 cell count
cannot be lower than the viable CD34 cell count.
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11. Result reports
Q. How can I add the patient’s body weight to a report?
Answer:
In the BD SCE module for BD FACSCanto clinical
software, information can be added to the Comments
section of the Lab Report.
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For additional troubleshooting assistance, see the Stem Cell
Enumeration Application Guide for the BD FACSCanto II
Flow Cytometer or the Stem Cell Enumeration Application
Guide for the BD FACSCalibur Flow Cytometer.
HESPAN is a registered trademark of B. Braun Medical.
Prepacyte is a registered trademark of Bio E Inc.
BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2012 BD
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If you have further questions, please visit:
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