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Presented by
Ellen Meinelt, MS MLS(ASCP)CM, Technical Applications Specialist and
Calin Yuan, Product Course Developer, BD Biosciences
Learning Objectives
ISHAGE
PROCOUNT VS SCE
5
Hematopoietic stem cells can generate all lymphoid and myeloid lineages.
7
Let’s Review. . .
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
FSC / SSC 1995 Owens and Loken
50K or 50 CD34+ events 7-AAD viability dye
Class III CD34 clone (1991)
History of CD34 Enumeration Protocols
1985 Siena, et al.
1994 Bender
Milan Protocol
CD45
Dual platform
FSC / SSC 1995 Owens and Loken
50K or 50 CD34+ events 7-AAD viability dye
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
FSC / SSC 1995 Owens and Loken
50K or 50 CD34+ events 7-AAD viability dye
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
Functions as
Instrument setup and performance
Process control for:
antibody staining for CD34+
7-AAD staining of non-viable cells
red blood cell lysis
Let’s Review. . .
Let’s Review. . .
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 Peripheral Blood (PB)
6 Specimen Type
Bone Marrow Leukapheresis
Cord Blood
24 hours for PB
7 Specimen Stability 24 hours for all samples
6 hours for Leukapheresis
EDTA
Heparin
8 Anticoagulant EDTA
ACD-A
CPD
BD FACSCalibur
9 Cytometer (OS 9 and OS X) BD FACSCalibur OS 9 only
BD FACSCanto II
The Winner: BD SCE Kit
BD SCE KIT
26
27
Shut Down System Perform daily clean and shut down system.
30
Plot 1
Use high stem cell control
(w/o 7-AAD)
• Adjust Threshold.
Plot 6
Plot 8
Use high stem cell control
(w/o 7-AAD)
• Adjust compensation.
Plot 7
33
Acquire Data
Start Up Perform Optimize Acquire Analyze Shut Down
System QC Settings Data Data System
• Gently vortex sample and acquire. Do not use the BD FACS TM Loader.
2 Expression Editors*
Enter values prior to acquisition.
- Trucount
- Dilution factor 3
- Sample volume
3 Gates & Statistics
Statistics are provided for
additional optional analysis.
4 SCE Results*
CQP automatically
provides SCE results.
4
3
• Expression editors and automatic
results are not available in BD
CellQuest software.
SCE Equations for BD CellQuest Software
Statistics provided in the Equations provided in the BD Stem Cell Enumeration
BD SCE CellQuest Template Application Guide for BD FACSCalibur Flow Cytometers
39
g. Adjust R4
46
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. . .
Analyze
Analyze data and adjust gates as needed.
Data
Shut Down
Perform daily clean and shut down system.
System
54
Workflow
Start Up Perform Optimize Acquire Analyze Shut Down
System QC Settings Data Data System
Workflow
Start Up Perform Optimize Acquire Analyze Shut Down
System QC Settings Data Data System
Workflow
Start Up Perform Optimize Acquire Analyze Shut Down
System QC Settings Data Data System
Workflow
Start Up Perform Optimize Acquire Analyze Shut Down
System QC Settings Data Data System
Tip: Confirm that the high and low process control results meet the
expected values for absolute CD34+ and %CD34+.
58
Workflow
Start Up Perform Optimize Acquire Analyze Shut Down
System QC Settings Data Data System
Acquire specimens.
Workflow
Start Up Perform Optimize Acquire Analyze Shut Down
System QC Settings Data Data System
• Check each plot and verify that the gates are properly placed.
Review Gates
• Plot numbers (1 – 8) in orange are shown for reference and do not appear in the
actual SCE module.
1 2 3 4 5
e h
d f
g
6 7 8
c b
a
61
1. Identify debris in plot 6.
5. Identify beads
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. . .
Let’s Review. . .
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
March 7, 2012
73
Q & A by Topic
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;4:449-
458.
3. BD SCE kit contents 76
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 78
Answer:
Freshly collected samples can be stored at 2° to 8°C for
up to 24 hours before staining.
4. Sample types and processing 80
Answer:
Yes, the kit is designed and used for this process.
4. Sample types and processing 81
Answer:
There are several protocols available for thawing frozen
stem cell specimens. See the Clinical and Laboratory
Standards Institute (CLSI) H42-A2 guidelines.*
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.
4. Sample types and processing 84
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.
4. Sample types and processing 85
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
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.
6. Instruments cleared for the BD SCE kit 89
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.
6. Instruments cleared for the BD SCE kit 90
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.
7. BD SCE software 92
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.
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.
9. Instrument setup 95
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.
9. Instrument setup 96
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.
9. Instrument setup 97
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.
9. Instrument setup 98
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.
9. Instrument setup 99
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.
9. Instrument setup 100
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, re-
acquire the sample with the collection criterion set to 100
viable CD34. There is a maximum acquisition time of 900
seconds (15 minutes).
9. Instrument setup 101
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).
9. Instrument setup 102
Answer:
The threshold in the BD FACSCanto II SCE module is set
to FITC 400 and cannot be changed.
10. Gating 103
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 104
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.
10. Gating 106
Answer:
R7 is an exclusion gate; events in R7 are excluded from
the data file. R7 should not exceed FSC and SSC 200.
10. Gating 108
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.
11. Result reports 109
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.
11. Result reports 110
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.
11. Result reports 111
Answer:
In the BD SCE module for BD FACSCanto clinical
software, information can be added to the Comments
section of the Lab Report.
112