Analyzing Samples for CD34 Enumeration Using the … · 2014-02-07 · Analyzing Samples for CD34 Enumeration Using the BD™ Stem Cell Enumeration Kit Presented by Ellen Meinelt,

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February 29, 2012

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

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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.

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BD Stem Cell Enumeration Kit Highlights

ISHAGE

PROCOUNT VS SCE

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Hematopoietic stem cells can generate all lymphoid and myeloid lineages.

Hematopoietic Stem Cells Neutrophil

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Sources & Phenotype of Hematopoietic Stem Cells

Hematopoietic stem cells are CD34+ / CD45dim / SSClow / FSClow to intermediate.

bone marrow cord blood mobilized peripheral blood

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To determine when to perform bonemarrow stem cell transplants.

To determine whether to continueapheresis.

To assess bone marrow and cord bloodviability.

The viable CD34+ absolute count is a critical parameterfor rapid and sustained engraftment.

Why Perform Stem Cell Enumeration?

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Let’s Review. . .

Why are stem cells enumerated?

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ISHAGE

History of CD34 Enumeration Protocols1985 Siena, et al. Milan Protocol Dual platform FSC / SSC 50K or 50 CD34+ events Class III CD34 clone (1991)


1994 Bender CD45


1994 Bender CD45

1995 Owens and Loken 7-AAD viability dye


1994 Bender CD45


1994, 1996 Sutherland, et al. Original ISHAGE Protocol Dual platform 4 Parameter, 2 Colors: FSC / SSC CD34 PE / CD45 FITC

Requires isotype control


1994 Bender CD45


1998 BD Procount KitSingle platform with BD™ Trucount beads Nucleic acid staining Isotype control included Lyse/no wash methodology Leukopheresis and Peripheral Blood




1994 Bender CD45


1998 Keeney, et al. Modified ISHAGE Protocol Single platform method with

counting beads 75K or 100 CD34 7-AAD viability dye 4 Parameter, 2 Colors: FSC / SSC CD34 PE/ CD45 FITC

Sequential gating eliminates use of isotype control.

1998 BD Procount KitSingle platform with BD™ Trucount beads Nucleic acid staining Isotype control included Lyse/no wash methodology Leukopheresis and Peripheral Blood



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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

Single platform withBD Trucount Tube

Kit contents (50 tests/kit)• CD34 PE/ CD45 FITC• 7-AAD Viability Dye• NH4Cl Lysing Solution• Trucount Tubes

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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.

A FEW SIMPLE STEPS IN A SINGLE TUBE

BD TrucountTM

tube

BD SCE Kit Sample Preparation* * Dilute samples with > 40,000 WBC/uL in PBS with 0.5% BSA.

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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.

BD Stem Cell Control Kit

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Let’s Review. . .

How are stem cell controls used in the BD SCE Assay?

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Let’s Review. . .

What are the critical aspects of sample handling and preparation?

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PROCOUNT VS SCE

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5

4

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2

1

2 (requires isotype)1No. of Tubes per Sample

None7-AADViability Dye

BD FACSCanto IIBD FACSCalibur OS 9 only

BD FACSCalibur (OS 9 and OS X)Cytometer

EDTA

EDTAHeparinACD-ACPD

Anticoagulant

6 hours for Leukapheresis

24 hours for PB24 hours for all samplesSpecimen Stability

Peripheral Blood (PB)Leukapheresis

Peripheral BloodLeukapheresisBone MarrowCord Blood

Specimen Type

5 months20 monthsReagent Stability

45 minutes (15 + 30)30 minutes (20 + 10)Incubation Time

No YesMeet ISHAGE Guidelines

BD Procount KitBD SCE KitComparison of BD SCE Kit and BD Procount Kit

The Winner: BD SCE Kit

BD SCE KIT

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Analyzing samples for CD34 enumeration using BD FACSCalibur

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Workflow overview using BD FACSCalibur

Perform daily clean and shut down system.Shut Down System

Analyze data and adjust gates as needed.Analyze Data

Acquire process controls and verify results.Stain and acquire samples.

Acquire Data

Perform instrument optimization with specific application setup (using a stem cell control).Optimize Settings

Stain the stem cell controls for instrument optimization and process control.Perform cytometer QC.

Perform QC

Start up the system.Start Up System

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Instrument Start Up and Perform QC

PerformQC

• Set up the cytometer using BD Calibrite™ beads and BD FACSComp™ software with3- or 4-color lyse/no-wash assay (Calib.LNW settings).

• Verify that all parameters pass.

Start UpSystem

Shut DownSystem

AnalyzeData

OptimizeSettings

AcquireData

Optimize Instrument Settings

Use high stem cell control (w/o 7-AAD)

•Adjust Threshold.

•Adjust FSC gain.

Plot 1

Plot 6

PerformQC

Start UpSystem

Shut DownSystem

AnalyzeData

OptimizeSettings

AcquireData

Use high stem cell control (w/o 7-AAD)

• Adjust compensation.

PerformQC

Start UpSystem

Shut DownSystem

AnalyzeData

OptimizeSettings

AcquireData

Plot 7

Optimize Instrument Settings

Plot 8

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• Gently vortex sample and acquire. Do not use the BD FACSTM Loader.

• Check that 100 viable CD34 events have been acquired.

PerformQC

Start UpSystem

Shut DownSystem

AnalyzeData

OptimizeSettings

AcquireData

Acquire Data

• Verify settings for acquisition and storage.

• Add custom keywords: Dilution Factor, Trucount, and Sample Volume.

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Analyze the Acquired Data

BD CellQuestTM Pro Software

• Enter the sample volume, dilution factor, and BD Trucount bead value in the expression editors to obtain results.

PerformQC

Start UpSystem

Shut DownSystem

AnalyzeData

OptimizeSettings

AcquireData

BD CellQuestTM Software

• Calculate SCE results using the statistics and equations provided.

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Analyze the Acquired Data

PerformQC

Start UpSystem

Shut DownSystem

AnalyzeData

OptimizeSettings

AcquireData

• 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.

Gates & StatisticsStatistics are provided for additional optional analysis.

SCE Results*CQP automatically provides SCE results.

Expression Editors*Enter values prior to acquisition.- Trucount - Dilution factor- Sample volume

SCE Template in BD CellQuest Pro Software

Plots and GatesFor every sample, gates must be checked and adjusted.

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2

1

4

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• Expression editors and automaticresults are not available in BDCellQuest software.

Gates & StatisticsStatistics are provided for additional optional analysis.

CD34+ SCE EquationThe equation enables calculation of CD34+ cells/L .

• Expression editors and automaticresults are not available in BDCellQuest software.

SCE Template in BD CellQuest Software

Plots and GatesFor every sample, gates must be checked and adjusted.

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3

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Equations provided in the BD Stem Cell Enumeration Application Guide for BD FACSCalibur Flow Cytometers

Statistics provided in the BD SCE CellQuest Template

SCE Equations for BD CellQuest Software

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Gate List for Acquisition Template

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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.

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Verifying Viability Gate in Plot 8a. Adjust R8:

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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.

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Verifying Gate in Plot 2

e. Adjust R2 to include all CD34+ events.

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f. Adjust R3

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Verifying Gates in Plots 4 and 6

g. Adjust R4

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Red events are 7-AAD positive (dead cells)

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

SCE Report in BD CellQuest Pro Software

SCE Report in BD CellQuest Software

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Let’s Review. . .

What are the main steps in stem cell enumeration on the BD FACSCalibur?

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Analyzing samples for CD34 Enumeration using BD FACSCanto II

Workflow Overview using BD FACSCanto II

Perform daily clean and shut down system.Shut Down System

Analyze data and adjust gates as needed.AnalyzeData

Acquire process controls and verify results.Stain and acquire samples.

AcquireData

Perform instrument optimization with specific application setup (using a stem cell control).

Optimize Settings

Stain the stem cell controls for instrument optimization and process control.Perform cytometer QC.

PerformQC

Start up the system.Set up the software for SCE acquisition.

Start Up System

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• Perform fluidics startup in BD FACS Canto clinical software.• Enter or confirm reagent lot IDs and acquisition targets.

PerformQC

Start UpSystem

Shut DownSystem

AnalyzeData

OptimizeSettings

AcquireData

Workflow

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Perform QC with BD FACS 7-Color Setup Beads

PerformQC

Start UpSystem

Shut DownSystem

AnalyzeData

OptimizeSettings

AcquireData

• 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.

Workflow

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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.

PerformQC

Start UpSystem

Shut DownSystem

AnalyzeData

OptimizeSettings

AcquireData

Workflow

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Acquire process controls and confirm that they pass.

PerformQC

Start UpSystem

Shut DownSystem

AnalyzeData

OptimizeSettings

AcquireData

Tip: Confirm that the high and low process control results meet the expected values for absolute CD34+ and %CD34+.

Workflow

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Acquire specimens.

Tip: Vortex each tube just prior to acquiring manually.

PerformQC

Start UpSystem

Shut DownSystem

AnalyzeData

OptimizeSettings

AcquireData

Workflow

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PerformQC

Start UpSystem

Shut DownSystem

AnalyzeData

OptimizeSettings

AcquireData

Workflow

• 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.

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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.

1 2 3 4

6 7

5

8

a

fd

c

hg

b

e

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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.

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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.

Dot PlotsDisplay populations and gates

Results

QC Messages and Comments

HeaderProvides information on sample ID, acquisition and analysis times, and status.

SCE Report in Canto Clinical Software

NOTE:For process control lab reports, the two viability data plots (outlined here) are omitted.

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Let’s Review. . .

What are the main steps in stem cell enumeration on the BD FACSCanto II?

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Let’s Review. . .

What are key advantages of the BD SCE Kit?

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Let’s Review. . . What are key advantages of the BD SCE Kit?

ISHAGE

PROCOUNT VS SCE

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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

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March 7, 2012

Q & A from the BD™ Stem Cell Enumeration (SCE) Webinar

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Q & A by Topic

1. BD Procount™ Kit vs BD SCE Kit2. BD SCE Kit US Clinical Trial and EU Performance 3. BD SCE Kit Contents 4. Sample Types and Processing 5. Controls for the SCE Assay6. Instruments Cleared for the SCE Kit7. BD SCE Software 8. BD SCE Assay 9. Instrument Setup10. Gating11. Result Reports

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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.

1. BD Procount kit vs BD SCE kit

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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;4:449-458.

2.BD SCE kit US clinical trial and EU performance

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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.

3. BD SCE kit contents

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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).

3. BD SCE kit contents

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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.

4. Sample types and processing

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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.


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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.


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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.


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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.


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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.

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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.


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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).


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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

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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.


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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.


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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.

6. Instruments cleared for the BD SCE kit

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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.

6. Instruments cleared for the BD SCE kit

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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.

7. BD SCE software

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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 andBD 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.

7. BD SCE software

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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.

8. BD SCE assay

*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.

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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.

9. Instrument setup

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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.

9. Instrument setup

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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™ softwarecan 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

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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.

9. Instrument setup

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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.

9. Instrument setup

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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.

9. Instrument setup

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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, 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

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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).

9. Instrument setup

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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.

9. Instrument setup

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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

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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).

10. Gating

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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.

10. Gating

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Q. There are too many events to set the viability gate in plot 8. What should I do?

Answer:Display fewer % of events.

10. Gating

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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.

10. Gating

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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.

10. Gating

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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.

11. Result reports

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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.

11. Result reports

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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.

11. Result reports

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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: www.bdbiosciences.com/askbd

Documents

Analyzing Samples for CD34 Enumeration Using the … · 2014-02-07 · Analyzing Samples for CD34 Enumeration Using the BD™ Stem Cell Enumeration Kit Presented by Ellen Meinelt,