Analyzing Samples for CD34 Enumeration Using the BD Stem Cell Enumeration Kit

Transcription

1 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

2 Learning Objectives 3 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.

3 BD Stem Cell Enumeration Kit Highlights 4 ISHAGE PROCOUNT VS SCE

4 BD Stem Cell Enumeration Kit Highlights 5

5 Hematopoietic Stem Cells Neutrophil 6 Hematopoietic stem cells can generate all lymphoid and myeloid lineages.

6 Sources & Phenotype of Hematopoietic Stem Cells 7 bone marrow cord blood mobilized peripheral blood Hematopoietic stem cells are CD34 + / CD45 dim / SSC low / FSC low to intermediate.

7 Why Perform Stem Cell Enumeration? 8 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.

8 Let s Review... 9 Why are stem cells enumerated?

9 BD Stem Cell Enumeration Kit Highlights 10 ISHAGE

10 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)

11 History of CD34 Enumeration Protocols 1985 Siena, et al Bender Milan Protocol CD45 Dual platform FSC / SSC 50K or 50 CD34 + events Class III CD34 clone (1991)

12 History of CD34 Enumeration Protocols 1985 Siena, et al 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)

13 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) 1994 Bender CD Owens and Loken 7-AAD viability dye 1994, 1996 Sutherland, et al. Original ISHAGE Protocol Dual platform 4 Parameter, 2 Colors: FSC / SSC CD34 PE / CD45 FITC Requires isotype control

14 History of CD34 Enumeration Protocols 1985 Siena, et al 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 1998 BD Procount Kit Single platform with BD Trucount beads Nucleic acid staining Isotype control included Lyse/no wash methodology Leukopheresis and Peripheral Blood

15 History of CD34 Enumeration Protocols 1985 Siena, et al 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) 1998 Keeney, et al. Modified ISHAGE Protocol Single platform method with 1998 BD Procount Kit 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. 1994, 1996 Sutherland, et al. Original ISHAGE Protocol Dual platform 4 Parameter, 2 Colors: FSC / SSC CD34 PE / CD45 FITC Requires isotype control Single platform with BD Trucount beads Nucleic acid staining Isotype control included Lyse/no wash methodology Leukopheresis and Peripheral Blood

16 BD Stem Cell Enumeration Kit Highlights 17

17 Features of the BD Stem Cell Enumeration Kit 18 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 NH 4 Cl Lysing Solution Trucount Tubes Single platform with BD Trucount Tube

18 BD SCE Kit Sample Preparation* A FEW SIMPLE STEPS IN A SINGLE TUBE BD Trucount TM 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. * Dilute samples with > 40,000 WBC/uL in PBS with 0.5% BSA. 19 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.

19 BD Stem Cell Control Kit 20 BD Stem Cell Control Kit (Catalog # ) CD34 High (approximately 35 cells/ L) CD34 Low (approximately 10 cells/ L) Functions as Instrument setup and performance Process control for: antibody staining for CD AAD staining of non-viable cells red blood cell lysis Always run process controls prior to staining specimens.

20 Let s Review How are stem cell controls used in the BD SCE Assay?

21 Let s Review What are the critical aspects of sample handling and preparation?

22 BD Stem Cell Enumeration Kit Highlights 23 PROCOUNT VS SCE

23 Comparison of BD SCE Kit and BD Procount Kit Meet ISHAGE Guidelines No. of Tubes per Sample Viability Dye Incubation Time Reagent Stability Specimen Type Specimen Stability Anticoagulant Cytometer BD SCE Kit Yes 1 7-AAD 30 minutes ( ) 20 months Peripheral Blood Leukapheresis Bone Marrow Cord Blood 24 hours for all samples EDTA Heparin ACD-A CPD BD FACSCalibur (OS 9 and OS X) BD FACSCanto II BD Procount Kit No 2 (requires isotype) None 45 minutes ( ) 5 months Peripheral Blood (PB) Leukapheresis 24 hours for PB 6 hours for Leukapheresis EDTA BD FACSCalibur OS 9 only 24

24 The Winner: BD SCE Kit BD SCE KIT

25 26

26 BD Stem Cell Enumeration Kit Highlights 27

27 Analyzing samples for CD34 enumeration using BD FACSCalibur 28

28 Workflow overview using BD FACSCalibur 29 Start Up System Start up the system. Perform QC Optimize Settings Acquire Data Analyze Data Shut Down 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 process controls and verify results. Stain and acquire samples. Analyze data and adjust gates as needed. Perform daily clean and shut down system.

29 Instrument Start Up and Perform QC 30 Start Up System Perform QC Optimize Settings Acquire Data Analyze Data Shut Down System 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.

30 Optimize Instrument Settings Start Up System Perform QC Optimize Settings Acquire Data Analyze Data Shut Down System Use high stem cell control (w/o 7-AAD) Plot 1 Adjust Threshold. Plot 6 Adjust FSC gain.

31 Optimize Instrument Settings Start Up System Perform QC Optimize Settings Acquire Data Analyze Data Shut Down System Use high stem cell control (w/o 7-AAD) Plot 8 Adjust compensation. Plot 7

32 Acquire Data 33 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 TM Loader. Check that 100 viable CD34 events have been acquired.

33 Analyze the Acquired Data 34 Start Up System Perform QC Optimize Settings Acquire Data Analyze Data Shut Down System BD CellQuest TM Pro Software Enter the sample volume, dilution factor, and BD Trucount bead value in the expression editors to obtain results. BD CellQuest TM Software Calculate SCE results using the statistics and equations provided.

34 Analyze the Acquired Data 35 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. Identify viable cells. 2. Identify the leukocytes and lymphocytes. 3. Identify the CD34 + cluster among the viable cells. 4. Gate the Trucount Beads.

35 SCE Template in BD CellQuest Pro Software 1 Plots and Gates For every sample, gates must be checked and adjusted 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.

36 SCE Template in BD CellQuest Software 1 Plots and Gates For every sample, gates must be checked and adjusted. 1 2 Gates & Statistics Statistics are provided for additional optional analysis. 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.

37 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

38 Gate List for Acquisition Template 39

39 SCE Analysis 40 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.

40 Verifying Viability Gate in Plot 8 41 a. Adjust R8:

41 Verifying Gates in Plots 1 and 6 42 b. Adjust R1: c. Adjust R5 to include most of the lymphocytes. d. Adjust R4 to include viable lymphocytes.

42 Verifying Gate in Plot 2 43 e. Adjust R2 to include all CD34 + events.

43 Verifying Gate in Plot 3 44 f. Adjust R3

44 Verifying Gates in Plots 4 and 6 45 g. Adjust R4

45 Verifying Gate in Plot 5 h. Adjust R6: 46 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)

46 SCE Report in BD CellQuest Pro Software

47 SCE Report in BD CellQuest Software

48 Let s Review What are the main steps in stem cell enumeration on the BD FACSCalibur?

49 50

50 BD Stem Cell Enumeration Kit Highlights 51

51 Analyzing samples for CD34 Enumeration using BD FACSCanto II 52

52 Workflow Overview using BD FACSCanto II Start Up System Perform QC Optimize Settings Acquire Data Analyze Data Shut Down System Start up the system. Set up the software for SCE acquisition. 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 process controls and verify results. Stain and acquire samples. Analyze data and adjust gates as needed. Perform daily clean and shut down system.

53 Workflow 54 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.

54 Workflow 55 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.

55 Workflow 56 Start Up System Perform QC Optimize Settings Acquire Data Analyze Data Shut Down System 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.

56 Workflow 57 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 +.

57 Workflow 58 Start Up System Perform QC Optimize Settings Acquire Data Analyze Data Shut Down System Acquire specimens. Tip: Vortex each tube just prior to acquiring manually.

58 Workflow 59 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.

59 Review Gates 60 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 e d f g 5 h 6 7 c 8 b a

60 1. Identify debris in plot Identify debris in the lower left corner of the plot.

61 2. Identify viable events 62 Identify viable events in plot 8. Use plot 7 to confirm the placement of the viable gate.

62 3. Identify lymphocytes in plot Identify lymphocytes among CD45+ events.

63 4. Identify stem cells. 64 Use plots 2 and 3 to identify stem cells.

64 4. Confirm the Viable CD34 Gate 65 Plot 4 displays the viable CD34 population. Viable lymphocytes are shown in light blue and viable CD34 + cells are shown in red.

65 5. Identify beads 66 Confirm placement of the Trucount beads gate.

66 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

67 Let s Review What are the main steps in stem cell enumeration on the BD FACSCanto II?

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

69 Let s Review... What are key advantages of the BD SCE Kit? 70 ISHAGE PROCOUNT VS SCE

70 References 71 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: 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: 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 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: 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 Sutherland DR, Anderson L, Keeney M, et. al. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. J Hematotherapy. 1996;5: 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: 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: Lemarie C., et al. A new single-platform method for the enumeration of CD34+ cells Cytotherapy (2009) 11(6): 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 BD

71 72 Q & A from the BD Stem Cell Enumeration (SCE) Webinar March 7, 2012

72 Q & A by Topic BD Procount Kit vs BD SCE Kit 2. BD SCE Kit US Clinical Trial and EU Performance 3. BD SCE Kit Contents 4. Sample Types and Processing 5. Controls for the SCE Assay 6. Instruments Cleared for the SCE Kit 7. BD SCE Software 8. BD SCE Assay 9. Instrument Setup 10. Gating 11. Result Reports

73 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

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. 75 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:

75 3. BD SCE kit contents 76 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.

76 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).

77 4. Sample types and processing 78 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 79 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 80 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 81 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 82 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 83 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 CD45 dim, 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 84 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 85 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 ) when using the BD SCE kit.

86 5. Controls for the BD SCE assay 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. 87

87 5. Controls for the BD SCE assay 88 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 89 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 90 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 91 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 92 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 93 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:

93 9. Instrument setup 94 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.

94 9. Instrument setup 95 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.

95 9. Instrument setup 96 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.

96 9. Instrument setup 97 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.

97 9. Instrument setup 98 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.

98 9. Instrument setup 99 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.

99 9. Instrument setup 100 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).

100 9. Instrument setup 101 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).

101 9. Instrument setup 102 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.

102 10. Gating 103 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.

103 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). 104

104 10. Gating 105 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.

105 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. 106

106 10. Gating 107 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.

107 10. Gating 108 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.

108 11. Result reports 109 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.

109 11. Result reports 110 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.

110 11. Result reports 111 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.

111 112 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 BD

112 113 If you have further questions, please visit: