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InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 2020STEM CELLLABORATORY (STCL)DOCUMENT NUMBER: FLOW-GEN-040DOCUMENT TITLE:Using the BD Stem Cell Enumeration Kit for Hematopoietic Transplant Products and MobilizedPeripheral BloodDOCUMENTNOTES:Document InformationRevision: 03Vault: FLOW-General-relStatus: ReleaseDocument Type: FLOWDate InformationCreation Date: 23 Aug 2020Release Date: 19 Oct 2020Effective Date: 19 Oct 2020Expiration Date:Control InformationAuthor: REESE008Owner:Previous Number: FLOW-GEN-040Rev02REESE008Change Number: STCL-CCR-490CONFIDENTIAL - Printed by: BJ42 on 19 Oct 2020 12:39:21 pm
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 2020FLOW-GEN-040Using the BD Stem Cell Enumeration Kit for Hematopoietic TransplantProducts and Mobilized Peripheral Blood1PURPOSE1. 1The purpose ofthis process is to prepare specimens obtainedfrom qualified StemCell Laboratoryblood products for analysisby flow cytometry usingthe BD StemCell Enumeration assay method.2INTRODUCTION2. 1The BDT Stem Cell Enumeration (SCE) kit provides simultaneous enumerationofviable dual-positive CD45 /CD34 hematopoietic stem cell populations inCD34 absolute counts (cells/ 1) as well as the percentage of the total viableleukocyte count that is CD34 (%CD34). This kit is cleared for in vitrodiagnostic use on either a BD FACSCaliburT flow cytometer using BDCellQuestTorBD CellQuestTPro softwareor aBD FACSCantoT II flowcytometer using BD FACSCantoT software.3SCOPEAND RESPONSIBILITIES3. 1This kit is cleared for in vitro diagnostic (IVD) use and will be used by the STCLfor testingthe following specimens: Fresh andthawedumbilical cord blood, freshand thawed peripheral blood stem cells, mobilized peripheral blood, and freshbone marrow. It is the responsibility ofthe STCLdirector, manager, and flowcytometry staffto assurethat this procedure is followed.45DEFINITIONS/ACRONYMS4. 1UCB - Umbilical Cord Blood4. 2PB - Peripheral Blood4.3PBSC- Peripheral Blood Stem Cells4. 4BM - Bone marrow4. 5STCL - Stem Cell Laboratory4. 6BD - Becton Dickinson4. 7PBS- PhosphateBuffered Saline4. 8BSA - Bovine Serum Albumin4. 9MSDS - Material Safety Data Sheet4. 10SCE - Stem Cell EnumerationMATERIALS5. 1BD Stem Cell Enumeration Kit (Becton Dickinson) includes the following:5. 1. 1Trucount tubesFLOW-GEN-040 Using the BD Stem Cell Enumeration Kit for Hematopoietic Transplant Products andMobilized Peripheral BloodSTCL, DUMCDurham,NCPage 1 of 14CONFIDENTIAL-Printed by: BJ42 on 19 Oct 2020 12:39:21 pm
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 2020675. 1. 2CD45 FITC/CD34PE cocktail5. 1. 37AAD reagent5. 1. 4Ammonium chloride lysing solution (1Ox)5. 2PBS/1% BSA (Gibco BRL)5. 312x75 test tubes, Fisher brand5. 4TrucountT tubes (Becton Dickinson)5. 5Flow Cytometry WorksheetEQUIPMENT6. 1Vortex mixer6. 2BD FACSCaliburflow cytometer system6. 3BD FACSCantoII flow cytometer system6. 4Calibrated 10, 20, 100, 1000 microliter adjustable pipettes and tips (Rainin)6. 5Calibrated 2000 microliter automated pipette and tips (Rainin)6. 6Calibrated 20 ml automated pipette and tips (Rainin)SAFETY7. 1Review MSDS for sodium azide contained in BD antibodyreagents.7. 2Review MSDS for BD Trucount tubes.7. 2. 17.38Cobalt chlorideUse universal precautionswhenworkingwithbiological material.PROCEDURE8. 1Caution: Do not prepare more test samplesthan can be acquiredwithin 1 hourofcompletion of stainingas this is a viable cell assay.8.2Use the following pipetting guidelineswhenaddingantibodiesand specimen:8. 2. 1Adjust the pipette volume setting to the correct volume.8.2.2Keepthe pipette upright and hold the Trucounttube in the handopposite the pipetting handwhile dispensingthe antibodybeing sure toavoidtouchingthe beadpellet withthe pipette tip.8.2.3Use the sametip whenaddingthe same antibodyreagentto multipletesting tubes if the addition is to a clean tube.8.2.4Use a cleantip betweendifferentantibodyvials or if the tube receivingantibody contains another agent.8.2. 5Touchthe end ofthe tip to an interior side ofthe tubejust abovethemetal barrierto dispensethe reagent so that it runs downand into thebottom ofthe tube. Normal pipetting may be used.FLOW-GEN-040Usingthe BD Stem Cell Enumeration Kit for HematopoieticTransplant ProductsandMobilized Peripheral BloodSTCL, DUMCDurham, NCPage 2 of 14CONFIDENTIAL - Printed by: BJ42 on 19 Oct 2020 12:39:21 pm
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 20208.2. 6Use the reverse pipetting method when making specimen dilution oradding 100 1 of specimen (or specimen dilution if required) to Trucounttubes. Refer to FLOW-GEN-023 Specimen Dilution Protocol forspecimen dilution guidelines.8. 2. 6. 1Hold the Trucount tube in the opposite hand to the pipettinghand.8.2.6.2Touch the pipette tip opposite the interior side that wasusedto add antibody reagent approximately 2/3rds the distancedown the tube (i. e. 1,3rd from the bottom ofthe tube) anddispense.8.2. 6. 38. 2. 7If blood adheresto the side ofthe tube around the top, use acotton swabto wipe clean.Residual specimen may be dispensed back into the original specimentube unless the tip wascontaminated. In this casethe tip with residualspecimen should be disposed of.8.2. 8Use a cleantip eachtime specimen is addedto a Trucounttube.8.2. 9Disposeofthe dilution tube, if applicable.8.2. 10Record the stainingtime information on the Flow Cytometry Worksheetbased on the timer setting compared to the cytometer computer clocktime.FLOW-GEN-040Usingthe BD Stem Cell EnumerationKit for HematopoieticTransplantProductsandMobilized Peripheral BloodSTCL, DUMCDurham, NCCONFIDENTIAL- Printed by: BJ42 on 19 Oct 2020 12:39:21 pmPage 3 of 14
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 20208. 3Staining: Refer to Table 1 below to prepare process controls and test specimens.Table 1: Use the followingguidelineswhen preparing process controls and test samplesas recommended by the manufacturer:1. Preparethe process controls or test sample accordingto the following table.Prepare and run the process controls prior to staining test samples to completethe instrument setup and assure reagent performance qualification.Cells (\t\}Reagent (til)Tube TypeSample TypeReverse pipettingNormal pipetting7-AAD controlPolystyreneCD3420For Canto onlyHigh ControlTrucount20Low ControlTrucount20Test SampleTrucount207-AADHigh20Low100 of either10010020100 test sample2. Cap the tubes, vortex gently, and incubate for 20 minutes in the dark at roomtemperature.3. Add 2 ml of IX ammonium chloride lysing solution to each tube.Make IXsolution by using 2 mis oflOX ammonium chlorideto 18mis Dl waterfromSTCLspigot 1 (deionizedandfiltered). Doubleto ratio of 4 and 36 if test volume callsfor more lysing solution.4. Cap the tubes, vortex gently, and incubate for 10 minutes in the dark at roomtemperature.5. Immediately put the tubes on wet ice and acquirewithin 1 hourof lysing.NOTE: Store BD Trucount tubes at 2-25 C. These tubes must be used within 1 hour ofremoval from the foil pouch. Remainingtubes must be usedwithin 1 month of openingthe foil pouch. Carefully reseal the foil pouch immediately after removing a tube.Examinethe desiccanteachtime the pouch is opened to determine if color has changedfrom blue to lavender. Discard tubes if color has changed.8. 4Optimizing for BD SCEtest acquisitions on the FACSCalibur instruments:8. 4. 1Perform instrument startup and daily quality control using BDCalibriteT beads and FACSComp software. See SOPFLOW-GEN-014.8.4.2Verifythat all parameters PASSprior to initiation oftestmg.8.4. 3Refer to section 8.4 in procedure FLOW-GEN-038to optimize theFACSCalibursettings in orderto acquirethe SCEassay controls andtest samples. Savethe optimized settings as BD SCEin the instrumentsettings folder on the FACSCaliburhard drive.8. 5Acquiring SCE assay samples on the FACSCalibur Flow Cytometer:8. 5. 1Control cell testing must be completed, acquiredon one oftheFACSCaliburflow cytometers or on the FACSCantoflow cytometer,FLOW-GEN-040 Using the BD Stem Cell Enumeration Kit for Hematopoietic Transplant Products andMobilizedPeripheralBloodSTCL, DUMCDurham, NCPage 4 of 14CONFIDENTIAL- Printed by: BJ42 on 19 Oct 2020 12:39:21 pm
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 2020and the results must fall within the assayed range of values for thecontrol cell lot prior to staining test samples.8. 5.2In the parameter description window, specify the storage location anddata file name. The file naming convention for control cells is asfollows: LotID (H/L) mmddyy (eg. FC012H012212). The file namingconvention for test samples is as follows:8. 5. 2. 1When relevant enter pt. ID using the first initial of first nameand first 3 letters of last name, abbreviation for sample type,date as mmddyy.8. 5. 2. 2For fresh umbilical cord blood the file name structure isUCB (A, B, C etc. ) mmddyy.8. 5. 2. 3Specimen designations are as follows:8. 5.2.3. 1PeripheralBlood PB8. 5. 2. 3. 2Leukapheresis (Peripheral blood stem cells) L1, L2, L3 and so on. Add letters A, B, Cwhen dealing with selected products orproducts for which multiple stages inprocessing are monitored. Add MP formidpoint tests.8. 5.2.3.3Peripheralblood stem cell reinfusion LR.Add number designation if multiplereinfusions are performed.8. 5. 2. 3.4Umbilical cord blood reinfusion CBR8. 5. 2. 3.5Bone Marrow BMOR for samples taken fromthe OR bag or BMPO for post processedmarrow.5.3Create a data storage folder on the specified drive locationcorrespondingto the current date.5.4Enter operator name or initial in the Operator field of the parameterdescription window.5.5Use the following table list for entering specimen ID and patient IDinformation in the Parameter description window:SpecimenSample IDPatient IDControlsLot number(not required)PBPatient history numberLast name. First nameMidpoints (leukapheresis)Patient history numberLast name. First nameReinftisions and fresh productsBar codeLast name. First nameFresh UCBLab ID (UCBA, B, C)Bar codeFLOW-GEN-040Using the BD Stem Cell EnumerationKit for HematopoieticTransplantProductsandMobilized Peripheral BloodSTCL, DUMCDurham, NCCONFIDENTIAL - Printed by: BJ42 on 19 Oct 2020 12:39:21 pmPage 5 of 14
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 20208. 5.6Go to the Acquire menu headingand scroll to open the Acquisition andStorage window.8. 5. 7Whenpreparing for control cell testing, verify that the acquisitionwillReject Debris (R7) and the Event Count or Time stop criteria is set at75000 of viable 45 events or after 900 seconds.8. 5. 8Fortest samples changethe Eventcountto 300,000 and leave all otherparameters the same as the control setup.8. 5.9Openthe custom keywordswindow(from the Acquire menu).8. 5. 10In the Value fields, enter the Trucount bead count, Dilution Factor (1 ifnot diluted), and Sample staining volume (100 1)8. 5. 11Click to check the SETUP box in the Acquisition Control window (alsofound in the browser window).8. 5. 12The flow cytometer must be placed in RUN mode prior to installing thetubes on the SIP when using the loader system, otherwise an alarm willsound.8. 5. 13Mix the test tube on the vortex at medium (see mark on mixer) speed 24 seconds.8. 5. 14Remove the cap and load the tube on the SIP. (Ifusingthe loader, placethe tube on the carousel and replace the loader cover).8. 5. 15The carousel will circle around to the number 1 slot locationautomatically. NOTE: Only 1 tube at a time may be placed on theloader since samples must be kept on ice prior to acquisition.8. 5. 16Ifperforming workwithout a loader, move the tube guide arm to the leftand install the testing tube on the SIP making sure the tube is pushedsecurely onto the SIP.8. 5. 17Click Acquire in the acquisitionwindow(optionally use the browseracquu-e window).8. 5. 18Observethe acquisitionplots to make surethe events are showingonthe screen display as they should be.8. 5. 19If no events showup on the display, an air bubble or clog may bepreventing flow oftest sample and the testing tube should be uninstalledwhile troubleshooting the problem.8. 5. 20Return to the acquisition window and proceed as follows:8. 5.21Click on STOP,then, ABORT.8. 5.22Remove the check from the SETUPbox.8. 5. 23Click on ACQUIRE, to begin saving data.8. 5. 24When the test tube has been acquired, remove the tube, replace thecap(s) and place the tube on the "To be analyzed"rackuntil the analysisfor the batch is complete.FLOW-GEN-040 Using the BD Stem Cell Enumeration Kit for Hematopoietic Transplant Products andMobilized Peripheral BloodSTCL, DUMCDurham, NCCONFIDENTIAL - Printed by: BJ42 on 19 Oct 2020 12:39:21 pmPage 6 of 14
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 20208. 5.25Place a tube with about 1 milliliter ofDI water on the SIP and place thecytometer in standbymode by pressingthe STANDBYbutton on thefront ofthe cytometer.5. 26On page 2 of the Flow Cytometry Worksheet complete the entries fordata collection through acquisition oftesting samples.FLOW-GEN-040Usingthe BD Stem Cell Enumeration Kit for HematopoieticTransplantProductsandMobilized Peripheral BloodSTCL, DUMCDurham, NCCONFIDENTIAL - Printed by: BJ42 on 19 Oct 2020 12:39:21 pmPage 7 of 14
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 20208. 6Use the guidelinesprovided below for the BD SCECQ Pro analysis ofthe dataacquired from the SCE testing.8.6. 1Analysis guidelines for BD SCECQ Pro method:ExplanationDot PlotFC012CALH))1012.001§. ?yF-. 0000.(*; 1!-i o:0 0.'3 i»t ::' .I'f;:.'' J'-''M. Ill 1 1 - 'r:WTwos--.This plot contains the first gate to be adjusted and isused to identify viable cells (7-AADneg.)Adjust R8 to enclose the 7-AAD"eg events. Theregion extends beyond the top of the plot on the yaxis, beyond the far left of the plot on thex-zxis, andexcludes positive-stained cells.Caution: If there are a large number ofred(dead) events outside R8, see page 34 ofapplicaton guide for troubleshooting''&information.0i6 "'io'' i62 i63 K7.AADPlot 8: 7-AAD vs SSCThis plot is gated on the Total CD34 gate (Rl andR2 and R3) and is used during compensationoptimization (FL3%-%F12).FC012CALW11012.0Q1§gwThis plot displays total CD34 cells and confirmspaper adjustment of region R8 on Plot 8.co.Sow oJ8R8Ifthe instrument is appropriately set up andcompensated,the gatedviable CD34 cells willU)Vwoexhibit the same level offluouescence as the viableslymphocytes in Plot 8.010Uio310210110"7.AADPlot 7: 7-AAD vs SSCThis plot is ungated and is used to include all CD45d""to CD45 bright events and excludes debris,o FC022HCAL20217t2.00100» yA lf V , 00t§ .'W D0 0 ovare all CD45 neg.& coplatelets, andunlysed red blood cells (RBCs), whichAdjust the Rl leucocyte gateto extend abovetherR1B :top of the plot to include high SSC events that areCD45 . Adjust the left side ofthe gateto includeall CD45 cells, including dim CD45 events thatare CD34 . The right side can extend to the edge ofthe plot.wo0 Adjust polygon region R5 aroundthe lymphocytes.0'I-''""I**'"-'I ' .". i6 16' i62 i63 i6'1C&45 FITCPlotl:CD45vsSSCInclude only as many events as necessary to definethe viable lymphocyte population displayed in Plot6.Tip Display fewer events on this plot so that thelimits ofthe lymphocytes are easier to define.FLOW-GEN-040Using the BD Stem Cell EnumerationKit for HematopoieticTransplantProductsandMobilized Peripheral BloodSTCL, DUMCDurham, NCCONFIDENTIAL - Printed by: BJ42 on 19 Oct 2020 12:39:21 pmPage 8 of 14
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 2020Dot PlotFC022HCAL2021712.COIExplanationThis plot displays viable CD45 (Gl) cells and isusedto identify CD34 cells. Adjust R2 to includeall CD34 /low SSC (below 400)events.NOTE This region should exclude any plateletsthat form a streak between neutqihils and CD34 events. See examples of this on page 3 of theapplication guide.i6 i61 io2 i63 i64co irePlot 2: CD34 vs SSCFC022HCAL202t712.0C.IThis plot displays 02 (R2 and viable CD45 ) cellsand is use to further define the viable CD34 CD45 cell populationAdjust R3 to include only those events that form acluster with low to intermediate SSc and Cd45 dimexpression. Adjust R3 to exclude any lymphocytesor monocytes seen to the right of the stem cellcluster.00-, 01--, 02--, Q3-, 10'CD45FITCPlot 3: CD4S vs SSCThis plot displays Viable Lymphs (R5 and R8) andis usde to establish the minimum FSC and SSCranges for R4.Display region R4 on this plot and adjust theposition os that only viable lymphs from R5 areincluded.R7 is an acquisition exclusion gate used to excludedebris. Region 7 should not exceed FSC channel0200 -100 600 800 1000FSC-Height200 and SSC channel 200 and shold not encroachthe lymphcytes population because events in R7will be excluded from the data file.Plot 6: FSC vs SSCFLOW-GEN-040Usingthe BD StemCell Enumeration Kit for HematopoieticTransplant Products andMobilized Peripheral BloodSTCL, DUMCDurham, NCCONFIDENTIAL- Printed by: BJ42 on 19 Oct 2020 12:39:21 pmPage 9 of 14
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 2020ExplanationThis plot displays G3 (R3 and G2) cells and is usedto identify viable stem cells (CD34 ).Adjust R4 to include only those events that form aDot PlotFC022HCAL2021712.001cluster with low to intermediate SSC and medium tohigh FSC.The gate serves to exclude platelets and debris thatcan show weak, nonspecific binding ofCD34 andCD45. Its Lower FSC boundary is adjusted in Plot6. Region R4 is the lymplVblast region and will beadjusted by scatter oflymphocytes.0200 4CO 6CO 800 \QQQTip This plot can be gated on Rl and R2 and R3to display Total CD34 cells instead of ViableFSC-HeightCD34 cells.Plot 4: FSC vs SSCThis ungated multicolor plot is used to identifybeads (R6).Optional: Adjust the quadrant marker to establish avisual lower limit ofCD45 expression by theFC012CALHO11012.001CD34 events, as in Plot 1.The bead population appears in the top right comer(aqua). Allow R6 to extend beyond the plotboundary (x and y axes) to include all bead events.Verify that there are no cellular events in the beadgate.)6 16' t62 i63 i64CD45FFTCPlot 5: CD45 FITC vs CD34 PE8.6.2BD Stem Cell Enumeration CQ Pro Gating definitions:000WindowUS Gate ListBD SCt CQ Pro Template 1. 0 RevAf Set All to Default ''Multillihte color Color v"'.y ' 1syt, r- «. r-l . 4, r-- V. . --, 8. 7---'. .c3IDV. . eC.'. -. !cf.' . ;'! -Rtd-i d RSR2 and . Vt. jblt. CC ! 4 r.R3 . nd C2.03. »R-) nd C3th.-y , -.,s'DefinitionLabel 1-v Tn 'P-;r, y r lp \;(- ';'-'1 ; '-.:i .\:-t.*»-.t A?-'.-. . fi; -sPrint the results of testing to keep with the flow worksheet and save a PDF byperforming the following steps:8.7. 1Choose file print.FLOW-GEN-040 Using the BD Stem Cell Enumeration Kit for Hematopoietic Transplant Products andMobilized Peripheral BloodSTCL, DUMCDurham, NCPage 10 of 14CONFIDENTIAL - Printed by: BJ42 on 19 Oct 2020 12:39:21 pm
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 20208. 7.2In the bottom left corner of the print window choose PDF and scroll toSave PDF.8.7.3Enter the file name as it is for the data file and add the suffix PDF on theend. Example: JDOEPBMMDDYY.PDF.8. 8If required for the specimen type, calculate the total viable CD34 cells or Totalviable CD34/kgvalues by multiplying the viable CD34/ ilby the volume inmicroliters and divided by the recipient weight using the calculation fieldsprovided on the flow cytometry worksheet.8. 9Calculations must be reviewed by a second tech prior to reporting results.8. 10 Acquiring SCE assay samples on the FACSCanto II Flow Cytometer usingFACSCanto software and BD Stem Cell application:8. 10. 1Power up the FACSCanto II flow cytometer and computer.8. 10.2Log into windows:Administrator and enter the requiredpassword.8. 10.3Launch BD FACSCanto Clinical software from the desktop icon.JlBD FACSCantOSoftware8. 10. 4From the drop down window find your name as user and enter yourpassword.8. 10.5Refer to sections 8.3. 6-8.4 in procedure FLOW-GEN-039forinstructions on instrument setup, optimization, and process controltesting.8. 10. 6Instrument performance checks must pass, control cell testing must becompleted, and the results must fall within the assayed range of valuesfor the control cell lot prior to staining test samples.8. 10.7In the FACSCantoworklist "NAME"column enter SPECIMEN.8. 10. 8In the FACSCantoworklist "SAMPLEID" column use the followingguideline for entering sample identification information:8. 10. 8. 1When relevant specimen type, enter pt. ID using the firstinitial of first name and first 3 letters of last name,abbreviation for sample type, date as mmddyy.8. 10. 8. 2For fresh umbilical cord blood the file name structure isUCB (A, B, C etc. ) mmddyy.8. 10. 8. 3Specimen designations are as follows into the :8. 10. 8. 3. 1Peripheral Blood PB8. 10. 8. 3. 2Leukapheresis (Peripheral blood stem cells) LI, L2, L3 and so on. Add letters A, B, Cwhen dealing with selected products orproducts for which multiple stages inFLOW-GEN-040Usingthe BD Stem Cell EnumerationKit for HematopoieticTransplantProductsandMobilized Peripheral BloodSTCL, DUMCDurham, NCCONFIDENTIAL-Printed by: BJ42 on 19 Oct 2020 12:39:21 pmPagellofl4
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 2020processing are monitored. Add MP formidpoint tests.8. 10. 8. 3. 3Peripheral blood stem cell reinfusion LR.Add number designation if multiplereinfusions are performed.8. 10. 8. 3. 4Umbilical cord blood reinfusion CBR8. 10. 8.3. 5Bone Marrow BMORfor samples taken fromthe OR bag or BMPO for post processedmarrow.8. 10.9In the "CASENUMBER"column enterthe product bar code or pt.history number (if no bar code assigned as with PB specimens).8. 10. 10 Select "BD StemCell 7AAD" from the Paneldrop downmenu.1 8. 10. 11Click Run Vto start the run.8. 10. 11. 1 A prompt appears asking to save the worklist; Choose No.8. 10. 11.2 A prompt appears asking if manual run is OK; Choose OK.8. 10. 11. 3 At the next prompt choose Ignore.8. 10. 12 Vortex the test tube on medium speed 2-4 second (vortex dial should beplaced where arrow is drawn) before acquisition.8. 10. 13 When prompted, manually install tube on the cytometer by doing thefollowing:8. 10. 13. 1 Push the aspirator arm to the left.8. 10. 13. 2 Place the tube on the SIT, ensure that the tube is straight,and firmly push up until the tube comes to a complete stopand is fully seated.8. 10. 13. 3 Center the aspirator arm under the tube so that the bottom ofthe tube sits centered above the three sensor pins on theaspiratorarm.8. 10. 14 After acquisition is complete the lab report appears on the screen.8. 10. 15 Refer to the FACSCanto II BD Stem Cell Enumeration ApplicationGuide for examples of various product types to help determineappropriate region placement. See report example 1 as a guide.FLOW-GEN-040 Using the BD Stem Cell Enumeration Kit for Hematopoietic Transplant Products andMobilized Peripheral BloodSTCL, DUMCDurham, NCPage 12 of 14CONFIDENTIAL - Printed by: BJ42 on 19 Oct 2020 12:39:21 pm
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 2020Example 1:Chapter6: Gating guideline and ex.amples 41Example Lab ReportPlot no.The plots arc displayed on the Lab Report in the following order,Gaf(l)Plot no.0CD45Pos, ;, Lymphs CE 34P CD45Poi Viable CD34" CD45Dimj G«t«(a)CDBeads Debris cg , ViableViable8. 10. 16 To make adjustments to regions, click on the plot to be changed (thisaction activates the plot and allows the user to modify gates) and clickon the region to make adjustments as needed.8. 10. 17 Once the analysis is complete, click on the Review button Choose theappropriate User ID click OK.8. 10. 18 Print report to keep with the flow worksheet.8. 11 If required for the specimen type, calculate the total viable CD34 cells or Totalviable CD34/kg values by multiplying the viable CD34/ il by the volume inmicroliters and divided by the recipient weight using the calculation fieldsprovided on the flow cytometry worksheet.8. 12 Calculations must be reviewed by a second tech prior to reporting results.FLOW-GEN-040 Using the BD Stem Cell Enumeration Kit for Hematopoietic Transplant Products andMobilized Peripheral BloodSTCL, DUMCDurham, NCPage 13 of 14CONFIDENTIAL - Printed by: BJ42 on 19 Oct 2020 12:39:21 pm
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 2020RELATED DOCUMENTS/FORMS9. 1FLOW-GEN-012 FRM5 Stem Cell Laboratory Flow Cytometry Worksheet9.2FLOW-GEN-038Enumeration of Viable CD34 Stem Cells in Fresh UmbilicalCord Blood Usingthe BD Stem Cell EnumerationKit: BD FACSCaliburand BDCellquest Pro Version9. 3FLOW-GEN-039Enumeration of Viable CD34 Stem Cells in Fresh UmbilicalCord Blood Using the BD Stem Cell Enumeration Kit: FACSCanto II ClinicalSoftware Version9.4FLOW-GEN-023Specimen Dilution Protocol (JA1)10 REFERENCES10. 1BD Stem Cell Enumeration Kit product insert: catalog # 344563.10. 2 BD Stem Cell Enumeration Application Guide for BD FACSCanto II FlowCytometer and for BD FACSCalibur Flow Cytometer. Electronic version storedon the cytometer workstation instrument workstation desktop or on sharednetwork drive.11 REVISIONHISTORYRevision No.Author03M. ReeseDescription of Change(s)1. Modified document title2. Made corrections to document names in section 9FLOW-GEN-040Usingthe BD Stem Cell Enumeration Kit for HematopoieticTransplantProductsandMobilized Peripheral BloodSTCL, DUMCDurham, NCCONFIDENTIAL- Printed by: BJ42 on 19 Oct 2020 12:39:21 pmPage 14 of 14
InfoCard #: FLOW-GEN-040 Rev. 03 Effective Date: 19 Oct 2020Signature ManifestDocument Number: FLOW-GEN-040Revision: 03Title: Using the BD Stem Cell Enumeration Kit for hlematopoietic Transplant Products and Mobilized PeripheralBloodEffective Date: 19 Oct 2020All dates and times are in Eastern Time.FLOW-GEN-040 Using the BD Stem Cell Enumeration Kit for Hematopoietic Transplant Products andAuthorf Name/Signature; TitleDate29 Sep 2020, 03:44:43 PMMelissa Reese (REESE008)j Meaning/ReasonApprovedManagementI Name/SignatureTitleDateBarbara Waters-Pick29 Sep 2020, 05:28:21 PM(WATER002)r Meaning/ReasonApprovedMedical DirectorName/Signature! TitleJ Date Joanne Kurtzberg (KURTZ001)29 Sep 2020, 06:35:23 PMMeaning/ReasonApprovedQualityj Name/SignatureI Titlel2at Meaning/Reason.30 Sep 2020, 12:44:49 PMIsabel Starch (IMS19)ApprovedDocument Releasej Name/SignatureSandy Mulligan (MULL1026)I TitleDate12 Oct 2020, 06:39:53 PMCONFIDENTIAL - Printed by: BJ42 on 19 Oct 2020 12:39:21 pmMeaning/ReasonApproved
8.5 Acquiring SCE assay samples on the FACSCalibur Flow Cytometer: 8.5. 1 Control cell testing must be completed, acquired on one of the FACSCalibur flow cytometers or on the FACSCanto flow cytometer, FLOW-GEN-040 Using the BD Stem Cell Enumeration Kit for Hematopoietic Transplant Products and Mobilized Peripheral Blood STCL, DUMC
