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BD FACSCanto临床软件进行仪器日常质控（QC）无论在BD FACSCanto临床软件或者是在BD FACSDiva软件中运行样本，都需要使用BD FACSCanto临床软件来运行自动化设置和QC。
利用BD FACS 7色设置微球，对检测器电压进行调节，把设置微球调整在预定的靶值位置，对检测灵敏度值进行测量，并且对荧光补偿值进行计算并保存荧光补偿的资料。
使用BD FACSCanto临床软件中的Levey-Jennings功能，按时间追踪并监测流式细胞仪性能，留下参数的变化和趋势。
1．准备BD FACS 7色设置微球：取一只BD FACS 7色设置微球样本管，加稀释液至刻度线（约1ml），充分混匀。
注意：不要使用过期微球。
如果使用过期微球可能导致错误的设置结果。
2．按每日开机程序打开流式细胞仪主机和电脑，打开FACS Canto临床软件并登陆，启动液路。
3．选择“Cytometer（流式细胞仪）”→“Setup（设置）→Standard Setup（标准设置）。
4．在“Setup Lot Information(设置批号信息)”对话框中点击“New Lot ID （新批号）”。
5．输入相应微球批号及失效期，并点击“OK”。
6．按照BD FACS 7色校准微球标签上所显示的信息输入或检查Lot ID(批号)、targets(靶值)及spectral overlap factors(光谱叠加因子)，输入完毕后点击“Next”。
7．仪器显示Save Setup Bead Lot Info (保存校准微球批号信息)对话框，点击“Yes”。
（注意：该对话框仅在更换批号时显示。
）8．选择“Run setup in Manual mode(手动执行校准)”，点击“Next”。
9．仪器显示如下提示框，充分摇匀微球样本管，将废液吸引臂推到最左边，上样，废液吸引臂推回中间位置后，点击“OK”。
10．校准过程自动进行，待软件出现如下提示框时，将废液吸引臂推到最左边，取下样本管，废液吸引臂回到中位。
流式細胞儀---FACSCanto之基本原理與應用產品專員劉益年美商必帝公司E-mail : kenny_liu@大綱•實驗設計原理•流式細胞儀運作原理•儀器設定調整•開、關機流程實驗設計原理•藉由螢光抗體標識細胞之抗原特性•藉由螢光化合物標識細胞特性•藉由螢光染劑標識細胞特性•Cytometry Beads Array單一細胞特性之偵測單一細胞特性之偵測cytokinesystructureenzymesAdhesionReceptorsMetabolic淋巴球免疫分型FITC PE使用原理CD45CD14儀器可自動依據CD45與CD14之免疫螢光染色，測量三群白血球的比例，淋巴球(CD45Bright/CD14-/Low FSC/Low SSC)，單核球(CD14+/CD45Intermediate/High FSC/Intermediate SSC)，顆粒性球(CD14dim/CD45dim/High FSC/High SSC)，細胞碎片與紅血球應是(CD14-/CD45-g g)細胞碎片與紅血球應是(/Low FSC/Low SSC)。
IgG1IgG1根據陰性對照組之染色程度來劃分陽性/陰性之界線，依此界線在陰性對照的樣品中，假陽性(Quardrant1+2+4)不得超過百分之二。
CD3CD19T 細胞必須表達有CD3+ 抗原，B 細胞表達有CD19+ 抗原。
此兩群細胞與NK 細胞之總和應約略等於淋巴球的總數，可作為品管的指標。
CD3CD4CD4+T細胞必須同時表達有CD3+與CD4+抗原。
CD3CD8CD8+T細胞必須同時表達有CD3+與CD8+抗原。
CD3CD16+or CD56+NK細胞必須不表達有CD3抗原，同時表達有CD16+或CD56+抗原。
NK細胞與T&B兩群細胞之總和應約略等於淋巴球的總數，可作為品管的指標。
訊息傳導實驗設計原理•藉由螢光抗體標識細胞之抗原特性•藉由螢光化合物標識細胞特性•藉由螢光染劑標識細胞特性•Cytometry Beads ArrayAnnexin V AssayAnnexin V-FITCC a++C a++C a++ C a++conjugateExternalization ofphosphatidylserinePlasmaApoptosismembraneCytoplasm Cytoplasm實驗設計原理•藉由螢光抗體標識細胞之抗原特性•藉由螢光化合物標識細胞特性•藉由螢光染劑標識細胞特性•Cytometry Beads ArrayDNA 特異性染劑EthidiumPropidiumNH 2NH 2NH 2NH 2p N +C 2H 5N +C 2H 2)3N +C 2H 5CH 3C 2H 5細胞周期位相的決定G 2MG 0G 1sG 0G 1Co sG 2Mu n t2004006008001000DNA content2N4NSub G1Sub G1細胞增殖反應實驗設計原理•藉由螢光抗體標識細胞之抗原特性•藉由螢光化合物標識細胞特性•藉由螢光染劑標識細胞特性•Cytometry Beads ArrayCytometric Beads Array (CBA)Cytometric Beads Array (CBA)Single StepIncubationTwo-Step IncubationorBeads Provide a Flexible PlatformBeads provide an expandable assay platform for use with a flow cytometer platform for use with a flow cytometerMultiple Different Different colors Multiple sizes Different intensities*Different colors with different intensitiesProteins MeasuredProteins MeasuredA. Interleukin (IL)-2B. IL-4C. IL-5D. IL-10E. Tumor Necrosis Factor-αF. Interferon-γBD FACSCanto TMBD FACSCanto TM流式細胞儀流式細胞儀的工作原理的工作原理dynamic Focusing)---液流聚焦(Hydro (Hydro--dynamic Focusing)偵測區-螢光激發光源-散射光樣品流動區緩衝液緩衝液液流區液流區流式細胞儀能測量:•散射光–細胞大小(前方散射光, 2-16°)–細胞折射率(側方散射光, 90)90°•各色螢光綜合三個系統的功能:‧液流系統:將細胞依序送到測量區受檢。
BD FACSCanto II TM 中文培训手册目 录录FACSCa FACSCanto nto (II)培训所需设备培训所需设备//试剂试剂 (3)FACSCanto(II)开机程序开机程序 (4)FACSCanto(II)关机程序关机程序 (6)BD FACSCanto 临床软件进行仪器日常质控临床软件进行仪器日常质控（（QC QC））........................ 7 HLA HLA--B27校准校准 (99)FACSCanto Clinical 六色免洗淋巴细胞亚群检测六色免洗淋巴细胞亚群检测 (10)FACSCanto Clinical 软件的获取分析的具体操作软件的获取分析的具体操作 (11)FACSCanto Clinical 软件进行HLA-B27分析分析 (14)Diva 软件进行HLA-B27手动获取和分析手动获取和分析 (18)Calibrite 四色小球模拟细胞演示四色实验演示四色实验样本获取和分析样本获取和分析样本获取和分析 (20)Diva 软件进行单色&双色获取和分析双色获取和分析 (23)Cytometer Setup and Tracking （CS&T ）操作流程操作流程 (24)FACSCanto (II)流式细胞仪HTS 操作规程 (31)FACSCanto(II)日常保养和维护日常保养和维护 (33)FACSCa FACSCanto nto (II)培训所需设备培训所需设备//试剂1. 普通低速离心机（实验要求为300g,相当于1000～1500rpm，可调转速调时间，能离心12x75mm 的5ml 试管)；2. 固定或可调节的加样器及相应加样头若干： 20ul,50ul, 100ul, 200ul, 250ul，450ul，1ml；3. 旋涡混匀器；4. 烧杯，量筒或移液管，三个清洁的玻璃或塑料带盖试剂瓶(50-100ml)，一个棕色瓶，漏斗，滤纸；5. 300目过滤尼龙网；6. 可放置12x75mm 的5ml 试管的试管架2－3个；7. PBS 溶液和蒸馏水：0.22µm 滤膜过滤；8. NaClO：分析纯，有效氯浓度10%；9. 每天需要一管2ml 的抗凝的人血样本 (最好是EDTA-K 3抗凝)；10.多聚甲醛。
碧迪FACS CantoⅡ流式细胞仪性能评价及实验室应用方佩琪;孙林;顾梅秀;吴蕙;潘柏申;郭玮;王蓓丽【摘要】目的评估碧迪FACS CantoⅡ(BD FACS CantoⅡ)流式细胞仪的性能及实验室应用.方法依据中华人民共和国医药行业新版标准《YY/T0588-2017流式细胞仪》[1]对BD FACS CantoⅡ流式细胞仪进行性能评价,评价内容包括:荧光检出限、荧光线性、前向角散射光(forward scatter resolution,FSC)检出限、仪器分辨率、FSC分辨率、侧向角散射光(side scattering resolution,SSC)分辨率、倍体分析线性、表面标志物检测的准确度、表面标志物检测的重复性、携带污染率和仪器稳定性.完成性能评价后,分别用BD FACS CantoⅡ流式细胞仪及其配套的BD 六色淋巴细胞亚群试剂盒和BD FACS Calibur流式细胞仪及其配套的BD四色淋巴细胞亚群试剂盒分别检测20份全血标本,比较两台仪器在标本前处理、上机检测、分析结果所用时间和结果比较的差异.结果该流式细胞仪对异硫氰酸荧光素(fluorescein isothiocyanate,FITC)通道荧光检出限为40.18等量可溶性荧光分子数(molecules equiva-lent soluble fluorochrome,MESF),对藻红蛋白(P-phycoerythrin,PE)通道荧光检出限为23.77MESF,两者的决定系数(R square,r)均为0.999 9;FSC最小可检测0.22 μm微球;仪器分辨率FSC,FITC和PE荧光通道的变异系数(coeffivirnt of cariation,CV)分别为2.3％,2.4％和2.4％;FSC/SSC散点图可明显区分外周血中红细胞和血小板,同时能区分白细胞中的淋巴细胞、中性粒细胞、单核细胞;倍体分析线性中G2/M期(四倍体细胞峰)与G0/G1期(二倍体细胞峰)的平均荧光强度比值为1.98;表面标志物检测值的平均值均在质控品说明书给定的靶值参考范围内;表面标志物检测重复性CV分别为CD3∶2.75％,CD4∶1.86％,CD8∶ 5.14％,CD16/CD56∶7.33％和CD19∶11.26％;携带污染率最大为0.05％;该仪器开机8h检测FSC及各荧光通道峰值荧光道数偏差值(B)分别为-0.40％,-0.55％,1.57％,1.88％,1.66％,7.44％,7.23％,6.63％和6.21％;BD FACS CantoⅡ流式细胞仪及其配套试剂盒较BD FACS Calibur流式细胞仪及其配套试剂盒在总体检测速度上有显著提高;两台仪器所得的检测结果差异无统计学意义(P＞0.05),且前者检测结果的离散程度小于后者.结论 BD FACS CantoⅡ流式细胞仪各项性能均符合行业标准,仪器各项技术指标达标,可有效保障实验室检测结果的准确性,能为临床诊疗提供可靠支持.【期刊名称】《现代检验医学杂志》【年(卷),期】2019(034)002【总页数】6页(P94-99)【关键词】BD FACS CantoⅡ;流式细胞仪;性能评价【作者】方佩琪;孙林;顾梅秀;吴蕙;潘柏申;郭玮;王蓓丽【作者单位】复旦大学附属中山医院检验科,上海200032;复旦大学附属中山医院检验科,上海200032;复旦大学附属中山医院检验科,上海200032;复旦大学附属中山医院检验科,上海200032;复旦大学附属中山医院检验科,上海200032;复旦大学附属中山医院检验科,上海200032;复旦大学附属中山医院检验科,上海200032【正文语种】中文【中图分类】R446流式细胞术(flow cytometry，FCM)是一种利用流式细胞仪对单细胞或生物颗粒进行多参数、快速分析的技术[2]。
The BD FACSCalibur™ platform offers a unique modular approach to flow cytometry and allows users to perform both cell analysis and cell sorting in an innovative single bench-top system. Designed with multicolor application support in mind, the BD FACSCalibur flow cytometer provides flexibility to support a wide variety of research and clinical applications.Engineered to meet evolving clinical and research requirements, the user-friendly BD FACSCalibur system can be easily upgraded to address emerging automation or sorting requirements in a laboratory. Walkway automation can be achieved by the BD FACS™ Loader option that handles tube-based assays or the BD™ High Throughput Sampler that handles assays in 96 or 384-well microtiter plates.* In addition, the BD FACSCalibur system can be upgraded to support cell sorting to allow for the isolation of a population of interest.*Built-in modularity and simple upgrades make the BD FACSCalibur an excellent long-term investment for today’s cost conscious laboratories.The BD FACSCalibur system is compact and easy to use. It is complemented by a broad suite of intuitive software solutions to streamline analysis for a wide range of applications, including enumeration of lymphocyte subsets, stem cells, residual white blood cells, reticulocytes, DNA analysis,* immune function studies,* bead based immunoassays,* and multiplexed analysis of signal transduction* and phosphorylation targets.*Enabling fast, easy, and accurate results forroutine applications with a versatile, modulardesign has made the BD FACSCalibur theautomated benchtop cytometer of choicefor cell analysis in research and clinicallaboratories worldwide.*Some options and applications are for Research Use Only.Rare-event analysis A growing number of flow cytometry–based assays depend on rare-event detection. For example, in the characterization of subpopulations of stem cells, millions of cells are analyzed to achieve a statistically significant sampling of the subset of interest.Advanced applications like this require a flow cytometer capableof high-speed analysis. The BD FACSCalibur system features amultiparameter triggering system that allows a user to acceleratethe analysis by electronically eliminating events from onboardprocessing.A R egion R3 defines basophils and region R4 defines CD123+ DCs(0.14% of total).B Region R5 defines CD11c + DCs (0.21% of total).A B CD123 PE 1010101010A n t i –H L A -D R P e r C P Gated on R1 and R2R4R3a CD11c APC Gated on R1 and R2R5b 4R E S U L T SDual-laser design for multicolor analysisThe BD FACSCalibur dual-laser design provides the flexibility and sensitivity needed for multicolor analysis. Two lasers, an air-cooled argon laser and a red diode laser, are spatially separated for high sensitivity, minimal compensation, and maximum flexibility in fluorochrome selection. This maximizes the information obtained from limited samples and increases the cost-effectiveness of each assay.A fully integrated multiparameter system, the BD FACSCalibur system ensures objective and reproducible results, which are vital to today’s laboratories.Unique easy-to-use sorting technology (optional)The BD FACSCalibur cell sorting option allows users to identify and isolate a population of interest. Sorting on the BD FACSCalibur system occurs in a completely enclosed, aerosol-free environment for enhanced safety, which is especially important when processing biohazardous samples. The sorting process is designed to be straightforward. After the population of interest is gated, simply click “Acquire” to begin the sort. After the sample is acquired and cells pass through the laser, a unique catcher tube mechanism moves in and out of the sample core stream at a rate of roughly 300 times per second to capture designated cells and direct them to a collection tube or to an optional cell concentrator module for further processing. (The BD FACSCalibur cell sorting and cell concentrator options are for Research Use Only)BD FACSCalibur sorting mechanism5 BD FACSCalibur optical benchP R O D U C T I V I T YFlexibility for sample introductionand enhanced productivityBD FACS Loader optionThe BD FACS Loader option available for theBD FACSCalibur system provides walkaway automationfor routine applications. Featuring a removable 40-tubecarousel, onboard mixing, and BD™ Worklist Managersoftware, the BD FACS Loader option offers superiorflexibility to efficiently address the workflow demandsof the busy laboratory.BD High Throughput Sampler (HTS) optionThe BD High Throughput Sampler (HTS) option providesflexible, easy-to-use automation. For those who prefer towork with multiwell plates, BD Biosciences offers the firstwalkaway sample introduction device for 96- and 384-well plates. The HTS option allows users to rapidly acquiresamples with the BD FACSCalibur flow cytometer in amicrotiter plate. As a modular option for new or existingBD FACSCalibur flow cytometers, the HTS option is designedto speed through a variety of microtiter plates in less than15 minutes* and support a wide range of applicationsincluding the BD™ Cytometric Bead Array assay. The HTSoption supports 96 U-, V-, and flat-bottom plates as well as384-well microtiter plates.BD™ Plate Manager software allows you to createcustomized delivery protocols to be used repeatedlywith user-defined mixing, wash, and analysis parameters.(The BD High Throughput Sampler option and BD PlateManager are for Research Use Only)Efficient residual white blood cell enumerationThe BD Leucocount™ system is the first automated, IVD-cleared product for monitoring leucoreduced blood products. Together with BD Trucount™ tubes andBD Leucocount process controls, the BD Leucocount system provides excellent accuracy, precision, and linearity. For high-throughput walkaway capability, the BD FACS Loader can also be added to your base BD FACSCalibur system.BD Leucocount kitThe BD Leucocount system provides excellentaccuracy, precision, and linearity at clinicallysignificant levels of detection for RBC (red) andplatelet (blue) samples.10001001010.1MeanObservedLeucocountWBC/µL0.1Expected WBC/µL1101001000<5×106<1×106Decision PointArea6* B ased on a 2-second sample acquisition.100101102103104100101102103104C D 4-P ECD3-FITCD A T A M A N A GE M E N T Delivering results with a powerful, easy-to-use data management system The BD FACSCalibur system is appreciated for its user-friendly design, high sample throughput, improved workflow management through automation, and simple yet sophisticated software applications that enhance routine analyses.While performing any of a wide range of supported applications, the BD FACSCalibur system provides the tools to obtain results quickly, easily, and accurately.The ease-of-use of the BD FACSCalibur system is enhancedby the BD FACStation™ data management system thathas become one of the most popular, user-friendly datamanagement systems in flow cytometry. The BD FACStationsystem automates many software functions and delivershigh-performance acquisition and analysis tools for creatingplots, gating, statistical analysis, and reporting.The BD FACStation workstation takes advantage of theintuitive Mac® environment by using simple pull-downmenus and icons. It performs all the computing tasks requiredfor fast, accurate results including instrument setup, as wellas data collection, analysis, and management.A comprehensive quality assurance systemBD Biosciences offers a complete family of productsdesigned to ensure the highest quality clinical results fromthe BD FACSCalibur system. BD FACSComp™ software,in conjunction with BD Calibrite™ beads, provides acomprehensive system to set up the instrument, assesssensitivity, and set compensation for immunofluorescenceapplications. For clinical applications, BD Biosciences offersa wide variety of cellular controls to verify performancefor immunophenotyping, reticulocyte analysis, stem cellenumeration, and rWBC applications.DNA Analysis The BD FACSCalibur system works together with BD DNA analysisquality control products to eliminate many of the variables asso-ciated with ploidy and cell-cycle analysis. The instrument’s opticaland fluidic design ensures that high-resolution DNA ploidy andS-phase determinations are easy to achieve. Electronic doubletdiscrimination, using the area and width of the fluorescence pulse,allows for the removal of artifacts—doublets and aggregates—from the analysis. High-resolution electronics and linearity ensureaccurate results. To ensure optimal performance, BD DNA QCproducts, including fixed cells and fluorescent particles, can beused for setup, verification of the doublet discrimination module,and monitoring daily operation. (BD DNA Analysis products are forResearch Use Only)DNA analysisPulse processing is a powerful tool used in the discrimination of doublets for optimal DNA analysis.0200400600800100002004006008001000FL2-Width R1F L 2-A r e a 02004006008001000060120180240300FL2-Area C o u n t s 8BD Cytometric Bead Array assaysThe BD FACSCalibur flow cytometer can beused to acquire data from BD CytometricBead Array (CBA) multiplexed assays.These assays are powerful, flexible toolsthat simultaneously quantify multiplecytokine, chemokine, immunoglobulin, or cell signaling targets from a single sample. Choose from preconfigured kits for maximum convenience in screening routine panels of Th1/Th2 or inflammatory cytokines and chemokines, or build your own multiplex assay using the open and configurable BD CBA Flex Set format.For all of their power, BD CBA assays are fast and remarkably easy to use. Pre-optimized protocols and one-step detection reagents minimize hands-on time and maximize reproducibility. (BD Cytometric Bead Array assays are for Research Use Only)A B C DE F Beads Wash Detector antibodiesSample N I RRedAnalyze by flow cytometry Concentration M F IConcentration (pg/mL)9BDIS HLA-B27 Laboratory Report000CD3/CD16+56/CD45/CD19 TruCMulti-tube QCBDIS MultiSET™ Lab ReportCD3/CD8/CD45/CD4 TruCS E R V I C E A N D S U P P O R T Service and SupportBD Biosciences instruments and reagents are backed by a world-class service and supportorganization with unmatched flow cytometry experience.For over twenty-five years, BD has actively worked with researchers to develop tools that help improve workflow, ease of use, and performance. 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BD FACSCanto II TM 中文培训手册目 录录FACSCa FACSCanto nto (II)培训所需设备培训所需设备//试剂试剂 (3)FACSCanto(II)开机程序开机程序 (4)FACSCanto(II)关机程序关机程序 (6)BD FACSCanto 临床软件进行仪器日常质控临床软件进行仪器日常质控（（QC QC））........................ 7 HLA HLA--B27校准校准 (99)FACSCanto Clinical 六色免洗淋巴细胞亚群检测六色免洗淋巴细胞亚群检测 (10)FACSCanto Clinical 软件的获取分析的具体操作软件的获取分析的具体操作 (11)FACSCanto Clinical 软件进行HLA-B27分析分析 (14)Diva 软件进行HLA-B27手动获取和分析手动获取和分析 (18)Calibrite 四色小球模拟细胞演示四色实验演示四色实验样本获取和分析样本获取和分析样本获取和分析 (20)Diva 软件进行单色&双色获取和分析双色获取和分析 (23)Cytometer Setup and Tracking （CS&T ）操作流程操作流程 (24)FACSCanto (II)流式细胞仪HTS 操作规程 (31)FACSCanto(II)日常保养和维护日常保养和维护 (33)FACSCa FACSCanto nto (II)培训所需设备培训所需设备//试剂1. 普通低速离心机（实验要求为300g,相当于1000～1500rpm，可调转速调时间，能离心12x75mm 的5ml 试管)；2. 固定或可调节的加样器及相应加样头若干： 20ul,50ul, 100ul, 200ul, 250ul，450ul，1ml；3. 旋涡混匀器；4. 烧杯，量筒或移液管，三个清洁的玻璃或塑料带盖试剂瓶(50-100ml)，一个棕色瓶，漏斗，滤纸；5. 300目过滤尼龙网；6. 可放置12x75mm 的5ml 试管的试管架2－3个；7. PBS 溶液和蒸馏水：0.22µm 滤膜过滤；8. NaClO：分析纯，有效氯浓度10%；9. 每天需要一管2ml 的抗凝的人血样本 (最好是EDTA-K 3抗凝)；10.多聚甲醛。
