CD4 Immunophenotyping for HIV Monitoring Flow Cytometry
3 Introduction Absolute CD4 T-lymphocyte counts are used to evaluate the immune status of patients with the human immunodeficiency virus (HIV). CD4 antigen is the receptor for HIV. The absolute number of CD4 T lymphocytes is closely associated with HIV progression.
5 Flow Cytometry What Can a Flow Cytometer Tell Us About a Cell? Its relative size (Forward Scatter- FSC) Its relative granularity or internal complexity (Side Scatter-SSC) Its relative fluorescence intensity (FL1,FL2,FL3, and FL4)
13 Flow Cytometry System Fluidics To introduce and focus cells for analysis. Optics To generate and collect light signals. Electronics To convert optical signals to digital electronic signals for computer analysis.
17 Optics Excitation optics Laser(s) Lenses to shape and focus the laser beam Collection optics A collection lens to collect light emitted from the particle-laser beam interaction A system of optical mirrors and filters to route specified wavelengths of the collected light to designated optical detectors.
23 PMT Dichroic Filters Bandpass Filters Example Channel Layout for Laser-based Flow Cytometry Laser 1 2 3 4 Flow cell original from Purdue University Cytometry Laboratories; modified by R.F. Murphy Flow Layout
24 Fluidics and Optics Review Created an illumination region with the excitation optics Passed the cells precisely through the illumination region using hydrodynamic focusing Routed the generated light signals to the specific detectors by collection optics
25 Electronics Converts optical signals to proportional electronic signals (voltage pulses) Analyzes voltage pulse height, area, or width Interfaces with the computer for data transfer
26 SIGNAL AND PATTERN GENERATION PMT 1 LASER PMT 1 LASER PMT 1 LASER VOLTAGE TIME
27 PMT 1 LASER FLUORESCENCE INTENSITY PATTERN FROM A CELL POPULATION Number of events Relative Fluorescence Intensity lowmediumhigh
28 DETECTION OF THREE FLUORESCENCE INTENSITY PATTERNS FROM CELL SURFACE PMT 1 LASER Relative Fluorescence Intensity Number of events lowmediumhigh
29 Number of events PMT 1 LASER Relative Fluorescence Intensity FLUORESCENT SIGNAL PATTERN COLLECTION
45 CD3 CD56 CD8 CD4CD8 CD56 CD8CD4 FOUR COLOR PATTERN
46 FOUR COLOR DUAL LASER IMMUNOPHENOTYPING Antibodies labeled with fluorescein Antibodies labeled with phycoerythrin (PE) Antibodies labeled with PE/CY5 or PerCP Antibodies labeled with APC, CY5 or CY7
47 CD45 BASED HETEROGENEOUS GATING FOR T-CELL SUBSETS CD8 FL4 CD4 FL2 SS CD45 FL1 CD45-Gating Protocol HC, NIH & CDC GUIDELINES Bergeron et al. 2002
48 Topics of Discussion Testing Platforms Single Dual Instrumentation Reagents
49 Testing Platforms Single platform instrument Single platform testing can be performed on flow cytometer using calibration beads. Cost per test is relatively higher. Dual platform testing relies on a Hematology Analyzer. Hematology cost per test is relatively inexpensive. Comparison of both platforms.
50 Instrumentation Flow Cytometers Beckman Coulter FC500, MCL-XL, Elite, Profile, Point Care Becton Dickinson Canto, FACSCalibur, FACSCan, FACSort, FACSCount Guava Technologies Inc. Personal Cell Analyzer System (PCA) Partec - CyFlow Accuri Cytometers Hematology Analyzers Coulter, Sysmex, Cell Dyn Pipetters
51 Flow Cytometry Software Beckman Coulter EPICS System II and Expo 32 Becton Dickinson Simultest, Multitest, and Cell Quest (Pro) for BD FACS Becton Dickinson FACSCount Guava PCA System Partec FloMax Accuri CFlow Plus TreeStar, Inc. FlowJo Verity Software House - WinList
52 Reagents There are many manufacturers of monoclonal antibodies. Select IVD reagents to ensure quality and reliability. Cytometer manufacturers are a good source for flow reagents.
54 Instrument Maintenance Daily start-up and shut down Daily calibration Monthly and periodic maintenance Troubleshooting
55 BD FACSCount Procedures Instrument Start Up Preparing and Running Controls Preparing and Running Samples Instrument Cleaning and Maintenance Troubleshooting
56 BD FACSCount Sample Prep Procedure Material Whole blood sample collected in anticoagulant. BD FACSCount Reagents BD FACSCount Control Beads BD FACSCount Fixative BD Reverse Pipetter Procedure Aliquot 50ul whole blood sample to FACSCount Reagent tubes Incubate for 1 hour at RT Add 50 ul of fixative Run sample on instrument
57 BD FACSCalibur Procedures Instrument Start up FACSComp Calibration Daily Controls Patient Samples Instrument Cleaning and Maintenance Troubleshooting
58 BD MultiTest Reagent Staining Procedure (Lyse No Wash) Materials Whole blood collected in anticoagulant. MultiTest Reagent Panel BD FACSLyse Solution BD Falcon 12 x 75mm test tubes Procedure Add 50ul of blood sample to 10ul of antibody reagent. Incubate for 15 minutes at RT Add 450ul of a working dilution of BD FACSLyse solution. Incubate for 15 minutes at RT Acquire samples on flow cytometer
60 Beckman Coulter Cyto-Stat Reagent Staining Procedure (Lyse No Wash) Materials Whole Blood collected in anticoagulant. Coulter Cyto-Stat Reagent Panel Coulter T-Q Prep 12 x 75mm test tubes Procedure Add 100ul of blood sample to 10ul of antibody reagent. Incubate for 15 minutes at RT Place tubes in T-Q Prep and start sample processing run. Acquire samples on flow cytometer.
61 Quality Control and Quality Assurance Controls Reagents Instruments Proficiency Testing Training and Competency External Quality Assessment UKNEQAS samples
62 Patient Reporting and Data Management Patient Confidentiality Reference Ranges Data List Mode Files Data Storage Devices
63 Acknowledgements Beckman Coulter Reagents & Training Material Becton Dickinson Reagents & Training Material Health Canada, Francis Mandy, PhD Training Material (PPT slides) Purdue U. Cytometry Laboratories Training Material (PPT slides) Roswell Park Cancer Institute Laboratory of Flow Cytometry, Carleton C. Stewart, Ph.D Training Material (PPT slides)
64 Conclusion Thank you for your participation.