Welcome IQAC at DHVI

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.

4 Flow Cytometry Laser based high speed electronic cell analyzer Fluorescent conjugated monoclonal antibodies Analyze surface (and cytoplasmic) cellular antigens. Flow rates 500 –700 cells /second

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)

6 Light Scatter Properties

7 Blood Cells

8 Flow Light Scatter Pattern

9 Fluorescence

10 Fluorescence Emission

11 Fluorescence Intensity

12 2-parameter Dot Plot

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.

14 Fluidics

15 Injector Tip Fluorescence signals Focused laser beam Sheath fluid Purdue University Cytometry Laboratories Flow Cell

16 Sample Flow

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.

18 Forward Angle Light Scatter FALS Sensor Laser Purdue University Cytometry Laboratories

19 90 Degree Light Scatter FALS Sensor 90LS Sensor Laser Purdue University Cytometry Laboratories

20 Laser Fluorescence Detectors Fluorescence FALS Sensor Fluorescence detector (PMT3, PMT4 etc.) Purdue University Cytometry Laboratories

21 Optical Filters

22 Optics Scheme

23 PMT Dichroic Filters Bandpass Filters Example Channel Layout for Laser-based Flow Cytometry Laser 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

30 SINGLE COLOR HISTOGRAMS

31 Dot Plot

32 FL1 FL2 Uncompensated vs. Compensated

33 Emissions Spectra

34 Fluorescence Overlap

35 Fluorescence Compensation

36 FITC Compensation

37 Compensation Examples

38 A spectral image generated by fluoro- chromes G and O Spectral energy from fluorochrome G is subtracted from O FUNDAMENTAL ASPECT OF COLOR COMPENSATION HOW TO REMOVE GREEN (G) FROM ORANGE (O) GO

39 THREE PART DIFFERENTIAL ANALYSIS OF WHOLE BLOOD Light Scatter Cell Volume Lymphocytes Granulocytes Monocytes BECTON DICKINSON

40 BIVARIATE QUADRANTS FOR T-CELL SUBSET MARKERS FSC SSC AB FL2 FL Mandy et al., 2001

41 COMPONENTS OF BIVARIATE QUADRANT DISPLAY DUAL T-CELL MARKERS: A=CD4 and B=CD3 A B A B A B

42 BIVARIATE QUADRANT HISTOGRAM FOR CD3 AND CD4 POSITIVE CELLS

43 TWO COLOR PATTERN FL1-CD3 FL2-CD4 color CD3-CD4-black CD3+CD4-blue CD3-CD4+cyan CD3+CD4+green

44 THREE COLOR PATTERN CD3 CD4 CD3 CD4 CD8

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.

53 Multi-color Antibody Panels 2-color panels (Leucogate CD45/CD14, CD3/4, CD3/8, CD3/19, CD3/16+56) 3-color panels (CD3/4/8; CD3/4/45, CD3/8/45, CD3/19/45, CD3/16+56/45) 4-color panels (CD3/4/8/45, CD3/19/16+56/45)

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

59 Beckman Coulter Epics / FC500 Procedures Instrument Start up FlowCheck & FlowSet Calibrations Fluorescence Compensation Daily Controls Patient Samples Instrument Cleaning and Maintenance Troubleshooting

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.