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IQA. Welcome. IQAC at DHVI. CD4 Immunophenotyping for HIV Monitoring. Flow Cytometry. Introduction. Absolute CD4 T-lymphocyte counts are used to evaluate the immune status of patients with the human immunodeficiency virus (HIV).

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  1. IQA Welcome IQAC at DHVI

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

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

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

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

  21. Optical Filters

  22. Optics Scheme

  23. Example Channel Layout for Laser-based Flow Cytometry Flow Layout PMT 4 PMT Dichroic 3 Filters Flow cell PMT 2 Bandpass Filters PMT 1 Laser original from Purdue University Cytometry Laboratories; modified by R.F. Murphy

  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. LASER LASER LASER PMT 1 PMT 1 PMT 1 TIME SIGNAL AND PATTERN GENERATION VOLTAGE

  27. FLUORESCENCE INTENSITY PATTERN FROM A CELL POPULATION low medium high LASER Number of events PMT 1 Relative Fluorescence Intensity

  28. low medium high DETECTION OF THREE FLUORESCENCE INTENSITY PATTERNS FROM CELL SURFACE LASER Number of events PMT 1 Relative Fluorescence Intensity

  29. FLUORESCENT SIGNAL PATTERN COLLECTION LASER Number of events PMT 1 Relative Fluorescence Intensity

  30. SINGLE COLOR HISTOGRAMS

  31. Dot Plot

  32. Uncompensated vs. Compensated FL2 FL1

  33. Emissions Spectra

  34. Fluorescence Overlap

  35. Fluorescence Compensation

  36. FITC Compensation

  37. Compensation Examples

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

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

  40. BIVARIATE QUADRANTS FOR T-CELL SUBSET MARKERS A B + + + FL1 SSC + FSC FL2 Mandy et al., 2001

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

  42. BIVARIATE QUADRANT HISTOGRAM FOR CD3 AND CD4 POSITIVE CELLS + + + + - - +

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

  44. THREE COLOR PATTERN CD4 CD4 CD3 CD8 CD8 CD3

  45. FOUR COLOR PATTERN CD8 CD4 CD56 CD3 CD3 CD3 CD8 CD4 CD4 CD56 CD56 CD8

  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. SS CD45 FL1 CD4 FL2 CD8 FL4 CD45 BASED HETEROGENEOUS GATING FOR T-CELL SUBSETS 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

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