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Overview of Flow Cytometry and FACS: Principles, Applications, and Future Directions

This comprehensive overview covers flow cytometry and fluorescence-activated cell sorting (FACS), detailing its history, principles, and procedures. It explores the applications of FACS in hematology, including the analysis of cell morphology, size, granularity, and specific markers such as T-lymphocytes and myeloid markers. Additionally, the text discusses advancements in monoclonal antibodies, challenges in data interpretation, and the relevance of multi-parameter analysis in the detection of minimal residual disease. The piece concludes with insights into future directions for FACS technology.

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Overview of Flow Cytometry and FACS: Principles, Applications, and Future Directions

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  1. Flow cytometry Fluorescence Activated Cell Sorter ד”ר בנצי כץ המעבדה ההמטולוגית מרכז רפואי ע”שסוראסקיתל-אביב

  2. Flow cytometry History Principles Procedures Applications Future directions

  3. Morphology Size granularity nucleus size and form nucleoli ratio nucleus/cytoplasm cytoplasm color vacuoles granulopoiesis Multi-parameter analysis

  4. FACS was invented in late 60`s - early 70`s (Herzenbergs) A surface membrane determinant shared by subpopulations of thymocytes and B lymphocytes.Stout RD, Yutoku M, Grossberg A, Pressman D, Herzenberg LA. J Immunol. 1975 Aug;115(2):508-12. Leukemia-associated antigens in man.Brown G, Capellaro D, Greaves M.J Natl Cancer Inst. 1975 Dec;55(6):1281-9. Characterization of subpopulations of T lymphocytes. I. Separation and functional studies of peripheral T-cells binding different amounts of fluorescent anti-Thy 1.2 (theta) antibody using a fluorescence-activated cell sorter (FACS).Cantor H, Simpson E, Sato VL, Fathman CG,Herzenberg LA.Cell Immunol.1975Jan;15(1):180-96.

  5. One color, no monoclonal antibodies, no CDs, no literature ~ 30 years 5 color (commercial) and more Directly conjugated, calibrated and controlled monoclonal antibodies More then 250 CDs >61,000 publications

  6. FACS (Coulter, BD)

  7. In fact, FACS is a trade mark of BD what we do is: Flow cytometry FACS sorting module (optional)

  8. Physical parameters Side scatter (SSC) Forward scatter (FSC)

  9. Fluorescent labels Y FL1(FITC) A Y FL2(PE) B Y FL3(PerCP) C D Y FL4(APC) Expanding possibilities...

  10. FACS reagents Monoclonal antibodies Calibrated, FDA approved Continuously monitored (QA, proficiency) …expensive ! Y

  11. CD (cluster of differentiation) >300 Myelo/mono markers Lymphoid markers CD1a CD2 CD3 CD4 CD7 CD8 CD10 CD19 CD20 CD22 CD13, CD33, CD15, CD11b CD14, CD64 MK and platelet markers: CD41a, CD62p Plasma cells markers: CD38, CD138 Red blood cells markers: CD71, GlyA NK cells marker: CD56

  12. Immature cells markers CD117 c-kit (SCF-R) tyrosine kinase drug target CD34 Function ? CD133 ?

  13. CD (cluster of differentiation) >300 Aberrant markers CD5 Positive B cells in CLL CD10 (ALL) in AML CD56 (NK) in multiple myeloma Important for follow-up and minimal residual disease

  14. DNA labels - cell cycle PI

  15. G1/G0 S G2/M DNA content apoptosis

  16. II 1. 50 ml blood 2. Add antibodies 3. Incubate 45 min 4. Lyse RBC 5. Wash 6. Analyze I 1. Blood 2. Phycoll purification 3. Add antibodies 4. Incubate 45 min 5. Wash 6. Analyze FACS- sample processing

  17. FACS- acquisition I SSC Laser (s) FL1 FL4 FSC III II II I ~ 30,000 events

  18. FACS - analysis

  19. 1-dimensional analysis 2-dimensional analysis No of events FL2 FL1 FL1

  20. l l l l l l k k k k k k k k k k k k k k k k k k k B-cell lymphocytosis A polyclonal process l l k k l A monoclonal process l k

  21. 2-dimensional analysis Clonality l l Non B Non B k k Mono clonal Poly clonal

  22. FACS - need to be an expert... How to define a population ? ? ?

  23. 101 102 103 100 What is a positive population ? Negative Other methods Relative – ZAP70 # of events Positive FL Two main values 1. Mean FL 2. % positive cells Isotype control sample

  24. CD45 pan-leukocyte marker Leukocyte membrane phosphatase

  25. Side scatter (SSC) CD45 “Blast hole”

  26. Normal bone marrow Gr Mo NRBC Ly

  27. Multi-dimensional analysis 2 1

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