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F luorescence A ctivated C ell S orter

Flow cytometry. F luorescence A ctivated C ell S orter. ד”ר בנצי כץ המעבדה ההמטולוגית מרכז רפואי ע”ש סוראסקי תל-אביב. Flow cytometry. History Principles Procedures Applications Future directions. Morphology. Size granularity nucleus size and form nucleoli ratio nucleus/cytoplasm

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F luorescence A ctivated C ell S orter

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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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