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Issues in Multicolor Flow Cytometry: Beyond 6 Colors

Issues in Multicolor Flow Cytometry: Beyond 6 Colors. Brent Wood MD PhD Department of Laboratory Medicine University of Washington. The Power of Flow Cytometry. Single cell analysis Multiparametric Rapid Quantitative Flexible. 18%. 66%. 80%. 0.4%. Inferential reasoning. Insensitive

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Issues in Multicolor Flow Cytometry: Beyond 6 Colors

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  1. Issues in Multicolor Flow Cytometry: Beyond 6 Colors Brent Wood MD PhD Department of Laboratory Medicine University of Washington

  2. The Power of Flow Cytometry • Single cell analysis • Multiparametric • Rapid • Quantitative • Flexible

  3. 18% 66% 80% 0.4% Inferential reasoning • Insensitive • Misattribution if assumptions incorrect 07-08646

  4. Analytical Approach • Antigen expression is present/absent • Antigen intensity is minimally used • Can be expressed as a table of percentages • Focus is on abnormal immunophenotype • Presence or absence of expression of antigens

  5. Direct Observation • Combination of reagents uniquely identifies cell type, lineage and maturational stage • Emphasize normal maturational patterns • Direct determination of immunophenotype without inference • Improvement in sensitivity and specificity • More simultaneous fluorochromes improves

  6. Multiparametric Flow Cytometry • More accurate population identification • Greater informational content • Make better use of small specimens • Fewer cells, more information • Process fewer tubes • Save on reagents, tech and instrument time • Collect large number of events efficiently • Allow standardized reagent combinations

  7. Instrumentation Becton-Dickinson FACSCanto I - 6 colors, 2 lasers II - 8 colors, 3 lasers Beckman-Coulter FC500 5 colors - 1 or 2 lasers Beckman-Coulter Gallios 10 colors - 3 lasers Becton-Dickinson LSRII ~20 color, up to 7 lasers

  8. How Many Colors are Enough? • Ideal • Add all reagents of interest into single tube • Real • Too many parameters of interest

  9. Define Purpose of Assay • Most important question • What information is required? • What information is most important? • Prioritize • Compromises are inevitable • Simplest assay is best

  10. Bethesda International Consensus Conference N = 35

  11. Euroflow

  12. Panel Design

  13. Cell Type Identification Borowitz et al (1993) AJCP 100:534-40. Steltzer et al (1993) Ann NY Acad Sci 667:265-280

  14. Normal Blast Maturation Wood (2004) Methods Cell Biology 75:559-576

  15. Acute Myelomonocytic Leukemia Wood and Borowitz (2006) Henry’s Laboratory Medicine

  16. Hodgkin Lymphoma

  17. ALL MRD 0.1% abnormal immature B cells 06-01469

  18. Tandem Fluorochromes Phycoerythrin (PE) PE-Texas Red

  19. Tandem Breakdown NH4Cl prelyse and wash 1 hour prior Whole blood lysis Cytometry Part A (2009) 75A:882-890

  20. Compensation • Spectral overlap between fluorochromes • Critical to success of method • For 10 color experiment • Need to determine 90 values for Comp Matrix • Software compensation required • Maximum flexibility • Non-destructive

  21. FITC = Green PE = Orange Excitation = Dotted Emission = Solid

  22. Compensation - Method • Single stained controls used • One for each individual fluorochrome • One for each individual tandem • As bright as brightest reagent to be used • Samples run without compensation • Compensation calculated in software • Applied either at acquisition or analysis

  23. Compensation Correct Undercompensated Overcompensated

  24. Compensation • Don’t worry unduly about PMT voltage 245% 103% 47.5% 23.0% 12.2% 1.9% 4.7% 10.3% 21.0% 41.0% 500 volts 550 volts PE = 400 volts 450 volts 600 volts PE-TR = 550 volts Compensation values should reflect relative spectral overlap, i.e. detector gains should be equal

  25. Compensation Validation Fluorescence minus one “FMO” controls

  26. Compensation Validation Fluorescence minus one “FMO” controls

  27. Compensation

  28. Compensation

  29. Compensation Background • Avoid increased background due to fluorochromes • Adjacent with longer wavelength emission • PE / PE-TR, PE-TR / PE-Cy5, PE-Cy5.5 or PerCP-Cy5.5/ PE-Cy7 • APC / APC-A700, APC-A700 / APC-Cy7 • Primary fluorochrome of tandem • PE and PE-TR, PE-Cy5, PE-Cy5.5, or PE-Cy7 • APC and APC-A700 or APC-Cy7 • Interlaser excitation and emission • PE-Cy5 and APC • PE-Cy5.5 or PerCP-Cy5.5 and APC-A700 • PE-Cy7 and APC-Cy7 • PE-TR and A594

  30. Adjacent fluorochromes

  31. Primary of Tandems 10.5% 1.6%

  32. Interlaser compensation

  33. Strategies to deal with compensation background • Avoid bright fluorescence • Put fluorochromes on different populations • Put fluorochromes brightly on same population • Avoid detection of dim expression in presence of high background

  34. Avoid bright fluorescence

  35. Different populations

  36. Bright dual positive

  37. Compensation Compromises 09-13546

  38. Infinicyte - Cytognos • Nearest neighbor estimate of relationship between parameters in different tubes Pedreira, et al. Cytometry (2008) 73A:834-846

  39. Mass Cytometry 30-100 simultaneous antigens

  40. Mass Cytometry Bendall, et al (2011) Science 332:687

  41. Mass Cytometry Bendall, et al (2011) Science 332:687

  42. Mass Cytometry 13 parameters Bendall, et al (2011) Science 332:687

  43. Mass Cytometry 18 functional markers 13 conditions Bendall, et al (2011) Science 332:687

  44. Conclusion • Multicolor flow cytometry • Powerful tool • Purpose must be primary consideration • Simpler is better • Fluorescent spectral overlap is major limitation • Mass Cytometry • Allows high level multiparameter measurements • Eliminates fluorescent spectral overlap • Detection sensitivity and reagent availability are concerns Both require improved data analysis tools

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