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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 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 • Misattribution if assumptions incorrect 07-08646
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
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
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
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
How Many Colors are Enough? • Ideal • Add all reagents of interest into single tube • Real • Too many parameters of interest
Define Purpose of Assay • Most important question • What information is required? • What information is most important? • Prioritize • Compromises are inevitable • Simplest assay is best
Cell Type Identification Borowitz et al (1993) AJCP 100:534-40. Steltzer et al (1993) Ann NY Acad Sci 667:265-280
Normal Blast Maturation Wood (2004) Methods Cell Biology 75:559-576
Acute Myelomonocytic Leukemia Wood and Borowitz (2006) Henry’s Laboratory Medicine
ALL MRD 0.1% abnormal immature B cells 06-01469
Tandem Fluorochromes Phycoerythrin (PE) PE-Texas Red
Tandem Breakdown NH4Cl prelyse and wash 1 hour prior Whole blood lysis Cytometry Part A (2009) 75A:882-890
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
FITC = Green PE = Orange Excitation = Dotted Emission = Solid
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
Compensation Correct Undercompensated Overcompensated
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
Compensation Validation Fluorescence minus one “FMO” controls
Compensation Validation Fluorescence minus one “FMO” controls
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
Primary of Tandems 10.5% 1.6%
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
Compensation Compromises 09-13546
Infinicyte - Cytognos • Nearest neighbor estimate of relationship between parameters in different tubes Pedreira, et al. Cytometry (2008) 73A:834-846
Mass Cytometry 30-100 simultaneous antigens
Mass Cytometry Bendall, et al (2011) Science 332:687
Mass Cytometry Bendall, et al (2011) Science 332:687
Mass Cytometry 13 parameters Bendall, et al (2011) Science 332:687
Mass Cytometry 18 functional markers 13 conditions Bendall, et al (2011) Science 332:687
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