issues in multicolor flow cytometry: beyond 6 colors
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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Issues in Multicolor Flow Cytometry: Beyond 6 Colors
Brent Wood MD PhD
Department of Laboratory Medicine
University of Washington
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The Power of Flow Cytometry
• Single cell analysis• Multiparametric• Rapid• Quantitative• Flexible
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Inferential reasoning
• Insensitive• Misattribution if assumptions incorrect
07-08646
18%
80%
66%
0.4%
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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
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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
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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
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Instrumentation
Beckman-Coulter FC5005 colors - 1 or 2 lasers
Becton-Dickinson FACSCantoI - 6 colors, 2 lasersII - 8 colors, 3 lasers
Beckman-Coulter Gallios10 colors - 3 lasers
Becton-Dickinson LSRII~20 color, up to 7 lasers
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How Many Colors are Enough?
• Ideal - Add all reagents of interest into single tube
• Real - Too many parameters of interest
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Define Purpose of Assay
• Most important question– What information is required?– What information is most important?
• Prioritize• Compromises are inevitable
– Simplest assay is best
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Bethesda International Consensus Conference
N = 35
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Euroflow
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Panel Design
PB FITC PE PE-TR PE55 PE7 A594 APC A700 APC7
B cells 45 k l 19 34 20 38 10 - 5
T cells 45 2 7 34 8 3 4 56 - 5
Blasts DR 15 33 19 117 13 38 34 71 45
Myeloid DR 64 123 4 14 13 38 34 16 45
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Cell Type Identification
Borowitz et al (1993) AJCP 100:534-40.Steltzer et al (1993) Ann NY Acad Sci 667:265-280
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Normal Blast Maturation
Wood (2004) Methods Cell Biology 75:559-576
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Acute Myelomonocytic Leukemia
Wood and Borowitz (2006) Henry’s Laboratory Medicine
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Hodgkin Lymphoma
CD15 APC CD71 APC-A700 CD20 PerCPCv55SSC-H
CD15 APC CD40 PE SSC-HSSC-H
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0.1% abnormal immature B cells
ALL MRD
06-01469
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Tandem Fluorochromes
Phycoerythrin (PE) PE-Texas Red
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Tandem Breakdown
Whole blood lysis NH4Cl prelyse and wash1 hour prior
Cytometry Part A (2009) 75A:882-890
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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
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FITC = Green PE = Orange
Excitation = DottedEmission = Solid
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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
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Compensation
Correct Undercompensated Overcompensated
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Compensation• Don’t worry unduly about PMT voltage
245%
1.9%
103%
4.7%
47.5%
10.3%
23.0%
21.0%
12.2%
41.0%
500 volts 550 volts 600 volts450 voltsPE = 400 volts
PE-TR = 550 volts
Compensation values should reflect relative spectral overlap, i.e. detector gains should be equal
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Compensation Validation
Fluorescence minus one “FMO” controls
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Compensation Validation
Fluorescence minus one “FMO” controls
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Compensation
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Compensation
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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
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Adjacent fluorochromes
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Primary of Tandems
10.5% 1.6%
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Interlaser compensation
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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
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Avoid bright fluorescence
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Different populations
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Bright dual positive
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Compensation Compromises
09-13546
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Infinicyte - Cytognos
• Nearest neighbor estimate of relationship between parameters in different tubes
Pedreira, et al. Cytometry (2008) 73A:834-846
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Mass Cytometry
30-100 simultaneous antigens
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Mass Cytometry
Bendall, et al (2011) Science 332:687
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Mass Cytometry
Bendall, et al (2011) Science 332:687
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Mass Cytometry
Bendall, et al (2011) Science 332:68713 parameters
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Mass Cytometry
Bendall, et al (2011) Science 332:68718 functional markers13 conditions
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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