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
Inferential reasoning
• Insensitive• Misattribution if assumptions incorrect
07-08646
18%
80%
66%
0.4%
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
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
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
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
Bethesda International Consensus Conference
N = 35
Euroflow
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
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
Hodgkin Lymphoma
CD15 APC CD71 APC-A700 CD20 PerCPCv55SSC-H
CD15 APC CD40 PE SSC-HSSC-H
0.1% abnormal immature B cells
ALL MRD
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Tandem Fluorochromes
Phycoerythrin (PE) PE-Texas Red
Tandem Breakdown
Whole blood lysis NH4Cl prelyse and wash1 hour prior
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 = DottedEmission = 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%
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
Compensation Validation
Fluorescence minus one “FMO” controls
Compensation Validation
Fluorescence minus one “FMO” controls
Compensation
Compensation
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
Adjacent fluorochromes
Primary of Tandems
10.5% 1.6%
Interlaser compensation
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
Avoid bright fluorescence
Different populations
Bright dual positive
Compensation Compromises
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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
Bendall, et al (2011) Science 332:68713 parameters
Mass Cytometry
Bendall, et al (2011) Science 332:68718 functional markers13 conditions
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