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

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

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