Beyond Colorful Science. A Practical Approach to Setting Up Multicolor Panels

Transcription

1 Beyond Colorful Science A Practical Approach to Setting Up Multicolor Panels ISCT Paris April 2014

2 Layout: Multicolor Panel Design Identify the pitfalls Have a look at issues with spectral overlap How to design a robust multicolor panel

3 Panel Design Objectives High sensitivity for dim antigens, independent of their expression pattern (discrete or modulated). Clear signal separation from unspecific background. Robust spillover patterns.

4 Panel Design Toolbox 6 rules based on Dye performance characteristics Spectral overlap Co-expression patterns

5 Panel Design Toolbox 6 rules based on Dye performance characteristics Spectral overlap Co-expression patterns

6 Dim Marker, Bright Fluorochrome?

7 Dye Brightness The requirement for brightness depends on the antigen density and expression characteristics.

8 Dye Spillover 488nm ECD Brightness is not always better but an ambiguous dye property as it may involve strong spillover and data spreading. KrO

9 Spectral Consistency APCA750 (CD27, CD45RA) PC5.5 (CD25, CD56, CD19) APCA700 (CD25, CD16) Overlays of separate stainings using a variety of specificities with identical conjugation Multicolor cytometry requires first class tandem dyes with high spectral consistency

10 Panel Design Toolbox 6 rules based on Dye performance characteristics Spectral overlap Co-expression patterns

11 Ideal World: No Spectral Overlap 488nm 638nm 405 nm APC Pacific Blue

12 Impact of Spectral Overlap HLA-DR - PE HLA-DR - PE Loss of sensitivity CD45-FITC CD45-FITC CD45-Pac Blue CD45-Pac Blue

13 Compensation

14 Channels on a Log Scale bit resolution is analogous to increasing the number of pixels in digital photography 9 channels 90 channels 900 channels channels channels channels

15 Channels on a Log Scale Compensation only corrects for mean fluorescence intensity!

16 Compensation and CV Spread FL3 FL3 FL3 FL3 FL3

17 Compensation and CV Spread

18 Visualization of Spectral Overlap Identification of 2 types of distortion: Silent Fluorochromes: Causing no/marginal distortion in other channels Ideal for bright markers (gating markers) Untouchable Channels No/marginal distortion from other dyes Ideal for dimly expressed antigens Gallios/Navios Flow Cytometers Distortion pattern depends on instrument type, filter combination, and choice of fluorochromes.

19 Panel Design Toolbox 6 rules based on Dye performance characteristics Spectral overlap Co-expression patterns

20 Co-Expression Patterns Modulated antigens Non-exclusive antigens Discrete antigens Exclusive Antigens Exclusive subpopulations Co-expressed, discrete antigens

21 Co-Expression of Modulated Antigens Crosstalk results in loss of sensitivity for the modulated antigen.

22 Co-Expression of Mutually Excluding Antigens Unaffected limits for CD4-/CD8- cells Mutually excluding antigens serve each other as dump channels, crosstalk has no impact on sensitivity

23 Co-Expression Patterns of Sub-Populations Compensated Uncompensated Parent / Descendent Rule : Sub-populations can spectrally overlap into gating markers, but not vice-versa.

24 Summary: The 6 Rules 1. Old School : Weakly expressed antigens work best with a bright dye, strongly expressed antigens work with all dyes. However, always take care of the neighbours! 2. Channels : weakly expressed antigen works best on untouched channels, strongly expressed antigens work best with silent dyes. 3. Exclusion : Allow spillover between excluding antigens (CD3/CD19, etc.). 4. Co-expression : Avoid spillover between non-exclusively co-expressed antigens. 5. Parent-descendant : Allow spillover from descendent antigens to parent antigens, avoid vice-versa. 6. Complexity : Minimize complexity of overspill patterns, in case not all fluorescence detectors are used in the experiment.

25 Summary: Determinants of Effective Sensitivity Pacific Orange Discrete vs Modulated Expression Krome Orange Fluorochrome Brightness vs Antigen Density Detection Limits Coexpression Pattern Sterical Hindrance Unspecific Staining Preparation Filter Configuration Dye Spectra

26 6 Rules Applied to ONE

27 6 Rules Applied to ONE Design: High sensitivity for weak antigens, robust spillover patterns, phenotype-independent detection limits. CD45-KrO: Bright antigen matched with a silent dye. CD45-RA: Modulated antigen uses untouchable channel.

28 6 Rules Applied to ONE

29 Questions / Comments? Marketing Manager Flow Cytometry Reagents and Applications Europe, Middle East, Africa & India aboehmler@beckmancoulter.com