The Panel Design Tool - UZHcc43871f-30aa-4f9e-9ec4... · Panel Design Tool. Running the PDT in R. Get the total spillover score of the panels: 01.06.2017 Stephan Benke. Select the

Flow Cytometry Facility

The Panel Design Tool

Automated design of multicolor flow cytometry panels.

01.06.2017

Flow Cytometry Facility Panel Design Tool

Contents

• The antibody – fluorophore panel

• Parameters & desired properties

• The Panel Design Tool

• What it can do

• The input needed

• Input processing

• Panel creation

• Output visualization

• Functionality summary and future implementations

01.06.2017 Stephan Benke


Flow Cytometry Panels


+

FluorophoresAntibodies

Detection

bdbiosciences.com

Cell types,markers of interest


Flow Cytometry Panels


Matching markers with fluorophores and instrument channels for optimal detection.

For the best resolution:

• Minimize spillover

• Fluorophore emission• Optical filters• Distribution of fluorophores

on cell types

• Equalize signal intensity

• Fluorophore brightness• Marker expression levels• Antibody quality

Challenge:

• Large number of combinations (6 markers: 720, 28 markers: 3 � 1029)

• Strong interplay of parameters


Computation Tool for Designing Panels

Input:• What do you want to measure (cells & markers)• How do you want to measure (instrument & possible dyes)

Output:• Spillover optimized panel propositions



Panel Design Tool: Calculation Steps

• Calculation of dye properties for specific instruments based on spectra.

• Instrument specific brightness

• Ranking of dyes by generated spillover

• Marker ranking according to co-occurrence with other markers.

• Initial matching of markers and dyes based on expression levels and dye brightness.

• Sampling of maker – dye assignments and sorting based on spillover criteria.



Input: Cell Types and Markers

Example: The HIPC T-cell panel


Finak et al., Scientific Reports 2016

Identification of 10 cell types using 7 markers.


Input: Cell Types and Markers

Input format:


Options for expression levels:• ? unknown level -> set to range from lowest to highest• +, ++, … relative level• + - ++… expression level range

Expression levels from Bausch-Fluck et al., PLOS 2015


Input Processing: Cell Types and Markers

Markers:


Markers are ranked according to their number of co-occurrences with other markers.

Cell Types:

Cell types are ordered by number of markers.


Input: Optical Layout of the Instrument

Instrument detection channels are calculated based on model functions of the LP and BP optical filters for each excitation wavelength.


José Duarte


Input: Instrument Example

The optical layout (lasers, optical filters and detection channels) is provided as an Excel sheet.


Canto @ USZ

José Duarte


Input: Dyes

List of dyes that can be used for detection.

For each dye the

• Excitation spectrum• Emission spectrum

• Relative brightness

is required.



Input Processing: Dyes

Excitation


Amount of Dye absorbance at each instrument excitation wavelength

Calculation of fluorescence signal in each instrument channel scaled by relative excitation and brightness.

Emission




For each dye the following is calculated:

• Fluorescence signalfor each channel, scaled by relative excitation and brightness.

• Main channel instrument channel that detects largest part of dye fluorescence

• Spillover score sum of detected fluorescence in all channels except the main channel relative to the main channel signal

• Relative brightness of the dye as measured on the instrument

• Fraction of total dye emission detected in the main channel

The dyes are ranked by their spillover score.




Summary of processed dye information is provided for the user to facilitate initial selection of dyes to use in the panel.


Input: Dyes


Free: dyes that can be used in the panel

LD: live/dead stains (optional)

Marker X: example for a restricted assignment (optional)

Marker Y: example for a fixed assignment (optional)

Free LD Marker X Marker Y ….AF488 LD_Aqua AF488 BUV395AF647 LD_Blue APCAF660 LD_FarRed BV421AF680 LD_GreenAF700 LD_NIR

… …

Excel file containing:


Input Processing: Dyes for Markers


Each marker receives a list of suitable dyes.

To equalize signal levels, marker expression level is matched with dye brightness (as measured on the instrument).

CD3 CD8 CD4 CCR7 CD45RA CD38 HLADRPerCP-Cy5.5 PE-Cy7 Cy3 AF488 AF488 APC PE-Cy7

PE-Cy5 PE-Cy5 Cy3.4 AF647 AF647 PE-Cy5.5 PE-Cy5PE-Cy7 PE PG FITC FITC PerCP PE

PE APC V500 PG PG PE APC… … … … … … …In

crea

sing

sp

illov

er

scor

e

Decreasing # of co-occurrences


Assignment Procedure


• Cell type with most markers

• Marker with highest number of co-occurrences

Choose dye for marker randomly from the top n suitable dyes for

that marker.

Remove dyes using the same detection channel as the chosen dye

from other dye lists.

Keep best ksamples

Calculate spillover to marker signal ratio for each

sample

Repeat process for unassigned markers on

remaining cell types keeping only best

assignment.

k different panels

Sampled m times?

Unassigned marker left?

Fixed assignment for marker?

Parameters• Number of dyes to

choose from (n)• Sampling number

per cell type (m)• Number of panels

to work with (k)

yes no

yesno

noyesNext marker

Restart


Assignment Example


T-cell CD8 activated


CD3 CD8 CD38 HLADR

PerCP-Cy5.5 PE-Cy7 APC PE-Cy7

PE-Cy5 PE-Cy5 PE-Cy5.5 PE-Cy5

PE-Cy7 PE PerCP PE

PE APC PE APC

APC PE-Cy5.5 AF488 PE-Cy5.5

… … … …

CD8 and HLADR have same expression level -> same dyes to chose from.

Incr

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ng s

pillo

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Assignment Example



CD3 CD8 CD38 HLADR



PE-Cy7 PE PerCP PE

PE APC PE APC


… … … …

Dye is chosen from the best n dyes (5 in this example).


Incr

easi

ng s

pillo

ver

scor

e


Assignment Example



CD3 CD8 CD38 HLADR



PE-Cy7 PE PerCP PE

PE APC PE APC


… … … …


Chosen dye is removed from all following lists

Incr

easi

ng s

pillo

ver

scor

e


Assignment Example



CD3 CD8 CD38 HLADR

APC PE-Cy7 PE-Cy5.5 PE-Cy7

PE-Cy5 PerCP PE-Cy5

PE PE PE

PE-Cy5.5 AF488 PE-Cy5.5

PerCP … …

…


Dyes are also removed when using the same detection channel as the chosen dye.

Incr

easi

ng s

pillo

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scor

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Assignment Example



CD3 CD8 CD38 HLADR

APC PerCP PE PE-Cy7

AF488 PE

FITC

PB

PE-CF594


When less than the desired number of dyes is in the list, dye is chosen from remaining dyes.

When only one dye left, that dye is assigned to marker. If list is empty, it is refilled from remaining free dyes.

Incr

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Assignment Example



CD3 CD8 CD38 HLADR

APC PerCP FITC PE

Assignment for cell type 1 repeated mtimes, best kcombinations kept.


CD3 CD4 CD38 HLADR

APC Cy3 FITC PE

Cy3.5

PG

…

Procedure repeated for next cell type, keeping already assigned markers fixed.


Assignment Example


Result:m different panels

Panel 1 Panel 2 Panel 3 …CD3 APC PE-Cy7 PE-Cy7

CD8 PerCP APC APC

CD4 V450 PacificBlue PacificBlue

CCR7 PE-Cy7 PE-CF594 PE-CF594

… … … …

The different panels can be sorted and selected by their overall spillover scores and further selected by the predicted resolution for certain markers.


Choosing a Panel

1) Select the best panels by total spillover score.2) Look at the spillover scores for each marker on each cell type.3) Also check the estimated signal levels.4) Select panel that best suits your experimental question.



A “Spectra Viewer” Look at Individual Panels

Generate spectra viewer like plots for a panel:Plots are made for each cell type and for each excitation wavelength.



Running the PDT in R


Generating the dye library for a specific instrument:

Processing the input:

make_dye_library(optical_filters, dye_spectra, brightness_data, instrument_name)

input_dat <- process_input(experiment_layout, dyes_for_markers, dye_library, min_nr_dyes)

Calculates dye emission detected on instrument, spillover and brightness.

Processes the user input, ranks markers and dyes, does brightness –expression level matching etc.




Generating the panels (stochastic search):

nr_dyes: number of dyes to randomly choose from during assignmentnr_sampling: number of times the assignment is repeated for one cell typenr_panels: number of panels generated

To get a reference value for the spillover score of a panel layout, run the assignment first with nr dyes, sampling and panels set to 1.

Increase parameters to sample more combinations and get higher chance for good results (can take a while).

panels <- make_panels(input_dat, nr_dyes, nr_sampling, nr_panels)




Generating the panels (thorough search):

Tries to test all possible dye – marker combinations. If no marker – dye restriction lists are given, dyes tested are taken from the marker expression levels – dye brightness matching lists.

For larger panels with many possibilities the computational costs will be huge! Estimate the number of possible combinations beforehand and do not run searches with more than a couple hundred thousands possibilities.

panels <- make_panels(input_dat, search_all = TRUE)



Get the total spillover score of the panels:


Select the best panels by their spillover score:

Save dye – marker assignments to excel file:

Save detailed spillover information of each panel to excel file:

get_total_so(panels)

panel_selection <- get_best_by_so(panels, nr_selection)

write.assignment(panel_selection, file_name)

write.panels(panel_selection, file_name)

Generate the spillover per marker plots:plot_spillover(panel_selection, file_name, input_dat)

Generate the “spectra viewer” plot for one panel:plot_spectra(output_name, panel_selection,

panel_nr, filter_list, input_dat)



A preliminary graphical user interface is available for a simple workflow.

The full functionality is currently only available from the R console.



Feature Summary – User Essentials

Input needed:

• Instrument configuration and dye properties (provided by the FCF for all facility machines)

• Experiment layout with cell types, markers and expression levels (template provided)

• List of available dyes / restrictions / fixed assignments (template provided)

What you get:

• List of panel propositions ready to be tested

• Spillover calculations and visualization for each marker on each cell type



Future Development

• Give us your feedback and send us your wishes for new features!

• Read in expression level data (if available).

• Get our own data for dye brightness.

• Proper documentation.


Documents

The Panel Design Tool - UZHcc43871f-30aa-4f9e-9ec4... · Panel Design Tool. Running the PDT in R. Get the total spillover score of the panels: 01.06.2017 Stephan Benke. Select the