CytoRSuite: Compensation of Fluorescent Spillover

Dillon Hammill

2019-03-07

1. Introduction

CytoRSuite provides some useful functions to aid in compensation of fluorescent spillover, including spillover_compute for automated compensation, spillover_edit to interactively modify spillover matrices and cyto_plot_compensation to visualise compensation. Here we aim to demonstrate the appropriate use of these functions to obtain accurate spillover values for compensation.

The CytoRSuite compensation workflow is as follows:

1. Save .fcs files to folder “Compensation Controls” and read into a flowSet.
2. Load in compensation controls and an unstained control into a GatingSet.
3. Gate controls using gate_draw to home in on a homogenous cell population (e.g. Single Cells).
4. Use spillover_compute to automatically calculate estimates for the spillover matrix.
5. Fine tune spillover values interactively using spillover_edit.
6. Visualise compensation using cyto_plot_compensation to identify any issues.
7. If issues exist continue to fine tune the spillover values using spillover_edit.
8. Apply the spillover matrix to samples prior to transformation of fluorescent channels and gating.

2. Demonstration

2.1 Load in Compensation Controls into flowSet

Traditionally, all compensation controls including an universal unstained control are stored in a folder called “Compensation Controls” in the current working directory. For demonstration purposes we will use the Compensation dataset supplied in CytoRSuiteData which is a flowSet containing 5 single stain compensation controls and a universal unstained control.

library(CytoRSuite)
library(CytoRSuiteData)

# Assign Compensation flowSet to fs
fs <- Compensation

2.2 Add Compensation Controls to GatingSet

gs <- GatingSet(fs)

2.3 Gate Compensation Controls

Let’s gate the samples to home in on the Single Cells population. Gating prior to using spillover_compute will yield much more accurate spillover values.

# Cells
gate_draw(gs,
         parent = "root",
         alias = "Cells",
         channels = c("FSC-A","SSC-A"),
         gtfile = "Compensation gatingTemplate.csv")

# Cells
gate_draw(gs,
         parent = "Cells",
         alias = "Single Cells",
         channels = c("FSC-A","FSC-H"),
         gtfile = "Compensation gatingTemplate.csv")

2.4 Automated Spillover Calculation Using spillover_compute

spillover_compute will guide the user through channel selection for each of the compensation controls and save these selections to “Compensation-Channels.csv” for downstream use. This file can be passed to the channel_match argument of cyto_plot_compensation to bypass the channel selection process. Next the user gates the positive signal for each single stain control using an interval gate. This population will be used internally to calculate the percentage spillover based on the unstained control. The calculated spillover matrix is returned and written to a csv file for downstream analyses. The name of this file is controlled by the spillover argument.

spill <- spillover_compute(gs, parent = "Single Cells")

2.4.1 Channel Selection Process

Users are prompted to select a fluorescent channel for each of the compensation controls from a drop-down menu. The results are saved to a csv file called “Compensation-Channels.csv” for later use. This channel selection process can be bypassed by supplying the name of a channel match file to the channel_match argument.

2.4.2 Gating Positive Signal

A density distribution of each control in the appropriate channel will be plotted for users to gate the positive signal to use for spillover calculation. The unstained control is overlaid in red as a reference for gating.

2.4.3 Reading Exported Spillover Matrix File

The calculated spillover matrix can be read into R using read.csv:

spill <- read.csv("Spillover-Matrix.csv", header = TRUE, row.names = 1)
colnames(spill) <- rownames(spill)
spill

##                   Alexa Fluor 488-A     PE-A PE-Texas Red-A  7-AAD-A
## Alexa Fluor 488-A          1.000000 0.000000       0.000000 0.000000
## PE-A                       0.091033 1.000000       0.651885 0.416292
## PE-Texas Red-A             0.000000 0.000000       1.000000 0.000000
## 7-AAD-A                    0.063728 0.064851       0.081134 1.000000
## PE-Cy7-A                   0.005500 0.012740       0.013858 0.019638
## Alexa Fluor 405-A          0.000000 0.000000       0.000000 0.000000
## Alexa Fluor 430-A          0.122271 0.103727       0.108006 0.104338
## Qdot 605-A                 0.000000 0.000000       0.000000 0.000000
## Alexa Fluor 647-A          0.000000 0.000000       0.000000 0.000000
## Alexa Fluor 700-A          0.000000 0.000000       0.000000 0.000000
## APC-Cy7-A                  0.020893 0.021993       0.025842 0.032989
##                   PE-Cy7-A Alexa Fluor 405-A Alexa Fluor 430-A Qdot 605-A
## Alexa Fluor 488-A 0.000000          0.000000          0.000000   0.000000
## PE-A              0.088103          0.119826          0.065562   0.404853
## PE-Texas Red-A    0.000000          0.000000          0.000000   0.000000
## 7-AAD-A           0.271757          0.099010          0.052167   0.210264
## PE-Cy7-A          1.000000          0.009957          0.005951   0.025511
## Alexa Fluor 405-A 0.000000          1.000000          0.000000   0.000000
## Alexa Fluor 430-A 0.035866          0.789992          1.000000   3.313563
## Qdot 605-A        0.000000          0.000000          0.000000   1.000000
## Alexa Fluor 647-A 0.000000          0.000000          0.000000   0.000000
## Alexa Fluor 700-A 0.000000          0.000000          0.000000   0.000000
## APC-Cy7-A         0.056412          0.028982          0.016159   0.064532
##                   Alexa Fluor 647-A Alexa Fluor 700-A APC-Cy7-A
## Alexa Fluor 488-A          0.000000          0.000000  0.000000
## PE-A                       0.063705          0.041998  0.010067
## PE-Texas Red-A             0.000000          0.000000  0.000000
## 7-AAD-A                    0.076405          0.143769  0.035942
## PE-Cy7-A                   0.005629          0.016500  0.054000
## Alexa Fluor 405-A          0.000000          0.000000  0.000000
## Alexa Fluor 430-A          0.060548          0.023957  0.006892
## Qdot 605-A                 0.000000          0.000000  0.000000
## Alexa Fluor 647-A          1.000000          0.000000  0.000000
## Alexa Fluor 700-A          0.000000          1.000000  0.000000
## APC-Cy7-A                  0.087923          0.219586  1.000000

2.5 Modify Spillover Values Interactively Using spillover_edit

spillover_edit leverages a shiny interface to facilitate realtime visualisation of changes to fluorescent spillover values.

spillover_edit(gs, parent = "Single Cells", spillover = "Spillover-Matrix.csv")

spillover_edit features:

• automatic plot updates with changes to spillover values
• matrix cell colour change indicates the degree of compensation required
• overlay unstained control as a reference
• red horizontal line indicating median fluorescent intensity of the unstained control
• purple median tracker line which monitors changes in the median fluorescent intensity of the stained population
• density distributions automatically update with changes and display median fluorescent intensities for unstained and single stain controls.
• save updates by clicking save button
• Plots tab displays compensation changes in all fluorescent channels

Modifying the spillover values will automatically result in the plot being updated. The correct spillover value is such that the median of the stained population is equal to that of the unstained population (i.e. red line and purple line should meet in the center of the stained population). Looks like the PE-Cy7 control is slightly over compensated in the APC-Cy7 channel, let’s modify the value and click save to correct this:

Clicking on the Plots tab will display fluorescence spillover in all fluorescent channels with options to overlay the unstained control and the un-compensated sample.

2.6 Visualise Compensation Using cyto_plot_compensation

cyto_plot_compensation will automatically plot each compensation control in all fluorescent channels to identify any potential compensation issues. To identify any issues simply scroll through the plots by clicking the arrows in the Plots window. For brevity only the plots for PE Cy7 compensation control are shown below:

# Un-compensated
cyto_plot_compensation(gs, 
                       parent = "Single Cells", 
                       channel_match = "Compensation-channels.csv")

# Compensated
cyto_plot_compensation(gs, 
                       parent = "Single Cells", 
                       channel_match = "Compensation-channels.csv",
                       compensate = TRUE,
                       spillover = "Spillover-Matrix.csv")

3. More Information

For more information on these compensation functions refer to the documentation for these functions in the Reference. To learn more about the gating functions included in CytoRSuite refer to Gating Functions vignette.