Advanced Topics in Compensation & Panel Design Katharine Schwedhelm January 30, 2020
O VERVIEW Ø Compensation Ø Spillover/spreading error Ø Panel Design
C OMPENSATION – W HY IS IT N ECESSARY ? • Light is not discrete Ø Spectral overlap between dyes results in the detection of the primary fluorochrome in one or more secondary detectors Ø Spillover fluorescence must be subtracted from the total fluorescence detected in the secondary detector(s) Spillover characteristics of dyes excited by the red laser
C OMPENSATION – U NCOMPENSATED D ATA Sample stained only with FITC Ø Spillover 10 4 fluorescence is % spillover of FITC into PE= 1º Detector: proportional to the FITC level of the primary 10 3 Fluorescence FL2 x100 fluorescence Fluorescence FL1 10 2 85 Ø The percent spillover MFI FL2 (pos) - MFI FL2 (neg) x100 of the primary MFI FL1 (pos) - MFI FL1 (neg) fluorescence is 10 1 subtracted from the 4 total fluorescence in 26 - 3 x100 = 28% the secondary 10 0 85 - 4 detector, on a per 3 26 10 0 10 1 10 2 10 3 10 4 cell basis 2º Detector: PE Data provided by J. Stucky, 041012 *we are only considering the signal of the FITC fluorochrome
C OMPENSATION – COMPENSATED DATA Sample stained only with FITC Ø 28% of the total signal measured in 10 4 the secondary (PE) detector is 1º Detector: removed Compensated FITC Ø After compensation, the median 10 3 fluorescence signal of the primary antibody as seen in the secondary 10 2 85 detector is the same for both the negative and positive populations 10 1 Ø Repeat for every detector 4 Ø Repeat for every single stained 10 0 antibody 10 0 10 1 10 2 10 3 10 4 2º Detector: PE 3 Data provided by J. Stucky, 041012
C OMPENSATION – EXAMPLE OF A 17 COLOUR COMPENSATION MATRIX Detector Fluorochrome
C OMPENSATION – PRACTICAL CONSIDERATIONS (1) • PMT voltages must be set properly before acquiring compensation samples and remain unchanged Ø Changing PMT voltages will change compensation requirements • Make single stained compensation controls – one for each fluorochrome in the panel Ø Each sample must be stained with ONLY ONE antibody Ø Control must be as bright or brighter than the experimental sample Ø Ideally use the same reagent as used in the staining panel • Utilize compensation beads (check species reactivity and isotype) Ø Stain comp control with CD4 in the same fluorochrome
C OMPENSATION – PRACTICAL CONSIDERATIONS (2) • Treat the compensation controls exactly like the experimental sample • Positive and negative populations within a compensation control must be of the same kind (i.e. have the same autofluorescence) Ø Do not use a positive bead and a negative cell in combination
C OMPENSATION – TYPES OF SINGLE STAINED CONTROLS • Antibody capture beads Ø Test ahead for intensity/binding – isotype and species reactivity Ø Titrate reagent on beads if too bright Ø Be sure that the negative beads are the same as the positive beads Ø Not all fluorophores accurately compensate on beads (see TDS from manufacturer) • Single stained cells Ø Give the most accurate compensation matrix (especially for larger panels) Ø Some markers may not stain “normal” cells – require stimulation (activation markers, cytokines) Ø Positive and negative cells must have the same autofluorescence • Proper negative for CD14 expressed on monocytes is the scatter gated monocytes on an unstained sample or the single stained control spiked with unstained cells prior to acquisition
C OMPENSATION – TANDEM DYES • A tandem dye consists of a donor and acceptor Emission Excitation Emission Excitation fluorochrome that are covalently bonded • Donor molecule transfers excitation energy to the acceptor molecule via FRET (fluorescence resonance energy transfer) • Acceptor molecule gives off light • Usually the first molecule excited by a laser line serves as the “base” for the remainder of the Excitation Emission fluorophores excited by the same laser • i.e.: Green laser fluorophores: PE, PE-CF594, PE-Cy5, PE- Cy5.5, PE-Cy7
C OMPENSATION – TANDEM DYES • Tandem dyes will differ: Ø In their spillover characteristic between different antibody conjugates Ø Lot to lot for the same antibody conjugate Ø Between manufacturers • Be aware that many BV (brilliant violet) and BUV (brilliant UV) dyes are also tandems! PE-Cy7 CD8 PE-Cy7 F4/80 PE-Cy7 IFNg PE-Cy7 TNFa 10 5 10 5 10 5 10 5 10 4 10 4 10 4 10 4 <PE-A> <PE-A> <PE-A> <PE-A> 10 3 10 3 10 3 10 3 10 2 10 2 10 2 10 2 0 0 0 0 10 3 10 4 10 5 10 3 10 4 10 5 10 3 10 4 10 5 10 3 10 4 10 5 0 0 0 0 <PE-Cy7-A>: CD8 <PE-Cy7-A>: F4-80 <PE-Cy7-A>: IFNg <PE-Cy7-A>: TNFa All are compensated with the single stained control for CD8-PE-Cy7 Images courtesy of Florian Mair
C OMPENSATION – ADDITIONAL NOTES ON FLUOROCHROMES • Spectral properties change over time due to exposure to light and fixation reagents Ø Tandem dyes are susceptible to degradation over time • Minimize exposure to light during staining and store stained samples in the dark • Minimize concentration of fixative in the final resuspension volume (0.5 to 1% PFA) or wash out and resuspend in wash buffer for longer term storage
C OMPENSATION – LOG SCALE VERSUS LINEAR SCALE • Fluorescence is usually displayed on a log scale • Log display may skew perception of the data and lead to manual overcompensation • Events at zero are squished against the axis (log scale does not go below zero) Uncompensated Log Linear 10 5 400 10 4 A B C D A B C D 200 10 3 Spillover 100 0 Fluorescence 10 -200 1 0.1 -400 10 4 10 4 10 3 10 3 1 10 100 1 10 100 Primary Fluorescence Primary Fluorescence Image courtesy of M. Roederer, NIH Compensated
B I - EXPONENTIAL OR LOGICLE TRANSFORMATION Ø Transforms the log scale to display values below zero Ø Allows for better visualization of populations centered around zero Ø Feature is available in most FACS analysis software Ø Always analyze properly transformed data!
T RANSFORMATION CONFIRMS CORRECT COMPENSATION Not 10 5 10 5 Transformed 10 4 10 4 10 3 10 3 Median 10 2 10 2 10 1 10 1 CD4 10 1 10 2 10 3 10 4 10 5 10 1 10 2 10 3 10 4 10 5 Many events are 10 5 10 5 squished on the x 10 4 10 4 and y axes Transformed 10 3 10 3 0 0 10 3 10 4 10 5 10 3 10 4 10 5 0 0 CD8 Slide courtesy of M. Roederer, NIH
R E - TRANSFORMATION • Cells with large negative fluorescence values affect transformation • Excluding these cells by drawing a temporary gate and retransforming produces better results Re-transform on cells 10 3 10 3 in this gate PerCP 10 2 Cy5.5 10 2 (Unstained) 10 1 0 0 10 1 10 2 10 3 10 1 10 2 10 3 0 0 PE-Cy7 CCR7 Data provided by Jeff Pufnock, 061307, CD8 FMO
R E - TRANSFORMATION – INCORRECT GATE CHOSEN IN F LOW J O 10 3 PerCP 10 2 Cy5.5 Unstained 0 10 1 10 2 10 3 0 PE-Cy7 CCR7 Transforming when this gate is selected produces this result Data provided by Jeff Pufnock, 061307, CD8 minus FMO
R E - TRANSFORMATION – CORRECT GATE CHOSEN IN F LOW J O 10 3 PerCP 10 2 Cy5.5 Unstained 10 1 0 10 1 10 2 10 3 0 PE-Cy7 CCR7 Transforming when this gate is selected produces this result Data provided by Jeff Pufnock, 061307, CD8 minus FMO
C OMPENSATION ERRORS - DIAGNOSIS • Consider an error in compensation in the following situations Ø Diagonal staining populations (except for two markers with correlated expression, e.g. IFNg vs TNFa Ø Unexpected positive populations (e.g. high frequency of cells expected at low frequencies, CD25, IL-4…) Ø Cells leaning over the axis
C OMPENSATION ERRORS – I NVESTIGATIVE OPTIONS • Steps to investigate potential compensation errors: Ø Apply custom transformation to visualize negative events and to assess medians Ø Visualize each parameter versus all others to search for unobserved compensation issues (multigraph overlay, N by N plot) Ø Apply compensation to the compensation samples • Does the compensation matrix need to be re-calculated or is the comp matrix not working for the test samples but okay for the comp samples Ø Ensure that the compensation sample is bright enough and/or that the gate is placed high enough • Calculation is based on the median fluorescence in the positive gated population • Higher gates useful for markers with continuous distribution Ø Ensure that there are enough events in the positive population Ø Use a compensation control(s) from another experiment and remake the matrix
E XAMPLE PLOTS OF OVER , UNDER , AND CORRECTLY COMPENSATED DATA Primary fluorochrome on x-axis, secondary detector on the y-axis Over Compensated Correctly Compensated Under Compensated V450 MFI on V610+ cells is less V450 MFIs of V610+/- cells are V450 MFI on V610+ cells is greater than V450 MFI on V610- cells idential than V450 MFI on V610- cells
M ANUAL ADJUSTMENT OF THE COMPENSATION MATRIX • Only to be done if the reason for the matrix failure cannot be identified AND • It is certain that there is an error in compensation Ø Do not overcompensate to attempt to correct for spreading error! • Each row shows the percent signal of a fluorochrome subtracted from each detector, listed in columns Detector Ø Overcompensated – decrease number Ø Undercompensated – increase number Fluorochrome
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