Fluorescence intensity normalisation: correcting for time effects in large-scale flow cytometric analysis

Abstract

A next step to interpret the findings generated by genome-wide association studies is to associate molecular quantitative traits with disease-associated alleles. To this end, researchers are linking disease risk alleles with gene expression quantitative trait loci (eQTL). However, gene expression at the mRNA level is only an intermediate trait and flow cytometry analysis can provide more downstream and biologically valuable protein level information in multiple cell subsets simultaneously using freshly obtained samples. Because the throughput of flow cytometry is currently limited, experiments may need to span over several weeks or months to obtain a sufficient sample size to demonstrate genetic association. Therefore, normalisation methods are needed to control for technical variability and compare flow cytometry data over an extended period of time. We show how the use of normalising fluorospheres improves the repeatability of a cell surface CD25-APC mean fluorescence intensity phenotype on CD4(+) memory T cells. We investigate two types of normalising beads: broad spectrum and spectrum matched. Lastly, we propose two alternative normalisation procedures that are usable in the absence of normalising beads.

Figure 1

(a) Black crosses show nonnormalised MFIs in the CD4⁺ memory T cell population as a function of time. The back line was fitted line to these MFI values using a loess procedure. The red line shows the normalisation coefficient estimated from the beads. (b) Repeatability plots (n = 15 pairs) for MFIs of CD25-APC cell surface expression in the CD4⁺ memory T cell population. (c) Repeatability plots (n = 15 pairs) for CD25-APC MEF (normalised MFI) in the same cell population. For (b) and (c), each individual's blood donations were separated by at least 3 months.

Figure 2

(a) Correlation between the fraction of CD25-positive cells in the CD4⁺ memory T cell population and the CD25-APC MEF in this population. (b) Repeatability (n = 15) of the estimated fraction of CD25-positive cells in the CD4⁺ memory T cell population obtained by background subtraction of the isotype control distribution.

Figure 3

Variability across time of the normalisation coefficient for broad spectrum beads (black) and APC spectrum matched beads (red). The blue line shows the APC photomultiplier tube voltage setting used to measure the beads MFI.

Figure 4

Variability across time of the isotype control MFIs (red crosses, one point per sample) and the normalising beads MFIs (black line, one point per experimental day). MFIs are scaled such that the value is equal to one for the first day, and a logarithmic scale is used for the y-axis.

Figure 5

(a) Repeatability for the CD4⁺ memory T cell population CD25-APC MFI normalised using a multiplicative correction factor estimated by a regression analysis on the set of 149 samples with identical T1D susceptible genotype. (b) Repeatability for the CD4⁺ memory T cell population CD25-APC MFI divided by the CD25-APC MFI in the full CD4⁺ T cell gate for the same sample/analysed tube.