Data-Driven Compensation For Flow Cytometry Of Solid Tissues

Data-Driven Compensation for Flow Cytometry of Solid Tissues
Nickolaas Maria van Rodijnen,1 Math Pieters,1 Sjack Hoop,1 and Marius Nap1,2
1Department of Pathology, Atrium Medical Centre, P.O. Box 4446, 6401 CX Heerlen, The Netherlands
2TiCC, Tilburg University, Warandelaan 2, 5037 AB Tilburg, The Netherlands
Received 3 February 2011; Revised 20 May 2011; Accepted 8 June 2011
Academic Editor:Shandar Ahmad
Copyright © 2011 Nickolaas Maria van Rodijnen et al. This is an open access article distributed under theCreative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract
Propidium Iodide is a fluorochrome that is used tomeasure the DNA content of individual cells, taken from solid tissues, with a flow cytometer. Compensation for spectral cross-over of this fluorochrome still leads to compensation results that are depending on operator experience. We present a data-driven compensation (DDC) algorithm that is designed to automatically compensate combined DNA phenotype flow cytometry acquisitions. The generatedcompensation values of the DDC algorithm are validated by comparison with manually determined compensation values. The results show that (1) compensation of two-color flow cytometry leads to comparable results using either manual compensation or the DDC method; (2) DDC can calculate sample-specific compensation trace lines; (3) the effects of two different approaches to calculate compensation values canbe visualized within one sample. We conclude that the DDC algorithm contributes to the standardization of compensation for spectral cross-over in flow cytometry of solid tissues.
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1. Introduction
Multiparameter flow cytometry (MP-FCM) of solid tumors is a powerful tool for quantification of antigen expression and DNA content, based on largenumbers of individual mammalian cells. However, simultaneous application of different fluorochromes introduces spectral cross-over. Spectral cross-over is the acquisition of fluorochrome intensities from a primary fluorochrome in the detector(s) used to acquire the intensity of secondary fluorochromes. Compensation is the estimation of the amount of fluorochrome intensity that needs to be subtractedfrom the acquired intensities to correct for spectral cross-over [1–4]. Proper compensation is achieved when the compensated data in the cross-over detector has no bias in the fluorescence distribution that is related to the intensity measured in any other detector [5]. To achieve proper compensation, the amount of spectral cross-over of each fluorochrome in a flow cytometry panel can be estimatedwith a single stained control (SSC). An SSC consists of a single cell suspension of which the individual cells are labeled with only one fluorochrome. This fluorochrome, of which the intensity is acquired in its primary detector, exhibits spectral cross-over in other secondary detectors. The cross-over of the SSC in each secondary detector is expressed as a percentage of the intensity acquired inthe primary detector. This percentage is based on the correlation coefficient between the fluorochrome intensity of a SSC in the primary and secondary detector(s) [1, 3]. The combination of all the percentages cross-over for each SSC, in each secondary detector, is expressed in a compensation matrix. It is common to calculate this compensation matrix once or twice a day and to use it for allfollowing acquisitions.
The problem of flow cytometry when working with cells originating from solid tissues is the use of propidium iodide (PI). PI is a dye that binds to DNA, and the acquired intensity is proportional to the amount of DNA in a (tumor) cell. The major advantages of PI are that (1) the DNA profile of the acquired (tumor) cells can be studied in relation to their phenotype, (2) it is...