Quality assurance in DNA image analysis on diploid cells

Abstract

DNA image analysis is presently performed in many laboratories. Before general extrapolation of results between different laboratories, validation has to be performed for interlaboratory studies on DNA image analysis. The aim of this study was to establish the performance of different DNA image analysis instruments when measuring different diploid cells. On three separate parts of the same object slide, human liver cells, white blood cells, and imprints of a breast fibroadenoma were sampled. In this quality assurance interlaboratory study, 13 laboratories participated voluntarily. Two slides were sent to each participating laboratory: one Feulgen and one unstained to be stained according the participating laboratory in‐house procedure. The features integrated optical density (IOD) and object AREA were recorded for each nucleus. For calculation of the results, the average IOD value of liver diploid cells was set at 2c. A striking difference was observed between the different presumed diploid cell types, from almost 1c to 3c. This variation was not dependent on central or in‐house staining. Although in‐house calibration was performed for each image analysis system, a surprisingly large variation existed in the reported object AREA, irrespective of the cell type. These results clearly demonstrate that measurements performed in one laboratory cannot be extrapolated to the outcome of others and support the need for standardization. The use of external control cells works well for comparison of instruments. In conclusion, in DNA image analysis quality control is necessary, standardization is obligatory, and the use of an internal control for determination of the diploid peak in a histogram of patient samples is recommended. Cytometry 27:21–25, 1997. © 1997 Wiley‐Liss, Inc.

From Pixels to Picograms

“…Recent advances in computing and image processing technology, combined with proven methods of histochemical staining, allow the reliable and rapid estimation of nuclear DNA contents. Image analysis densitometry has been accepted as an accurate means of quantifying DNA for clinical applications (Bertino et al 1994;Borgiani et al 1994;Fischler et al 1994;Pindur et al 1994;Yamamoto et al 1994;Böcking et al 1995;Thunnissen et al 1996Thunnissen et al ,1997Reeder et al 1997;Marcos et al 1998;Puech and Giroud 1999), and its broader utility in the measurement of both plant and animal genome sizes has been demonstrated (Vilhar et al 2001; and the present study). In this review we have provided the necessary background information, as well as a straightforward guide to the use of image analysis technology for genome size determination using this method.…”

“…Most software packages include data filters to automatically eliminate objects touching the edge of the field or falling outside a specified size range. Consistency in the criteria employed for exclusion is important for minimizing variability in results obtained by different investigators (e.g., Thunnissen et al 1996Thunnissen et al ,1997Reeder et al 1997;Vilhar et al 2001).…”

“…As with flow cytometry, the use of image analysis technology in DNA quantification began in cancer diagnosis (e.g., Borgiani et al 1994;Fischler et al 1994). Given the extreme importance of accuracy in such an application, it is not surprising that the fidelity of the technique has been scrutinized with exceptional rigour (e.g., Böcking et al 1995;Thunnissen et al 1996Thunnissen et al ,1997Reeder et al 1997;Puech and Giroud 1999). Comparisons have shown that flow cytometry and image analysis provide similar efficacy of DNA quantification for diagnostic purposes (e.g., Bertino et al 1994;Borgiani et al 1994;Marcos et al 1998).…”

Standardisation of DNA quantitation by image analysis: Quality control of instrumentation

“…Precision and reproducibility of the measurements depend, in part, on instrumentation quality. It has been already reported that image analysis systems used for DNA quantification differ widely in their characteristics (1). Thus, quality control procedures should be developed and implemented that are efficient for measurements using image analysis systems.…”

DNA degradation in nuclei of muscle cells followed by ischemic injury in a rabbit amputation model

“…Recently, with the development of ICM technology, the measurement of DNA content is expected to be more rapid and accurate. 22,23 The DNA contents in the cells of heart, liver, and kidney with different ischemia intervals were studied with Feulgen staining by the cytometry technique. 24 The DNA contents of these cells were found to be at 99.5% of normal after 6 h of ischemia, 91.3% after 12 h of ischemia, and 81.3% after 24 h of ischemia.…”