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

Abstract

Background: DNA image analysis is frequently performed in clinical practice as a prognostic tool and to improve diagnosis. The precision of prognosis and diagnosis depends on the accuracy of analysis and particularly on the quality of image analysis systems. It has been reported that image analysis systems used for DNA quantification differ widely in their characteristics (Thunissen et al.: Cytometry 27: 21–25, 1997). This induces inter‐laboratory variations when the same sample is analysed in different laboratories. In microscopic image analysis, the principal instrumentation errors arise from the optical and electronic parts of systems. They bring about problems of instability, non‐linearity, and shading and glare phenomena. Methods: The aim of this study is to establish tools and standardised quality control procedures for microscopic image analysis systems. Specific reference standard slides have been developed to control instability, non‐linearity, shading and glare phenomena and segmentation efficiency. Results: Some systems have been controlled with these tools and these quality control procedures. Interpretation criteria and accuracy limits of these quality control procedures are proposed according to the conclusions of a European project called PRESS project (Prototype Reference Standard Slide). Beyond these limits, tested image analysis systems are not qualified to realise precise DNA analysis. Conclusions: The different procedures presented in this work determine if an image analysis system is qualified to deliver sufficiently precise DNA measurements for cancer case analysis. If the controlled systems are beyond the defined limits, some recommendations are given to find a solution to the problem. Cytometry 36:11–17, 1999. © 1999 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.…”

“…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).…”

Automatic Segmentation and Classification of Cells From Broncho Alveolar Lavage

“…To determine the best suited aperture level of the condenser, a calibration slide with a set of neutral density filters with known transmissions (5, 10, 30, 50 and 80%) was used. Theoretically, photodiodes have a linear response with respect to incident light intensity (Puech and Giroud, 1999). We used this assumption to determine the correct aperture.…”

“…For the evaluation of linearity, the mean luminance of a flat field image has been determined according to the transmission of neutral density filters for several condenser apertures within its allowed range. According to medical standards, the coefficient of determination of the regression line plotted over a set of measurements with different neutral density filters should be close to 0.99 (Puech and Giroud, 1999). We have then chosen the regression line having the closest determination factor which corresponds to an aperture of 0.25.…”

Raman spectroscopy for DNA quantification in cell nucleus

“…Yet another approach involving image analysis of nuclei was developed for clinical practice (6). The chemistry of DNA-Feulgen staining relies on high-stoichiometry binding of the dye to DNA, which results in more accurate data compared to the DNA staining with fluorochromes (3,4).…”