An automated scoring procedure for the micronucleus test by image analysis

Abstract

The micronucleus assay (MNT) in human lymphocytes is frequently used to assess chromosomal damage as a consequence of environmental mutagen exposure, to assess the effect of mutagens or to search for reduced DNA repair capacity after a mutagenic challenge. We have established an automated scoring procedure for the cytokinesis blocked MNT based on computerized image analysis (Metasystems Metafer 4 version 2.12). To evaluate the results we used the reproducibility of counts, established a dose-response curve for gamma-irradiation and used the ability of the system to differentiate between breast cancer patients and controls as a biological reference, a difference which we had observed before by visual counting. Blood cultures were irradiated with gamma-rays (2 Gy) at the beginning and treated with cytochalasin B during the last 24 h. The slides were stained with Giemsa for visual counting and with DAPI for automated analysis. Our test sample consisted of 73 persons (27 with breast cancer and 26 female and 20 male controls). A comparison between visual counting (controls, mean MN frequency 313) and automated counting (mean MN frequency 106) in slides from the same culture revealed a large drop for the automated counts. However, the automated counts were as reproducible as the visual counts [coefficient of variation (CV) on the sample approximately 20%; CV on repeated counts of the same slides approximately 5%] and both counts were highly correlated. Furthermore, the discrimination between cases and controls improved for automated counting of slides from the same cultures [visual odds rato (OR) < or = 4.0, P = 0.009; automated OR > 16, P < 0.0001], with a strong dependence on the set of parameters used. This improvement was confirmed in a validation sample of an additional 21 controls and 20 cases (OR = 11, P = 0.0018) performed as a prospective or diagnostic test.

Laboratory Intercomparison of the Cytokinesis-Block Micronucleus Assay

“…The resulting calibration curves show a good regression, but in general curves of manual scoring were steeper by a factor of about 2 in relationship to the curves generated by automated scoring procedures, which can probably be explained by the better detection efficiency of human scorers (25,28,31).…”

“…Several automated systems are available on the market and have been tested by human monitoring (28,(31)(32)(33)(34). In our study the scoring system of MetaSystems (Altlussheim, Germany) was utilized.…”

Cytokinesis-block micronucleus cytome assay

“…For this goal to be achieved, it is essential that scoring criteria are well developed and that a robust slide preparation protocol be put in place and that slide preparations be permanent so that they can be re-examined visually if necessary. Automated image analysis systems have recently been developed for scoring of MNi in mammalian cells [66][67][68][69][70][71] (see http://www.imstar.fr/applications/genotoxicity/micronucleiassay/; http://www.metasystems.de/products/metafer/ metafer_msearch.htm#Micronucleus; http://www.biocompare.com/techart.asp?id=1247) but these systems do not take into account other important events such as NPBs, NBUDs, necrosis, apoptosis and cytostasis, which are essential for the correct interpretation of the result obtained. In the future, we should expect to have an automated system that can score reliably the various end points possible with the CBMN Cyt assay outlined in this paper.…”

“…This provides the opportunity to test, using the same system, the extent to which the in vitro test is predictive of in vivo genotoxic events. The adoption of the CBMN Cyt assay by the pharmaceutical industry 57,58,63 , its application in human biomonitoring of genotoxic exposures 24,60,61 and its increasing application in preventive medicine and nutrition 62,64,65 [66][67][68][69][70][71] .…”

Reduced DNA repair in BRCA1 mutation carriers undetectable before onset of breast cancer?

“…The MNT was carried out exactly as described by Varga et al (2006). Counting was performed by an automated system (Varga et al, 2004) to avoid any variation possibly introduced by different observers and to eliminate imperfect reproducibility (coefficient of variation of the machine o5% for counts on the same slide). As obvious from Figure 2, the deviation from the final mean decreases with an increasing number of BNCs and becomes sufficiently small only when more than 1000 BNCs are counted.…”