A flow cytometry protocol was applied for the detection of neoplasia in Macoma balthica L. from the Gulf of Gdansk (Baltic Sea, Poland). A simple method, based on an osmotic shock, was used to permeabilise gill cells. The cytometric pattern of normal clams consisted of 2 peaks, a major peak B and a smaller peak C. The cytometric pattern of affected clams consisted of 2 peaks named B' and C'. Two parameters were used to define the stages of abnormalities in M. balthica clams based on the percentage of cells in peaks B, C, B' and C' and on the ratio between the fluorescence value of peaks B, C, B' and C' in all individuals. Three stages of neoplasia were clearly distinguished by flow cytometry considering peak C'. Stage 1 was characterised by a major population of cells in peak B' and more than 10% of cells in the C' peak. Stage 2 consisted of a lower percentage of cells in peak B' and more than 25% of cells in peak C'. Stage 3 of the neoplasia was characterised by a further reduction in peak B' and more than 40% of cells in peak C'. Flow cytometry allowed for objective detection of neoplasia and provided a rapid method for measuring the DNA content of thousands of cells per individual. The accuracy of flow cytometry was assessed by comparing with standard histological techniques, used here as a reference technique for the detection of neoplasia, and with chromosome analysis. All individuals were analysed in parallel using the 3 techniques. The proportion of normal and affected individuals diagnosed using flow cytometry was comparable to the proportion determined by histology and chromosome analysis.
KEY WORDS: Flow cytometry · Histology · Cytogenetics · Neoplasia · Macoma balthica · Baltic Sea
Resale or republication not permitted without written consent of the publisherDis Aquat Org 65: [187][188][189][190][191][192][193][194][195] 2005 can neoplasia has been motivated by the question as to whether bivalves can be used as bioindicators in environmental monitoring (Roy et al. 1998, Brousseau et al. 2000, Fournier et al. 2001, Sauve et al. 2002.High prevalence of neoplasia can be found in Macoma balthica L. sampled from industrialised waterways around the Gulf of Gdansk in Poland (Thiriot-Quiévreux & Wolowicz 1996). Clams at these sites are known to be exposed to a range of chemical carcinogens and pollutants (Wolowicz 1994, Szefer et al. 1996, Sokolowski et al. 1999, Hummel et al. 2000. Neoplasia in M. balthica have also been reported from the Chesapeake Bay (Christensen et al. 1974) and Finnish coast (Pekkarinen 1993).This report describes a flow cytometry protocol for the detection of gill neoplasia in Macoma balthica from the Gulf of Gdansk (Baltic Sea, Poland). Flow cytometry has already been used to investigate disseminated neoplasia in molluscs (Elston et al. 1990, Moore et al. 1991, Reno et al. 1994. In the present study, a new method of cell lysis based on a hyposmotic shock has been developed and a combined comparison of the flow cytometry method, histology and chromosome analysis is reporte...