Neoplasia detection in Macoma balthica from the Gulf of Gdansk: comparison of flow cytometry, histology and chromosome analysis

Abstract

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.