Influence of section thickness, mean nuclear diameter and nuclear crowdingon DNA ploidy in histologic sections of melanocytic skin lesions

OBJECTIVE: To determine the influence of section thickness, nuclear diamete r (MND) and area percentage of nuclei (a measure of nuclear crowding) on hi stologic DNA ploidy assessed by image cytometry (ICM) of primary melanocyti c skin neoplasms (MSNs). STUDY DESIGN: Initially a feasibility study wns per formed to determine if comparable DNA ploidy histograms could be obtained from cell disaggregates and tissue sections. Following this, DNA ICM was performed oil Feulgen-stai ned tissue sections (4, 6, 8 and 10 mu m thick) from 30 primary MSNs (20 be nign, 10 malignant) with nuclear diameters from 5.6 to 8.6 mu m. Area perce ntage of nuclei was assessed in all cases at all section thicknesses. RESULTS: The feasibility study produced comparable results far cytocentrifu ge and tissue section preparations. For sectioned MSNs, DNA ploidy histogul zms from 4-mu m sections had a higher coefficient of variation of the 2c pe ek than those from 6-, 8- and 10-mu m sections. Ten-micrometer sections had marked overlapping of nuclei, and only small numbers of cells could be mea sured, giving inadequate results. MND and area percentage of nuclei did not have an important influence on the results. CONCLUSION: Adequate DNA ploidy profiles can be obtained by DNA ICM on 6- a nd 8-mu m-thick histologic sections of MSNs, provided that a strict measure ment protocol is followed.