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

OBJECTIVE

To determine the influence of section thickness, nuclear diameter (MND) and area percentage of nuclei (a measure of nuclear crowding) on histologic DNA ploidy assessed by image cytometry (ICM) of primary melanocytic skin neoplasms (MSNs).

STUDY

DESIGN

Initially a feasibility study was performed to determine if comparable DNA ploidy histograms could be obtained from cell disaggregates and tissue sections. Following this, DNA ICM was performed on Feulgen-stained tissue sections (4, 6, 8 and 10 microns thick) from 30 primary MSNs (20 benign, 10 malignant) with nuclear diameters from 5.6 to 8.6 microns. Area percentage of nuclei was assessed in all cases at all section thicknesses.

RESULTS

The feasibility study produced comparable results for cytocentrifuge and tissue section preparations. For sectioned MSNs, DNA ploidy histograms from 4-micron sections had a higher coefficient of variation of the 2c peak than those from 6-, 8- and 10-micron sections. Ten-micrometer sections had marked overlapping of nuclei, and only small numbers of cells could be measured, 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- and 8-micron-thick histologic sections of MSNs, provided that a strict measurement protocol is followed.