Volume-weighted mean nuclear volume and nuclear area in advanced ovarian carcinoma: An investigation of sampling methods, sample size and reproducibility

The influence of sampling issues on the reproducibility of volume- weighted mean nuclear volume (v̄(v)) and mean nuclear area (MNA) assessments in patients with International Federation of Gynecology and Obstetrics stage III and IV ovarian carcinoma was evaluated. Ten cases representing tile whole range of MNA values were selected from a population of 131 cases. The MNA and v̄(v), of the same tumor cell nuclei were determined in one session by switching between the stereologic module and the morphometric module of the video overlay program used. For both MNA and v̄(v) in one series of measurements, tumor nuclei were sampled from the whole tumor area and in a second series from the most poorly differentiated part (the measurement area) in each section, thus giving four series of measurements per case. For all four series, 500 nuclei were point sampled from approximately 100 systematically randomly selected fields of vision, using the automated scanning stage controlled by the morphometry program. These large samples, containing 500 nuclei for each case, were regarded as representative in each case. To investigate the susceptibility of MNA and v̄(v) to variance at lower sampling levels (fields, nuclei), a nested analysis of variance was performed. Then the influence of sample size and sampling method was evaluated by drawing subsets from these 500 nuclei in each case in three different ways (cluster, systematic or random) with four different sample sizes (50, 100, 125, 250). It was shown that for MNA assessed in the measurement area, the variance between patients contributed the most to the total variance. In the sample size experiment, MNA assessments with systematic sampling from all over the measurement area appeared to be reliable (coefficient of variation <5%) at a sample size of 50 nuclei, while for v̄(v) a sample size of 250 nuclei was needed. The time required for tracing 50 nuclei to assess MNA was 10 minutes and for measuring 250 intercepts for the estimation of v̄(v), 30 minutes, on average. With a sample size of 100, intraobserver and interobserver reproducibility for MNA were better than for v̄(v).