The application of design-based stereological methods for estimating nuclear features quantitatively in invasive ductal breast cancer is described. Nuclear number, size and size variability are explored in relation to the tumour grade and patient prognosis. The study includes an examination of the efficiency in estimating different nuclear volumes, and two different estimators of the nuclear size variability are contrasted. Forty-two invasive ductal breast carcinomas diagnosed and graded by two pathologists were used. Both 5-microns and 25-microns-thick sections were obtained from paraffin blocks for stereological study. More undifferentiated tumours show significantly larger nuclei than low-grade tumours. The estimates based on the disector method demonstrate a decrease in the number of tumour cell nuclei per unit volume of tissue from grades 1 to 2 and especially from grades 2 to 3. The univariate survival analysis shows a high prognostic value of the nuclear volume estimates. The study shows that an efficient sampling procedure was performed, particularly when estimating volume-weighted mean nuclear volume using the point-sampled intercepts method. This method is more efficient than estimation of the number-weighted mean nuclear volume using the selector method; however, the latter provides paired estimates of volume- and number-weighted mean nuclear volume, as well as an estimate of the coefficient of variation of nuclear volume in the number distribution of the same cells.
A new stereological method is proposed which combines vertical slice projections with the fractionator to estimate the total capillary length in a skeletal muscle. The method was demonstrated on the soleus muscle of a Wistar rat. The implementation required capillary highlighting, tissue sampling, and data acquisition in the form of intersection counts between capillary projections and cycloid test lines. The capillaries were demonstrated using vascular perfusion (with gelatine) of the hind leg of the rat. The sampling procedure followed the fractionator design, namely a multistage systematic sampling design with a known sampling fraction at each stage. To make the design unbiased, vertical slices were used ; for efficiency, the vertical axis was chosen parallel to the main axis of the muscle. As prescribed to avoid bias, the cycloid test lines were superimposed on the slice projections, viewed under the light microscope, with their minor axes normal to the vertical axis. The estimation precision was compared for different sampling and subsampling fractions. The proposed method was globally highly efficient, unbiased, and easy to implement.
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