COUNTING OF APOPTOTIC CELLS - A METHODOLOGICAL STUDY IN INVASIVE BREAST-CANCER

Aims-To arrive at a reproducible sampling technique for counting apopt otic cells in tissue sections of invasive breast cancer that can serve as a protocol for further clinical studies. Methods-In 4 mu m thick h aematoxylin and eosin stained tissue sections of 12 breast cancers, ap optotic cells, recognised by strict morphological criteria, were count ed in consecutive fields of vision at x1000 magnification in a marked area in the most poorly differentiated region of tumour. These counts were regarded as the gold standard. Subsequently, in a systematic samp ling experiment, the number of fields that had to be counted to derive an acceptable coefficient of variation (CV) was determined. To compar e counts at different magnifications, all fields of vision were also c ounted at x630 and x400. The intra- and inter-observer reproducibility was tested by repeated measurements at these magnifications in 10 sys temically selected fields of vision. Results-Apoptosis seemed to be a rare event, affecting, on average, about 1% of tumour cells. Noticeabl e clustering of apoptotic cells was observed. The systematic sampling experiment showed that at x1000 magnification, the CV was improved by counting up to 20 fields. When comparing x400 and x630 magnifications with the x1000 magnification, the correlation coefficients were 0.88 a nd 0.87, respectively. However, the lower magnifications yielded lower counts. With regard to reproducibility, the intra-observer correlatio n coefficient was 0.91 at x630 and 0.76 at x400. The inter-observer co rrelation coefficient was 0.77 at x630 and 0.68 at x400. Conclusions-A poptotic cells can be counted readily in haematoxylin and eosin staine d tissue sections. However, a systematic sampling protocol must be fol lowed and cells should be counted at a relatively high magnification t o obtain acceptable reproducibility. The suggested protocol will permi t further correlative and prognostic studies and the monitoring of the effects of treatment.