Assessing Lymph Node Metastasis

Lymph node samples were cut in 4 equal slices (a, b, c, d) with a special cutting device.18 Two of these slices (a&c) were snap-frozen in liquid nitrogen and stored at −80°C until OSNA analysis was performed. The remaining 2 slices (b&d) were fixed in 4% buffered formaldehyde and embedded in a single paraffin block for histological examination at 5 levels since this was the standard in-house method for sentinel node investigation in both breast cancer and melanoma patients (Fig. 1).20 (link)
Figure 1

Study design: Histology versus OSNA. 346 lymph node samples were cut into 4 pieces. Slices “a” and “c” were subjected to the OSNA method, slices “b” and “c” to histological work-up consisting of 5 levels of H&E, CAM5.2 and CK19 staining. In 120 histologically negative lymph node samples, as determined by the 5 level method, the remainder of the block was completely cut into further levels to assess specificity. 18 samples with differing results as obtained by the 2 methods (discordant cases) were also cut into further levels.

In 346 lymph node samples concordance, sensitivity and specificity were determined based on the comparison of these 2 methods. To investigate whether these figures might be influenced by a sampling bias caused by limited investigation of the material the histologic work-up was extended to all levels in the first 120 histologically negative lymph node samples. The same was done for paraffin blocks of discordant cases. In addition, the homogenised lymph node lysates of samples with discordant OSNA versus histology results were subjected to quantitative reverse-transcriptase polymerase chain reaction (QRT-PCR) and Western Blot analysis. In case these investigations yielded a result compatible with a positive OSNA result these samples were excluded from the final analysis because of a strong indication for sampling bias.