Histopathological evaluation
After surgery, all SLNs and other surgical specimens were fixed in 10% neutral-buffered formalin, cut into 2-mm blocks, and embedded in paraffin. Sections (4 µm) obtained from each block of lymph nodes were then submitted for a routine pathological assessment after haematoxylin and eosin staining (HE), examined by pathologists specialising in gynaecological oncology. False-negative cases were defined as the hemi-pelvis being negative for SLNs but positive for non-SLNs in the same pelvic side. Since the goal of our study was not to provide deeper insight into the problems associated with pathologic ultrastaging on low-volume metastasis, if an SLN had metastasis in the initial HE-stained section, no further work-up was performed on that lymph node. If SLN was negative in the initial section, but with metastasis in non-SLN, then the ideal ultrastaging protocol was carried out on the whole SLN, which included consecutive sections (4 μm) obtained in regular 150-μm intervals, and immunohistochemical staining using antibodies against cytokeratins (AE1/AE3, 1:50 dilution; Dako, Glostrup, Denmark) to identify micrometastasis14. For other cases with SLNs that were metastasis-free in the initial HE, an additional simple ultrastaging protocol was performed to examine the SLN for low volume metastatic disease: two consecutive sections (4-μm thick) in regular 150-μm intervals and six additional sections from each block were stained with HE and immunohistochemical staining (Figure 2)15.