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.