Double Staining of Elastic Fibre and Immunohistochemistry is Helpful to Differentiate Pleural Invasion of Lung Cancer

Visceral pleural invasion (VPI) is one of the critical determining factors for pathological stage of lung cancer. The pleural invasion (PL) status can be divided into four categories: PL0, PL1, PL2 and PL3, depending on the spatial relationship between the tumour tissue and outer elastic layer of pleura, visceral pleura surface and parietal pleura, respectively. Elastic stains are recommended if it is difficult to distinguish PL status by H&E staining. As reported, 19 of 100 lung cancer specimens were redefined as PL1 which were formerly deemed as PL0 by HE stain only, changing the stage from T1 (stage IA) to T2 (stage IB), which is essential for chemotherapy after surgery. Nonetheless, even assisted by elastic stains, VPI assessment is still difficult in some scenarios marked by multiple layers of elastic fibres, inflammatory infiltrates, fibrotic proliferation or carbon deposition which makes the location of scattered tumour cells barely discernible. To solve this technical problem, we developed a double staining method to show the spatial relationship between tumour cells and elastic fibres. Specifically speaking, the elastic fibres and tumour cells are stained and displayed by different colours on one slide, to determine whether the pleura is invaded. Double staining process: FormalinFixed, ParaffinEmbedded (FFPE) blocks were sectioned by thickness 3–5 um for double staining. Step one is routine immunohistochemistry staining, including: (1) antigen retrieval for 23 min in 97°C, (2) washing with distilled water, (3) 3% hydrogen peroxide solution incubated for 10 min to remove endogenous peroxidase, (4) rinsing with phosphate balanced solution (PBS) 3 min for three times, (5) add primary antibody (thyriod transcription factor1 (TTF1), and PanCK (AE1/AE3), (Zhongshan, Beijing, China)) and incubate at room temperature for 40–60 min, (6) rinsing with PBS, add the secondary antibody (Horseradish Peroxidase (HRP)) and incubate for 10–15 min at room temperature, (7) rinsing with PBS 3 min for three times and then diaminobenzidine (DAB) colour development. Second step is double dyeing of elastic fibre using a Fuxi Assay (Fuxi Bio, Guiyang, China) according to the instruction for 10–15 min, then 70% ethanol is used for counterstaining with haematoxylin. Finally, dehydrate and transparently seal after washing. TTF1 or AE1/AE3 shows a dark brown immunohistochemistry (IHC) signal, with the former exclusively nuclear stained and the latter localised to the cytoplasm. Elastic fibres are stained in purple while all the nuclei are stained blue with haematoxylin, including tumour cells, stromal cells, lymphocytes and so on. Figure 1 shows a nodule scanned in CT with slight pleural stretch, which is hard to discern in routine H&E slides since the elastic fibres are intact stained in an ambiguous pink architecture, but with the help of double staining, we can easily recognise tumour cells attached on the surface of pleura to a correct diagnosis for PL2. Figure 2 shows Correspondence