Autostainer link 48

IHC analysis was performed in paraffin-embedded tissue samples using CD45 antibodies (IR75161-2, Agilent Technologies) and CXCR4 antibodies (ab124824, Abcam). IHC staining was performed in an Autostainer Link 48 (Agilent Technologies), following the manufacturer’s instructions. AML organ infiltration by CD45 and CXCR4 expression was evaluated and quantified using an Olympus BX53 microscope and Image J Fiji software v.1.8.0.172 (NIH), choosing six fields randomly for each tissue. The results were expressed as stained total area ± SEM.

After the pathologist reached a diagnosis, the number of tumor cells was counted manually by one cytoscreener and one pulmonologist in a blinded manner using HE staining slides. Then, the average number of tumor cells was calculated.
After sectioning of samples to 4–5 μm, PD-L1 staining was performed with 22C3 antibodies (rabbit monoclonal, clone 22C3; Agilent Dako, Glostrup, Denmark) using autostainer (Autostainer Link 48, Agilent Dako). PD-L1 positivity was defined as membranous staining in at least 1% of cells [10 (link)], regardless of staining intensity and proportion in the membrane. PD-L1 was evaluated by experienced pathologist, and the cut off values were classified as ≥50% and ≥ 1%. The number of tumor cells by a single biopsy, total number of tumor cells, average number of tumor cells, and PD-L1 expression for each patient were compared between Cryo and TBB.

Deparaffinised and rehydrated tissue sections were processed for epitope retrieval using EnVision FLEX target retrieval solution (Agilent, K8004), as per manufacturer’s instructions. Samples were processed and stained for TRIM22 (Proteintech, 13744-1-AP; 1/50 dilution) and FLEX haematoxylin (Agilent, K8008) using a Autostainer Link 48 (Agilent), as per manufacturer’s instructions. TRIM22 antibody specificity was validated by staining sectioned MRC5 (positive control) or A549 (negative control) cell pellets. Tissue sections were independently assessed for TRIM22 expression by a qualified pathologist. Automated quantitation of TRIM22 expression levels was performed using whole-slide scans and Image-Pro Premier (Media Cybernetics), as previously described (Leeming et al., 2015 (link); Akram et al., 2018 (link)).

Immunohistochemical assays were carried out using 4-μm-thick paraffin sections in an automatic immunostainer (Autostainer Link 48; Agilent, Santa Clara, CA, USA) equipped with a 2-step immunohistochemical staining system (EnVision FLEX/HPR; Dako, Glostrup, Denmark) that uses a peroxidase-labelled polymer conjugated to the secondary antibody. Before the immunostainer, the samples were treated for antigenic retrieval according to the manufacturer’s protocol in the pre-treatment module (PT link, Dako; Agilent). Samples were incubated with a primary antibody against TDG (thymine-DNA glycosylase) (polyclonal, pH6, dilution: 1:100, 40 min, Sigma Life Science, St Louis, MO, USA). Nonimmune serum samples were substituted for the primary antibodies as negative controls. Normal colonic mucosa was used as a positive control.

The same cell staining reagents and the same reaction time were used for the CTC-FIND method and the RareCyte method. Specifically, cell staining was performed using a proprietary custom reagent kit developed by RareCyte. This included the following: 4′,6-diamidino-2-phenylindole (DAPI) to stain the cell nucleus) and antibodies specific for the epithelial marker CK, the epithelial marker EpCAM, and the cell surface antigen CD45/CD66b (used for staining leukocytes). We modified the assay by adding additional counter-stain reagent with the following phycoerythrin (PE)-conjugated antibodies: anti-CD14-PE (Clone M5E2, 1:200, BioLegend, San Diego, CA, USA), anti-CD34-PE (Clone 581, 1:200, BioLegend, San Diego, CA, USA), and anti CD11b-PE (Clone M1/70, 1:200, BioLegend, San Diego, CA, USA). For the CTC-FIND method, all staining was done with the slit-filter isolation unit of CTC-FIND. For the RareCyte method, all staining was done with the AutoStainer Link 48 (Agilent Technologies, Santa Clara, CA, USA) and according to manufacturer’s protocol.

In melanoma samples, PD-L1 expression was assessed using the Dako Omnis platform (Agilent, Santa Clara, CA) and the 28–8 pharmDx antibody (Agilent, Santa Clara, CA), which is the FDA-approved complementary diagnostic for nivolumab. For RCC and NSCLC samples, the 22c3 pharmDx antibody (Agilent, Santa Clara, CA) was employed on Autostainer Link 48 (Agilent, Santa Clara, CA), which is the FDA-approved companion diagnostic for pembrolizumab. Established cutoffs for the diagnostics in each histologic type were used to score PD-L1 IHC tumor proportion score (TPS) and immune cell staining (ICS) as follows: melanoma TPS, 1%, [13 ] NSCLC TPS, 50 and 1%, [7 ] RCC TPS, 1%; RCC ICS, 1%.