How to Mimic a Histological Sample Slide for RNAscopeTM Applications from BAL Cytological Specimens.

The recent publication by Canini et al.[1] highlights the usefulness of bronchoalveolar lavage (BAL) to provide further insights for the management of patients with COVID-19 admitted to Intensive Care Units, despite some controversial studies that appeared during the last months.[23]

Our group contributed to this study by performing in situ hybridization with RNAscopeTM technique[4] in combination with BOND-III Automated stainer (Leica Biosystems) testing cell-blocks of selected patients for SARS-CoV-2 probe (catalog number 848568; Advanced Cell Diagnostics, Newark, CA).

In November 2020, during the second wave of COVID-19 pandemic, to allow easier and safer processing of BAL samples aimed at obtaining slides suitable for RNAscopeTM analysis, we decided to improve the methodology used during the first pandemic wave and we elaborated a novel procedure.

After the macroscopic description of the BAL samples, including volume, appearance, and turbidity, we performed a fresh cell count transferring about 10 μl of sample in the Thoma cell-counting chamber, proceeding the count of the elements without red blood cells and epithelial cells.

Depending on the elements count, we then pipetted 200 μl (≤500.000 cells) or 150 μl (≥500.000 cells) in the concentrator filters of a cytocentrifuge (Statspin Cytofuge 12), then samples were centrifuged for 5 minutes at 500X rpm to spot cells on glass slides. Slides were then fixed in 10% neutral buffered formalin for 24 hours, rinsed for 10 minutes in running tap water, and then placed in distilled water.

Despite Canini et al.[1] proposed their protocol for cell-block preparation[5] we strongly suggest avoiding the passage on ethanol as suggested by the technical note of the RNAscope manufacturer's handbook[6] because this could compromise RNAscopeTM positive dots visualization [Figure 1]. Afterward, spotted cells were covered with a 4 μm thin paraffin section to mimic histologic tissue slides ready to be processed on BOND III stainer and then slides were incubated for 10-15 min at 60°C in the oven.

Figure 1

(a) Images showing positive control probes expression (Hematoxylin staining, magnification 40X). (1) Clustered brown dots in macrophages cytoplasm of high level of UBC mRNA. (2) PPIB gene expression is positive and represented by fewer clustered brown dots. (b) Images showing positive control probes expression (Hematoxylin staining, magnification 40X). (1) Clustered brown dots in macrophages cytoplasm of high level of UBC mRNA. (2) PPIB gene expression is positive and represented by fewer clustered brown dots

Using this approach, we can obtain suitable samples from BAL in a standardized, easier, and reliable manner. Using this method, the time of cell fixation is 24 hours, time needed to guarantee the high sensitivity and specificity of hybridization in situ methodology with the use of RNA and the concomitant inactivation of the SARS-CoV-2.

We, therefore, recommend using this standardized method to quickly and reliably obtain cytospins of lung-derived cells, that can be used for immunocytochemistry and RNAscopeTM investigations not only for the detection of SARS-CoV-2 proteins but also as standard procedure for the detection of other proteins, such as tumoral markers or nucleic acids for diagnostic purposes.

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