Journal of Cytology
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 Table of Contents    
IMAGES IN CYTOPATHOLOGY  
Year : 2021  |  Volume : 38  |  Issue : 4  |  Page : 231-232
How to mimic a histological sample slide for RNAscopeTM applications from BAL cytological specimens


1 Department of Pathology, Papa Giovanni XXIII Hospital, Bergamo, Italy
2 Department of Molecular Medicine, University of Padova, Padova, Italy

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Date of Submission30-Mar-2021
Date of Decision06-Apr-2021
Date of Acceptance03-Oct-2021
Date of Web Publication12-Nov-2021
 

How to cite this article:
Morotti D, Caroli E, Forlani V, Cadamuro M, Gianatti A. How to mimic a histological sample slide for RNAscopeTM applications from BAL cytological specimens. J Cytol 2021;38:231-2

How to cite this URL:
Morotti D, Caroli E, Forlani V, Cadamuro M, Gianatti A. How to mimic a histological sample slide for RNAscopeTM applications from BAL cytological specimens. J Cytol [serial online] 2021 [cited 2021 Nov 29];38:231-2. Available from: https://www.jcytol.org/text.asp?2021/38/4/231/330390




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.[2],[3]

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

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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.

Financial Support and Sponsorship

Nil.

Conflicts of Interest

There are no conflicts of interest.



 
   References Top

1.
Canini V, Bono F, Calzavacca P, Capitoli G, Foti G, Fraggetta F, et al. Cytopathology of bronchoalveolar lavages in COVID-19 pneumonia: A pilot study. Cancer Cytopathol 2021;129:632-41.  Back to cited text no. 1
    
2.
Wang W, Xu Y, Gao R, Lu R, Han K, Wu G, et al. Detection of SARS-CoV-2 in different types of clinical specimens. JAMA 2020;323:1843-4.  Back to cited text no. 2
    
3.
Geri P, Salton F, Zuccatosta L, Tamburrini M, Biolo M, Busca A, et al. Limited role for bronchoalveolar lavage to exclude COVID-19 after negative upper respiratory tract swabs: A multicentre study. Eur Respir J 2020;56:2001733.  Back to cited text no. 3
    
4.
Wang F, Flanagan J, Su N, Wang L-C, Bui S, Nielson A, et al. RNAscope: A novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 2012;14:22-9.  Back to cited text no. 4
    
5.
Aisagbonhi O, Birungi A, Atwine R, Behayo P, Ayebaziwe B, Roberts D, et al. Modified plasma-thrombin method of cell block preparation for fine-needle aspiration biopsies in resource-limited settings. Am J Clin Pathol 2018;150:137-45.  Back to cited text no. 5
    
6.
Preparing FFPE Cell Pellets for the RNAscopeTM and BaseScopeTM Assays, ACD Technical Note, SOP 45-008/Rev A/Draft Date 06062017.  Back to cited text no. 6
    

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Correspondence Address:
Dr. Denise Morotti
ASST Papa Giovanni XXIII, Piazza OMS 1, 24127 Bergamo
Italy
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JOC.JOC_61_21

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