Multiplex fluorescence in-situ hybridisation testing for anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) gene rearrangements on cytology smears in lung adenocarcinomas: comparative study with formalin fixed paraffin embedded sections

Background: Anaplastic lymphoma kinase (ALK) immunohistochemical staining on formalin-fixed paraffin-embedded tissue or cell blocks (CB) has been reported as an effective alternative to fluorescence hybridization in situ (FISH) for the detection of ALK gene rearrangement. However, CB frequently lack adequate cellularity even when the direct smears are cellular. This study aims to assess the utility of ALK immunocytochemical (ICC) staining on direct smears using the cell transfer (CT) technique for the detection of ALK rearrangement. Methods: Fine-needle aspiration (FNA) cases of lung adenocarcinoma in which the ALK status had been determined by FISH on CB or a concurrent biopsy were identified. ICC staining for ALK was performed on alcohol-fixed Papanicolaou-stained direct smears using the CT technique. ALK immunoreactivity was evaluated using a modified semiquantitative scale. Results were compared with those of FISH. Results: A total of 47 FNA specimens were included. Five of 7 FISH-positive cases showed positive ALK ICC staining (71.4%), and 39 of 40 FISH-negative cases were negative on ALK ICC staining (97.5%). The overall correlation between ALK ICC and FISH was 93.6%. Conclusion: ICC performed on FNA smears using the CT technique is an alternative method for the assessment of ALK rearrangement, especially when CB lack adequate cellularity.

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Development of immunohistochemistry and in situ hybridisation for the detection of SARS-CoV and SARS-CoV-2 in formalin-fixed paraffin-embedded specimens

Scientific Reports ◽

10.1038/s41598-020-78949-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Fabian Z. X. Lean ◽

Mart M. Lamers ◽

Samuel P. Smith ◽

Rebecca Shipley ◽

Debby Schipper ◽

...

Keyword(s):

Animal Models ◽

In Situ Hybridisation ◽

Virus Antigen ◽

Control Material ◽

Live Virus ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Infected Cells ◽

Formalin Fixed

AbstractThe rapid emergence of SARS-CoV-2, the causative agent of COVID-19, and its dissemination globally has caused an unprecedented strain on public health. Animal models are urgently being developed for SARS-CoV-2 to aid rational design of vaccines and therapeutics. Immunohistochemistry and in situ hybridisation techniques that facilitate reliable and reproducible detection of SARS-CoV and SARS-CoV-2 viral products in formalin-fixed paraffin-embedded (FFPE) specimens would be of great utility. A selection of commercial antibodies generated against SARS-CoV spike protein and nucleoprotein, double stranded RNA, and RNA probe for spike genes were evaluated for the ability to detect FFPE infected cells. We also tested both heat- and enzymatic-mediated virus antigen retrieval methods to determine the optimal virus antigen recovery as well as identifying alternative retrieval methods to enable flexibility of IHC methods. In addition to using native virus infected cells as positive control material, the evaluation of non-infected cells expressing coronavirus (SARS, MERS) spike as a biosecure alternative to assays involving live virus was undertaken. Optimized protocols were successfully applied to experimental animal-derived tissues. The diverse techniques for virus detection and control material generation demonstrated in this study can be applied to investigations of coronavirus pathogenesis and therapeutic research in animal models.

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