A Narrative Review of Lung Cancer Cytology in the Times of Coronavirus

What Physicians Should Know

Pasquale Pisapia; Umberto Malapelle; Maria Salatiello; Rafael Rosell; Giancarlo Troncone

Disclosures

Transl Lung Cancer Res. 2020;9(5):2074-2081. 

In This Article

Cyto-preparation: Biosafety Procedures

According to the international consensus, SARS-CoV-2 is classified as a hazard group 3 organism, being able to cause severe human diseases, representing a serious hazard to employees and community and currently lacking effective prophylaxis and treatment.[12–15] Processing pulmonary cytological specimens, including sputum, bronchoalveolar lavage (BAL) and washing, fine needle aspiration (FNA) and pleural effusion, requires different levels of laboratory biosafety measures (Figure 1A)[15,16] These should be able to mitigate the not negligible risk of handling fluids and cells potentially carrying viable and thus transmissible SARS-CoV-2.[16] However, cytopathology diagnostic activities are less dangerous than viral culture, isolation or neutralization assays; thus, procedures equivalent to biosafety level (BSL) 2 are generally sufficient.[14,15,17] As a matter of the facts, pleural effusions were processed during the healthcare emergency in our laboratory without reduction.[18] Nevertheless, the risk assessment depends on whether cytological samples are properly fixed or are unfixed and fresh. Usually cytological fluids, received unfixed at the laboratory, are mixed with >70% alcohol solution in a range from 1:1 to 2:1; the virus is inactivated and these specimens can be considered as a low-risk samples and processed according to good microbiological practices and procedures (GMPP).[19] These latter include the use of appropriate disinfectants with proven activity [including, hypochlorite (bleach), alcohol, hydrogen peroxide and phenolic compounds] against SARS-CoV-2 in any laboratory area and surface.[16,19] However, immediate fixation may not well preserve morphological details; to improve cyto-preparation quality, cytological fluids can be centrifuged fresh; then, the cell pellet is used to prepare smears, cytospins and cell blocks.[20] These procedures carried out on fresh and unfixed cells, however, require appropriate personal protective equipment (PPE), to be worn by all laboratory personnel handling these specimens and a number of more stringent BSL-3 measures.[21] Centrifuges should have safety buckets or sealed rotors and should be placed in a biosafety cabinet (BSC). This is key to contain aerosols and respiratory droplets.[14] Any procedure initial processing (before inactivation) of all specimens, should take place in an appropriately maintained and validated BSC, in particular when there is the potential to generate aerosols such as slides smearing and air-drying, removing tube caps, blending, shaking, mixing, vortexing, pipetting, aliquoting, and diluting.[9,17] In particular, class II BSC are preferable since provide not only personnel and environmental protection but also product protection.[9,17] As an alternative class I BSC, frequently used in pathology laboratories, with high efficiency particulate air (HEPA) filters are also appropriate for risk mitigation.[9,17] Cytological samples should be delivered by hand rather than by pneumatic tube systems; in fact, pneumatic pods propelled by air pressure may generate aerosols in the case of sample leaking and specimens from suspected SARS-CoV-2 positive patients require a triple leak-proof package.[22]

Figure 1.

In the times of COVID-19 pandemic, processing pulmonary cytological specimens requires different levels of laboratory biosafety measures (A). As a matter of the facts, rapid on site evaluation, usually performed by using Diff Quik staining (B), and the different cyto-preparations (C) protocols should be modified to prevent the transmission of viable SARS-CoV-2, before microscopic evaluation (D). Also molecular pathology required reshaping and reorganizing of the genotyping workflow, to guarantee laboratory staff security. In particular, to reduce hands-on work and to limit the amount of time spent by the laboratory staff to process lung cancer samples (E), timely procedures (F,G) have been replaced by fully automated technologies (H). (Credit: Created with Biorender). COVID-19, coronavirus disease 19; SARS-CoV-2, syndrome coronavirus 2.

Persistence of viable and thus transmissible SARS-CoV-2, due to suboptimal fixation of cells obtained from the oropharyngeal and respiratory tract and processed as LBC samples, represents an additional relevant biosafety issue that needs to be mitigated.[23,24] Indeed, LBC methodology is widespread in many laboratories and very often used to prepare respiratory cytological specimens. The reasons are several. First, respect to conventional smears, that may display large amounts of obscuring mucus and/or blood, in the LBC preparations, cells are rinsed in fixative solutions rich in mucolytic and haemolytic agents obtaining slides with a clean background, devoid of obscuring blood and exudates elements.[25] Secondly, when a pathologist is not available to prepare on site, LBC simplifies the handling of cytological material, avoiding the need of training interventional radiologists and/or bronchoscopists to prepare high-quality smears.[26] Thirdly, LBC limits the exposure to potentially infected fresh cells obtained from pulmonary lesions, as the cells, instead of being smeared, are flushed into a low alcoholic concentration collection medium such as PreservCyt and CytoLyt (Hologic, Inc, Marlborough, MA) and SurePath (BD, Franklin Lakes, NJ) and processed on automated devices.[24] Unfortunately, it is uncertain whether these weak alcohol solutions, are adequately inactivating the virus thus that additional precautions, like the use of gloves, may be indicated when preparing and observing LBC slides.[24] In order to overcome this issue, a new virus-inactivating method was suggested by the Hologic for all LBC specimens.[24] This novel method is based on a preliminary fixation step in >70% ethanol, then followed by centrifugation and sequential cell pellet suspension first in CytoLyt and then in PreservCyt.[24] This modified technique might increase the amount of fibrin in the background, especially for fine-needle aspiration biopsies, probably related to the sudden fixation of the haemorrhagic material in a large volume of ethanol.[24] Due to the increased ethanol concentration, the cells are also smaller and more scattered than in samples processed with the original technique. Although, the distinction between normal, reactive, and atypical cells may be on occasions more difficult, the nuclear details of the neoplastic cells are generally well preserved and the immune reactivity of the majority of the cells is also maintained.[24]

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