Reply to Rapid on-site evaluation and the COVID-19 pandemic.

We thank Cozzolino et al for their comments and interest in our article. The medical professionals who perform fine‐needle aspirations or rapid on‐site evaluation (ROSE) are believed to be at relatively high risk for exposure to coronavirus disease 2019 (COVID‐19). Cozzolino et al shared their internal protocol for biosafety during ROSE, which generally was consistent with the procedures and personal protective equipment recommended in our recent publication.

It is interesting to note that Cozzolino et al kindly provided details of their approach to using air‐dried smears during ROSE, which was not mentioned in our article but deserves some discussion. During ROSE, Cozzolino et al described using air‐dried smears fixed in a methanol‐based solution (MGG Quick Stain Reagent A; Bio‐Optica) for 20 to 40 seconds, but they have extended the fixation time to 60 to 90 seconds to increase the potential disinfectant effect of the fixative. According to the public safety data sheet from Bio‐Optica, MGG Quick Stain Reagent A contains at least 80% methanol. Because fixation with 100% methanol for at least 5 minutes was able to inactivate severe acute respiratory syndrome coronavirus (SARS‐CoV), and fixation with 50% methanol took at least 60 minutes to inactivate Middle East Respiratory Syndrome coronavirus (MERS‐CoV), we cannot conclude that fixation with 80% methanol for 90 seconds would be entirely adequate for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). However, a higher concentration of methanol or ethanol would likely dehydrate cells and could result in cellular distortion, especially with longer fixation times. This leads to a potential dilemma regarding the use of air‐dried smears.

It is interesting to note that Cozzolino et al mentioned that for Papanicolaou staining, they would use a prolonged fixation with 95% alcohol for over 30 minutes for biosafety purposes. It has been shown that fixation with ethanol (78%‐95%) for at least 30 seconds will inactivate SARS‐CoV with a ≥4.3 log10 reduction of viral titer in suspension tests. Because the first step of standard Papanicolaou staining is to fix slides in 95% alcohol for 15 minutes, we believe that the standard protocol for Papanicolaou staining adequately inactivates SARS‐CoV‐2. In addition, Cozzolino et al shared that they have extended the formalin fixation time for cell blocks to at least 24 hours. A well‐fixed sample is definitely necessary both for inactivation and for good preservation of morphological structures. Formalin is a 37% aqueous solution of formaldehyde and 10% neutral buffered formalin (ie, 3.7% formaldehyde); Kumar et al reported that a minimum 30‐minute treatment time at room temperature with either 10% neutralized formalin or 4% paraformaldehyde would be adequate to ensure complete inactivation of MERS‐CoV–infected cultured cells. The aldehyde cross‐linking is required to inactivate a virus particle. Kariwa et al found that fixation of SARS‐CoV–infected cells with a fixative including 100% acetone, 100% methanol, 3.5% paraformaldehyde, and 2.5% glutaraldehyde for ≥5 minutes eliminated all infection. Table 1 summarizes the published data for coronavirus infectivity using different fixatives. , ,

TABLE 1

Summary of the Inactivation of Coronaviruses by Different Types of Fixatives , ,

Fixatives	Viral Type	Method	Time for Inactivation
10% Formalin	MERS‐CoV	Cell culture	≥30 min, no infectivity
4% Paraformaldehyde	MERS‐CoV	Cell culture	≥30 min, no infectivity
Methanol: acetone (50:50)	MERS‐CoV	Cell culture	≥60 min, no infectivity
100% Acetone	SARS‐CoV	Cell culture	≥5 min, no infectivity
100% Methanol	SARS‐CoV	Cell culture	≥5 min, no infectivity
3.5% Paraformaldehyde	SARS‐CoV	Cell culture	≥5 min, no infectivity
2.5% Glutaraldehyde	SARS‐CoV	Cell culture	≥5 min, no infectivity
Ethanol 78%, 80%, 85%, and 95%	SARS‐CoV	Suspension tests	30 s, ≥4.3 log10 reduction
Ethanol 80%	MERS‐CoV	Suspension tests	30 s, >4 log10 reduction
Ethanol 70%	MHV, CCV	Suspension tests	10 min, >3.7 log10 reduction
Formaldehyde 1%, 0.7%	SARS‐CoV	Suspension tests	2 min, >3 log10 reduction
Formaldehyde 0.7%	MHV	Suspension tests	10 min, >3.5 log10 reduction
Formaldehyde 0.7%	CCV	Suspension tests	10 min, >3.7 log10 reduction
Formaldehyde 0.009%	CCV	Suspension tests	24 h, >4 log10 reduction
Glutaraldehyde 0.5%, 2.5%	SARS‐CoV	Suspension tests	2 min, >4 log10 reduction

Abbreviations: CCV, canine coronavirus; MERS‐CoV, Middle East Respiratory Syndrome coronavirus; MHV, mouse hepatitis virus; SARS‐CoV, severe acute respiratory syndrome coronavirus.

We agree with Cozzolino et al that the performance of ROSE during the COVID‐19 pandemic should be discouraged to reduce exposure. The preparation of air‐dried smears during ROSE is best performed using a certified class II biosafety cabinet, especially for airway samples. In addition, the concentration of fixatives, characteristics of the samples, and fixation times all are important aspects for COVID‐19 inactivation in cytology samples.

Funding Support

No specific funding was disclosed.

Conflict of Interest Disclosures

The authors made no disclosures.