Safety during crisis: Rapid on-site evaluation at the time of COVID-19 pandemic.

The COVID-19 pandemic is posing a worldwide challenge to control and contain. SARS-CoV-2 is a highly infectious virus. Health care providers at the front lines are at high risk of getting the infection and the risk applies also to laboratory personnel as they deal with specimens that might be contaminated with infectious materiel. Cytopathology teams specifically are at high risk of dealing with contaminated material because of patients encounter during fine-needle aspiration biopsies or Rapid On-Site Evaluation (ROSE) for adequacy. In our article, we discuss alternative safer staining methods to the widely used Diff-Quick stain that can be utilized for ROSE to decrease the risk of viral exposure during the current COVID-19 pandemic.

INTRODUCTION

In late December 2019, an outbreak of a novel coronavirus disease (COVID‐19) caused by highly infectious virus (SARS‐CoV‐2) was reported in Wuhan, China. Subsequently, and due to the highly infectious nature of the virus, the disease became a pandemic in a relatively short period of time with over 1.6 million cases and 97 000 deaths worldwide (as of 10 April 2020). 1, 2, 3 In general, most patients with COVID‐19 present with mild symptoms such as fever, shortness of breath and dry cough. However, it can also be deadly due to acute severe alveolar damage and progressive respiratory failure which may require hospitalization and intensive care. The virus can easily spread from person‐to‐person interacting in close proximity through speaking, coughing, or sneezing even if the carrier is not having any symptoms. 1, 2, 3 The virus can spread through both direct (droplets) as well as indirect contact (contaminated objects and airborne aerosolized droplets transmission). Until now, it is unclear how long the virus could stay on surfaces, but preliminary studies suggest that coronaviruses including (SARS‐CoV‐2) may remain infectious on surfaces for a few hours to several days depending on the different conditions including type of surface, humidity, and temperature. 1, 2, 3

DISCUSSION

Rapid on‐Site Evaluation (ROSE) is an important part of daily cytopathology practice. In most practices, air‐dried slides stained with Diff–Quick (Romanowsky) stain are a widely accepted method for ROSE and adequacy assessment. Theoretically, air‐drying of slides carry a risk of aerosolizing infectious droplets and immediate immersion in alcohol to prepare Papanicolaou (Pap) stained smears is a safer method of fixation with lower risk of contamination. Conventional Pap staining method is time‐consuming and cannot be utilized for ROSE. Alternative methods for rapid cytology smears preparation are available and can be utilized during this pandemic to decrease the risk of viral exposure. One fast staining method is a rapid Pap stain. The staining steps of this technique are shown in Table 1.

TABLE 1

Rapid Pap staining steps

Alcohol fixation of smears

1% acetic acid: 10 dip

Hematoxylin stain: 10 dips

Tap water: 10 dips

1% acetic acid: 10 dips

OG‐6:10 dips

1% acetic acid: 10 dips

EA‐50:10 dips

1% acetic acid: 10 dips

Methanol: 10 dips

Xylene: 10 dips and then mounting media for coverslipping

Another method for rapid staining is ultrafast Pap stain which is a combination of air‐dried preparation and wet fixed Pap preparation. It includes the basics of air‐drying of cells, followed by rehydration with normal saline and fixation in alcohol. The purpose of air‐drying is to enlarge the cells and thus increase the resolution of cellular details. Rehydration of cells is achieved with normal saline so that clarity is regained plus hemolysis of the background blood. The staining steps shown in Table 2.

TABLE 2

Ultrafast Pap staining steps

Air‐drying of smears

Six slow dips under tap water

Staining with hematoxylin for 30 s

Six slow dips under tap water

Six dips in 95% Isopropyl alcohol

Staining with EA‐36 for 15 s

Six dips in 95% isopropyl alcohol

Six dips in 100% Isopropyl alcohol

Ten slow dips in xylene

Mounting media for coverslipping

In Ultrafast Pap stain, air‐drying is used to minimize the artifactual changes seen in wet fixed smears due to poor fixation plus cell loss with wet fixation is avoided. But interpretation of cytoplasmic keratinization with ultrafast Pap stain is not possible due to the omission of Orange‐G from the staining protocol. Another method for fast staining is using a rapid H&E stain. This technique is used universally in neuropathology smears and is also a widely accepted technique for staining frozen sections during intraoperative consultation. The staining steps are shown in Table 3.

TABLE 3

Rapid H&E staining steps ,

Alcohol fixation of smears

Rinse with water for 5‐7 s

Stain with hematoxylin for 60 s

Ammonia: 4‐5 dips

Eosin stain for: 20‐30 s

95% alcohol for 10 s

100% alcohol for 10 s

Xylene for 10 s

Mounting media for coverslipping

The last substitution to Diff–Quick stain that we are mentioning is to use Toluidine blue stain which is a supra‐vital stain that enhances good morphology of nuclear details and cells in a wet mount film. It is cheap, available, and used for quick reporting. It also has the flexibility of de‐staining and re‐staining with permanent stains and can be used for intra operative consultations. Toluidine blue staining process is the shortest and the steps of staining are shown in Table 4.

TABLE 4

Toluidine blue staining steps

Air‐drying of smears

Cover the slides with toluidine blue stain

Rinsed gently with tap water

Examine the slides under the microscope

CONCLUSION

In conclusion, based on the preliminary data and the current information, we know about SARS‐CoV‐2 virus and COVID‐19 disease, for ROSE, we recommend any staining technique that requires no/minimal air‐drying plus alcohol fixation. In our opinion and during the current pandemic, rapid pap stain and rapid H&E stains are preferred substitutes to Diff–Quick staining for ROSE as those two techniques require no air‐drying of smears at all plus alcohol fixation is part of the staining protocol. Ultrafast Pap stain would have the same safety profile as Diff–Quick stain because it requires air‐drying at the beginning of the process which carry the risk of aerosolizing any infectious droplets even if that will be followed by alcohol fixation of smears which helps with virus decontamination. Although Toluidine blue staining is faster than Diff–Quick staining method, it will still be the least preferred substitute as it still includes air‐drying of slides and alcohol fixation is not part of the staining process.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.