Diagnostic accuracy of fine needle aspiration cytology in comparison to open biopsy for lesions of oral cavity in COVID-19 era

Objective: To compare the diagnostic accuracy and uses of fine needle aspiration cytology with histopathology in the diagnosis of lesions of oral cavity. Method: The single-centre, prospective, non-randomised controlled trial was conducted at the departments of Oral and Maxillofacial Surgery and Pathology of Allama Iqbal Medical College/Jinnah Hospital, Lahore, Pakistan, from January 10 to August 10, 2021, and comprised patients of either gender aged >10 years having superficial or deep well-established mass, palpable, nodular, ulcerative lesions, swellings of the oral cavity or of major or minor salivary glands having intraoral presence or manifestation with size >1 cm. Data was analysed using SPSS 20. Results: Of the 43 patients, 22(51.2%) were males and 21(48.8%) were females. The overall mean age was 40.5±15 years (range 13-70 years). On cytological examination, 6(14%) samples were excluded for being insuﬃcient aspirate. Of the remaining 37(86%) samples, 20(51.4%) were detected with malignancy on histopathology. The accuracy of fine needle aspiration cytology was 33(89.19%). Conclusion: Fine needle aspiration was found to be a reliable and cost-eﬀective technique in the diagnosis of oral cavity lesions.


Introduction
The term biopsy has its origin from the Greek words 'bios' , meaning life, and 'opsis' , meaning a sight.It was first conceived in 1879. 1 It involves the removal of a small piece of a tissue from the body of a living organism to examine it under the microscope or some form of magnification. 1 But the history of the origin of the technique in literature dates back to the 10 th century when Arab physician Abulcasis (1013-1107) used a needle puncture on thyroid to differentiate between various forms of goiter. 1 The modern way of removing the tissue by aspiration by using fine needles is attributed to Martin and Ellis who founded this method in early 20 th century. 1 In the later part of the same century, the Swedish School of Cytopathology embraced this technique and extended to the world. 1 There are various ways to perform these biopsies.4][5] Incisional or excisional biopsy for the purpose of definite diagnosis are considered the gold standard. 3,4But the drawback is that it causes a lot of discomfort to the patient and may produce unwanted bleeding due to the vascularity of the oral mucosa. 3,4It is also a more invasive method requiring time to perform. 3,4here is a significant difference in the cost and processing time when compared to a fine needle aspiration cytology (FNAC), [3][4][5] resulting in delays in the planning of definitive treatment. 3,4It is very important to select the most appropriate site for the biopsy and requires proper skills to perform the procedure in the oral cavity. 3,4When the lesions are large and located in areas with poor accessibility, surgeons face difficulties in collecting the most appropriate specimen. 3,4Moreover, sometimes a diagnostician is also reluctant to interrupt an encapsulated tumour to prevent an extra-capsular spread. 3,4There are cases where it is not possible to even carry out biopsy as the patients may be medically compromised, using anticoagulants, or have limited mouth opening that limits the access. 3,4Also, some patients with asymptomatic lesion may not give their consent for biopsy. 3,4There are times when patient needs to be hospitalised before or after the procedure, and subsequent emergency visits with complaints of bleeding from the biopsy site is a routine experience. 3,4The high patient turnover in the public facilities of developing and under-developed countries causes delays in getting an appointment for biopsy and then in reporting of histopathological diagnosis, which allows the disease to progress to a more advanced stage, and even increasing morbidity and mortality in patients. 3,4,63][4] It is commonly used as a diagnostic modality in the diseases of the salivary glands and in the diagnosis of lumps in the body, including the head and neck region. 3,4,7But the technique is not a common practice for the diagnosis of lesions of oral cavity, where biopsy and histopathological diagnosis is the gold standard. 3,4,7It could be because of inexperience, variation or heterogeneity in the nature of lesions with respect to their anatomic locations, and the inconvenience in fixing the lesion for aspiration. 3,4There are very few studies available correlating the accuracy of cytological diagnosis with the histological one using FNAC and incisional or excisional biopsy for the lesions of oral cavity. 4,7They have advocated that FNAC can be used as an alternative procedure. 4,5,7e coronavirus disease-2019 (COVID-19) has increased the risk of contracting the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) among oral and maxillofacial surgeons as the specialty deals in the region where the viral load is significant. 8Removal of a specimen along with the achievement of haemostasis requires time and is a hazard for operating surgeons and their teams.When FNAC is compared with open biopsy, it takes less time and minimal aerosol generation. 3,43][4] If the accuracy mentioned in other studies is considered acceptable, the use of the technique on an emergency basis to evaluate and diagnose cases of malignancy or oral pathologies requiring management should be a priority. 3,4nother advantage is that it can be performed quickly with less exposure time for the surgeons and least discomforting to the patients with low cost and burden on any country with a struggling healthcare system. 3,4,8e current study was planned to compare the diagnostic accuracy and uses of FNAC with histopathology in the diagnosis of lesions of oral cavity.It was hypothesised that FNAC will fail to accurately diagnose lesions of oral cavity when compared to histopathological diagnosis.

Patients and Methods
The single-centre, prospective, non-randomised controlled trial was conducted at the departments of Oral and Maxillofacial Surgery and Pathology of Allama Iqbal Medical College/Jinnah Hospital, Lahore, Pakistan, from January 10 to August 10, 2021.The sample was raised using non-probability purposive sampling technique.The patients were enrolled from the outpatient department (OPD) of Jinnah Hospital, Lahore, after sample size was calculated using confidence level of 95% and margin of error 5%.Those included were patients of either gender aged >10 years having superficial or deep well-established mass, palpable, nodular, ulcerative lesions, swellings of the oral cavity or of major or minor salivary glands having intraoral presence or manifestation with size >1cm, and having no contraindications for performing fine-needle aspiration or an excisional or incisional biopsy. 3,4Those excluded were pregnant women, children age 10 years or below, patients with the bleeding disorders or on anticoagulant therapy. 3,4Patients who refused to participate or were uncooperative were also excluded, and so were those with superficial flat lesions, like macules, papules, port-wine lesions and vascular lesions.
Approval was obtained from the institutional ethics review committee, and informed consent was taken from all the participants.All demographic details and data of detailed clinical examination were collected.Bias and confounders were controlled by exclusion criteria.All baseline investigations were done for surgical procedure, including reverse transcription polymerase chain reaction (RT-PCR) for COVID-19. 9Clinically, swellings, exophytic growths and lesions were examined and findings were documented. 3,4amples were collected using intraoral approach. 3,4,5Slides were marked by patient name and identification number.FNAC was performed by disinfecting the oral cavity by using chlorhexidine gluconate 0.12-0.2%oral rinses commercially available. 10A 22-23 gauge needle connected to a 5cc disposable plastic syringe was used for all needle aspirations. 3,4Using the thumb and the index finger, the desired area from where the aspiration was planned to be taken was immobilised when it was necessary. 3,4The needle was inserted without the use of any local anaesthesia into the mass or lesion with the single and slow motion without any negative pressure. 3,4When the needle was inside the targeted area, negative pressure in the syringe was generated that helped in withdrawal of the material into the needle.The needle was moved a few times in a back and forth motion and in different directions within the localised lesion or mass.The negative pressure on the syringe was released before the needle was withdrawn.This reduced the chance of seeding the path of syringe with the aspirated cells and also to prevent aspiration of cells from healthy area or area not under consideration.The aspirate was forced out on to the slides, and smears were prepared.Standard cytological staining techniques were used and the slides were inspected by two double-blinded pathologists.The cytological aspirates were broadly classified as adequate or inadequate, depending on the amount of material aspirated.Adequate specimens were further classified and sub-classified as neoplastic (benign, atypical cells, suspicion of malignancy, malignant) and non-neoplastic. 3,4r the purpose of analysis, atypical cells were considered negative, and suspicion of malignancy or where pathologist expressed suspicion for a diagnosis or malignancy was considered positive.The samples in which the aspirated material was insufficient or only the blood was present were considered inadequate and they were excluded from the final analysis. 3,4r excisional or incisional biopsy, the relevant procedure was performed and the specimen was fixed in 10% formalin solution and evaluated by the standard histopathological tissue examination techniques.Histopathological report was the gold standard against which cytological results were compared. 3,4 some patients, minor complications, like gag reflex, were observed and managed.Haemostasis was achieved before discharging the patient in a vitally stable state with proper verbal and written instructions.
The researchers wore proper and recommended personal protective equipment (PPE) along with N95 masks and eye protection to prevent themselves from SARS-CoV-2. 8,9The procedure was conducted in a well-ventilated room with minimal use of oral suctioning to avoid excessive aerosol generation. 8,9The contact or working time was kept to the minimum, but necessary to ensure safety of the operator along with proper completion of the procedure. 8ta was analysed using SPSS 20.Qualitative variables were presented as frequencies and percentages.

Discussion
The study showed that the accuracy of FNAC was 89.19%, sensitivity was 91.67% and specificity was 97.06%.5]7 They also suggested that when the aspirates are enough, one can even correctly diagnose the pathology. 3,4,7More than half (54.1%) of specimen were of malignant lesions predominantly squamous cell carcinoma. 3,4The significant percentage of squamous cell carcinoma was also observed in other studies which might be due to its high prevalence. 6round 657,000 new cases of oral cancers are diagnosed every year around the world and the majority of them are from south and central Asian countries, including Pakistan where it is the second most common cancer and accounts  for 10.9% of all the new cancers and 11.3% of cancerrelated deaths. 11[5]7 The oral lesions and pathologies are as diverse as the oral cavity itself. 3,4Proper diagnosis relies on good history, clinical examination and proper investigations. 3,4Among the variety of investigations available, histopathological examination is considered the gold standard for definitive diagnosis. 3,4The most commonly used method is incisional/ excisional or open biopsy for oral lesions, ulcers or masses. 3,4The procedure is technique-sensitive, requires local anaesthesia and needs good patient cooperation. 3,4or the preparation of histological slides, there are five basic steps involved; fixation, histoprocessing, embedding, sectioning and staining.The time required for these steps results in delayed final reporting of histopathology results.The cost of procedure and reporting is sometimes beyond the affordability of many patients, especially in poor and developing countries. 3,4The dilemma of such countries is that the oral pathologies and conditions are highly prevalent due to poor socioeconomic conditions of the masses, poor oral hygiene and lack of proper awareness. 5,6,8he healthcare system is also quite weak and is not enough to accommodate the increasing needs of the population. 8arious habits, diets, misconceptions and myths among majorly illiterate societies and communities further add to the problems of delays or reluctances to timely seek expert guidance or help, with the net outcome of increase in morbidity and mortality of the patients. 3,4,6,8,12,13ometimes the very minor oral condition or disease turn into a life-threatening conditions or situations. 3,12,13Delays could be sometimes due to the limited facilities available, overrun by patients more than their capacities. 6Setting patient appointments for biopsy involves delays as the procedure requires special setting, skills and equipment. 3,4e COVID-19 pandemic further escalated the problem as it spread by aerosol, and oral cavity and its vicinity carry high viral load, exposing the surgeons to the risk of contracting SARS-CoV-2. 8,9Procedures like biopsy require time, use of electric cautery and also cause aerosol generation.To minimize the risk of the virus and efficiently manage and diagnose the increasing number of patients, alternative techniques need to be explored. 3,44][5] One of such procedures is FNAC, which has many of these advantages. 3,4In case of biopsy, sometimes it is very important to take a proper   sample in the first go because of the irreversible damage the procedure causes to the tissue. 3,4With fine needle aspiration, one can also repeat the procedure if the aspirates collected are not enough. 3,4On average, FNAC requires from a few seconds to <5 minutes to perform, while biopsy can take 15-60 minutes.It takes a few hours to a maximum of 48 hours to report cytology results, while a histopathology report can take 7-14 days.The use of FNAC in the diagnosis of the salivary gland pathologies is very common and very well established. 3,4,7But FNAC is not commonly used for the diagnosis of lesions and masses of the oral cavity, such as squamous cell carcinoma, where histopathology is the gold standard. 3,4,7In the light of the findings, it is FNAC seems to be good enough for use as a regular or emergency procedure in separating the malignant lesions from the benign ones, and nonneoplastic pathologies from the neoplastic ones, especially when limitation of resources in proportion to number of patients is an important concern, or there is respiratory pandemic situation. 3,4,8It can help in early and timely management of malignant lesions or masses from the benign ones. 3,4,12In case of some non-neoplastic pathologies, early and quick diagnosis can help in the timely management without wasting time on a surgical procedure. 3,4,7,12e current study had some limitations, like a small sample size, which was enough from the statistical point of view. 3,4 larger sample size would have given a better insight or accurate results.The samples were collected in the procedure room and were transported to the pathologist.The unavailability of light microscopy and pathologist in the procedure room when the samples were being collected resulted in some samples having insufficient material for cytology which later had to be excluded.There was no repeat sampling, and no immediate examination of samples using microscopy for blood or insufficient material.
To collect good sample, immobilisation was necessary. 3,4mmobilisation of the area of interest was difficult in some cases while collecting the aspirates which affected the amount of material collected and could have also impacted the results.Sufficient quantity of material would have given more accurate results. 3,4It is important to observe caution while immobilizing the tongue or soft tissue using fingers while trying to get good sample as the risk of needle prick is high while collecting cytological aspirates.Some of the current limitations were common with earlier studies. 3,4

Conclusion
Fine needle aspiration was found to be a very rapid, reliable, cost-effective procedure.It can be deployed for regular or emergency purposes to differentiate between various benign and malignant pathologies, and also between neoplastic and non-neoplastic ones.It can be helpful and a repurposed tool in the armamentarium of oral surgeons during the COVID-19 or other respiratory pandemics.

Table - 1
: Intra-oral locations from where the specimen were collected.

Table - 2
: Comparison between histological and cytological diagnosis and distribution according to the nature of the lesion.