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Abstract
Background: The Fine needle aspiration cytology (FNAC) is considered as a valuable and distinguished diagnostic test in the initial assessment of the patients presenting with a mass in the head and neck region or when a recurrence is suspected after previous treatment. Aims: This study was therefore designed to elucidate the efficacy of FNAC as an alternate diagnostic tool to histopathology in head and neck swellings and evaluation of staining efficacy of PAP and MGG stain over Haematoxylin and eosin (H and E) in routine cytopathological smears. Settings and Design: The study was conducted in the Department of Oral and Maxillofacial Pathology, where FNAC samples were collected from 150 patients with head and neck swellings. Materials and Methods: All the slides were stained with H and E, Papanicolaou (PAP), and May Grunewald Giemsa (MGG) stains. The cytopathological diagnosis was compared with histopathological diagnosis based on H and E stained sections obtained from paraffin-embedded formalin-fixed biopsy specimen of benign and malignant neoplasms. Statistical Analysis Used: The resulting data were analyzed using SPSS software version 19. Differences between the variables were analyzed using Pearson Chi-square test and Kruskal–Wallis test wherever applicable. Results: The FNAC as a diagnostic tool has sensitivity of 84.8%, 72.72%, and 78.78%, specificity of 62.5%, 75%, and 75%, and accuracy of 80.48%, 73.14%, and 78.04% in H and E, MGG, and PAP stain, respectively. PAP stain was the most efficient stain when all qualitative parameters are taken into consideration with maximum sensitivity and specificity for achieving definitive cytodiagnosis. Conclusions: The FNAC is an inexpensive and minimally invasive technique to diagnose different types of head and neck swellings and complement histopathological diagnosis.
Keywords: Cytology, FNAC, MGG stain, PAP stain
How to cite this article: Introduction 
The Fine Needle Aspiration Cytology (FNAC) is important for pre-operative and pre-treatment diagnosis of diverse benign and malignant lesions. Palpable head and neck swellings may involve multiple anatomic sites such as lymph nodes, thyroid, salivary glands, adipose tissue, blood vessels, and neural tissues. These may be attributed to multitude of etiologic factors such as cysts, thyroid masses, salivary gland pathologies, and enlargement of lymph nodes due to varied reasons. These pathologies commonly require biopsy from the affected site for histopathology diagnosis which would aim to determine the prognosis and therapeutic approach toward their management.[1]
The FNAC is a technique which involves acquisition and study of cells and tissue fragments aspirated through a fine needle introduced into the tissue in order to obtain cells and tissue fragments for diagnosis. The FNAC is widely accepted and integrated in the diagnostic routines and has gained great value because of the ease of access, non-invasiveness of the technique, rapidity, and heterogeneity in diagnosis.[2],[3] The clinical value of FNAC is not limited only to neoplastic conditions but also for the diagnosis of inflammatory, infectious, and degenerative conditions, in which the samples can be used for microbiological and biochemical analysis in addition to cytological preparations.[4],[5]
It is often speculative to learn that a confounding diagnostic and therapeutic dilemma is often presented by masses involving glandular and nodal structures of the head and neck—lymph nodes, thyroid glands, and salivary glands. Undoubtedly, a formal open surgical biopsy yields a definitive histopathologic diagnosis but may mandate an extensive operative approach which may be inappropriate for best management. Furthermore, when a malignant lesion is discovered during an open biopsy, the surgeon may be unprepared or be incapable of proceeding with adequate management.[4] FNAC of such masses can usually be an indispensable aid in surgical histopathology.[2]
The FNAC may employ the use of routine stains such as Haematoxylin and eosin (H and E) or specific stains such as Papanicolaou (PAP) stain which better aids in differentiating different layers of oral epithelium in the smear. It stains the nuclear chromatin well and also gives a subtle range of green, blue, and pink hues to the cell cytoplasm, giving a good cytoplasmic transparency. May Grunewald Giemsa (MGG) stain is also often used as a cytological stain which is easy to prepare, less time consuming, reduces the effects of poor techniques and increases cell yield.[6] The present study elucidates the efficacy of fine needle aspiration cytology (FNAC) as an alternate diagnostic tool to histopathology in head and neck swellings and evaluation of staining efficacy of PAP and MGG stain over H and E in routine cytopathological smears.
[TAG:2]Materials and Methods[/TAG:2]
Patient and tissue sample
The study was conducted in the Department of Oral and Maxillofacial Pathology, after gaining approval from the Institutional Review Board. The procedure was explained and written informed consent was obtained from the patient/guardians in case of minors after which a detailed clinical history was noted.
Sample collection
FNAC samples were collected from 150 patients with head and neck swellings. All the slides were stained with H and E, PAP and MGG stains. The study cases were analyzed for their distribution and frequency. All cases were categorized into five groups based on FNAC diagnosis, that is, Inflammatory/Reactive, Infectious, Traumatic, Cystic, Benign, and Malignant. Ethical approval from the institutional review board was obtained for this study.
The cases which were inflammatory and reactive were excluded from histopathology evaluation after thorough clinical follow-up. As per the pathological recommendations, lymphadenopathies of infective origin do not require histopathological evaluation; hence, such cases were not biopsied. Histopathology evaluation was done on those cases which were cystic, benign, or malignant after FNAC examination. The biopsy specimens were fixed in 10% neutral buffered formalin and processed using standard tissue processing protocol; 4 μ thick sections were obtained and stained using routine H and E stain. Based on histopathology examination, the cases were categorized into infection, inflammations, cystic, benign, and malignant categories [Figure 1].
Figure 1: (a) H and E stained cytosmear of squamous cell carcinoma showing clusters of atypical cells with crisp cellular details (40×). (b) May Grunewald Giemsa stained cytosmear of squamous cell carcinoma showing clusters of atypical cells with fair cytoplasmic and nuclear details (40×). (c) Papanicolaou stained cytosmear of squamous cell carcinoma showing clusters of atypical cells with excellent preservation of cytoplasmic and nuclear details (40×)Procedure for FNAC smear preparation and preservation
Standard disposable 22-24-gauge with 1-1½-inch needles were used for performing FNAC along with standard disposable plastic syringes of 5 ml volume. A syringe piston handle was used, leaving one hand free to immobilize the lesion. Plain glass slides of good quality which were clean, dry, transparent, and grease free were used and fixed using 95% ethyl alcohol, except one slide which was air dried to perform MGG stain directly.
Aspiration procedure
Relevant history and clinical details, radiological findings, and provisional diagnosis were entered in the requisition form. The site of FNAC was clearly stated. The procedure was clearly explained to the patient, and consent and cooperation was ensured. All universal precautions were followed during the procedure. The lesion to be aspirated was palpated and its suitability for aspiration was assessed.
A comfortable position was chosen depending on the convenience to palpate the lesion and the comfort of the patient. Skin was cleaned firmly with an alcohol swab. The lesion was fixed between the thumb and index finger of the left hand, with the skin stretched. Fixing the lesion with one hand, the syringe was grasped with the needle attached (with or without syringe holder) by the dominant hand and introduced through the skin into the lesion, carefully and swiftly. The angle and depth of entry varied with the type of lesion. For small lesions, aspiration of central portion was indicated. For larger lesions that may have necrosis, cystic change, or hemorrhage in the center, aspiration was done from the periphery. When material was seen in the hub of the needle, procedure was discontinued. Before withdrawing the needle, suction was released and the needle was pulled straight out. The piston was just allowed to slowly fall back by itself. Slides were fixed with 95% alcohol for PAP, H and E, and MGG staining using the standard technique.
Observations of the stained slides
All the stained FNAC smear slides were observed under bright field microscope (Olympus BX53) Under low and high magnification and were assessed for cytological parameters such as background, cytological details, and nuclear details for each stain (H and E, PAP, and MGG stain) [Figure 2]. The cytopathological diagnosis were compared with histopathological diagnosis based on haematoxylin and eosin stained sections obtained from paraffin-embedded formalin-fixed biopsy specimen of benign and malignant neoplasms for the following subjective parameters—Cytoplasmic details (Not preserved - 0, Non transparent markings of nuclear details - 1, Non transparent with intact cell membrane - 2, Transparent, intact cell membrane without masking of nuclear details - 3); Nuclear details (Poor preservation - 0, Smudgy - 1, Fair preservation but chromatin granularity not appreciable - 2, Excellent preservation with crisp chromatin - 3); and Background staining (Intensely stained obscuring cellular details - 0, Moderately stained with better cellular details - 1, Less intense staining with crisp cellular details - 2).
Figure 2: H and E stained (a) Cytosmear showing malignant cells, (b) Cytosmear of tubercular lymphadenitis showing necrotic material, (c) Section of moderately differentiated Squamous cell carcinoma (SCC) showing islands of tumor cells surrounding keratin pearls (10 × with inset 40×), (d) Section of tubercular lymphadenitis with central Caseous necrosis surrounded by lymphocytes, plasma cells, and Langhans type giant cells (10 × with inset 40×)Statistical analysis
The resulting data were analyzed using SPSS software version 19. Descriptive statistics including the mean values, standard deviations, ranges (minimum and maximum) were calculated for each variable. Differences between the variables were analyzed using Pearson Chi-square test and Kruskal–Wallis test wherever applicable. Besides this, areas under the curve values were calculated by applying the receiver operating characteristic (ROC) curve analysis. The qualitative data were compared by linear coefficient depicting the trend in various stains and parameters under variable time period. P ≤ 0.05 was taken as significant at 95% confidence intervals.
Results Demographic data
This study comprised of FNAC examination of 150 cases of head and neck swellings, in which maximum incidences were observed in the age group of 41 to 50 years. The mean age of the study cases was 39.9 years. There were 22.7% cases in 41 to 50 years age group, followed by 18% in 21 to 30 years age group, 16.7% in 51 to 60 years age group, and least 1.3% in 81 to 90 years age group. Among the study cases, head and neck swellings were found to be more common in neck with the incidence of cervical lymph node swellings being the most common with 37 cases out of 150 (24.4%), followed by angle of mandible swellings comprising of 26 cases (17.33%) and submandibular lymph nodes, that is, 15 cases out of 150 (10%). Other sites included thyroid, salivary gland, soft tissues, and subcutaneous tissues. The overall number of head and neck swellings were more commonly seen in males with 94/150 (63%) of the study cases whereas females being 56/150 (37%) of total cases.
On the basis of the FNAC diagnosis, the majority of the head and neck masses were malignant, and the other categories included inflammatory, infectious, cystic, traumatic, and benign lesions. Out of the 41 cases subjected for histopathologic examination, 7 were benign (which included inflammatory, infectious, benign cystic, and benign lesions) and 34 were malignant in final diagnosis after biopsy. The histopathologic diagnosis was considered as definitive diagnosis and was used to compare FNAC diagnosis to calculate sensitivity, specificity, Positive predictive value (PPV), Negative predictive value (NPV), and accuracy of FNAC in the diagnosis of palpable head and neck swellings.
Amongst 10 cases diagnosed as benign lesions by cytopathological examination using H and E stain, 5 cases were diagnosed as benign lesions (true negative), whereas 5 cases were diagnosed as malignancy (false positive) on histopathologic examination. Out of the 31 cases diagnosed as a malignant lesion by cytopathological analysis using H and E stain, 28 cases were diagnosed as malignancy (true positive) and 3 cases were diagnosed as benign lesions (false negative) on histopathologic examination. However, amongst the 15 cases diagnosed as benign lesions by cytopathologic examination using MGG stain, 6 cases were diagnosed as benign lesions (true negative), whereas 9 cases were diagnosed as malignancy (false positive) on histopathologic examination. Out of the 26 cases diagnosed as a malignant lesion by cytopathology using MGG stain, 24 cases were diagnosed as malignancy (true positive) and 2 cases were diagnosed as benign lesions (false negative) on histopathologic examination. Under PAP stain, out of the 13 cases diagnosed as benign lesions by cytopathologic examination, 6 cases were diagnosed as benign lesions (true negative), whereas 7 cases were diagnosed as malignancy (false positive) on histopathologic examination. Out of the 28 cases diagnosed as a malignant lesion by cytopathology using PAP stain, 26 cases were diagnosed as malignancy (true positive) and 2 cases were diagnosed as benign lesions (false negative) on histopathologic examination [Table 1]. On the basis of the number of true positive, false positive, true negative, false negative cases, sensitivity, specificity, NPV, PPV, and accuracy of FNAC in three different stains were calculated. The FNAC as a tool for diagnosis has the highest sensitivity of 84.8% using H and E stain followed by 78.78% using PAP stain and 72.72% using MGG stain. The specificity of FNAC was highest and comparable for both PAP and MGG stains being 75% followed by H and E stain being 62.5%. The positive predictive value was highest for PAP stain (92.85%), followed by MGG stain (92.3%) and H and E (90.3%). The negative predictive value for FNAC was highest using H and E stain (50%), followed by PAP stain (46.14%) and lowest for MGG stain (40%). The accuracy of H and E stain (80.48%) was best compared with PAP stain (78.04%) and MGG (73.17%) stain when histopathologic diagnosis is taken as gold standard [Table 2].
Table 1: Correlation of FNAC diagnosis (H and E stain, MGG stain, and PAP stain) with histopathology diagnosis
Table 2: Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of FNAC diagnosis in H and E, MGG, and PAP stainsThe comparative analysis of the three stains, that is, H and E, PAP, and MGG, for assessing the qualitative parameters of the FNAC smears showed that PAP stain was the most efficient stain to visualize the nuclear details followed by H and E stain and the MGG stain. On evaluation of the cytoplasmic details, PAP was the most effective stain followed by H and E stain and MGG stain. On assessment of the background details, H and E stain was a superior stain compared with PAP stain or MGG stain. The results were statistically significant (P ≤ 0.05, Kruskal–Wallis test) [Table 3].
Table 3: Qualitative analysis of H and E, MGG, and PAP stain in observing nuclear details, cytoplasmic details, and background in FNAC smearsThe ROC curve plotted with sensitivity along Y axis and 1–specificity along X axis showed PAP stain has a maximum area under the curve (0.769). The result was statistically significant, thus indicating that PAP has the best sensitivity and specificity values when all qualitative parameters were evaluated for assessing the FNAC smears. Area under the curve for H and E stain was comparable to MGG stain [Figure 3] and [Figure 4].
Figure 3: (a) Cytosmear showing atypical cells in mucinous background, (b) Cytosmear of angulated hyperchromatic atypical cells in a mucinous background, (c) Section of metastatic prostrate carcinoma showing tumor islands and bony trabeculae (10 × with inset 40×), (d) Section of Adenocarcinoma showing islands of tumor cells with necrosis and pseudo ductal pattern (10 × with inset 40×)
Figure 4: ROC curve of different stains (H and E, MGG, and PAP stains) in evaluating FNAC smears Discussion Head and neck masses are the common clinical conditions seen by clinicians in routine practice. It is of utmost importance to assess the conditions properly to decide the correct management protocol as the differential diagnosis of head and neck swellings cover a broad spectrum of diseases. The FNAC is a valuable diagnostic test in the initial assessment of the patients presenting with a mass in the head and neck region or when a recurrence is suspected after previous treatment. The most commonly described advantages of the FNAC method are the pre-operative diagnosis of the lesions, clinical follow-up, the ability to avoid unnecessary damage to crucial structures, low discomfort to the patient, and low risk of infection or tissue damage.
In this study, out of the 150 fine needle aspiration procedures, 66 cases (44%) were from lymph nodes, which were mostly inflammatory in nature (reactive hyperplasia and tubercular lymphadenitis), while the rest of the sites constituted 84 cases (56%) which included oral cavity, mandible, neck, and other anatomical areas of craniofacial complex. These results were in accordance with Sousa et al.,[7] who reported that lymph nodes are the most common site of such swellings. In a similar study by Khetrapal et al.,[8] the largest number of aspirates were from lymph nodes, 185 (64.1%), followed by thyroid lesions, 49 (16.9%). The most common site of presentation of these malignancies in head and neck region were cervical lymph nodes. It usually presents late and with nodal metastasis. Metastatic squamous cell carcinoma was found to be the most common pathology in our study accounting for 24% of cases followed by tubercular lymphadenitis constituting 21.3% of cases. El-Hag et al.[9] carried out a similar study in Saudi Arabia over a period of 5 years which included 225 patients. Their results showed that tuberculous lymphadenitis was the most common pathology constituting 21% of cases followed by malignant swellings found in 13% of cases.
Histopathological correlation was done for comparison in 41 of the cases with FNAC diagnosis. However, after histopathology, it was revealed that 34 cases from these 41 cases were reported as malignancies, while 7 reported as benign neoplasms with metastatic squamous cell carcinoma being the most general malignancy. In a study on FNAC of lymph nodes of head and neck region by Modi et al.,[10] aspirations were done in 958 cases out of which 108 cases were inconclusive and were not taken into account, whereas in 170 cases, histopathological examinations were available for correlation with a cytological diagnosis.
In this study, the overall accuracy of FNAC was calculated under H and E, MGG, and PAP which revealed highest sensitivity, specificity, and accuracy of H and E (84.8%, 62.5%, and 80.48%, respectively) in comparison to MGG (72%, 75%, and 73%, respectively) and PAP (78%, 75%, and 78%, respectively) [Table 2]. Moreover, Moghadam et al.[11] reviewed similar studies to evaluate the accuracy of FNAC in diagnosis of head and neck masses, and the range of diagnostic accuracy was between 56% and 100%, the range of sensitivity 55% to 100%, and the range of specificity was 59% to 100%. As per literature review, accuracy of FNAC depends upon different factors such as location and pathologic type of mass, experience of pathologist, sample adequacy, disease endemic in that country (e.g., TB), technology used to aid in diagnosis (ultrasonography, computed tomography scan), and age of the patient (patients with mass diagnosed as malignant neoplasm were older than those diagnosed as non-malignant). The FNAC as an atraumatic method can help to design an effective surgical plan in addition to identifying the tumor characteristics.
A study conducted by Maniyar et al.[12] showed 10 benign and 7 malignant cases of soft tissue lesions. All the 14 cases available for histopathology showed consistent findings. In a review of 341 cases of salivary gland cytologies, Stewart et al.[13] reported that most false negative results were caused by sampling error especially in cystic tumors or due to a misinterpretation of uncommon neoplasms. Other workers have also shown that FNAC is an unsatisfactory technique in low grade neoplasms. In 42/150 cases of head and neck swellings in this study, cytological diagnoses consistent with the histological findings were seen in 35 (80.4%) cases and inconsistent findings in 7 (8.2%) cases. Maniyar et al.[12] reported cytohistopathological consistent diagnoses in 85.87% cases and 14.13% cases in which the cytological and histopathological diagnoses were different, which is comparable. Also, 42 cases were available for histopathological correlation in this study and the sensitivity and specificity of the same were evaluated to be 87.7% and 57.1%, respectively.
The three stains used in our study were evaluated for cytoplasmic details, nuclear details, and background in FNAC smears. The nuclear details were best seen in PAP-stained smears with excellent preservation with crisp chromatin followed by H and E and MGG, respectively. Similarly, the cytoplasmic details were also best visualized in PAP stain with transparent, intact cell membrane without masking of nuclear details followed by H and E and MGG stains, respectively [Table 3]. These results are in accordance with the study conducted by Ayyad et al.,[14] where it was found that PAP stain is better stain than other stains. Also, in PAP stain, 95% alcohol is used for fixation, which has a bactericidal effect. The alcohol allows the permeability of dyes across cell boundaries, permits cell adhesion to the glass slides, and replaces cellular water. It also penetrates the cell rapidly and maintains morphologic integrity. PAP is considered to be a good nuclear stain with nuclear transparency. The results of our study were also in accordance with Sujathan et al.,[15] who found PAP to be better than MGG stain for nuclear staining. According to Idris and Hussain,[16] for routine diagnostic cytology, the PAP stain is and can be used, as it stains nuclear chromatin well, gives good differential cytoplasmic counterstaining and produces good cytoplasmic transparency.
According to Belgaumi and Shetty,[17] MGG stain has the tendency for high background staining leading to obscuring in identifying the background material and also the cellular details. MGG needs preparation of fresh solution every day. Palaskar and Jindal[18] conducted a study to evaluate the micronuclei count in PAP and MGG stain. According to their study, stains like MGG where the smears were air dried resulted in a background which was full of cell debris and cellular proteins, thus masking the counting of micronuclei.
For visualizing the cytoplasmic details, PAP stain is comparatively an inferior stain to MGG stain in the study cases. Similar study conducted by Sujathan et al.[15] suggested MGG stain to be a better stain than PAP stain. According to Baker et al.,[19] the utility and adaptability of MGG are defined in cytologic specimens which gives excellent cytoplasmic details of the cell with limited nuclear details.
Conclusion The search for an inexhaustible source of high-quality pathological samples with matching clinical data for cutting-edge research is the major prerequisite for diagnosis. The FNAC is an excellent first line method for investigating the patients presenting with palpable head and neck lesions. Future studies should aim at prospective exploration to formulate an ideal cytological stain which could emphasize the nuclear details in a more accurate manner. This could be achieved by modifications of various staining components and staining methodologies to incorporate excellent features of each stain.
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References 
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CheckDOI: 10.4103/ijpm.ijpm_1254_21
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