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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 14  |  Issue : 2  |  Page : 99-103

Ultra fast papanicolaou stain versus conventional papanicolaou stain in oral cytology smears: A comparative study


1 Department of Oral Pathology, Government Dental College and Hospital, Hyderabad, Telangana, India
2 Department of Oral Pathology and Microbiology, Government Dental College and Hospital, Hyderabad, Telangana, India

Date of Submission27-Oct-2021
Date of Decision21-Dec-2021
Date of Acceptance03-Jan-2022
Date of Web Publication01-Jul-2022

Correspondence Address:
Paremala Konda
Room No: 331, Department of Oral Pathology, Government Dental College and Hospital, Afzalgunj, Hyderabad - 500 012, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jorr.jorr_70_21

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  Abstract 


Background: Exfoliative cytology is the technique of microscopic examination of shed or desquamated cells from the epithelial surface of mucous membrane. Papanicolaou stain (PAP) is the common stain used for these cytological smears. Conventional PAP (cPAP) stain has undergone many modifications, of these ultrafast PAP (UFPAP) is a popular stain used as it shortens the staining time and quality. UFPAP was discovered as a fusion of Romanowsky and PAP.
Aim and Objectives: To evaluate and estimate the quality of UFPAP and c PAP stains in oral cytology smears of normal mucosa (NM), oral squamous cell carcinoma (OSCC), and oral potentially malignant disorders (OPMDs).
Materials and Methods: 50 samples/smears were collected from outpatients from the Government Dental College and Hospital, Hyderabad. Samples include 10 cases of NM, 20 cases each of OPMDs (leukoplakia) and OSCC. Two cytological smears of representative areas were obtained and each smear is stained by the cPAP and UFPAP stains, respectively.
Results and Observation: The quality index is obtained for the comparison of UFPAP and cPAP stains by using four parameters like - nuclear details, over all staining, background staining, and cell morphology. QI for all parameters of UFPAP staining was good compared to cPAP and percentage of UFPAP for NM, OPMDs and OSCC was 92.7%, 88.1%, 66.3% respectively.
Conclusion: The study concludes that UFPAP staining is used for diagnosis in a regular oral cytology smears to offer immediate diagnosis of OPMDs and OSCCs.

Keywords: Conventional-papanicolaou stain, exfoliative cytology, leukoplakia, normal mucosa, oral squamous cell carcinoma, ultrafast papanicolaou stain


How to cite this article:
Rani KS, Konda P, V. N. Shyam N D, Kumar G K, Narayen V, Reshma Y. Ultra fast papanicolaou stain versus conventional papanicolaou stain in oral cytology smears: A comparative study. J Oral Res Rev 2022;14:99-103

How to cite this URL:
Rani KS, Konda P, V. N. Shyam N D, Kumar G K, Narayen V, Reshma Y. Ultra fast papanicolaou stain versus conventional papanicolaou stain in oral cytology smears: A comparative study. J Oral Res Rev [serial online] 2022 [cited 2022 Dec 3];14:99-103. Available from: https://www.jorr.org/text.asp?2022/14/2/99/349715




  Introduction Top


Cytology is a study of the cells. Exfoliative-cytology is defined as the knowledge of microscopic study of desquamated/shed cells from an epithelial surface of the mucous membrane. It has helped in identifying and preventing about 80% of mortality and 60% of morbidity.[1]

In normal epithelium, the cells of basal layer are tightly attached to each other by junctional complexes whereas in malignancy, these complexes are lost and cells become loose, to examine these exfoliated cell morphology by making smears helps in initial identification of oral disorders, where some lesions could be innocuous and asymptomatic which are difficult to find and diagnose it as precancerous and cancerous lesions.[2] Hence the Papanicolaou stain (PAP) stain helps in cytomorphometric analysis for these cells by measuring the nuclear- cytoplasmic ratio and helps in initial identification of oral lesions.

In cytology, the atypical features observed are altered nuclear-cytoplasmic ratio, budding, nuclear pleomorphism, hyperchromatism and micronuclei, indented cellular outline, inflammatory cells and intracytoplasmic vacuoles.[3] PAP is the common stain used for study of these oral cytology smears. PAP is a polychromatic cytological stain which helps in identification of oral potentially malignant disorders (OPMDs) and oral squamous cell carcinoma (OSCCs). It was initially described by George Papanicolaou 1943.[4] The modified pap method was intended to reduce cost and time of staining without reducing the quality or the cytodiagnosis of the smear.[5]

Many modifications have come from cPAP from its time of invention. Ultrafast pap (UFPAP) is the one of the modifications which increase staining quality. It was described by Alvarez and Yang in 1995 has the combination of Romanowsky preparation and wet fixed PAP preparation.[6]

UFPAP incorporates air drying of cells, followed by rehydration in saline and alcohol mixed formalin fixation. The Richard Allan hematoxylin and cytostain are used. Due to suboptimal cell morphology obtained by conventional PAP (cPAP), UFPAP stain was developed to get better staining quality and also reduce the staining time.[7]

Transparency in nuclear details and cytoplasm when stained with UFPAP helps in study of changes in cytoplasm which suggests that increase in nuclear diameter and reduction in the mean cytoplasmic area in exfoliated cells acts as an initial indicator of dysplastic changes.[8] Their are no studies done to compare these two stains in oral cytology smears. The present study was done to evaluate the quality of staining among UFPAP and cPAP stains in OPMDs and OSCC cases.


  Materials and Methods Top


This study was undergone using 50 cytological smears which is collected from the Oral pathology and microbiology department. Cases included in the study are 10 normal mucosa (NM) cases, 20 cases each of OSCC and leukoplakia. Two smears from lesional areas were obtained and each smear was prepared with cPAP and UFPAP stains. The smears collected from the patients reported to Oral and maxillofacial Pathology and microbiology department, Government Dental College and Hospital, Afzalgunj, Hyderabad.

The patients were histopathologically diagnosed as OPMDs and OSCC after taking the smears of the patient. UFPAP kit obtained from Micro express Tulip diagnostics (P) Ltd., which includes Micro-fix fixative spray, Ultra-pap nuclear stain, Ultra-pap Cytostains, Scott's buffer, Dehydrant, Xylene, DPX mounting media.

The staining quality of UFPAP and cPAP was assessed by using four parameters. The quality index (QI) for both stains is calculated with the formula- QI = original score obtained/maximum score possible. UFPAP staining is performed with the procedure described by Yang -Alvarez in 1995.

Inclusion criteria

  1. Clinically diagnosed cases of leukoplakia and OSCC
  2. Patients with habit history
  3. Patients with good health conditions.


Exclusion criteria

  1. Patient history of general illness
  2. Patients without habit history.


Methodology

UFPAP stain

Each smears prepared on dirt-free glass slides and are fixed with micro fix spray, wash in tap water-10 passes, nuclear stain-45 s, water wash for 10 passes, add few drops of Scott's buffer-10 s, water wash for 10 passes, add dehydrant-10 s, add mixture of cytostain A and B-15 s, water wash for 10 passes, add dehydrant-20 s, dip in xylene-2 dips, DPX mounting.

Conventional PAP staining

Smears are to fix in alcohol – 10 min, slowly clean in tap water – 1 to 2 min, hematoxylin – 2 to 4 min, slowly clean in water – 3 min, OG 6 – 10 to 15 min, alcohol I – 10 to 15 dips, alcohol II – 10 to 15 dips, EA 36 – 10 to 15 min, Alcohol I – 10 to 15 dips, alcohol II – 10 to 15 dips, xylene + alcohol – 5 to 10 dips, xylene, cleaning and mounting with DPX.

Statistical analysis

The data were analyzed with the Statistical Package for the Social Sciences (SPSS) for windows 25.0. The observed data were analyzed by one-way ANOVA and Chi- square test.


  Results and Observation Top


The obtained data assessed with SPSS for windows 25.0. The observed data was assessed by Fischer's exact test. In this study, the statistics showed 100% staining quality for UFPAP. QI for UFPAP and cPAP stain is calculated for each smears, cPAP stain in NM, leukoplakia, and OSCC was 63.6%, 54.5%. 42.7%, respectively [Table 1].

For UFP stain, QI was 92.7%, 88.1%, 66.3%. When comparison was done between UFPAP and cPAP stains of NM, the QI showed 92.7% and 63.6%, respectively. Fisher exact test is done to compare these stains where P < 0.05 was considered statistically significant [Figure 1] and [Figure 2].
Figure 1: Photomicrograph showing conventional papanicolaou stain staining of normal oral mucosa with moderate overall staining (conventional Papanicolaou stain ×20)

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Figure 2: Photomicrograph showing ultrafast Papanicolaou stain staining in normal oral mucosa with good overall staining and individual cellular details (ultrafast Papanicolaou stain ×20)

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The comparison between UFPAP and cPAP stain was done in leukoplakia the quality measurement showed 88.1% and 54.5%, respectively. Fischer exact test was performed to compare these stains where P value was 0.000 considered as statistically significant [Figure 3] and [Figure 4].
Figure 3: Photomicrograph showing conventional Papanicolaou stain staining in leukoplakia with poor cell morphology (conventional Papanicolaou stain ×40)

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Figure 4: Photomicrograph showing ultrafast Papanicolaou stain staining in leukoplakia with good cell morphology (Ultrafast Papanicolaou stain ×40)

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In OSCC, the QI was compared between UFPAP and cPAP stains which showed 66.3% and 42.7%, respectively. Fisher exact test is done in comparison of these stains where P value was 0.005 considered statistically significant [Figure 5] and [Figure 6].
Figure 5: Photomicrograph showing conventional Papanicolaou stain staining in oral squamous cell carcinoma with poor nuclear details (conventional Papanicolaou stain ×40)

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Figure 6: Photomicrograph showing ultrafast Papanicolaou stain staining in oral squamous cell carcinoma with clear nuclear details (Ultrafast Papanicolaou stain ×40)

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  Discussion Top


Oral exfoliative cytology (OEC) is a simple, sensitive, and noninvasive staining technique used in samples where biopsy is not feasible.[9] The initial diagnosis of OPMDs and OSCC by using UFPAP helps in preventing moratlity of patients. The staining quality of UFPAP stain was good compared to cPAP. The clear background caused by removing RBCs, overall staining, retained cell outline, and retained nuclear details with UFPAP stain helps in the identification of cellular changes. These parameters help in quick identification of OPMDs and OSCCs.



The transparency of cytoplasm helps in study of Micronucleus refers to the small nucleus serve as marker for increased cancer risk which form in response to DNA damaging agents.[10] The frequency of micronuclei is three to four times higher in patients with OSCC compared to OPMDs, indicating the increased cytogenetic damage of epithelial cells in OPMD to OSCC. The probable reasons for MN formation in preneoplastic conditions include chromosomal aberrations, chromosome loss/breakage, mitotic apparatus dysfunction, aneuploidy, and genetic instability. Based on these, they suggested that gradual increase in micronnuclei from precancerous to cancerous lesions suggested a link of this biomarker with neoplastic progression.

Our study compared the staining quality in UFPAP and cPAP stains in oral cytology smears and the parameters taken was in accordance to the study done by Thakur and Guttikonda in fine needle aspiration cytology (FNAC) collected in head and neck swelling.[7]

This is the first study to compare cPAP and UFPAP stains in oral cytology smears. Background and overall staining done in NM, leukoplakia, and OSCC using UFPAP and cPAP stains showed statistically insignificant results where no much difference in quality of staining was observed.

In this study, comparison was done between UFPAP and cPAP stains in NM, leukoplakia, and OSCC the morphology of cell, nuclear features were retained when stained with UFPAP stain and results showed statistically significance.

These findings are similar with the study done by Thakur and Guttikonda in which modified ultrfast MUFP stain were compared to routine PAP stains in FNAC samples collected from swellings of maxillofacial regions. The FNAC collected from thyroid swellings when stained with MUFP showed better staining pattern compared with other stains.[7]

The nuclear findings in NM, leukoplakia, and OSCC using UFPAP and cPAP stains show statistically significant results. In which the nuclear staining was well preserved in leukoplakia and OSCC.

Our study is in accordance to the study conducted by Arul et al. where FNAC samples and body fluids were collected and these samples stained with MUFP stain which showed good nuclear and cellular details in smear done with body fluids when compared with FNAC collected from other swellings of head and neck.[4]


  Conclusion Top


UFPAP stain plays a crucial role in fast diagnosis of oral cytological smears by reducing time and improving staining quality which helps in early identification of oral cancers. It is a modified stain in which the reagents are easily available and help in fast diagnosis. As UFPAP staining provides transparency of cytoplasm and good cellular details help in study of cytomorphometric analysis and micronucleus.

Limitations

  1. Technique sensitive procedure.
  2. Commercially available only
  3. Problems in storage with Indian setup


Future prospects

Further such studies on bigger sample size.

Ethical clearance

ECR/300/Inst/AP/2013/RR-16(GDCH -IEC/PG/1934-dated 13-11-2020).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Vidya S, Shashi Kiran M, Rahul R. Exfoliative cytology in every day practice. J Dent Orofacial Res 2019;15:63-8.  Back to cited text no. 1
    
2.
Chitturi RT, Rathinam E, Santo R, Yoithapprabhunath TR. The role of exfoliative cytology and molecular biology in oral potentially malignant disorders. J Health Res Rev 2017;4:43-6.  Back to cited text no. 2
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3.
Kabiraj A, Khaitan T, Bhowmick D, Ginjupally U, Bir A, Chatterjee K. Screening of oral potentially malignant disorders using exfoliative cytology: A diagnostic modality. J Cancer Epidemiol 2016;2016:8134832.  Back to cited text no. 3
    
4.
Arul P, Eniya S, Pushparaj M, Masilamani S, Kanmani P, Lingasamy C. Comparative assessment of conventional papanicolaou and modified ultrafast papanicolaou Stains in fine needle aspiration samples and body fluids. J Cytol 2018;35:46-50.  Back to cited text no. 4
[PUBMED]  [Full text]  
5.
Gachie RN, Muchiri LW, Ndungua JR. A comparison of modified and standard papanicolaou staining methods in the assessment of cervical smears at Kenyatta national hospital. East Afr Med J 2011;88:244-50.  Back to cited text no. 5
    
6.
Yang GC, Alvarez II. Ultrafast papanicolaou stain. An alternative preparation for fine needle aspiration cytology. Acta Cytol 1995;39:55-60.  Back to cited text no. 6
    
7.
Thakur M, Guttikonda VR. Modified ultrafast papanicolaou staining technique: A comparative study. J Cytol 2017;34:149-53.  Back to cited text no. 7
[PUBMED]  [Full text]  
8.
Singh A. Role of exfolaitive cytology in oral lesions: With special reference to rule out malignancy. J Coll Med Sci 2010;6:29-37.  Back to cited text no. 8
    
9.
Hassan SA, Bhateja S, Arora G, Prathyusha F. Exfoliative cytology application in dentistry – A review. IP Arch Cytol Histopathol Res 2020;5:116-9.  Back to cited text no. 9
    
10.
Jaitley S, Agarwal P, Upadhyay R. Role of oral exfoliative cytology in predicting premalignant potential of oral submucous fibrosis: A short study. J Cancer Res Ther 2015;11:471-4.  Back to cited text no. 10
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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