INTRODUCTION:

Exfoliative cytology deals with study of superficial and deeper cells of epithelium that are exfoliated from the malignant and premalignant lesions in the body. Cytology smears that are collected from the site of interest are immediately fixed to prevent withdrawal of cells from the glass slide. These smears are then stained with various stains like hematoxylin and eosin, papanicolaou, periodic acid Schiff, silver salts and are observed under microscope to interpret the dysplastic nature of cells. Commonly used instruments to collect cytological smear includes wooden blade, metal spatula but oral brush is proved to be more efficient as cytological smear collected by latter is more thick and effective.

Evolution of exfoliative cytology:

The seminal work that was done by Papanicolaou and Traut in studying cells from precancerous and cancerous lesions of cervical mucosa led to the development of oral cytology⁽³⁾.(Table-1)

Table 1: Initial developments in oral exfoliative cytology

YEAR	AUTHOR	CONTENT
1860	Beale	Cytological review of sputum for pharyngeal cancer
1940	Wienmann	Cytological viewing of keratinisation of oral cells.
1941	Ziskin et.al	Menstrual cycle influence on oral cells morphology
1942	Papanicolaou	Staining techniques for cytological smears.
1943	Papanicolaou and Traut	Diagnosis of uterine carcinomas
1949	Morrison et.al	Diagnosis of nasopharyngeal malignant conditions.

Over a period of time, investigators Montgomery et.al and von Haam et.al wanted to overcome the limitations in the field of oral cytology and this led to various modification methods⁽³⁾. (Table 2)

Table 2: Later modifications in exfoliative cytology.

YEAR	AUTHOR	MODIFICATIONS
1951	Gladstone	Usage of “sponge biopsy”
1952	Schneider	Modifications in staining techniques
1960	Cawson	Changes in staining techniques
1963	King	Usage of frosted glasses as slides
1963	Staats and Goldsby	Comparing wooden and metal spatula usage in smear collection
1964	Sandler	Removing keratotic layers with sharp scalpel
1981	Dumbach et.al	Smear by using curettage.

These modifications did improve the sensitivity of cytology, however the main advantage of cytology is lost as these sharp scalpels and smear curettage became invasive. Main disadvantage was that minimally invasive techniques were not able to sample the deeper layers of epithelium.

Uses of oral brush:

Conventional oral brush cytology has sensitivity from 79% to 97% and specificity ranging from 95.1% to 99.5%. Use or oral brush is better that wooden spatula with the advantages of ease of use, painless, with the better cell spreading capacity in objective slides than wooden spatula⁽⁴⁾. Based on the study performed by Orellana Bustos et.al, Espinoza Martinez et.al to determine the depth of cytological smears of oral mucosa collected with wooden spatula and cytobrush and to compare the degree of keratinisation, they concluded that cytobrush allows to sample deeper cells of buccal mucosa⁽⁵⁾

Stains used in the study of exfoliative cytology:

PAP (Papanicolaou) Staining⁽¹⁾:

This method of staining was first developed by “George Papanicolaou” – the father of cytopathology. “Pap” stain was introduced with three main objectives, definition of nucleus, and transparency of cytoplasm and proper differentiation of cells. The dyes that were employed in papanicolaou stain includes Hematoxylin, Orange green 6, Eosin azure, Eosin y, Light green SF, Bismarck brown Y. Both progressive and regressive staining techniques are indicated in Papanicolaou stain. Before staining the smears are fixed with cytology fixation spray(90% ethanol for a minimum 30 minutes).

· Progressive method- wherein the nucleus is stained with hematoxylin to a needed intensity.

· Regressive method- wherein the nucleus is purposely overstained with non-acidified hematoxylin.

Hematoxylin and eosin staining⁽¹⁾:

Hematoxylin and eosin staining is the most commonly employed technique in histopathology.

The reagents that were used in H and E stain

· Harri’s hematoxylin solution (A=1gm hematoxylin in 10ml of ethanol; B=20gms of ammonium alum in hot distilled water. Mix A and B boil and add 0.5gm of mercuric oxide and filter)

· Eosin solution (yellow eosin=1gm; distilled water=80ml; ethanol=320ml; glacial acetic acid=2 drops.)

· 0.5%Hcl and Dilute ammonia water.

The principle employed in H and E staining is that the alum acts as a mordant (substance in a dye or stain that is used to fix a material) and stains nucleus light blue. This turns red in the presence of acid. Bluing converts initial soluble red color within the nucleus into an insoluble blue color. Counterstaining is done with eosin that stains cytoplasm pink⁽¹⁾.

Procedure in h and e staining:

Deparaffinize by flaming the slide on the burner and place in xylene and repeat the treatment. Hydrate the section by passing it through decreased concentrations of water and alcohol; Stain in Hematoxylin for 3-5 minutes; Wash in running tap water until it becomes blue, this process is called “bluing”; differentiate in 1% acid alcohol; wash in running water until it becomes blue again; stain using 1% Eosin Y for 10 minutes; wash again in running water, dehydrate, mount and observe under microscope.

INTERPRETATION:

· Nucleus-stained blue / black

· Cytoplasm-stained pink

· Muscle fibres-stained deep red.

AgNORs (Argyrophilic Nucleolar Organizer Regions):

Nucleolar organizer regions are basically proteins present within the nucleus of the cells and these NORs are responsible for the formation of RNA. Larger the number of nucleolar organizer regions in nucleus, greater is the replication capacity of cells⁽⁶⁾. Since the nucleolar organizer regions are stained with silver salts (Argyrum-Ag) they are called as AgNORs⁽⁷⁾ that was first used by Howell et al in the year 1975. Studies performed by Schawarzacher and Ploton⁽⁸⁾ used silver stained AgNORs technique and concluded in 1983 that silver stained proteins were formed by RNA polymerase enzyme that are involved in nucleolus DNA and ribosomic RNA copy.

PAS (periodic acid Schiff) staining:

This staining is used to determine the polysaccharides such as glycogen and muco substances glycoproteins, glycolipids and mucins in tissues.

SITES OF SMEAR TAKING:

Most of the cytological smears are collected from two anatomical sites floor of mouth, lateral borders of tongue as these sites are highly affected and shows cellular changes caused due to effects of smoking. More than the lateral borders of tongue, floor of mouth is most affected because less keratinized regions are highly affected⁽⁹⁾. Apart from these sites, mucosa of lip is also an included site as smokers develop hyper and hypo pigmented patches around upper and lower lip due to prolonged placement of cigarette ⁽¹⁰⁾.

Different methodologies and studies performed using exfoliative cytology:

Detection of micronuclei using exfoliative cytology:

Micronuclei are small nuclei that are formed during stages of anaphase. These are extra nuclear cytoplasmic inclusions. These micronuclei formation is induced by genotoxic and carcinogenic chemicals from betel nut, tobacco and also from Ca(OH)₂content of lime in betelquid⁽¹¹⁾ and are considered as biomarkers to assess DNA damage. Thus oral exfoliative cytology showing the presence of micronuclei as extra cytoplasmic inclusion confirms dysplastic changes of oral mucosa.

Cytomorphological study using exfoliative cytology:

According to the study conducted by Mustafa et .al, Hayati Murat et.al, Cemal et.al⁽¹²⁾, the following parameters were included in their study:- NA(nuclear area), minimal and maximal ND(nuclear diameter), CA(cytoplasmic area), NA/CA in both study group(included smokers) and control groups(non smokers). Cytological study proved that a statistical difference existed in NA, minimal and maximal ND, ratio between NA and CA among study and control groups with increased NA(nuclear area) and ND(nuclear diameter) among smokers. This also proves the dysplastic changes in oral mucosa of smokers.

According to studies conducted by Renata patella cancado et.al, Liliane Soares et.al, Manoel et.al, cytological smears were collected from two anatomical sites floor of mouth and lateral border of tongue from smoking and non smoking patients. Study was done to detect the presence of silver stained NORs(AgNORs) responsible for RNA replication. They concluded that smears collected from smoking patients when stained and studied had more than 3 AgNORs contained within the nucleus than smears collected from non smoking patients proving the proliferative nature of oral mucosal cells⁽¹³⁾.

Based on the cytological studies performed by Renuka verma et.al, ratio of NA(nuclear area) and CA (cytoplasmic area) and ratio of NA and CA of cells were studied⁽¹⁴⁾. Smears for cytological study were collected from a normal buccal mucosa, from OLD (oral leukoplakia with dysplasia) and from OSCC (oral squamous cell carcinoma). The cytological study gave a conclusion that an “increased nuclear area(NA) and decreased CA(cytoplasmic area)’ is seen in OSCC smears followed by OLD smears and nuclear, cytoplasmic area is found to be normal in a normal mucosa thus proving the dysplastic nature of cells in patients with OSCC and OLD⁽¹⁵⁾.

Present scenario in exfoliative cytology:

Exfoliative cytology is considered as most dependant and trustworthy tool in the diagnosis of malignancies in the body. Exfoliative cytology is employed to detect carcinomas in oral cavity, uterine cervix, urinary bladder, lungs, oesophagus, stomach, rectum and sigmoid colon. Exfoliative cytological study on breast secretions are done as latest advances. Lots of technical improvements have also been done recently. Immediate fixation of smear is of utmost importance. Improper fixation or excessive drying of smear can lead to withdrawal of cells from slide resulting in false diagnosis. Various developments in stains and staining methods is also done that aids in clear nuclear identification, cytoplasm transparency, accurate cell differentiation which positions exfoliative cytology as a standardized diagnostic tool for pathologists all over the world.

Recent advancements in diagnosis:

The ultimate aim of WHO is to reduce the incidences of oral cancer to a third by diagnosing and treating at the pre malignant state itself.

Current advancements include the use of the following,

· ViziLite^R(Zila pharmaceuticals USA) is an alternative to white light illumination. A disposable chemiluminescent source is illuminating the oral tissues with a blue light. Studies conducted by Epstein et.al⁽¹⁶⁾ and Kerr et.al ⁽¹⁷⁾ ViziLite helped in the identification of oral premalignant lesions by increasing brightness and sharpness.

· Wide field fluorescence imaging is also used as a latest technique. It works on the principle that presence of fluoropores⁽¹⁸⁾ namely NADH(nicotinamide adenine dinucleotide )and FAD(flavin adenide dinucleotide) in oral epithelium and stroma absorb UV and visible light and re-emits in the form of fluorescence.

· VELscope^R(LED dental , Canada) is the instrument that is designed to detect tissue auto fluorescence from the oral cavity. According to Lane et.al ⁽¹⁹⁾ VELscope detects abnormal tissues brown to black in color due to decreased auto fluorescence and normal healthy tissues in pale green auto fluorescence. This helps to detect premalignancies in oral cavity.

· Some of the molecular biomarkers that are used in the early diagnosis of premalignancies includes DNA content, chromosomal polysomy, loss of heterozygosity(LOH), nucleolar organizer regions, histo- blood group antigens, proliferation markers, increased EGFR(epithelial growth factor receptor)⁽²⁰⁾.

· Usage of optical spectroscopy using a fibreoptic probe is also a bloodless diagnostic method for detecting oral premalignancies⁽²¹⁾.

· Carlson et.al made a dual mode reflectance and fluorescence confocal microscope to view the molecular properties of tissues and to view tissue morphology and cellular morphology using specific contrasting agents and dyes which is a “high resolution technique” to view oral cancer⁽²²⁾.

Advantages in employing exfoliative cytology:

Exfoliative cytology is a simple, non invasive, bloodless diagnostic procedure because of which it is indicated in systemic diseases like diabetes, haemophilia etc. It is easy to perform and it can be repeated multiple times with better sensitivity and specificity. This aids to differentiate pre malignancies and malignancies. There is no use of sharp scalpels for smear collection which is a great advantage over biopsy. The technique is comfortable for both patient and performer. Mainly false negative and post biopsy complications are eliminated in exfoliative cytology.

Limitations in exfoliative cytology:

Smears collected from keratinized sites of leukoplakia are called “negative smears” and gave false negative grading whereas smears collected from ulcer or non keratinized areas are called “positive smears” and gave positive grading. This hence shows that, in 60% cases of premalignancies like leukoplakia exfoliative cytology mostly gives a false negative grading. Also performing cytology multiple times did not give approximate results as it varied with quantity and quality of smears collected. Due to these limitations, biopsy is proved to be more sensitive and specific than exfoliative cytology.

CONCLUSION:

Exfoliative cytology has evolved with various staining methods and techniques from the time it was introduced till date. Having lots of advantages over biopsy it is still an adjuvant to biopsy. Results obtained from cytology are always confirmed by a definitive biopsy. Modifications and advancements are done in cell counting, smear collecting methods but still exfoliative cytology has its own standard in the diagnosis of dysplastic changes of oral cavity.

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Received on 22.07.2017 Modified on 23.08.2017

Research J. Pharm. and Tech 2017; 10(12): 43930-4396.

DOI: 10.5958/0974-360X.2017.00809.5