1. P R E S E N T E D B Y
D R . M . R A J A N N A V E N K A T R A M A N
1 S T Y E A R P O S T G R A D U A T E S T U D E N T
U L T R A ’ S B E S T D E N T A L S C I E N C E C O L L E G E
DR.A.KENNATH J.ARUL, MDS., MBA., PHD.
DR.P.SHANMUGA PRIYA, MDS.
History of EC
History of EC in oral cavity
Sites for smears
Preparation of the tissue
Systemic study of smears
Recent trends in EC
Exfoliative cytology (EC) is the
microscopic examination of shed,
desquamated cells from body
surfaces or cells harvested by
rubbing or brushing a lesional
It also includes cells harvested
from mucus membranes and
Deep lying cells adherent to
Cohesiveness is lost in malignancy and some benign
Analyzed quantitatively and qualitatively
5. HISTORY OF EXFOLIATIVE CYTOLOGY
1843 – Walshe – Cancer cells in sputum
1851 – Lebert – altered size of cells in diagnostic
1860 – Beale – cancer cells drawn from oropharynx
1927 – Dudgeon – direct smear technique for rapid
1943 – Papanicolaou and Traut – cytodiagnosis as a
routine to diagnose cervical cancers
6. HISTORY OF ORAL EXFOLIATIVE CYTOLOGY
1890 – Miller - epithelial cells and leucocytes in
1939 – Orban & Weinmann – cellular contents of
saliva in patients with dental caries
1940 – Ziskin Kamen et al – use of EC in oral cavity
1951 – Miller & Montgomery – EC in normal mucosa
1951 – Montgomery & Hamm – EC as a method to
diagnose oral cancer
1963 - Sandler – various methods of obtaining
The lesion is innocuous as not to arouse suspicion
There is hesitancy on part of dentist or the patient for
Large or multiple red lesions
Lesion located in region that presents surgical difficulty
When Herpes or Candida is suspected
As a follow-up for detection of recurrent cancer.
Unavailability of embedding & sectioning technology.
An obvious cancer that would
justify a biopsy
A sub-mucosal lesion
A dry or crusted lesion as may be
seen on the lips
A white lesion that does not rub
9. METHODICAL MODIFICATIONS
Gladstone (1951) - use of sponge biopsy
Schneider (1952) and Cawson (1960) - variants of
King (1963) - use of frosted glass slides
Staats and Goldsby (1963) recommended the metal
Sandler (1964) - removal of keratotic layers with a
Dumbach (1980) - smear curettage
Mehrotra (2008) - tooth brush to harvest cells in
resource challenged setting.
10. SITES FOR SMEARS
Junction of the hard & soft
Dorsum of the tongue
Floor of the mouth and
The lower labial region.
11. MATERIALS REQUIRED
Microscopic slides stored in
A cell harvesting instrument
(wooden spatula, metal
spatula, cytobrush, oralCDX
brush, tooth brush)
Clinical report form
Slide marking pencil simurg-mp.com
12. PREPARATION OF THE TISSUE SITE
Debris or slough – wet gauze used to clean
Tender lesion – local anesthetic application
Keratotic lesions – curette’s or small diamond
stone used to remove keratin layer. Smear taken
from pink tissue.
Exudates – treated like blood smears
13. SMEAR TAKING/HARVESTING CELLS
Identifiers written on slide with lead pencil
Suitable wet instrument is vigorously scraped against
the lesion in one direction
Scrapings picked up are spread evenly and rapidly
over an empty slide
Specimens fixed with ethanol or isopropyl alcohol
Stained with Papanicolaou/Gram/PAS/H&E stains
Clinical requisition form completed
Mounted and studied under microscopy
15. PAPANICOLAOU STAINING
10% aqueous Orange G – 50 mL
Alcohol – 950 mL
Phosphotungstic acid – 15 g
0.04 M Light Green SF – 100 mL
0.3 M Eosin – 20 mL
Phosphotungstic acid – 2 g
Alcohol – 750 mL
Methanol – 250 mL
Glacial acetic acid – 20 mL
16. STAINING PROCEDURE
Remove polyethylene glycol fixative in 50% alcohol,
Hydrate in 95% alcohol, 2 minutes, and 70% alcohol,
Rinse in water, 1 minute.
Stain in Harris’s hematoxylin, 5 minutes.
Rinse in water, 2 minutes.
Differentiate in 0.5% aqueous HCl, 10 seconds
Rinse in water, 2 minutes.
17. STAINING PROCEDURE – CONTD.,
Dehydrate, 70% alcohol, for 2 minutes.
Dehydrate, 95% alcohol, 2 minutes.
Dehydrate, 95% alcohol, 2 minutes.
Stain in OG 6, 2 minutes.
Rinse in 95% alcohol, 2 minutes.
Rinse in 95% alcohol, 2 minutes.
Stain in EA 50, 3 minutes.
Rinse in 95% alcohol, 1 minute.
Mount coverslip with DPX (Distyrene, plasticizer and
19. SYSTEMIC STUDY OF SMEARS
Class I (normal): only normal cells are observed.
Class II (atypical): presence of minor atypia due
to inflammation. No signs of malignancy.
Class III (intermediate): wider atypia suggestive
of severe dysplasia, carcinoma-in-situ or cancer.
Class IV (suggestive of cancer): shows few
epithelial cells with malignant changes. Biopsy is
Class V (positive for cancer): cells show
characteristic malignant changes. Biopsy is
20. ORAL CYTOLOGIC GRADING SYSTEM
Adequate for evaluation (note the presence of basal/parabasal cells)
Inadequate for evaluation (specify reason, e.g. obscuring elements,
B: Reactive - hyperkeratosis, inflammatory, infective, repair &
C: Atypical - probably reactive/low grade including low grade
squamous intraepithelial lesion (LSIL)
D: Atypical - Probably high grade
E: High grade squamous intraepithelial lesion
F: Invasive squamous cell carcinoma
G: Other neoplasms: Specify
21. NORMAL CELL CYTOLOGY
Cytoplasm stains orange to
Cytoplasm is eosinophilic
Superficial cells show
22. ATYPICAL CELL CYTOLOGY
Proportionate enlargement. Bacterial colonization in
cytoplasm. Indistinct cell outline. Perinuclear halo
Viral infection – ballooning degeneration and
inclusion bodies. Giant nuclei and multinucleated
Fungal infections – yeast cells and hyphae.
Benign acanthosis and pemphigus – rounded and
Benign dyskeratotic cells in oral lesions associated
with dermal conditions.
23. ATYPICAL CELL CYTOLOGY - INFECTIONS
Simonsiella infection in Pap smear Herpes simplex virus in oral smear
Oral Candidiasis in Pap smear
Mehrotra et al Mehrotra et al
24. CYTOPATHOLOGY OF ORAL CARCINOMA
Increased nuclear size
Aberrant chromatin pattern
Altered nuclear cytoplasmic
Degenerative changes of
26. RECENT TRENDS IN EXFOLIATIVE CYTOLOGY
ViziLite Plus with
Hitachi data systems
27. VIZILITE PLUS WITH TBLUE
detection system developed
from predicate devices to
detect cervical neoplasia.
Sites of epithelial
reflect the low energy blue-
white light generating an
Acetic acid rinse required
before the procedure.
28. MICROLUX DL SYSTEM
Microlux DL system is
developed from a
diode (LED) and a
diffused fiber-optic light
guide that generates a
low-energy blue light.
This system also uses
acetic acid gargle. gabinetyka.pl
29. ORASCOPTIC DK SYSTEM
handheld LED instrument
An oral lesion screening
Mild acetic acid rinse
promoted to improve
visualization of oral
30. VELSCOPE SYSTEM
Multiuse device with a hand-held scope
To scan the mucosa visually for changes in tissue
fluorescence. The wavelength used is 430 nm.
Principle - tissues of the
oral cavity have variable
fluorescence which is
altered by structural
changes and metabolic
31. DIAGNOSTIC TESTS
Diagnostic tests and
newer smear collecting
instruments were devised
to aid in harvesting
adequate cells for
Liquid based cytology
The brush - rotated under
slight pressure several
times on the suspicious
Immediately smeared on
glass slides and fixed with
Brush biopsy - as an
additional diagnostic tool
for oral lesions that are
not highly suspicious for
33. LIQUID BASED CYTOLOGY
used on oral smears
collected by cytobrush
Thinprep, Surepath and
smear thickness and
cellular distribution -
easier identification of
sensitivity of 95.1% and
specificity of 99%.
34. ORAL COMPUTER ASSISTED BRUSH CYTOLOGY
OralCDx - brush designed to obtain
a complete transepithelial
Stained using modified
Papanicolaou method & scanned in
OralCDx computer system.
OralCDx computer system - neural
specifically designed to detect oral
epithelial precancerous and
specificity and sensitivity over 90% cdxdiagnostics
36. METHODS IN DEVELOPMENT
Laser capture microdissection (LCM)
Lab-on-a-chip (LOC) sensor technique
DNA image cytometry
Saliva based oral cancer diagnosis
Optical coherence tomography (OCT)
37. LASER CAPTURE MICRODISSECTION
LASER coupled with microscope
An element is cut out from the tissue using LASER.
Non contact microdissection is also used.
Also to detect
models for early
38. LAB-ON-A-CHIP SENSOR TECHNIQUE
Utilizes membrane-associated cell proteins
expressed on the cell membranes of dysplastic and
cancer cells and their unique gene transcription
LOC sensor - embedded track-etched membrane,
which functions as a micro-sieve, to capture and
enrich cells from brush cytology suspensions.
Immunofluorescent assays reveal - presence and
phenotype of interrogated cells via automated
microscopy and fluorescent image analysis.
39. DNA IMAGE CYTOMETRY
Measures the malignant
potential of cells by DNA
Test group compared with
controls (normal epithelial
cells) after Feulgen dye stain.
A program identifies the
deviations in the cellular DNA
This method has 100%
sensitivity and specificity. www.cytopathologie-dna-icm.uni-duesseldorf.de
40. SALIVA BASED ORAL CANCER DIAGNOSIS
Effective modality for
prognosis of oral cancer
and for monitoring
Used to measure specific
and proteomic or
41. MOLECULAR ANALYSIS
Combined with liquid based cytology - visualization
of malignant cells using antibodies against
cytokeratin AE1 and AE3.
Nuclear organizer regions (NOR) measures the
cellular proliferation and thereby differentiates a
reactive lesion from nonneoplastic lesion.
Protein- Chip arrays (SELDI) is a recent technique of
monitoring oral lesions based on expression of
42. SPECTRAL CYTOPATHOLOGY
Technique for diagnostic differentiation of disease in
individual exfoliated cells.
Multispectral digital microscope acquires in-vivo
images in different modes i.e. fluorescence, narrow-
band reflectance, & orthogonal polarized reflectance.
Deviations from natural composition produced
specific spectral patterns.
Unique spectral patterns were analyzed to detect
cells in dysplasia, neoplasia, or viral infection.
43. OPTICAL COHERENCE TOMOGRAPHY
imaging to detect areas of
reflections to build a cross-
Contrast enhanced by
surface plasmon resonant
gold nanoparticles. ophthalmologymanagement.com
44. USES OF ORAL EXFOLIATIVE CYTOLOGY
Early detection and control of oral cancer, microbial
diseases (candidiasis and viral infections) and
dermatological lesions (pemphigus)
Assessment of nutritional iron deficiency
Forensic dentistry (age and sex determination)
Study of conditions like diabetes mellitus, smoking,
alcoholism, pregnancy, and ageing.
Predicting the cellular response of a tumour to
Evaluation of some hereditary disease, for toxic
reaction subsequent to cancer
45. ADVANTAGES AND DISADVANTAGES
Non-invasive and painless
Performed in large numbers
Early diagnosis of lesions
Can be used in patients with
systemic disorders where
biopsy is contraindicated
Easily done at the chairside
False negative results
Only an adjuvant
Not usable in non epithelial
Exfoliative oral cytology is a simple, pain-free, non-
invasive, non-aggressive and rapid technique.
Any dentist could perform an oral brushing.
Because of the continuing development of cytological
techniques and improvements in cell collecting
instruments and methods, there is now a big
challenge for oral cytology to become a routine
procedure in patients with oral mucosa problems.
Babshet M, Nandimath K, Pervatikar SK, Naikmasur VG.
Efficacy of oral brush cytology in the evaluation of the
oral premalignant and malignant lesions. J Cytol 2011;
Bancroft J, Layton C, Suvarna S. Theory and Practice of
Histological Techniques, 7th Edition.
Beale LS. Examination of sputum from case of cancer of
the pharynx and adjacent parts. Arch Med. 1860; 2:44–6.
Bernstein ML, Miller RL. Oral exfoliative cytology. J Am
Dent Assoc 1978; 96:625-9.
Johnston D G. Cytoplasmic: nuclear ratios in the
cytologicai diagnosis of cancer. Cancer 1952: 5: 945-9.
48. REFERENCES CONTD
Jones A, Stewart C, Baughman R. The cytobrush plus cell
collector in oral cytology. Oral surg Oral med Oral Pathol
Kaur M, Saxena S, Samantha YP, Chawla G, Yadav G.
Usefulness of Oral Exfoliative Cytology in Dental Practice. J
Oral Health Comm Dent 2013;7(3)161-165.
Kazanowska K, Hałoń A, Radwan-Oczko M. The Role and
Application of Exfoliative Cytology in the Diagnosis of Oral
Mucosa Pathology – Contemporary Knowledge with Review of
the Literature. Adv Clin Exp Med 2014, 23, 2, 299–305
Mehrotra and Gupta: Exciting new advances in oral cancer
diagnosis: avenues to early detection. Head & Neck Oncology
49. REFERENCES CONTD
Mehrotra R, Singh MK, Pandya S, Singh M. The use of an oral
brush biopsy without computer-assisted analysis in the
evaluation of oral lesions: a study of 94 patients. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod. 2008; 106:246–53
Mehrotra R. Textbook of Oral Cytology, First edition.
Montgomery, P. W.: A Study of the Exfoliative Cytology of
Normal Human Oral Mucosa, J. D. Res. 30: 12, 1951.
Ogden GR, Cowpe JG, Wight AJ. Oral exfoliative cytology:
review of methods of assessment. J Oral Pathol .Med 1997;
Papanicolaou GN, Traut HF. The diagnostic value of vaginal
smears in carcinoma of the uterus. Am J Obstet Gynecol 1941;
50. REFERENCES CONTD
Papanicolaou, G. N., and Traut, H. F.: Diagnosis of
Uterine Cancer by the Vaginal Smear, New York, 1943,
Commonwealth Fund, p. 46.
Sandler HC. Oral exfoliative cytology: Veterans
Administration Cooperative Study, 1962. Acta Cytol
Scheifele C, Schmidt-Westhausen AM, Dietrich T,
Reichart PA: The sensitivity and specificity of the
OralCDx technique: evaluation of 103 cases. Oral Oncol
2004, 40, 824–828.
Ziskin DE, Kamen P, Kitley I. Epithelial smears from oral
mucosa. J Dent Res. 1941; 20:386–7.