Presentation on Exfoliative cytology related to dentistry. I have covered recent advances as well.

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
Exfoliative Cytology
GUIDED BY:
DR.C.R.MURALI, MDS.
DR.A.KENNATH J.ARUL, MDS., MBA., PHD.
DR.SONIKA, MDS.
DR.S.SOUNDARYA, MDS.
DR.P.SHANMUGA PRIYA, MDS.

2. CONTENTS
 Definition
 Rationale
 History of EC
 History of EC in oral cavity
 Indications
 Contraindications
 Methodological
modifications
 Sites for smears
 Materials required
 Preparation of the tissue
site
 Smear procedure
 Papanicolaou staining
 Systemic study of smears
 Recent trends in EC
 Advantages
 Disadvantages
 Conclusion
 References

3. DEFINITION
 Exfoliative cytology (EC) is the
microscopic examination of shed,
desquamated cells from body
surfaces or cells harvested by
rubbing or brushing a lesional
tissue surface.
 It also includes cells harvested
from mucus membranes and
body fluids.
Gene Boyer

4. RATIONALE
 Epithelial physiology
 Epithelial turnover
 Deep lying cells adherent to
one another
 Cohesiveness is lost in malignancy and some benign
conditions
 Analyzed quantitatively and qualitatively
Wikipedia

5. HISTORY OF EXFOLIATIVE CYTOLOGY
 1843 – Walshe – Cancer cells in sputum
 1851 – Lebert – altered size of cells in diagnostic
cancer cytology
 1860 – Beale – cancer cells drawn from oropharynx
 1927 – Dudgeon – direct smear technique for rapid
diagnosis
 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
saliva
 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
smears

7. INDICATIONS
 The lesion is innocuous as not to arouse suspicion
 There is hesitancy on part of dentist or the patient for
biopsy
 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.

8. CONTRAINDICATIONS
 An obvious cancer that would
justify a biopsy
 Unreliable patient
 A sub-mucosal lesion
 A dry or crusted lesion as may be
seen on the lips
 A white lesion that does not rub
off.

9. METHODICAL MODIFICATIONS
 Gladstone (1951) - use of sponge biopsy
 Schneider (1952) and Cawson (1960) - variants of
staining techniques.
 King (1963) - use of frosted glass slides
 Staats and Goldsby (1963) recommended the metal
spatula.
 Sandler (1964) - removal of keratotic layers with a
curette.
 Dumbach (1980) - smear curettage
 Mehrotra (2008) - tooth brush to harvest cells in
resource challenged setting.

10. SITES FOR SMEARS
 Buccal mucosa
 Junction of the hard & soft
palate
 Dorsum of the tongue
medind.nic.in
 Floor of the mouth and
 The lower labial region.
healthadviceforlife.com

11. MATERIALS REQUIRED
 Microscopic slides stored in
containers
 A cell harvesting instrument
(wooden spatula, metal
spatula, cytobrush, oralCDX
brush, tooth brush)
 Fixative
 Clinical report form
 Slide marking pencil simurg-mp.com
medscape

12. PREPARATION OF THE TISSUE SITE
 No wiping/drying
 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

14. ILLUSTRATION

15. PAPANICOLAOU STAINING
 Composition:
 Harris hematoxylin
 Orange G6
 10% aqueous Orange G – 50 mL
 Alcohol – 950 mL
 Phosphotungstic acid – 15 g
 EA 50
 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
pocdscientific.com.au

16. STAINING PROCEDURE
 Remove polyethylene glycol fixative in 50% alcohol,
2 minutes.
 Hydrate in 95% alcohol, 2 minutes, and 70% alcohol,
2 minutes.
 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
approx.
 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
xylene)

18. PAPANICOLAOU STAIN - OUTCOMES
 Hematoxylin - Nuclei - blue/black
 Light green SF - Cytoplasm - blue/green
 OG-6 - Keratinizing cells - pink/orange
 EosinY – Squamous cell, nucleoli, RBC’s – Red/Pink
www.polysciences.com

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
mandatory.
 Class V (positive for cancer): cells show
characteristic malignant changes. Biopsy is
mandatory.

20. ORAL CYTOLOGIC GRADING SYSTEM
Specimen adequacy
 Adequate for evaluation (note the presence of basal/parabasal cells)
 Inadequate for evaluation (specify reason, e.g. obscuring elements,
broken slides)
General categorization
 A: Normal
 B: Reactive - hyperkeratosis, inflammatory, infective, repair &
chemo/radiation changes
 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
 Anucleated
orthokeratinized
squamous cells
 Polygonal.
 Cytoplasm stains orange to
yellow
 Parakeratotic cells
 Polygonal.
 Cytoplasm is eosinophilic
 Superficial cells show
pyknotic nuclei
www.glowm.com

22. ATYPICAL CELL CYTOLOGY
 Proportionate enlargement. Bacterial colonization in
cytoplasm. Indistinct cell outline. Perinuclear halo
evident.
 Viral infection – ballooning degeneration and
inclusion bodies. Giant nuclei and multinucleated
cells seen.
 Fungal infections – yeast cells and hyphae.
 Benign acanthosis and pemphigus – rounded and
small cell.
 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
Wikipedia
Oral Candidiasis in Pap smear
pinterest.com
Mehrotra et al Mehrotra et al

24. CYTOPATHOLOGY OF ORAL CARCINOMA
 Nuclear abnormalities
 Increased nuclear size
 Irregular shapes
 Multinucleation
 Abnormal mitosis
 Nuclear hyperchromatism
 Aberrant chromatin pattern
 Altered nuclear cytoplasmic
ratio
 Degenerative changes of
the nuclei
sphweb.bumc.bu.edu
Hopkins medicine

25. CYTOPATHOLOGY OF ORAL CARCINOMA cont
 Cytoplasmic abnormalities
 Scantycytoplasm
 Vacuolizationandinclusions
 Alteredstaining
 Cellasawhole
 Enlargement – Anisocytosis &
anisonucleosis
 Bizarreshapes

26. RECENT TRENDS IN EXFOLIATIVE CYTOLOGY
 ViziLite Plus with
Tblue
 Microlux DL
 Orascoptic DK
 VELscope
 OralCDx
Hitachi data systems

27. VIZILITE PLUS WITH TBLUE
 Chemiluminescent light
detection system developed
from predicate devices to
detect cervical neoplasia.
 Sites of epithelial
proliferation preferentially
reflect the low energy blue-
white light generating an
“acetowhite” change.
 Acetic acid rinse required
before the procedure.
denmat.com

28. MICROLUX DL SYSTEM
 Microlux DL system is
developed from a
blue-white light-emitting
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
 Three-in-one,
battery-operated,
handheld LED instrument
 An oral lesion screening
instrument attachment
 Mild acetic acid rinse
promoted to improve
visualization of oral
lesions.
moorefamilydentist.com

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
activity.
http://ritadarghamdentist.com/

31. DIAGNOSTIC TESTS
 Diagnostic tests and
newer smear collecting
instruments were devised
to aid in harvesting
adequate cells for
microscopy.
 Cytobrush
 Liquid based cytology
 OralCDX
cynthiaskibadds.com

32. CYTOBRUSH
 The brush - rotated under
slight pressure several
times on the suspicious
lesion.
 Immediately smeared on
glass slides and fixed with
alcoholic spray.
 Brush biopsy - as an
additional diagnostic tool
for oral lesions that are
not highly suspicious for
malignancy. actaodontologica.com

33. LIQUID BASED CYTOLOGY
 used on oral smears
collected by cytobrush
 Thinprep, Surepath and
Shandon PapSpin
 smear thickness and
cellular distribution -
easier identification of
abnormal cells.
 sensitivity of 95.1% and
specificity of 99%.
mdlab.com

34. ORAL COMPUTER ASSISTED BRUSH CYTOLOGY
 OralCDx - brush designed to obtain
a complete transepithelial
specimen.
 Stained using modified
Papanicolaou method & scanned in
OralCDx computer system.
 OralCDx computer system - neural
network-based image-processing
system
 specifically designed to detect oral
epithelial precancerous and
cancerous cells.
 specificity and sensitivity over 90% cdxdiagnostics

35. ORAL CDx TECHNIQUE

36. METHODS IN DEVELOPMENT
 Laser capture microdissection (LCM)
 Lab-on-a-chip (LOC) sensor technique
 DNA image cytometry
 Saliva based oral cancer diagnosis
 Molecular analysis
 Microscopy
 Spectroscopy
 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
biomarkers &
protein
fingerprint
models for early
SCC detection
http://pubs.niaaa.nih.gov/

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
profiles.
 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
ploidy.
 Test group compared with
controls (normal epithelial
cells) after Feulgen dye stain.
 A program identifies the
deviations in the cellular DNA
content
 This method has 100%
sensitivity and specificity. www.cytopathologie-dna-icm.uni-duesseldorf.de

40. SALIVA BASED ORAL CANCER DIAGNOSIS
 Effective modality for
diagnosis, determining
prognosis of oral cancer
and for monitoring
post-therapy status.
 Used to measure specific
salivary macromolecules
and proteomic or
genomic targets.
http://perirx.com/

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
protein levels.

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
inflammation, dysplasia
and cancer.
 records subsurface
reflections to build a cross-
sectional architectural
image.
 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
irradiation
 Evaluation of some hereditary disease, for toxic
reaction subsequent to cancer

45. ADVANTAGES AND DISADVANTAGES
 Advantages
 Non-invasive and painless
 Minimal skills
 Patient compliance
 Cost effective
 Performed in large numbers
 Minimal instruments
 Early diagnosis of lesions
 Can be used in patients with
systemic disorders where
biopsy is contraindicated
 Easily done at the chairside
 Disadvantages
 False negative results
 Only an adjuvant
 Contamination
 Low sensitivity
 Inadequate sampling
 Not usable in non epithelial
lesions

46. CONCLUSION
 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.

47. REFERENCES
 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;
28:165-72.
 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
1994;77:101-4.
 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
2011 3:33.

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;
26: 201-5.
 Papanicolaou GN, Traut HF. The diagnostic value of vaginal
smears in carcinoma of the uterus. Am J Obstet Gynecol 1941;
42:193-205.

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
1963; 7:180-2.
 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.