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Wishing you all a very

Happy & Prosperous
New year 2013..!!

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EXFOLIATIVE CYTOLOGY

Dr. Muhsina Mahmood

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CONTENTS
o Introduction
o Definition
o History
o Indications
o Contraindications
o Technic
o Advantages
o Limitations
o References

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 Introduction
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 Diagnostic cytology is the process of studying cells to

identify diseases.

 Methods to obtain cell samples are varied

 Involve taking either a sample of body fluids or a scra

ping of cells from body tissue.

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Cytological methods
---Rubin,1994

Exfoliative Cytology

Abrasive Cytology

Fine Needle Aspiration

Cytology

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Definition
 The microscopic examination of shed or desquamated cells
from the epithelial surface usually the mucous membrane.
It also includes the study of those cells that have been col
lected by scraping the tissue surface or collected from bo
dy fluids such as sputum, saliva etc

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 Histor
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y
 1843 – Walsh – first to describe cancer cells in patient’s sput
um

 1940- Ziskin et al – exfoliated cytology in oral cavity.

 1941 – George N Papanicolau- introduced cytodiagnosis as ro

utine procedure in detection of cervical cancer. ‘PAP Test’

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 1951 – Miller et al – studied on exfoliative cytology of norma

l epithelium.

 Montgomery & Von Hamm – use of cyology in diagnosis of ora

l cancer

 1981 – Couwpe et al – quantitative techniques to cytology cou

ld markedly improve diagnostic sensitivity.

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Rationale
 Continuous exfoliation of epithelial cells is a part of physiologi
cal turnover.

 Deeper cells, which are strongly adhered in normal conditions,

become loose in the case of malignancy and exfoliate along wit
h superficial cells.

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 Uses
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 Early detection & control of oral cancer

o Dental clinic --- cytology of oral precancerous lesions
o Mass screening --- Ideal tool for sorting out the lesions at
an initial stage in camps and epidemiological studies.
 Microbial disease
o viral & fungal infections
 Dermatological lesion
o e.g. pemphigus
 DNA studies

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 Forensic dentistry
o Sex determination
o Identification of individuals from cells retrieved from crime
scene

 Hormonal evaluation in females

o E.g. Study of gingival smears shows variation to cyclic chang
es during menstruation
 Assessment of nutritional status
o E.g. iron deficiency anemia

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‘Estimation of antioxidants in oral exfoliated cells’

N Gururaj, B Sivapathasundharam, S Sumathy

 Department of Oral and Moxillofacial Pathology, Meenakshi Ammal Dental Co
llege & Hospital, Chennai, India

- JOMFP

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‘Sex determination from buccal mucosa scrapes’ LOGO

Tushar M, K Saralaya et al
Kasturba Medical College, Mangalore

ABSTRACT:
Establishing individuality is an imperative aspect in any investigation procedure. At times, it becomes
necessary to determine the sex of the individual to establish identity, and saliva stains found at the
scene of crime are of major help in such cases. In the present study, we have determined the sex of the
individual from buccal mucosal scrapings. Buccal smears prepared from 100 men and 100 women were stained
by the Papanicolaou staining method. Cells were observed forBarr bodies under oil immersion with a
compound microscope, and the percentage of Barr-body-positive cells was determined. It was observed that
1.14% ofbuccal mucosal cells in men (range = 0-4%) and 39.29% of buccal mucosal cells in women (range = 20-
78%) showed Barr bodies. Inferences from the study show that the presence of Barr body in buccal mucosal
cells can be demonstrated with a fair degree of accuracy using Papanicolaou staining. The sex of the individual
can be determined accurately, as two non-overlapping ranges for the percentage of Barr-body-positive cells
has been obtained for men and women. This method not only proves to be accurate but is also simple and
economic

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 Indications
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 Mucosal lesions – clinically innocuous

Analysis of extensive mucosal lesion where multiple biopsies
may not be possible.
 Patient preference

Follow up
 Debilitating patient

As an adjuvant test
 Rapid evaluation of a lesion

To study & confirm false negative biopsy result.

 Population screening.

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 Contraindications
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 Obvious malignant lesions

 Lesions with intact normal epithelium

 Sub-mucosal lesions

 Pigmented lesions

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Smear Technic

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Requirements
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 Microscopic slides

 Sampling method:
o saliva collection
o wooden/plastic/sterile metal spatula
o oral cytobrush / tooth brush

 Fixative

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 Preparation of the lesion LOGO

Do not wipe or dry the lesion

Debris/slough -- cleanse with gauze moistened in normal saline

Topical anesthesia for tender lesions

Fissures- suitable site for sampling in keratotic lesions

keratotic lesions, scrape off white keratinized surface

with curette

Remove encrustations of lip

Exudates are treated like blood smears

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 PROCEDURE LOGO

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 Drawbacks
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 Only superficial cells are

obtained
 Inadequate sampling

 Poor access

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Oral Cytobrush

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 General Instructions LOGO

 Have patient rinse with water prior to brush biopsy.

 Topical anesthesia.

 A slight bend of brush handle–pressure indicator.

 Stop immediately if lesion bleeds.

 Transfer specimen to slide in a gentle rotating motion.

 Avoid re brushing of lesion to harvest more cells.

 Fix within 15 – 30 sec to prevent air drying.

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‘Cytobrush and wooden spatula for oral exfoliative cytology.

A comparison.’

Ogden GR, Cowpe JG, Green M

Department of Dental Surgery, Dundee University, Scotland.

Abstract; The Cytobrush has been used frequently in cervical cytology, but as yet its value in oral exfoliative cytology h
as not been assessed. A study was undertaken to compare the efficiency of the Cytobrush with that of the wooden tongue spatu
la. For 26 patients, two smears were collected from clinically normal mucosa from four sites in the oral cavity (buccal mucosa, do
rsal tongue, ventral tongue and hard palate). The smears were graded for cell yield and dispersion on a three-point scale. The re
sults were analyzed using the chi 2 test. The Cytobrush was found to be significantly more efficient than the wooden spatula, in
terms of both cell yield (P less than.005) and cell dispersion (P less than.005). When each site was examined separately, the Cyt
obrush produced significantly better dispersion for the dorsal tongue, ventral tongue and buccal mucosa and a better cell yield f
The study sh
or the tongue surfaces. No significant difference for cell yield or dispersion was found for the hard palate.
owed that the Cytobrush is an effective instrument for use in exfoliative cytology of
normal oral mucosa.

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 Technique suggestion for specific lesions LOGO

WHITE LESION
Brush until pink tissue or pin point bleeding
is seen

ULCERATED LESION
• Do not brush the center of ulcer
• Brush along the margin where healthy tissue
meets the lesion.

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RED LESIONS
 Do not apply much pressure.
 Brush along the margin.

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 THICK KERATINISED WHITE LESION / LOGO
LESIONS ON KERATINISED EPITHELIUM

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Specimen Preparation
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 Direct spread smears

 Spatula
 Simple technique – require skill & experience

 Need adequate sample

 Evenly distributed – uniform thickness

 Too thick – cells overlap – impossible to assess

 Too thin – unrepresentative smear

 Too pressure – cell distortion

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Liquid Based Cytology - 1999 LOGO

Brush is vigorously twirled in solution. Brush

head is placed in bottle.
Sampling- cytobrush

Slides
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centrifuged
 Advantages LOGO

 Good sample preservation

 Specimen adequacy – minimize hypocellularity

 Better visualization of cell morphology

 Improve specimen quality. – reduce mucus , blood , debris,

inflammatory components & less clumping

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Fixatives LOGO

 Ensure preservation of specimen

 Method of fixation depends on staining

 Wet fixed smears – e.g. H&E, PAP

 Air dried smears – Romanowsky stains

 Routine fixatives – 95% ethanol, 100% methanol, 95% denatu

red alcohol, 85% isopropanol

 Coating fixatives – aerosol sprays/ dropper. (Eg cytospray) –

for long distance transport
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STAINING LOGO

 Routine cytodiagnostic studies: PAP, H&E

• PAP : Harris’ hematoxylin – nuclear stain

orange G & eosin alcohol – cytoplasmic stain

• H & E : Harris’ hematoxylin – nuclear stain

eosin – cytoplasmic stain

Romanowsky stains – Leishman’s, Geimsa, & wright stains
o Nuclei – purple/ blue

o Cytoplasm – pink/ blue

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Special stains
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 PAS – fungal hyphae, glycogen

 Mucicarmine - mucin
 Gram stain - bacteria
 Prussian blue – iron deposits
 Feulgen - nucleus
 Sudan stains – lipid deposits
 Zeihl – Neelson - bacteria

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SCREENING OF SLIDES
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 Screening is done from left to right from higher area, then

at next lower level until all areas are covered.

 Magnification 150 times

 At least 10 minutes for each slide

Abnormal cells examined at higher

magnification

 Location marked with an ink dot

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Automated screening systems

 Neural network computer system & image analysis

 Image of cells are presented on screen

 Computer selects cells or group of cells available for viewin

g

 Cytologist makes decision whether smear as negative or to r

eview it manually.

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 ANALYSIS
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QUALITATIVE ANALYSIS QUANTITATIVE ANALYSIS

 CELLULAR ATYPIA  CELLULAR & NUCLEAR MORPHOLOGY

 CELLULAR & NUCLEAR DIAMETER &

 NUCLEAR ATYPIA
AREA

 STAINING PATTERNS  N/C RATIO

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 CYTOLOGICAL CLASSIFICATI LOGO
ONS
Papanicolaou’s classification (1960)

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NEGATIVE FOR MALIGNANCY

SUSPICIOUS FOR MALIGNANCY – biopsy recommended

POSITIVE FOR MALIGNANCY – biopsy is mandatory

N H Rickles. 1972 . CA: a cancer journal for clinicians

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 Class 1 – normal cytology LOGO

CRITERIA BASAL PARABASAL INTERMEDIATE SUPERFICIAL

Size (μ) 8-10 15 - 25 30 - 60 40 - 60

Shape round Round/oval polygonal polygonal
Cytoplasm scanty adequate abundant abundant
stain Deep blue Blue Blue/ pink Orange
border curl rare rare common rare
N/C ratio 8:10 5:10 2:10 1:10
Nuclear size μ 7-9 8-13 10 - 12 5-7
shape round Round/oval oval oval
chromatin pattern Coarse Granular Fine granular pyknotic

Nucleoli none Rare prominent small none

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Basal cell

Parabasal cell

Intermediate cell

Superficial cell

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 CLASS II – ATYPICAL CELLS
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Bacterial infection:
• Increase N/C ratio
• Bacterial colonization in cyto
plasm
• Severity – cell outline indisti
nct/ complete cytolysis – fre
e / naked nucleus.
• Perinuclear halo

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Viral infection:

• Ballooning degeneration
• Inclusion bodies
• Fusion of cells – hyperchromati
c giant nuclei/ multinucleated c
ells
• PAP, H&E stains

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Fungal infections:
• Presence of yeast cell or hypha
e

• Suspected candida: PAS – maje

nta colour

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Dermatological lesions:
• eg: pemphigus
• Acantholysis
• Greater % of basal/paraba
sal cells
• Small cell size
• Hyperchromatism of conde
nsed nucleus
• H & E, PAP, Leishman stains

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Inflammatory lesion:

• Eg: traumatic ulcer

• Inflammatory cells
• erythrocytes

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 CYTOPATHOLOGY OF ORAL CANCER
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 NUCLEAR ABNORMALITIES
o Size
o Irregular shape
o Multinucleation Multinucleation
o Nuclear hyperchromatism
o n/c ratio

28-12-2012 Hyperchromatic nuclei & inc.

47 n/c
 CYTOPATHOLOGY OF ORAL CANCER
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 NUCLEAR ABNORMALITIES

• Abnormal mitosis
• Aberrant chromatin pattern
• Prominent / multiple nucleoli
• Degenerative changes

Chromatin clumping with angular

borders

Degenerative changes
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 CYTOPATHOLOGY OF ORAL CANCER
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 CYTOPLASMIC ABNORMALITIES
• Scanty cytoplasm
• Vacuolization/ inclusion
• Altered staining characteristics

 CELL AS A WHOLE
• Enlargement
• Bizzare shapes

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Von Hamm – compared cytologic smears with surgical
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biopsy & concluded….

 Cytology is not a substitute but an adjunct to surgical biopsy.

 Quick, simple, painless & bloodless procedure.

 Helps as a check against false-negative biopsies.

 Helpful in follow-up detection of recurrent carcinoma in prev

iously treated cases.

 Screening lesions.
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ADVANTAGES
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 Quick & simple procedure

 Non invasive

 Fast result

 Greater patient acceptance

 Minimal risk

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 LIMITATIONS
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 False negative results

 Positive result results should be followed by biopsy

 Adjuvant for biopsy

 Cytological interpretation require fine knowledge & skill of

the cytopathologist.

 Not of much use in non epithelial lesions

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Possible factors for false negative results & errors
 SAMPLING ERROR
Smears from non representative sites.

 IMPROPER FIXATION
Air drying of smear or a wrong fixative – artifacts & alteration in
cellular morphology.

 CYTOPREPARATION
Staining & processing errors

 SUBJECTIVE ERRORS
Inexperience.
screen the slide completely.

LACK OF CLINICAL INFORMATION

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 REFERENCES LOGO

o Textbook of oral pathology – Shafer’s – 6th edition

o Theory & practice of histological techniques – John Bancroft.

Marilyn gamble – 5th edition

o Koss diagnostic cytology & its histopathologic bases – Leopold

G Koss . 5th edition

o Google images

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A R
W YE
N E
PPY
HA

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THANK YOU…!! 57