Exfoliative cytology

a branch of General Cytology which deals wit the microscopic study of
cells that have been desquamated from the epithelial surfaces.
recommended for :
1. detection of malignant cells or precancerous lesions in the
body
2. detection of asymptomatic or precancerous cervical lesions in
women
3. assessment of female hormonal status in case of sterility and
endocrine disorders
4. Determination of genetic (phenotypic) sex
5. Detection of the presence of infectious microorganisms

SPECIMENS FOR EXAMINATION
1. peritoneal, pericardial and pleural fluids
2. CSF
3. nipple discharge
4. bronchial brushings / washings
5. sputum
6. gastric washings
7. urine sediment
8. prostatic secretions / fluid
9. cervicovaginal (paps) smear

SPECIMENS THAT REQUIRE ADHESIVE AGENT
1. Urinary Sediment
2. Bronchial Ravage Specimen
3. Specimens that utilizes proteolytic enzymes during
processing (eg. Trypsin, conc. Sputum and enzymatic
lavage sample from the GIT)
• CHARACTERISTICS OF A GOOD ADHESIVE
1. must be permeable to both fixative and the stain
2. must not retain the stain
• GOOD ADHESIVES FOR CYTOLOGIC METHOD
1. Pooled human serum or plasma
2. Celloidin Ether-Alcohol
3. Leukonostoc culture

• Collection Procedure:
- Using standard paracentesis technique, obtain a fluid
specimen from the desired body cavity. A minimum of
10 mL of specimen is desirable for optimal cytologic
evaluation. Heparin may be added to the specimen to
reduce clotting.
- Place three (3) units of heparin per mL capacity of
the collection container. Gently agitate to thoroughly
mix the specimen and heparin. - - Submit the
specimen along with the completed cytology request
form.
- The specimen should be refrigerated or kept on wet
ice until transported to the lab.
COLLECTION OF SPECIMEN

A pleural smear examines a sample of pleural fluid under the
microscope to detect for abnormal organisms.

During a pericardial fluid culture, a small needle is inserted into the
chest between the ribs into the pericardium (the thin sac that
surrounds the heart) and a small amount of fluid is withdrawn.
Samples of the fluid are placed in various culture media in the
laboratory, and the media is observed for the growth of
microorganisms. The test is performed when an infection of the heart
is suspected or when a pericardial effusion is present.

A peritoneal culture is a procedure where peritoneal fluid is
withdrawn with a needle from the peritoneal cavity. The
peritoneal cavity is the space between the two membranes lining
the abdominal cavity. The test is done to determine the cause of
ascites, fluid accumulation in the peritoneal space.

CSF is usually obtained through a lumbar puncture
(spinal tap). During the procedure, a needle is
inserted usually between the 3rd and 4th lumbar
vertebrae and the CSF fluid is collected for testing.

A breast biopsy can help diagnose breast cancer early
in the disease process.

1. Gently strip the sub-areolar area and nipple with the
thumb and forefinger.
2. Place the slide upon the nipple and draw it quickly across
the nipple. ***If 1 drop is obtained, get another slide and
do the pull-apart technique.
3. Immediately immersed the slide into a bottle of 95%
isopropyl ROH or use spray fixative.
4. If the secretion is very little in amount, the smear should
be restricted to a small area only to prevent drying.
5. Label the secretions obtained indicating from where
they’re collected. (left or right breast)

a. bronchial washing
b. bronchial lavage
c. bronchial brushing

•Specimen:
- Bronchoscopically-directed brushing of the identified
lesion.
•Collection Procedure:
- Using standard bronchoscopy technique, identify the
lesion in question and obtain a brushing sample of the lesion.
- Gently apply the sample on to glass slide and
immediately immerse slide
into 3% glacial acetic acid alcohol fixative.
- The brush tip should also be cut off into the solution.
Bronchial Brushings

Bronchial Washings
•Specimen:
Bronchscopically-obtained washing (preferable at least
10 mL) of the bronchi in the region of the
suspected lesion.
•Collection Procedure:
-Using standard bronchoscopy technique, lavage the
distribution of the bronchus to be sampled.
-Collect the wash in a clean container. Label the
container with correct patient information and

NORMAL
-obtain at three consecutive morning
sputum specimens by deep cough
method.
-Collect the sample in a wide-mouth
container containing Saccomano fluid
(50% EtOH and 2% carbowax)
INDUCED
- Inhalation of aerosol solution for 20
mins to produce deep cough sample.
- Collect the sample in a wide-mouth
container containing Saccomano fluid
(50% EtOH and 2% carbowax)

Gastric suction is perform to empty the contents of the stomach
before it passes through the rest of the digestive tract

Washings (Esophageal, Gastric, Other)
•Specimen: Endoscopically obtained washing (preferably at least
10 mL) of the region of the suspected lesion.
Collection Procedure:
-Instruct the patient to fast overnight or for a minimum of
six hours prior to the
procedure.
- Using standard endoscopy technique, lavage the area of
interest using a physiologic solution. Aspirate the solution and
place in a clean specimen container.
- If transport of the specimen will be delayed more than
four (4) hours, the
specimen should be refrigerated or kept on wet ice until
transported to the lab.

Brushings (Esophageal, Gastroesophageal Junction, Gastric,
Duodenal, Bile Duct, Other)
•Specimen:
- Endoscopically-directed brushing sample of the identified
lesion.
•Collection Procedures:
-Instruct the patient to fast overnight or for a minimum of six
hours prior to the procedure.
- Using standard endoscopy technique, identify the lesion in
question and obtain a brushing sample of the lesion.
- Gently apply the sample on to glass slide and immediately
immerse slide into 3% glacial acetic acid alcohol fixative.
-The brush tip should also be cut off into the solution.

• Collection Procedure:
-Use an unlubricated speculum (saline or warm water may
be used).
-After visualization of the cervix is accomplished, insert
the sampling device (e.g. cytobrush®) into the
endocervical canal and rotate one complete turn.
-Withdraw the cytobrush and spread the collected
material quickly and evenly onto the slide opposite the
frosted end.

The endocervical mucus will prevent air-drying during
collection of the subsequent cervical component.
Using the extended-tip spatula ( e.g. Ayerst®), scrape
material with the spatula from the whole circumference of
the cervix.
Withdraw the spatula and spread the collected material
quickly and evenly onto the slide adjacent to the frosted
end.
Fix Immediately (drop slide into fixative or spray with
fixative, holding the spray bottleapproximately 8 to 12
inches from the slide).

Click to view the video:
•Insertion of speculum
•Physical examination of the genitalia
•Collection of cervico-vaginal specimen

 smears should be from fresh material
 see requisition form (patient’s ID: name, age; date and
type of specimen requested
 label the slide
-- Methods of Smear Preparation:
1. streaking
2. spreading
3. pull apart
4. touch or impression smear
SMEAR PREPARATION

- used for preparing mucoid secretions vaginal
secretions, sputum and gastric content)
- use a spatula, dissecting needle or applicator stick
and streak in a zigzag fashion
SMEAR PREPARATION

- used for thick mucoid secretions (smears of fresh
sputum and bronchial aspirates)
SMEAR PREPARATION

- for serous fluids, concentrated sputum, and enzymatic
lavage form the GIT, smears of urinary sediment, vaginal pool and
breast secretions
SMEAR PREPARATION

- for preparation of direct impression from the cut surface of
tissue like the lymph nodes and other surgical or autopsy secretions.
Impression cytology being collected From a patient with herpes simplex
keratitis, using a sterile glass slide with polished edges.
SMEAR PREPARATION

•WHY FIX?
- exfoliated cells decompose rapidly which may destroy cellular
and nuclear details, in turn will give inadequate results for diagnosis.
•COMMON FIXATIVES
1. equal parts of 95% EtOh and ether
2. 95% EtOH
3. Carnoy’s fluids
4.equal parts of tertiary butyl alcohol and 1 part 95% EtOH
5. SCHAUDINN’S FLUID – sat. aq. Hg2Cl, absolute HoAc
6. MeOH – for dried films

1. PAPANICOULAOU or Pap’s Smear
Advantage:
- transparent blue staining of cytoplasm is observed
- excellent nuclear staining
- color range is predictable and of great value in identification of
cells
Disadvantage:
- procedure is lengthy and complicated
- does not give accurate acidophilic index

Stains for Pap’s:
1. Harris Hematoxylin
2. OG 6 Stain
Orange Green 6, 0.5 solution in 95% ROH 100 ml
Phosphotungstic acid 0.015gm
3. EA 50
light green SF, yellowish 0.1% solution in 95% ROH 45ml
Bismarck brown 0.5 in 95% ROH 10 ml
Eosin Y, 0.5% in 95% ROH 45 ml
Phosphotungstic acid 0.2 gm
Lithium Carbonate. Sat. aq. Solution 1 drop
*** EA 50 is comparable to EA 36
*** EA 65 differs from EA 50 or EA 36 only with respect to the
concentration of the light green stock solution

Procedure for Pap’s Stain:
1. Fix in ether-ROH and pass thru 80% ROH, 40% ROH and
distilled H2O.
2. Stain in Harris Hematoxylin for 4-5 minutes.
3. Wash with H2O.
4. Pass thru 0.25% HCl in 50% ROH.
5. Immerse in 1.5% NH4OH in 70% ROH for 1 minute.
6. Rinse in 70% ROH and pass thru 80% and 95% ROH.

Procedure for Pap’s Stain:
7. Stain with OG 6 for 1.5-a minutes.
8. Pass thru 3 changes of 95% ROH.
9. Stain with EA 65 or EA 50 for 3 minutes.
10. Pass thru 3 changes of 95% ROH.
11. Dehydrate and clear in:
a. absolute ROH,
b. equal parts of ether and absolute ROH,
c. 2 changes of xylol
12. Mount in Canada Balsam.

Results:
Cytoplasm – either bright red or
greenish blue
vesicular nucleus – blue
pyknotic nucleus – dark blue to
black
bacteria – dark blue
mycelia – violet
Trichimonas vaginalis – pale
greenish blue blob of cytoplasm

Cytoplasm – either bright red or greenish blue
vesicular nucleus – blue
pyknotic nucleus – dark blue to black

Trichimonas vaginalis – pale greenish blue blob of cytoplasm

NUCLEI CHANGES
Altered nuclear-cytoplasmic ratios
– due to the enlargement of nuclei and a most important criteria of
malignancy. However this is benign in certain tissues, e.g. endocervical
and renal pelvic cells.
Hyperchromasia
– although common in malignant cells, one must be careful to exclude
overstaining and not to mistake pyknosis for hyperchromasia.
Increased mitotic activity
– frequently seen in reactive pleural and peritoneal mesothelial cells, in
histiocytes and in any tissue undergoing active repair and benign growths
Atypical mitoses
–triple or quantiple spindles are highly suspicious and important signs of
malignancy.

NUCLEI CHANGES
Multinucleate cells
– with irregular hyperchromatic or bizarre nuclei should be suspicious,
but caution is warranted in certain situations, e.g. after abortion, or
expulsion of H-mole, Herpes complex infection
Anisokaryosis
– considerable variation in nuclear size and shape is common in
malignant cells.
giant single nucleus (polyploidy)
– often seen in malignant cells, polypoidic cells may also occur in benign
conditions, especially in thyroids of older women and less in adrenal
cortex and hyperactive islets of Langerhans. In vaginal smears, they may
result from pregnancy as well as malignancy.

CYTOPLASMIC CHANGES
- cells of epidermoid carcinomas frequently show a
tendency to cytoplasmic eosinophila.
- adenocarcinoma cells may enclose PMNs, eg. endometrial
and colonic cancers)
- cytoplasmic vacuolation is common in adenocarcinoma
cells, but may be also be seen in endometrial cells following
cutterage.

APPERANCE OF WHOLE CELLS
In general:
- malignant cells show reduced cohesiveness, possibly related to a
defect of the intercellular “zippers” i.e., desmosomes.
-Cancer cells are larger than their normal counterparts and
frequently show bizarre and grotesque shape.
Occasionally:
- exfoliated cells from epithelial tumors assume a greatly
elongated, fibrocyte-like appearance.

CELL PATTERN
•Examine for the small groups or clusters of cells:
-Oblivious patterns
e.g. acini in adenocacinoma arising in glandular tissues
-Rosettes in neuroblastomas and ependymoma
-Stratification in squamous epithelial growths
-Whorls in mesotheliomas
•If there are no patterns revealed, focus up and down
the on cell clumps
-In strips epithelium, irregular stratification of
anisokaryotic, hyperchromatic cells are helpful in
diagnosing carcinoma of cervix and bronchus.

OTHER CRITERIA
1. In bronchial secretion smear, abundant lymphocytes
are common in presence of malignancy, but may also
be found in certain inflammatory conditions and in
leukaemia.
2. The presence of old blood and blood pigments is a
minor indirect clue, but has many other etiologies.

The vaginal epithelium is responsive to sex steroids, particularly
estrogen, and undergoes predictable changes through the cycle in
response to changes in blood concentrations of ovarian hormones.
Rising levels of estrogen cause the vaginal epithelium to become
"cornified" - the surface cells become large and flattened, with
small or absent nuclei.
In essence, vaginal cytology is a type of endocrine assay. Tracking
changes in the morphology of desquamated vaginal epithelial cells
provides a convenient means of assaying changes in estrogen
levels.

•Vaginal smears may be taken regularly and often.
•Hormonal changes are best mirrored in the upper third of
the vagina.
•They can also be taken from the lateral walls because their
more accessible and less likely to be contaminated by
cellular debris or discharge.

-Large (30-60u)
-Polyhedral flat cells
-Cytoplasm: may be acidophilic or basophilic
-Presence of small dark pyknotic nuclei (less than 6u)
SUPERFICIAL
CELLS
SUPERFICIAL
CELLS

SUPERFICIAL
CELLS
SUPERFICIAL
CELLS
Anucleate cells are abnormal which may be derived from:
1. smear contamination by the cells from the vulva
2. epidermization of the vagina or cervix resulting from
prolapse
3. leukoplakia of the cervix
4. ruptured membranes in pregnant women
5. marked hyper-estrinism

- medium large (20-30u)
- Polyhedral or elongated
- Cytoplasm: basophilic with vacuoles
- Vesicular nuclei (6-9u)
INTERMEDIATE
CELLS
INTERMEDIATE
CELLS

- Boat-shaped intermediate cells with a strong tendency to fold and
curl their edges.
- Expression of the combined estrogen-progesterone effect
-found in the latter half of menstrual cycle, during pregnancy,
menopause
- may also be found as a result of abnormal androgen stimulation,
either endogenous or exogenous
NAVICULAR
CELLS
NAVICULAR
CELLS

- Round or oval to oval
shaped cell
- has a translucent
basophilic cytoplasm (due to
glycogen accumulation)
- cytoplasm stains deep blue
or blue green + cell
membrane = a double cell
wall appearance
PREGNANCY
CELLS
PREGNANCY
CELLS

-Round to oval cells
-Smaller than intermediate (15-25 u)
-Thick
-“sunny-side up” like cells
-Have strong basophilc cytoplasm and vesicular nucei (6-9 u )
-Found from 2 weeks of age to puberty, after childbirth, abortion
or miscarriages and after menopause.
PARABASAL
CELLS
PARABASAL
CELLS

-Slightly cylindrical appearance
- occurs in groups and strips of three or more cells
- cytoplasm: deeply basophilic the that of the parabasal
cells
ENDOCERVICA
L CELLS
ENDOCERVICA
L CELLS

-small (13-20u)
-Round, slightly oval cells, with relatively large nucleus that
occupying half or more of the cell volume
-Cytoplasm: strongly basophilic
-Found in vaginal smears only before pregnancy and after
menopause
BASAL
CELLS
BASAL
CELLS

-Found during menstruation period ( in groups) and 1-4
days after the cessation of the period (single)
-Endometrial stromal cells: seen in tight clusters of
small, oval dark cells; Glandular cells: slightly larger.
-Nucleus: small and moderately dark
-Cytoplasm: basophilic and maybe vacuolated
ENDOMETRIAL
CELLS
ENDOMETRIAL
CELLS

- “Lactobacillus acidophilus”
-Gram + slender rod bacteria
- Predominant organism of the
vaginal normal flora: establishes the
low pH that inhibits the growth of
pathogens
-Stains pale blue to lavender
-Energy is obtained by the
fermentation of glycogen derived
from disintegrating epithelial cells
-Numerous in the luteal phase and
during pregnancy
DODERLEIN
BACILLUS
DODERLEIN
BACILLUS

CYTOLYSIS IN VAGINAL CYTOLOGY
- Infection
- estrogen
-low vaginal pH (below 4.2)
Occurs in the last trimester of
pregnancy, more common in
diabetic patients
 shows large numbers of
naked nuclei and very abundant
Doderlein bacilli.

QUANTITATION IN VAGINAL
CYTOLOGY
ACIDOPHILIC INDEX (A.I.) - percentage of cells that stain pink-orange to red
with Pap’s and red in Shorr method
PYKONTIC INDEX (P.I) – “karyo-pyknotic index”; percentage of cells having
shrunken, dark, small (less than 6μ) structures nuclei.
MATURATION INDEX (C.H.M.I.) – percentage proportion of cells from the three
layers of the vaginal epithelium.

CLASSIFICATION OF PAP TEST RESULTS
Class I Negative for Malignant cells
Class II
Atypical cells present, but Negative for
Malignancy
Class III Suspicious for Malignant Cells
Class IV Strongly Suggestive for Malignant Cells
Class V Conclusive for malignant Cells

The BETHESDA SYSTEM
Specimen Adequacy
Satisfactory
Limited
Unsatisfactory
General Categorization:
Negative for Intraepithelial lesion or malignant cell
Epithelial cell abnormality
Descriptive Diagnosis:
Atypical squamous cells of unknown significance
Low grade squamous intraepithelial lesion
High grade squamous intraepithelial lesion
Squamous Cell Carcinoma
Glandular cell abnormality
Atypical glandular cells
Adenocarcinoma
Others

Pap smear specimens are considered satisfactory for interpretation if
there are:
•Adequate numbers of well-visualized squamous cells present
•Adequate numbers of well-visualized endocervical cells or squamous
metaplastic cells (from the transformation zone).
•Less than 50% of the cells obscured by blood or inflammation
•Properly labeled specimens
Specimen SatisfactoryADEQUACY

Pap smear specimens are considered unsatisfactory for interpretation if
there are:
•Inadequate numbers of well-visualized squamous cells present
•Inadequate numbers of well-visualized endocervical cells or squamous
metaplastic cells (from the transformation zone).
•More than 75% of the cells obscured by blood or inflammation
•Improperly labeled specimens
Usually, these smears are recommended for repeat sampling.
Specimen
UnsatisfactoryADEQUACY

Specimen
Rejected/Not
Processed
ADEQUACY
Specimens to which the following conditions apply will be
rejected:
1. Specimen is submitted without a requisition.
2. Specimen is not labeled with the patient name.
3. The patient name (or other identifying information) on the
specimen and requisition do not correspond.
4. The specimen is labeled appropriately but the requisition is not
labeled.
5. The specimen slide(s) is (are) irreparably broken.
6. Specimen is submitted from an unauthorized source.

Negative for Intraepithelial
Lesion or Malignancy

* Atypical squamous cells
- of undetermined significance (ASC-US)
- cannot exclude HSIL (ASC-H)
EPITHELIAL CELL ABNORMALITIES
squamous
Atypical squamous cell (ASC-US) Atypical squamous cell (ASC-US),
favor mild dysplasia

* Low grade squamous intraepithelial lesion (LSIL)
(encompassing: HPV / mild dysplasia / CIN 1)
Mild Dysplasia Low Grade SquamousIntraepithelial
Lesion encompassing HPV cytopathic
effects
squamous

* High grade squamous intraepithelial lesion (HSIL)
(encompassing: moderate and severe dysplasia, CIS, CIN 2
and CIN 3)
- with features suspicious for invasion (if invasion
is suspected)
squamous
Severe Dysplasia or CIS Severe Dysplasia or CIS

* Squamous cell carcinoma
squamous

Atypical Glandular Cells
Adenocarcinoma in situ of the cervix
(upper left), next to normal glandular
epithelium (lower right).

Exfoliative cytology

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Exfoliative cytology

Similar to Exfoliative cytology (20)

More from Atifa Ambreen

More from Atifa Ambreen (20)

Recently uploaded

Recently uploaded (20)

Exfoliative cytology