Exfoliative Cytology

A branch of General Cytology which deals with the microscopic study of cells that have been desquamated
from the epithelial surfaces and mucous membranes.

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; and
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 (Pap) smear

Specimens that Requires Adhesive Agents:

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.

Nipple Discharge:
1. Gently strip the sub-areolar area and nipple with the thumb and forefinger.

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 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)

Bronchial Brushings:

• 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.

• 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

Collection Of Specimen

Sputum

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)

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.

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 - 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.

Routine Cervical/Endocervical (Pap) Smear

• 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).

Smear Preparation:
 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

Streaking
- 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

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

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

Touch or Impression Slide

- for preparation of direct impression from the cut surface of tissue like the lymph nodes and other
surgical or autopsy secretions.

Fixation

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

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:

• Harris Hematoxylin
• OG 6 Stain
Orange Green 6, 0.5 solution in 95% ROH 100 ml
Phosphotungstic acid 0.015 gm
• 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

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*** 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

Cytologic Criteria of Malignancies

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.

Multinucleate cells

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– 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.

Vaginal Cytology

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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.

Vaginal Cells

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

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

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

Navicular 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

Pregnancy 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

Parabasal Cells
- Round to oval cells
- Smaller than intermediate (15-25 u)

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 - 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.

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

Basal 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

Endometrial 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

Doderlein Bacillus
- “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

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.

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 • 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

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.

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.
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 5. The specimen slide(s) is (are) irreparably broken.
6. Specimen is submitted from an unauthorized source.

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