EXFOLIATIVE CYTOLOGY :

PAPANICOLAOU’S SMEAR
EXFOLIATIVE CYTOLOGY

- is a branch of general Pathology that deals with the

microscopic study of cells that have been desquamated
from epithelial surfaces.
EXFOLIATIVE CYTOLOGY
Is usually recommended for:
1. Assessing malignant or cancerous condition
2. detection of asymptomatic cancer in women
3. assessment of female hormonal activity (sterility and
endocrine disorders)
4. determination of genetic sex
5. determination of the presence of possible infection
EXFOLIATIVE CYTOLOGY: Specimens
1. Vaginal smears
2. Endometrial and endocervical smears
3. Prostatic and breast secretions
4. Gastric and bronchial secretions
5. Pleural and peritoneal fluids
6. Sputum
7. Smears of urine sediment
8. CSF
EXFOLIATIVE CYTOLOGY: Specimens

 Specimens that require an adhesive agent:

1. Urinary sediment
2. bronchial lavage specimens
3. Specimen that utilizes proteolytic enzymes during
processing (trypsin, concentrated sputum and enzymatic
lavage specimen from GI tract)
EXFOLIATIVE CYTOLOGY: Adhesive gents

CHARACTERISTICS:
1. Permeable to both fixative and stain
2. must not retain the stain

GOOD ADHESIVE AGENTS FOR CYTOLOGIC

METHODS:
1. Pooled human serum
2. Celloidin ether-alcohol
3. Leuconostoc culture
EXFOLIATIVE CYTOLOGY: Smear
Preparation
 SOP:
- smears should be from fresh material
- see requisition form (patient’s ID: name, age; date and
type of specimen requested
- label the slide
EXFOLIATIVE CYTOLOGY: Smear
Preparation
4 METHODS OF SMEAR PREPARATION:
1. Streaking
2. Spreading
3. Pull- Apart
4. Touch or Impression Smear
EXFOLIATIVE CYTOLOGY: Smear
Preparation
 1. 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 SMEAR
EXFOLIATIVE CYTOLOGY: Smear
Preparation
2. SPREADING
used for thick mucoid secretions (smears of fresh sputum
and bronchial aspirates)
- With the use of two applicator sticks or dissecting
needles, genty spread the specimen to a moderately thick
film by teasing the mucus strand part
EXFOLIATIVE CYTOLOGY: Smear
Preparation
3. PULL-APART
- for serous fluids, concentrated sputum, and enzymatic
lavage form the GIT, smears of urinary sediment, vaginal
pool and breast secretions
- Put a small amount of specimen on one slide then place
another slide on top of the specimen, slightly press and
pull the two slides on opposite side
EXFOLIATIVE CYTOLOGY: Smear
Preparation
4. TOUCH OR IMPRESSION SMEAR
-for preparation of direct impression from the cut surface
of tissue like the lymph nodes and other surgical or
autopsy secretions.
- Touch the cut portion of the specimen with the slide that
you will use
EXFOLIATIVE CYTOLOGY: 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 fluid
EXFOLIATIVE CYTOLOGY: STAINING
 PAPANICOLAOU METHOD (PAP’S SMEAR)
- considered to be the staining method of choice for
Exfoliative Cytology with the ff advantages:
1. Transparent blue staining of cytoplasm allows
overlapping cells to be seen and identified
2. Excellent nuclear detain is produced
3. Good differential coloring of basophilic and acidophilic
cells
4. Valuable in comparing cellular appearances in smears
with their counterpart in similarly stained sections
EXFOLIATIVE CYTOLOGY: STAINING
Disadvantages:
1. procedure is lengthy and complicated
2. does not give accurate acidophilic index
EXFOLIATIVE CYTOLOGY: GEORGE
PAPANICOLAOU

Georgios Nikolaou
Papanikolaou or George
Papanicolaou (May 13,
1883 – February 19, 1962)
was a Greek pioneer
in cytopathology and
early cancer detection, and
inventor of the "Pap smear".
-diagnosis of malignancy
thru microscopic
examination of a single or
group of isolated cells
EXFOLIATIVE CYTOLOGY: GEORGE
PAPANICOLAOU
- - Developed a staining method while examining vaginal
secretions from rodents and human subjects which
indentified stages in the maturation of exfoliated
squamous epithelial cells
- - In effect, formulated a method with which malignant
cells could be identified
- Originally indicated for vaginal smears to detect human
uterine and cervical cancers, Pap’s smear has been applied
for evaluation of sputum, urine, breast and other
secretions as well.
EXFOLIATIVE CYTOLOGY: Pap’s Smear
Staining Method
Stains for Pap’s:
- 1. Harris Hematoxylin
- 2. OG 6
Orange Green 6, 0.5 solution in 95% alcohol (100 ml)
- 3. EA 50 stain
- Light green SF, yellowish 0.1% soln in 95% alcohol (45ml)
Bismarck brown 0.5 in 95% alcohol (10ml)
- Eosin Y, 0.5% in 95% alcohol (45 ml)
- Phosphotungstic acid (0.2 g)
- Lithium Carbonate. Sat. aq. Solution (1 drop)
EXFOLIATIVE CYTOLOGY: Pap’s Smear
Staining Method
 Procedure for thr standard Pap’s staining method is as follows:
1. Fixation in 95% ethanol, then wash with water
2. Primary stain with Harris Hematoxylin
3. Differentiate with acid-alcohol, 1 quick dip, then wash in
water (regressive)
4. Blue ammonia water, then wash
5. Counterstain with OG-6
6. Wash in 2 changes of 95% ethyl alcohol
7. Counterstain with EA-36 or 50
8. Dehydrate
9. Clear with xylol
10. Mount with resinous media then coverslip
11. Label
EXFOLIATIVE CYTOLOGY: Anatomic Sites
For Preparation of Cytologic Samples
 In addition to cervix, other sites are examined as follows:
1. Upper (proximal) third of the vaginal wall
- Asses hormonal status of the woman, vaginitis, classify
microbiologic flora of the vagina, detection of malignant
lesions of the vagina
[Link]
- for cancer screening
3. Endocervix
- detection of some intrauterine lesions
Pap’s Smear: Normal Cellular Components
1. Neutrophils – increased just before, during and
shortly after menstruation. Suggests an inflammatory
response or an on-going infection
2. Red blood cells – seen during traumatic collection,
menstruation or post-menstrual period or because of
some pathologic bleeding
3. Lactobacillus acidophilus (Doderiein bacilli) – gram-
positive long, stubby bacillus that are part of the normal
flora of vaginal tract. Maintain the normal acid pH of
3.5-4.5 by glycogenolysis of glycogen into lactic acid
4. Leptothrix spp. – normal commensals, becoming
prolific if the vaginal pH increases.
Pap’s Smear: Normal Cellular Components
5. Talcum or starch Granules – usual contaminant that
may fall on the glass slide. Appear as ovoid bodies that
exhibits an apple-green birefringence
6. Endocervical Cells – columnar epithelial cells that
form part of the normal lining of the endocervical canal.
Indicates the clinical representation of the endocervical
canal.
7. Endometrial Cells – small epithelial cells coming from
the shedding of the endometrial lining or due to a
proliferating endometrial pathology. Indicate previous
menstruation or that menstrual bleeding is about to
occur
Pap’s Smear:
Abnormal Cellular Components
1. Candida albicans – budding yeast, which form branching
pseudohyphae that spears clusters of epithelial cells that
look like a “shish kebab.”
- seen in diabetic patients, patients taking oral contraceptives or
prolong steriod therapy, immunocompromised states, and in
malignancies such as leukemia and lymphoma
2. Trichomonas vaginalis – sexually transmitted pear-shaped
organisms. Flagellum is not preserved hence is not evident in
pap’s stain. Appear as ill defined blobs that can be readily
missed, especially in a background of dense, obscuring
inflammation
3. Gardnerella vaginalis – clings to the surface of the
cytoplsm of epithelial cells giving “moth-eaten” or “clue
cells” appearance. They indicate a shift in vaginal flora. They
are the cause of “non-specific bacterial vaginosis.”
Pap’s Smear:
Abnormal Cellular Components
4. Koilocytes – squamous epithelial cells that show the
cytopathic effects of HPV infection. Seen as a cell with an
atypical “wrinkled prune” nucleus surrounded by a
perinuclear halo.
5. Herpes Simplex Virus (HSV) – incites a cytopathic effect in
the infected cell characterized by macrosomia,
multinucleation, nuclear molding, and a ground-glass
chromatin pattern
6. High Grade Squamous Intraepithelial Lesions –
encompassing severe dysplasia, CIN III and carcinoma-in-situ
7. Invasive Squamous Cell Carcinoma – most common
from of cervical malignancy
The Bethesda system
 SPECIMEN TYPE

 SPECIMEN ADEQUACY

 GENERAL CATEGORIZATION

 INTERPRETATION/RESULT
The Bethesda system
 SPECIMEN TYPE
 Indicate
 conventional smear (Pap smear)
 vs. liquid-based preparation
 vs. other.
The Bethesda system
 SPECIMEN ADEQUACY
 Satisfactory for evaluation (describe presence or absence
of endocervical/transformation zone component and any
other quality indicators, e.g., partially obscuring blood,
inflammation, etc.)
 Unsatisfactory for evaluation …(specify reason)
 Specimen rejected/not processed (specify reason)
 Specimen processed and examined, but unsatisfactory for
evaluation of epithelial abnormality because of (specify
reason)
The Bethesda system
 GENERAL CATEGORIZATION (optional)

 Negative for Intraepithelial Lesion or Malignancy

 Other: See Interpretation/result (e.g., endometrial cells in
a woman >= 40 years of age)
 Epithelial Cell Abnormality: See Interpretation/result
(specify ‘squamous’ or ‘glandular’ as appropriate)
INTERPRETATION/RESULT
 NEGATIVE FOR INTRAEPITHELIAL LESION OR
MALIGNANCY,
 (when there is no cellular evidence of neoplasia, state this
in the General Categorization above and/or in the
Interpretation/Result section of the report, whether or
not there are organisms or other non-neoplastic findings).
INTERPRETATION/RESULT
 ORGANISMS:
 * Trichomonas vaginalis
 * Fungal organisms morphologically consistent with
Candida spp
 * Shift in flora suggestive of bacterial vaginosis
 * Bacteria morphologically consistent with Actinomyces
spp.
 * Cellular changes consistent with Herpes simplex virus
INTERPRETATION/RESULT
 OTHER NON NEOPLASTIC FINDINGS (Optional to report;
list not inclusive):
 * Reactive cellular changes associated with
 - inflammation (includes typical repair)
 - radiation
 - intrauterine contraceptive device (IUD)
 * Glandular cells status post hysterectomy
 * Atrophy

 OTHER
 - Endometrial cells (in a woman >= 40 years of age)
 (Specify if ‘negative for squamous intraepithelial lesion’)
EPITHELIAL CELL ABNORMALITIES
 - SQUAMOUS CELL
 * Atypical squamous cells
 - of undetermined significance (ASC-US)
 - cannot exclude HSIL (ASC-H)
 * Low grade squamous intraepithelial lesion (LSIL)
 (encompassing: HPV/mild dysplasia/CIN 1)
 * 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 cell carcinoma
- GLANDULAR CELL
 * Atypical
- endocervical cells (not otherwise specified (NOS) or
specify in comments),
- endometrial cells (NOS or specify in comments),
- glandular cells (NOS or specify in comments)
* Atypical
- endocervical cells, favor neoplastic
- glandular cells, favor neoplastic
* Endocervical adenocarcinoma in situ
* Adenocarcinoma:
- endocervical
- endometrial
- extrauterine
- not otherwise specified (NOS)
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