Though it is written on

the sign
“ DO NO PLUCK
THE FLOWER”
It is useless
against
the wind
who can’t read.
(By an unknown Japanese poet)
 Introduction.
• Pap-test is the procedure used for early
detection of intraepithelial lesions and
carcinoma of cervix.

• Pap-test is named after George

Papanicolaou as he developed the
procedure in the 1930’s
 Cervix
• Cervix is lower portion of uterus, roughly
cylindrical and measuring 3cm in all
directions.
• The stroma is consist mainly of fibrous
tissue and smooth muscle fibres with
branches of uterine vessels.
 Cervical lining epithelium.
• Vaginal aspects of cervix is lined with squamous
epithelium that turns back towards the posterior
vaginal wall, forming fornices.
• Endocervical canal is lined with mucosecreting,
monolayered columnar epithelium. Nuclei are
vesicular and often contain a small nucleoli and
X chromatin body affixed to nuclear membrane.
• The two layers meet at the squamocolumnar
junction which is situated at external os.
• Squamocolumnar epithelium is not fixed and
moves upward or downwards at different times.
 Histology of cervix
• At birth and infancy SC
junction is situated at
exocervix.
• After menopause it recedes
into the endocervical canal.
• At puberty columnar epithelium
of the exocervix is gradually
replaced by squamous
metaplasia. Hence squamous
metaplasia is a physiological
process and need not be
mentioned in diagnosis.

• (Red marked area is SC junction)

 Transformation zone
• The area of squamous metaplasia that
covers preexisting endocervical glands is
called transformation zone.
 Squamous epithelium
• Squamous epithelium consists of three
layers.
• - Parabasal cell layers consists of
immature cells
• - Intermediate cells
• - Superficial cells containing most mature
cells.
• Germinal or basal cells lie directly on
basement membrane and these are small
columnar shaped cells.
Parabasal cells
Intermediate and superficial
epithelial cells.
 Hormonal influence
• Thickness and maturation of the
squamous epithelium depends on the
hormonal stimulation:
• Estrogen causes maturation.
• Progesteron on the other hand, opposes
complete maturation, while causing
thickening of the intermediate layer.
 Endocervix
• Endocervical lining epithelium is of tall columnar
mucous secreting type with basal nuclei, often
contain a small nucleolus and the X-chromatin
body affixed to the nuclear membrane, which is
smooth and regular. When irritated, the
columnar cells may display cilia.
• Underneath the columnar layer are few reserve
cells which are not easily observed in histology.
These reserve cells can multiply and differentiate
to produce metaplasia.
Endocervical cells.
 Endometrial cells
• Endometrial cells are observed on the smear
during first ten days of the cycle. Those are
smaller than endocervical cells with scant,
sometimes vacuolated, cyonophilic cytoplasm
and grouped in tight three dimensional clusters.
Nuclei are vesicular with fine chromatin pattern.
From day 6th to day 10th of the cycle EGC form
tight balls surrounded with pale eosinophilic
cytoplasm, originating in endometrial stroma.
These has been called `exodus’ by
Papanicolaou.
Endometrial cells
 Histiocytes
• Small histiocytes are observed at the end of the
cycle and during first 10 days of the proliferative
phase. Thought to be originated in the
endometrial stroma. Equal to the size of
parabasal cells, cyaonophilic, foamy cytoplasm.
Nuclei are notched, or kidney or bean shaped
and there may be 2 or more nuceli. When nuceli
are numerous and histiocytes become large they
are known as multinucleated giant cells. In
absence of inflammation they are scavangers,
removes debris and blood after menstruation.
 Granulocytes
• Granulocytes are observed at the
beginning and end of the menstrual cycle
and do not indicate the presence of
inflammation.
• Lymphocytes and plasma cells are rarely
observed on normal smears.
• Psammoma like concretions are
occasionally observed and probably
represents inspissated mucus.
 When to collect sample :
• The sample to be collected 10-14 days
after the 1st day of the last menstrual
period.
• Things to be noted before collection
Vaginal medications, lubricants, vaginal
contraceptives, or vaginal douches to be
avoided 48 hours before sample
collection.
 Collection Procedure
(Conventional Pap smear)
• Supplies:
Vaginal speculum, plastic extended-tip or wooden
spatula, cytobrush, fixative or (spray fixative or bottle of
95% ethyl alcohol), clean glass slides.
PROCEDURE:
• Label one slide with the details of the patient, and
specimen source
• Place the patient in the lithotomy position.
• Examine the patient with a dry of slightly water
moistened speculum to give adequate exposure of the
entire cervix and upper vagina.
• Excessive mucous from the cervical lips and vaginal
canal to be cleaned.
 TYPES OF SPATULA

1. AYRE’S SPATULA
2. WOODEN
SPATULA
3. ENDOCERVICAL
BRUSH
 Technique

• In V-C-E technique:
• Full visualiztion of cervix and upper part of vagina
is necessary
• First sample obtained from the posterior fronix
with a wooden tongue depressor or an Ayre
spatula. The material is kept on the instrument.
• Then a scraping with a second spatula is made
from the exocervix, and special care is taken to
sample all of the transformation zone. Again
material is kept on the spatula.
• Lastly, the endocervical sample is secured with a
cotton tipped applicator or with an endocervical
brush.
• Now all these three samples are quickly
transferred to a slide.
 Technique

• In V-C-E technique:
• - First sample
obtained from the
posterior fronix with a
wooden tongue
depressor or an Ayre
spatula. The material
is kept on the
instrument.
 Technique

• Then a scraping with

a second spatula is
made from the
exocervix, and special
care is taken to
sample all of the
transformation zone.
Again material is kept
on the spatula.
 Technique

• Lastly, the
endocervical sample
is secured with a
cotton tipped
applicator or with an
endocervical brush.
 Preparation of smears.
• All these three samples are quickly
transferred to a slide.
• Endocervical sample is smeared from
edge to edge near the end, farthest from
the label.
• Exocervical sample is spread in the middle
of the slide.
• The posterior vaginal vault material is
placed nearest to the label.
 V-C-E smears.
• Now all these three samples are quickly transferred to a
slide.
• The V-C-E smear provides three distinct samples from
the female genital tract, thus reducing the chances of
missing a significant lesion.
• At the same time it is economical and practical :
• -Only one glass slide is to be stained, screened and
stored for each patient.
• Once the smear has been collected and adequately fixed,
it must be sent to cytopreparatory laboratory at earliest,
with completed requisition form.
 V-C-E smear.
• The sample next to
the label is from
posterior vaginal vault
(V).
• The middle sample is
from scraping of the
transfromation zone.
• Endocervical sample
is farthest from the
label.
 Specimen Adequacy
Satisfactory for evaluation" indicates that the specimen has all
of the following:
• Appropriate labeling and identifying information
• Relevant clinical information
• Adequate numbers of well-preserved and well-visualized squamous
epithelial cells.
• An adequate endocervical / transformation zone component (from a
patient with a cervix).
• Quality of the Pap smear will still be noted when:
• Less than 10 well preserved endocervical or metaplatic cells
are seen
• Blood or inflammation moderately obscuring the Pap smear
 "Unsatisfactory for evaluation"
It designates that the specimen is unreliable for the detection of
cervical epithelial cell abnormalities.
This term is used if any of the following apply:
• A broken slide that cannot be repaired.
• Scanty squamous epithelial component (less than 8,000
well
preserved and well visualized cells on conventional slides
or less
than 5,000 well preserved and well visualized cells on
liquid-based
preparations)
• Obscuring blood, inflammation, thick areas, poor fixation,
air-drying
artifact, contaminant, etc. that precludes interpretation of
approximately 75% of the epithelial cells.
Contradictions to perform a Pap Smear

1. Menstruation.
2. If a douche or vaginal medication has
been used in preceding 48 hours.
3. Cryosurgery or cautery within the
previous 3 months.
 Some causes of false positive
Pap Smears:
• 1. Recent cryocautery(within 3 months).
2. Folic acid deficiency.
3. Corticosteroid medication.
4. Digitalis.
5. Recent (3-6 months) pelvic irradiation.
6. Inflammation or repair related to
infection or recent therapy.
High risk factors related cervical
neoplasia:
1. Early age at first coitus.
2. Multiple partners, especially in the teens.
3. First pregnancy under age 20.
4. Multiple early pregnancies.
5. History of sexually transmitted diseases, e.g.
HPV, gonorrhea, syphilis, Herpes Type II,
Trichomonas, Chlamydia, Condylomata and
Haemophilus.
 High risk male exposure.

A. Occupation group of husband is

important. Mannual labours have three times
greater risk as compared with professionals.

B. Genital cancers found in other sites, i.e. penile

or prostratic malignancies.

C. Sexual promiscuity with or without history of

venereal disease.
 Factors unrelated to cervical
neoplasia:
1. Family history.
2. Menstrual history.
3. Parity per se.
4. Diet.
5. Chronic non-specific cervicitis.
6. Mechanical irritation.
7. Exposure to external carcinogen, e.g., coal tar
products, such as douches or vaginal
applications.
How often should be Pap Smear to
be done.
• After first sexual intercourse or at the age of 21 years,
any woman has to undergo Pap-test within 3 years.
• Annual Pap-test recommended for the age group of 21-
30 years old. After 30 years woman who test negative for
three years consecutively, can have the test after every 3
years.
• High risk patients, with dysplastic and CIN findings,
should be followed more closely, at least semi-annually.
 Liquid Based Pap smear
• Instead of "smearing“ the cells (as in
conventional type) onto a slide, the
collected cells are placed in a vial of
preservative solution and the vial is
sent to the laboratory. The collected
cells are placed onto a slide in the
laboratory using technology that
clears obscuring debris and prepares
a thin layer of cells for the
cytotechnologist to review under a
 Endocervical cells
• Naked nuclei of
endocervical cells in
mucus streaks.
 Endocervical cells.
• Endocervical cells
with anisokaryosis,
Thank you