GPHT1 Lab 3rd Shift Reviewer
Pap’s Smear
Prepared by: C3
PAPANICOLAU’S METHOD
• Screening test for the detection of
important and clinically significant
lesions of the uterine cervix
• Developed by George Nicolas
Papanicolau, an American anatomist of
Greek descent in 1928
• Collaborated with gynecologist Herbert
Traut in 1941 in using vaginal smear for
the diagnosis of cancer of the uterus
• In addition to the cervix, other sites are
examined such as:
o Upper third of the vaginal wall
o The ectocervix
o The endocervix
Anatomic Sites for the preparation of
cytologic samples
1. Upper (proximal) third of the vaginal
wall
• Especially valuable to assess
the hormonal status of the
woman, evaluation of possible
inflammatory conditions of the
vagina (vaginitis), classify the
microbiologic flora of the vagina,
and though rarely, for detection
of malignant lesions of the
vagina (clear cell
adenocarcinoma)
2. The ectocervix
• The ectocervical specimen is
most commonly used for cancer
screening
• The sample is collected using a
sterile wooden spatula that can
reach the transformation (T or
junctional) zone of the cervix,
where most of the malignancies
usually arises from
3. The endocervix
• The endocervical sample is
obtained by means of the Ayres
spatula or a premoistened
cotton tipped swab
• This specimen is not only useful
for the detection of endocervical
lesions (squamous cell
carcinoma and
adenocarcinoma), but may be
used also for the detection of
some intrauterine lesions, which
might be readily missed
clinically, if this sample is not
included in the Paps
• However, lesions of the
intrauterine cavity and corpus
uteri is best assessed by a
direct tissue sample of the site
or by diagnostic imaging studies
HORMONAL CYTOLOGY
• Based upon the specific response of the
vaginal epithelium to stimulation by
steroid hormones, mainly of the ovarian
origin
• These hormones, namely oestrogen, is
produced by the proliferating granulose–
theca cells of the developing ovarian
follicles
• The other hormone, progesterone, is
produced by the corpus luteum, formed
immediately after ovulation
Oestrogen
• Induces maturation of the vaginal
squamous epithelium
• The cells undergo maturation under the
influence of oestrogen, denoting
continuous growth and development of
the mucosa
• This is manifested by the thickening of
the epithelium due to the increase in
cellular layers, and at the same time,
differentiate to cells towards their most
evolved, matured form – superficial cell
Progesterone
• Responsible for the marked thickening
of the intermediate zone of the
epithelium. The cells of this layer are
referred to as the intermediate cells
• This hormone is an antagonist to the full
maturation of the cells. It therefore
impedes the maturation-promoting effect
of estrogen
TRIPLE C NOTES 1
Parabasal-Basal Cells 7. Counterstain in Eosin Azure (EA)-36 or
• Progenitor cells that remain as the only 50
intact layer of the vaginal epithelium 8. Dehydrate
• In the absence of both oestrogen and 9. Clear with xylol
progesterone, such as in the state of 10. Mount with resinous media then
menopause, the epithelium becomes coverslip
thin, the cells decrease in size (atrophy) 11. Label
and fails to progress to its terminal
differentiated form OG-6
• The exogenous intake of hormonal • Strong affinity for mature cells (i.e.
estrogen drugs in a menopausic woman, superficials, keratinizing malignant
however, will stimulate the parabasal squamous epithelial cells)
cells to once more respond to • This strong affinity imparts the so-called
maturation “cytoplasmic acidophilia” because the
• Conversely, intake of progestin drugs cytoplasm stains bright orange to
act similarly to that of progesterone, or yellow-orange
occasionally to testosterone • Alcohol-based solution, and notice that
the smears are washed in a 95% alcohol
Cytohormonal Maturation Index (CHMI) solution afterwards to remove the
• Helps in the evaluation of the hormonal excessive stain
status based on the distribution of these
cells in the vaginal sample of a paps EA-36 or EA-50
smear • Polychrome stain composed of 3 basic
• Numerical expression (in percent per stain components, namely:
100 cells) representing the relative o Eosin Y
proportion of the 3 vaginal cells types in o Bismarck brown
this order: o Light green SF
• Solution has an olive-green appearance,
𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 = 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝: 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖: 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 with a tinge of brown and red
• The presence of 3 stains imparts a
• This estimation is possible as a guess diverse display of vibrant colors,
estimate in percent, by glancing at the appreciated in paps stained smears, that
smears using the microscope gives the microscopist a crisp, diverse,
• The background of the smears must be brightly colored detail of the different
clean, that is, free from any obscuring cellular components
inflammation such as dense numbers of • It has a strong affinity to the immature
neutrophils, and obvious evidence of vaginal cell series, namely the
infection by various pathogens intermediates and parabasal cells,
• The sample must be adequate and the where their cytoplasm stain with a
cells well-distributed throughout the transparent blue-gray to brown-blue
smear • This color selectivity aids the
microscopist to better and easier identify
STAINING PROCEDURE and classify vaginal cells for CHMI
1. Fixation in 95% ethanol, then wash with determination
water • However, caution must be taken
2. Primary stain with hematoxylin because this is not always a reliable
3. Differentiate with acid-alcohol, 1 quick means for CHMI classification, due to
dip, then wash in water (regressive) factors such as staining technique, age
4. Blue in ammonia water, then wash of the stain, brand of stain, nature of the
5. Counterstain with orange-green (OG)-6 cells, presence of unwanted material on
6. Wash in 2 changes of 95% ethyl alcohol the smears, etc.

TRIPLE C NOTES 2
NORMAL CELLULAR COMPONENTS
1. Neutrophils
• As seen in a peripheral blood
smear
• Normally increased just before,
during, and shortly after
menstruation
• A persistence of a large number
of these cells suggest an
inflammatory response or an on-
going infection
o In the case of the latter,
a judicious search for
the causative agent
must be exhausted
2. Red Blood Cells (RBCs)
• Seen during traumatic
collection, menstruation, or
post-menstrual period, or
because of some pathologic
bleeding
• Ideally, the patient should be
schedule for Pap’s Smear 10
days after her LMP, to avoid
obscuring the smears due to
menstrual blood
3. Lactobacillus acidophilus (Doderlein
bacilli)
• These are gram-positive long,
stubby bacillus that are part of
the normal bacterial flora of the
female vaginal tract
• They maintain the normal acid
pH of 3.5-4.5 by glycogenolysis
of glycogen into lactic acid
• The loss of this normal flora
invites a potential for infection
by pathogenic organisms and
infection to set in
4. Leptothrix spp.
• These are long, thin,
filamentous, hair-like bacilli that
are normal commensals,
becoming prolific if the vaginal
pH increases
• It indicates the loss of the
normal bacterial population
responsible for the maintenance
of the acid milieu, and indirectly
tells the possibility of an
infection
• Under normal conditions, this
organism is observed in normal
healthy subjects usually prior
and shortly after their menses,
or during intake of antibiotics
5. Talcum or starch granules
• These are usual contaminant
that may fall on the glass slide
• These granules appear as ovoid
bodies that exhibit an apple-
green birefringence when
examined under the polarizing
microscope
• Talcum powder or starch
powder is used as dry lubricant
for donning latex surgical gloves
• In addition, the patient herself
may personally practice the
habit of powdering herself quite
thoroughly
6. Endocervical cells
• These are columnar epithelial
cells that form part of the normal
lining of the endocervical canal
• The cells have characteristic
basally oriented nuclei, and a
vacuolated to slightly granular
mucin-filled cytoplasm, and a
whispy surface
• The presence of these cells
indicates the clinical
representation of the
endocervical canal; and
together with the presence of
squamous epithelial cells, the
transformation zone
• Using the Bethesda system, the
presence of the transformation
zone components makes the
smears “adequate and
satisfactory for evaluation”
• Their mere absence on the
contrary, still constitute the
smears as satisfactory (provided
that there is no obscuring
inflammation, blood, or other
contaminants) but limited by the
factors that classify the smears
as less than optional
TRIPLE C NOTES 3
 7. Endometrial cells
• These cells are small epithelial
cells coming from the shedding
of the endometrial lining or due
to a proliferating endometrial
pathology
• These cells consist of the
glandular component, seen as
uniformly small, round cells
disposed in tight clusters, and in
acini or syncytial aggregates,
and/or as stromal cell
components, seen as spindly,
often degenerated to naked
nuclei or as histiocytic infiltrates
• The presence of these indicate
previous menstruation or that
menstrual bleeding is about to
occur
• This should be correlated with
the age of the woman and her
LMP history
• The findings of endometrial cells
in a post-menopausic woman,
or if, the menstrual cycle is “out-
of-phase” suggest a possible
endometrial pathology such as
endometrial hyperplasia,
endometrial polyps of
endometrial carcinoma
• The patient should be ideally
scheduled for a paps smear 10
days after her LMP
o This not only avoids the
presence of obscuring
blood, but avoids the
suspicious nature of
their presence
ABNORMAL CELLULAR
COMPONENTS
1. Candida albicans
• These are budding yeast, which
under the proper condition, form
branching pseudohyphae that
spears clusters of epithelial cells
that look like a “shish kebab.”
• Candidiasis is frequently seen in
diabetic patients, patients taking
oral contraceptives, or
prolonged steroid therapy,
immunocompromised states,
and in malignancies such as
leukemia and lymphoma
2. Trichomonas vaginalis
• These are sexually transmitted
pear-shaped organisms that
stain blue-gray to blue-green,
granular cytoplasm, with a slit-
like eccentric dark nucleus
• The flagellum is not preserved,
hence it is not evident in the
Paps stain
• These organisms appear as ill-
defined blobs that can be readily
missed, especially in a
background of dense, obscuring
inflammation
3. Gardnerella vaginalis
• These are tiny, pleomorphic
coccobacilli that clings to the
surface of the cytoplasm of
epithelial cells, giving a “moth-
eaten” appearance or the so-
called “clue cells”
• They indicate a shift in vaginal
flora, replacing the normal
lactobacilli
• These organisms are the cause
of “non-specific bacterial
vaginosis,” causing a thin-
watery, vaginal discharge
• Their presence may also
indicate a more significant
pathogenic agent that might be
lurking somewhere else, such
as Candida and Trichomonads
4. Koilocytes
• These are squamous epithelial
cells that show the cytopathic
effects of Human Papillomavirus
(HPV) infection
• The virus is the causative agent
of genital warts called
“condyloma acuminatum”
• A classic koilocyte is seen as a
cell with an atypical (wrinkled
prune) nucleus surrounded by a
perinuclear halo
TRIPLE C NOTES 4
 • The boundaries of the halo have
a sharp demarcation from the
rest of the normal cytosol
• The presence of koilocytosis is
diagnosed as a low-grade
squamous intraepithelial
lesion, using the Bethesda
system of reporting gynecologic
cytology
• They have a strong association
with the pathogenesis of
cervical carcinoma by the
mechanism of onocogenesis
that is brought about by the
incorporation of the virion DNA
into the host genome
5. Herpes Simplex Virus (HSV)-II
• The virus incites a cytopathic
effect in the infected cell
characterized by macrosomia,
multinucleation, nuclear
molding, and a ground-glass
chromatin pattern
6. High-grade squamous epithelium
intraepithelial lesions
• Encompass severe dysplasia,
CIN III, and carcinoma-in situ
7. Invasive squamous cell carcinoma
• Most common form of cervical
malignancy
• Developed at the National Cancer
Institute sponsored workshop in
December 1988 to provide uniform
diagnostic terminology that would
facilitate communication between the
laboratory and the clinician
• The format of the TBS report includes
the following:
o Specimen adequacy
o General categorization
o Descriptive diagnoses
• Specimen adequacy needs to be
overemphasized, especially for the
medical technologists because it
connotes the importance of proper slide
preservation, staining, and adequate
representation of the important anatomic
site of collection

SYSTEMS OF REPORTING
GYNECOLOGIC CYTOLOGY

The Class System Figure 1. Illustration of the technique used for

• No longer used for reporting gynecologic cytological cervical sampling.
cytology, but for cytologic specimens
other than Pap’s
• Manner of reporting is as follows:
o Class I – negative for malignant
cells
o Class II – atypical cells present
o Class III – suspicious but not
conclusive of malignant cells
o Class IV – strongly suggestive
of malignant cells Figure 2. Papanicolau stain.
o Class V – conclusive
(POSITIVE) for malignant cells

The Bethesda System (TBS)

TRIPLE C NOTES 5