SCHOOL OF MEDICAL

LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER LESSON 2: EXFOLIATIVE CYTOLOGY

● Assessment of female hormonal activity. (sterility &

OUTLINE endocrine disorders) - maturity index
I. Exfoliative Cytology → is achieved by microscopic evaluation for determination of
A. Purpose maturation index (MI), based on examination of smears
B. Preparation taken from the lateral vaginal walls
C. Smear Preparation → Especially as females, we can identify what are the cells
D. Adhesion present during menstrual phase, pre-menstrual phase, and
E. Specimens after the menstruation.
F. Staining Method ● Determination of genetic sex - Barr Bodies (conglomeration
G. Cells found in Cervico-Vaginal Smear of chromatin, XX chromosome)
H. Criteria for the Cytologic Diagnosis of Normal Pregnancy → most of the nuclei of females exhibit conglomeration of
I. Report for Cytologic Smear for Diagnosis of Cancer chromatin, representing XX chromosomes (Barr body), which
J. Other Microscopic Examination Techniques of Cytologic may be demonstrated in the smears from buccal or vaginal
Smears mucosa
● Detects the presence of possible infection.
I. EXFOLIATIVE CYTOLOGY
● CYTOLOGY
→ histology of exfoliated, abraded or desquamated cells
▪ This is the study or histology of the cells that are being
removed from the body. Cells that were scraped from the
body.
▪ Give an example of body fluid that is being removed from
the body:
− CNS - Cerebrospinal Fluid Figure 1. Barr body in Females
● IMPRINT/ABRADED CYTOLOGY
→ Study of cells directly taken from surfaces of excised B. PREPARATION
specimens by touching them to a clean glass slide ● Material from fluids of the body may be examined either by:
▪ To clean glass slides. So, we will go back a little bit to the → Preparation of Smears
different fresh tissue preparations from the last prelims. → Preparation of Tissue Blocks (Cell Blocks)
Most of the preparations that we are performing for
exfoliative cytology will include the use of slides,
smearing, spreading on the slides.
● EXFOLIATIVE CYTOLOGY
→ Microscopic study of desquamated cells from epithelial
surfaces.
→ Study of cells that have been shed or physically removed
▪ We will be studying those cells that are being removed
physically on the body

BOOK INFORMATION: EXFOLIATIVE CYTOLOGY

▪ Exfoliated cells may be found in smears that have been
Figure 2. Tissue/Cell Blocks
spontaneously shed or physically removed from epithelial and
mucous membranes. ● Various Regions
▪ Specimens can be collected from the epithelial surfaces by → Vaginal Smear
lightly scraping the surface, by swabbing, aspirating or → Endometrial and endocervical smear
washing the surfaces. → Prostatic and breast secretion
→ Gastric or bronchial secretions
A. PURPOSE → Pleural or peritoneal fluids
● Assessing cancerous conditions (staging) → Sputum
→ With these body fluids, we can identify what is the stage of → Smears of urine sediment
the cancer ng patient. If it is on the critical stage, or early → Cerebrospinal fluid
stage. ▪ (PPVE - PGP - SSC)
● Detection of asymptomatic cancers
→ Asymptomatic Cancer example - Cervical Cancer C. SMEAR PREPARATION
▪ Cervical cancer, upon diagnosis, usually it’s on stage ● Make smears from fresh and moist material on clean slide;
three. Usually, stage 1 and 2, it has no signs and should not have clumps
symptoms. So that is why. We are familiar with Pap Smear → When we are making a smear, make sure that it is not too
no? That is an example of the test to identify this thin or not too thick. It must be uniformly spread on our slide.
asymptomatic cancer. Because this pap smear class, it is It should not have clumps.
a screening test. So, it can identify early if you have this ● Diamond pen Is used to put Identifiers on the smear (name,
cancer - cervical cancer. age, date, and type of smear); frosted-end slides may also be
used
BSMLS – 2G Team Writers: Bersabe, Lasaga, Rodriguez, Zwijgers 1 of 11
 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER LESSON 2: EXFOLIATIVE CYTOLOGY
→ During labeling, diamond pen or lead pencil must be used
. by Saccamono’s preservative
Figure 3. Diamond Pen
● Immerse smears immediately in fixative by a single
uninterrupted motion (exfoliated cells decompose rapidly
● Spray fixatives: 12 Inches or 1 foot away
→ If it is used near the slide, there will be an increase of
pressure, then the tissue will be erased.
Figure 4. Spray Fixatives
● Avoid in the container to prevent the cells from dislodged
● Optimal time: 1 hour to allow dehydration; adhesion and
maximal penetration of the fixative
Figure 2. Smear Preparation
BOOK INFORMATION: (Smear Preparation)
▪ Smears should be made from fresh material, prepared in the
doctor's office or radiology units.
▪ Specimens can be collected from the epithelial surfaces by
lightly scraping the surface, by swabbing, aspirating or
washing the surfaces
▪ The commonly received cytoprep is that of the “thin prep.”
These smears are wet-fixed in 95% ethanol immediately after
preparation to preserve the fine structure of the chromatin and
help in the evaluation of nuclear changes.
BSMLS – 2G Team Writers: Bersabe, Lasaga, Rodriguez, Zwijgers
▪ Air drying is avoided with smears for cytological detection of
neoplasia because it changes the appearances of the cells.
▪ Slides bearing blood or bone marrow smears, on the other
hand, are usually air-dried. Marrow smears are stained in
parallel to sections of the bone marrow core biopsy.
SMEAR PREPARATION CONCENTRATION:
NOTE:
● Best (yet flammable) fixative - 95% ethanol-ether
● Routinely used fixative - 95% ethanol
If smears cannot be made immediately, place the collected
material in 50% alcohol (for all types of effusions) —> replaced
● Saccamono’s Components: 50% ethanol , 2% carbowax
There are instances that we cannot perform the smear
immediately, what would be our remedy for this? So we will place
it in a 50% alcohol and then replace it with Saccamono’s
Preservative
● Storage: add ROH & refrigerate. (If we are going to store
our sample, we will add alcohol and refrigerate it with the
following concentration.)
→ 50% - pleural / peritoneal
→ 70% - sputum
→ 95% - urine, bronchial, gastric
● Mailed Specimen: air-dry for 10-15 minutes after 2 hrs fixation
& place in a container
● If specimen Is more than a few drops:
→ Centrifuge at 2,000 rpm for 2 minutes.
→ Afterwards, decant the supernatant, and smear the sediment
directly to the glass slide, or cytocentrifuge directly on the
slide with albumin.
→ Extra sediment can be used for cell block technique.
Figure 4. Smear Preparation if specimen is more than a few drops
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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER LESSON 2: EXFOLIATIVE CYTOLOGY

GENERAL PROCESSING: A. RESPIRATORY SPECIMENS

General Processing ● SPUTUM
Viscous specimen Immerse in Ether ROH ASAP → Demonstrate abnormal cells early in disease
● Cervical, Vaginal, → Fresh, unifixed, at least 3 consecutive early morning
Prostate Secretion sputum
Mucoid Specimens Dry smear edges before fixing (to ▪ We must give clear instructions. Patients are the
● Sputum, Bronchial avoid runoff) ones that will collect the sample in their
and Stomach houses, early in the morning.
Secretions ▪ We must instruct the patient to collect three
Bloody specimens RBC can be destroyed by adding consecutive samples. The patient must have
2-5 mL of conc. Acetic acid per CONTAINER 1, CONTAINER 2, CONTAINER 3.
100 mL of the specimen ▪ So collection of this one must have 1 hour
For example, we received a interval
sample na CSF. To destroy RBC ▪ Since it is early morning, It is important to put the
present in the CSF (have time, so that we can identify what is the first one,
problems with the collection), add second, etc.
concentrated acetic acid per 100 → Patients unable to produce sputum: INDUCED
mL of the specimen. SPUTUM by inhalation of aerosol solution for 20
minutes
▪ If ever the patient has undergone repeat collection
many times, we can give them aerosol solution.
Inhalation lang for 20 minutes.
▪ Why aerosol solution? Because it can trigger
Water Specimens (urine, Specimen is centrifuged first and asthma. There will be sputum formation.
exudate, aspirate) the sediment is smeared in an ▪ Not recommended to everyone. Upon the
albumin coated glass slide. In the recommendation of the doctor.
event that there is excess → Wide mouthed bottle w/ Saccomanos's fluid
sediment, it may be subjected to ▪ The container must have a wide mouth
biopsy ▪ The sample needs to be preserved since the
sample came from the patient’s house.
D. ADHESION → After collection:
● For adequate adhesion of smeared material, ▪ Pour specimen in petri dish, examine for solid or
● Albumin not recommended because it will retain the OG of blood flecked particles
Pap stain ▪ Remove particles, place on slide, crush w/
● Specimens That Require Adhesives : another slide. Evenly distribute
→ Urinary sediment ▪ Fix for a minimum of 1 hour
→ BAL − After receiving the sample, we are going to
→ Specimens with proteolytic enzymes (saliva) prepare it using the crushing technique.
→ Concentrated sputum → Absence of histiocytes and alveolar macrophages
● Characteristics of Adhesives: indicate that only saliva was collected
→ permeable to both fixative & stain ▪ If saliva is collected, repeat the collection.
● Examples of Good Adhesives : ▪ How to collect the sample? It must be a deep
→ Pooled Serum / Plasma cough.
→ Celloidin Ether Alcohol ▪ We are going to look for histiocytes and alveolar
→ Leuconostoc Culture macrophages.

E. SPECIMENS BOOK INFORMATION: (sputum smear)

● Generally divided into two major areas. We have ▪ Collect early morning sputum by a deep cough in a wide
non-gynecologic specimens and gynecologic specimens. -mouthed jar containing Saccomanno fluid (50% ethyl alcohol
→ If gynecologic, they are samples from females. Other and 2% carbowax).
samples like sputum, urine, and others, non-gynecologic. ▪ If a more extensive study is to be made, it is recommended
● Non-gynecologic Specimens that a minimum of 2-4 slides be prepared, and one is air dried
→ Respiratory Specimens for Giemsa staining. At least two slides should be stained by
→ Gastrointestinal Specimens Papanicolaou method.
→ Peritoneal, Pleural & Pericardial Smears ▪ If no solid particles are found, an attempt should be made to
→ Urine secure representative samples from both thin and thick
→ Breast Secretions portions. With the end of a wooden applicator stick, these
→ CSF samples are evenly spread on the slides and immediately
→ Prostatic Secretion placed in fixative for a minimum of one hour.
● Gynecologic Specimens ▪ Sputum collected should have been coughed from
"down-deep" to ensure that the specimen is a true
I. NON-GYNECOLOGIC SPECIMENS representative of the sputum and not just saliva. The mucus

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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER LESSON 2: EXFOLIATIVE CYTOLOGY

present preserves the cell constituents; however, it is highly → smear must contain epithelial cells (ciliated bronchial
recommended that the specimen be as fresh as possible cells) + RBC and WBC
when examined, if better cellular detail is to be appreciated. → If specimen is scanty, prepare smear in OR to prevent
drying
● BRONCHIAL BRUSHING
→ Directly smear on two slides using Pull-apart
technique
→ Fix immediately to avoid production of Air drying
artifacts

Figure 7. Bronchial Aspirates

Figure 5. Bronchial Brushing

BOOK INFORMATION: (bronchial brushing)

▪ The slides are then fixed by a spray fixative or immediately
immersed in 95%alcohol. Failure to fix the slides within a few
seconds will produce unsatisfactory cytologic results due to
air drying artifact.

● BRONCHOALVEOLAR LAVAGE (BAL)/ BRONCHIAL

WASHING Has cilia; an indication of the doctor that is really a bronchial
→ Performed in patients with AIDS to rule out aspirate
Pneumocystis jirovecii infection Among these four, sputum lang ang patient ang mag collect.
▪ Also the causative agent of pneumonia The rest, si doctor na ang mag collect.

BOOK INFORMATION: (bronchial aspirate)

▪ The secretion obtained at bronchoscopy are collected either
by aspiration into a glass suction apparatus or by washing the
bronchi with 1-2 cc. of saline, into a cup, if sufficient in
amount, and sent immediately to the laboratory for
preparation and examination.

B. GASTROINTESTINAL SPECIMENS
● 2 methods: Through the mouth or Levine method -
through nasogastric
● GASTRIC LAVAGE, GASTRIC BRUSH, SUBMUCOSAL
LESION FNA
Figure 6. Bronchial Washing → Collection:
▪ Irrigation
BOOK INFORMATION: (bronchial washing) ▪ Aspiration
→ Difficult due to inaccessibility and presence of gastric
▪ Bronchial Washing specimens are freshly collected in the
fluid
bronchoscopy collection container and hand delivered to the
→ Examine ASAP
laboratory
▪ Sample must be processed as soon as possible
→ 8 hours fasting before gastric washing; even water is
● BRONCHIAL ASPIRATES
prohibited
→ Collection:
▪ Even water is prohibited
▪ aspiration (with glass suction apparatus) or
▪ washing (with saline)
→ Show evidence of malignancy in advanced stage for
20-30 minutes, medium speed

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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
SAN PEDRO COLLEGE – MAIN
CAMPUS
AY 2022 – 2023 - 2ND SEMESTER
Figure 8. Gastric Washing
BOOK INFORMATION: (gastric smear preparations)
▪ Specimen should be examined as soon as possible since any
delay of more than I /2 hour, before fixation, will digest the
cells and make the specimen unsatisfactory for evaluation.
▪ The patient should have fasted for at least 8 hours before
gastric washing is performed. Esophageal washings are to be
examined immediately
C. PERITONEAL, PLEURAL, & PERICARDIAL SMEARS
● Paracentesis
→ For the extraction of peritoneal, pleural, pericardial
fluid
● Collection: same as bronchial specimens
→ Increased fluid indicates pathologic process
→ Presence of malignant cells usually indicate
metastasis
→ Add 300 units of heparin per 100 mL of aspirate to
avoid clots
▪ Because peritoneal, pleural, and pericardial
samples are prone to clotting
Figure 9. Peritoneal, Pleural, & Pericardial Smears
BOOK INFORMATION: (serous effusion)
▪ The presence of malignant cells in serous effusions usually
indicate metastatic involvement and, therefore, a higher stage
of cancer
▪ They can be collected in tubes or syringes that may be either
plain or pre-heparinized, to prevent coagulation
▪ Freshly tapped specimens are preferred for cytology. If
immediate processing is not possible, it can be preserved in
the refrigerator for a period of 24-48 hours or pre-fixed in 50%
ethanol. Albuminized slides should be used to prepare
smears from prefixed sample.
D. URINE
● Collection:
→ Male - Second voided urine
▪ First morning is not recommended for cytologic
evaluation because the cells may degenerate
BSMLS – 2G Team Writers: Bersabe, Lasaga, Rodriguez, Zwijgers
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
LESSON 2: EXFOLIATIVE CYTOLOGY
after spending extended time period in acid
environment in the bladder
▪ In the morning, urine stays longer in the bladder
→ Female - catheterized
● At least 50 mL is needed
● For more reliable evaluation, urine may be collected and
examined twice: early morning and later in the day
● Use of preservative is not recommended
→ For diagnosis urothelial malignancy and not for
prostatic carcinomas
▪ Malignancy in the bladder, urethra, or renal pelvis
▪ Not on the prostate
Figure 10. Urine
BOOK INFORMATION: (urinary tract)
▪ Voided urine from males is usually sufficient for cytological
evaluation, but for female patients, a catheterized specimen is
recommended to prevent contamination of specimen with
vulvar cells.
▪ Early morning specimen yields the greatest number of cells,
but such cells are usually distorted due to prolonged bladder
retention
▪ Smears of sediments should be prepared and fixed as soon
as possible after collection. Since a great proportion of cells
become detached during fixation, unsatisfactory smears may
result in false negative diagnosis.
▪ The collection technique should be always mentioned on the
requisition form. The cytomorphologic features of low grade
urothelial carcinomas may be indistinguishable from those of
reactive urothelial hyperplasia.
E. BREAST SECRETION
● Collection: Spontaneous nipple discharge
● Materials: cotton swab or touch prep (Touch the nipple
directly during discharge using the slide or cotton swab)
→ For hormonal imbalance testing
▪ Not an indication or phase of cancer
→ Benign breast lesion or endocrine problems
→ Extremely low diagnostic yield for breast carcinoma
diagnosis
→ If bloody, benign intraductal papilloma
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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER LESSON 2: EXFOLIATIVE CYTOLOGY

BOOK INFORMATION: (CSF)

▪ Cells in cerebrospinal fluid (CSF) samples degenerate very
quickly and are usually present in very low numbers. Once the
slide has been prepared, it should be rapidly air dried before
staining. The addition of an equal amount of ethyl alcohol to
the cerebrospinal fluid (CSF) is recommended if a delay in
processing is anticipated. For CSF specimens, a minimum
amount of 1 cc. is necessary for cytologic evaluation

G. PROSTATIC SECRETION
● 3 Specimen:
Figure 11. Breast Secretion → Voided urine before massage
→ smear of prostatic secretion by massage
BOOK INFORMATION: (breast secretion) → urine after massage
▪ Spontaneous nipple discharge is usually a result of hormonal ▪ Label the container with ‘before’ and ‘after’ to
imbalance in young patients, and when the secretion is bloody distinguish the time of urine collection
a benign intraductal papilloma should be considered clinically. ▪ If unproductive massage = urine should still be
▪ The spontaneous nipple discharge should be smeared on a passed
clean glass slide, and immediately placed in fixative. ● Cytologic examination of 3rd specimen after
▪ In women, except during lactation and the immediate unproductive massage is recommended
post-lactation period, any discharge from the nipple is
abnormal .
▪ The nipple discharge is usually due to a benign breast lesion
such as duct ectasia and papilloma , or due to endocrine
problems.
▪ However the major value of cytologic examination of nipple
discharge is potential detection of malignant cells in a patient
with clinically undetected carcinoma.

Collection Technique:
▪ Gently strip the subareolar area and nipple using the thumb
and forefinger.
▪ Place the labeled slide upon the nipple and draw it quickly
across the nipple.
▪ If more than a drop is collected, use another slide to smear
with a pull-up technique.
▪ Immediately drop slide in a bottle of 95% isopropanol or use
spray fixative. Figure 13. Prostatic Secretion
▪ If the secretion is scanty in amount, smears should be
restricted to a small area of the slide to prevent drying. II. GYNECOLOGIC SPECIMENS
▪ Secretions obtained from both breasts should be properly A. VAGINAL AND CERVICAL SMEARS
identified as left or right. ● Screening test for precancerous condition
▪ For localized breast lesions producing discharge upon → Not an identification for cancer
pressure, the secretion may be smeared directly on the slide → Recommended for females (35 yrs old and above)
after expressing material from the breast ▪ If the female has a child = recommended to
undergo Pap Smear
F. CEREBROSPINAL FLUID ● Not diagnostic
● Minimum of 1 mL is necessary ● Transformation zone (T-zone: junction of the
● Manner of extraction: Lumbar Puncture endocervical mucosa)
● Collection: aspiration of posterior fornix, swabbing,
● Instruments:
→ 8 inch glass pipette & rubber bulb (for cancer
cytology)
→ Sterile tongue depressor (for hormonal studies)
→ Ayre’s spatula
→ Cervix brush

Figure 12. Cerebrospinal Fluid

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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER LESSON 2: EXFOLIATIVE CYTOLOGY

endocervix, smeared on glass slides and stained by the

Papanicolaou (Pap) method. The entire procedure is known
as Pap smear. Because cervical cytology is a screening test,
abnormal findings must be confirmed histologically.
▪ The patient should abstain from coitus, not douche the vagina
for at least a day, and not apply intravaginal preparations for
at least one week before the examination. Smears should not
be taken during menstrual bleeding, because of
contamination with blood, endometrial component, and tissue
debris that can obscure the cells during smear examination.
▪ Various collecting systems such as cotton swabs, wooden or
plastic spatula, and cervical brushes, so-called cytobrushes,
are generally used to collect gynecology cells. In brush
biopsies, more endocervical cells and blood may be present
in the smears, and this may lead to valuation difficulties. The
use of cotton swab for collection of cervical smear is to be
discouraged because of the drying artifacts and loss of cells
that are caused by this method. Wooden spatula is preferable
to plastic spatula, because of its mildly rough surface that can
collect more material. Endo-cervical brushing is used strictly
for taking materials from endocervix.
▪ After smear collection, the sample is evenly smeared on to
the center of the non-frosted area of the glass slide and fixed
immediately. Excessively thin or thick smears can result in
false-negative reports.

CYTOLOGIC COLLECTION AND PREPARATION:

Cytologic Collection and Preparation

Figure 14. Vaginal and Cervical Smears Instruments Endocervical brush samples of endocervical canal

● PRECAUTIONS FOR VAGINAL SMEAR PREP Vaginal scrape patients with hysterectomy
→ Patient must avoid vaginal exam’n or douching 24-48
hours before collection. Lateral vaginal scrape for hormonal evaluation
→ Spread smear thinly in a rotary motion
→ Glass pipette must be absolutely dry Four quadrant vaginal scrape for localization of vaginal
→ No lubricant or powder must be used adenosis

Vulvar scrape for detection of herpetic

lesions or carcinoma

Figure 15. Precautions Figure 16. Cytologic Collection and Preparation

BOOK INFORMATION: (cervical smear) F. STAINING METHOD

● PAPANICOLAU STAINING METHOD
▪ Cancer of the uterine cervix is the commonest cancer that can
→ Staining method of choice
be detected even at the pre-invasive stage. Cervical cytology
→ Developed by Dr. George Papanicolao (1940)
screening involves the microscopic examination of cell
→ Detecting human uterine & cervical CA.
samples that have been taken primarily from the ecto- and

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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER LESSON 2: EXFOLIATIVE CYTOLOGY

→ Cytologic evaluation of other secretions → Upper lateral 3rd of vaginal wall (less contamination &
→ Transparent blue staining of cytoplasm accessible)
▪ due to the action of high alcoholic content of the → Wooden spatula is recommended only in vaginal hormonal
cytoplasmic counterstain, allowing overlapping cells to be studies
seen and identified. → LPO:
→ Excellent nuclear detail is produced ▪ Assess smear & staining quality
→ Color range is predictable ▪ Detect RBC & WBC
▪ and of great value in identification and classification of ▪ Detect type of exfoliated cells
cells, producing a good differential coloring of basophilic ▪ Rough assessment of cell proportion
and acidophilic cells → HPO: Quantitate
→ Valuable in comparing cellular appearances in smears
▪ with their counterpart in similarly stained sections.

MAKE USE OF 3 STAINS:

Stain Cells stained

Hematoxylin Nuclear structure

OG6 (orange green) Cytoplasm of mature cell

Eosin Azure Cytoplasm of immature cell

Figure 17. Vaginal Hormonal Cytology
Components:
Eosin
PTA
G. CELLS FOUND IN CERVICO-VAGINAL SMEARS
Light green stain (36, 50, 65) 1. Mature superficial cells
Lithium carbonate 2. Intermediate cells
Bismarck brown 3. Parabasal cells
4. Basal cells
5. Endometrial cells
6. Endocervical glandular cells
7. Doderlein bacilli

Figure 17. 3 Stains

● MODIFIED PAP’S METHOD

→ It omitted the Bismarck brown dye from EA solution
→ Sharpness of color and brilliant staining reactions are
improved

BOOK INFORMATION: (papanicolaou stain)

▪ Papanicolaou's stain (also known as Pap stain) is a
multichromatic staining cytological technique that is used to
Figure 18. Cells found in cervico-vaginal smears
differentiate cells in smear preparations of various bodily
secretions, including gynecological smears (Pap smears),
sputum, brushings, washings, urine, cerebrospinal fluid, MATURE SUPERFICIAL CELLS
abdominal fluid, pleural fluid, synovial fluid, seminal fluid, fine ● Polygonal
needle aspiration material, tumor touch samples, or other ● Pale pink cytoplasm
materials containing cells. ● Dark pyknotic nuclei (< 6 µ)
● Abundant during proliferative phase
● VAGINAL HORMONAL CYTOLOGY → Before menstruation
→ Inexpensive ● True acidophilia (due to estrogen)
→ Not commonly used nowadays → Due to estrogen
→ performed regularly even in pregnant women without undue ● Pseudoacidophilia:
risk → Vaginal prolapse and drying
→ No prior vaginal examination or douching in the last 24 hours → Drying of smears
→ Infection
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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER LESSON 2: EXFOLIATIVE CYTOLOGY

→ Chemicals

Figure 19. Mature superficial cells.

INTERMEDIATE CELLS
● Medium sized polyhedral or elongated cells
● Basophilic cytoplasm with vacuoles
Figure 21. Parabasal Cells
● Abundant during secretory phase (influenced by progesterone)
A. NAVICULAR CELLS
BASAL CELLS
→ Boat shaped; folds / curls on edges ● Small, round to slightly oval cells
→ Estrogen progesterone effect ● Large nuclei (> half of cell vol.)
→ Latter half of menstrual cycle, pregnancy, & ● Strongly basophilic cytoplasm
menopause ● Found before puberty & after menopause
B. PREGNANCY CELLS
→ Round, oval cells
→ Translucent basophilic cytoplasm at the center of cell
d/t central glycogen accumulation
→ Eccentric nucleus
→ Double walled boundary with deep blue stain at the
cytoplasmic periphery

Figure 20. Intermediate cells. Figure 22. Basal Cells

PARABASAL CELLS ENDOMETRIAL CELLS

● Smaller than intermediate cells ● Like parabasal cells; slightly cylindrical
● Thick, round to oval ● Less basophilic cytoplasm
● Smaller strongly basophilic cytoplasmic area ● During & 1-4 days after menstruation
● Larger vesicular nucleus than intermediate cells
● 2 weeks of age to puberty, after childbirth, abortions & after
menopause

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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER LESSON 2: EXFOLIATIVE CYTOLOGY

Figure 25. Doderlein Bacilli

FERNING PHENOMENON
● “fern”/palm-leaf pattern of salt crystals
Figure 23. Endometrial Cells ● seen on dried cervical mucus
● signifies persistent estrogen effect
ENDOCERVICAL GLANDULAR CELLS ● basis for diagnosing early pregnancy
● Occur in large group or sheets
● Honeycomb appearance
● Finely vacuolated pale blue/gray cytoplasm

Figure 26. Ferning Phenomenon (positive & negative results)

H. CRITERIA FOR THE CYTOLOGIC DIAGNOSIS OF

NORMAL PREGNANCY
1. Marked progesterone effect
2. At least 50% of intermediate cells in cluster
3. At least some typical pregnancy cells are present
4. Less than 30% mature superficial cells
5. Doderlein-filled dirty background
Figure 24. Endocervical Glandular Cells
QUANTITATIVE EVALUATION FOR VAGINAL CYTOLOGY
Quantitative Evaluation for Vaginal Cytology
DODERLEIN CELLS
Maturation Index The percentage of cells from the main
● Lactobacillus acidophilus
(MI) layers of vaginal epithelium (superficial,
● most common normal vaginal flora
intermediate and deep)
● blue to lavender w/ Pap’s
Criterion: Pyknosis
● Increased number indicates healthy vagina
● Numerous in: last trimester of pregnancy, infection, estrogen Acidophilic Index (AI) The percentage of cells staining
deficiency, DM pink-orange to red with Pap’s smear.
● Decreased number leads to increase in population of invasive This is not a reliable index due to
Lepthothrix species; comes with presence of Trichomonas possible pseudoacidophilia
vaginalis Pyknotic Index (PI) The percentage of cells with shrunken,
dark and small structure-less nuclei

BSMLS – 2G Team Writers: Bersabe, Lasaga, Rodriguez, Zwijgers 10 of 11

 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER LESSON 2: EXFOLIATIVE CYTOLOGY

I. REPORT FOR CYTOLOGIC SMEAR FOR

DIAGNOSIS OF CANCER

CLASS I Absence of Atypical or abnormal cells

CLASS II Atypical Cytologic Picture but no evidence of
malignancy
CLASS III Cytologic picture suggestive but not conclusive
of malignancy
CLASS IV Cytologic picture strongly suggestive of
malignancy
CLASS V Cytologic picture conclusive of malignancy

J. OTHER MICROSCOPIC EXAMINATION

TECHNIQUES OF CYTOLOGIC SMEARS
1. ACRIDINE ORANGE FLUORESCENT TECHNIQUE
a. Binds DNA -> green/yellow
i. If increased (↑): MALIGNANCY
b. Binds RNA -> brick-orange red
i. If increased (↑): GROWTH
2. PHASE CONTRAST MICROSCOPY
a. The second best choice after Pap’s staining
b. Used for hormonal evaluation of gynecologic specimen
and for cancer detection
3. INTERFERENCE MICROSCOPY
a. Determines the dry weight of individual cells or cellular
constituents
b. Very expensive and complex

BSMLS – 2G Team Writers: Bersabe, Lasaga, Rodriguez, Zwijgers 11 of 11