BASIC DIAGNOSTIC

CYTOLOGY
 Basic Diagnostic Cytology
• Microscopic examination of cells from
different body sites
• Two divisions:
– Exfoliative Cytology
– Fine Needle Aspiration (FNA)

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EXFOLIATIVE CYTOLOGY

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 Exfoliative Cytology
• Microscopic study of cells that have
been desquamated from epithelial
surfaces.

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 Importance
• For assessing malignant or cancerous
conditions
• For detection of asymptomatic cancer in
women
• For assessment of female hormonal
activity in case of sterility and endocrine
disorders
• Determination of sex
• Determination of the presence of possible
infection
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 Specimen for Examination
• Vaginal smears
• Endometrial and endocervical smears
• Prostatic and breast secretions
• Gastric or bronchial secretions
• Pleural and peritoneal fluids
• Sputum
• Smears of urine sediment
• Cerebrospinal fluid
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 Adhesion
• Some specimens require addition of
adhesive agent to provide adequate
adhesion of smeared material
throughout fixation and staining.
– Urinary sediment
– Bronchial lavage specimen
– Specimen that utilizes proteolytic enzymes
during processing
• Trypsin, concentrated sputum and enzymatic
lavage specimen from GIT
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Characteristics of Adhesive Agents

• It must be permeable to both fixative

and stain
• It must not retain the stain

Egg albumin is NOT recommended as

an adhesive agent
Intensely stained by the basic light
green counterstain of Pap’s method.

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Adhesive Agents Used in Cytology

• Pooled human serum/plasma

• Celloidin ether alcohol
• Leuconostoc culture

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 Fixation
• Some can be fixed in 15 minutes
• Recommended to be left for a
minimum of 1 hour before staining
– Assure optimum dehydration and
adhesion permitting more complete
penetration of cells

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 Fixatives for Smears
– Equal parts of 95% Ethyl alcohol and
Ether
– 95% Ethyl alcohol
– Carnoy’s fluid

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 Fluid Specimen
• Fixatives:
– 50% alcohol (all types of effusion)
– Saccomano’s fixative (50?% ethanol and
2% carbowax)
• Centrifugation:
– 2000 rpm for 2 minutes

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 Precautions during
fixation
‼ Identify the slides before preparing smears.
‼ Use paper clips to the identified end of the slide
before preparing smears.
‼ Smears should be placed into the fixative
container immediately after preparations.
‼ Place each smear in fixative by single
uninterrupted motion to avoid rippling of
smeared material.
‼ Avoid striking the bottom of the fixative
container forcefully to prevent dislodging of the
cells.
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NON-GYNECOLOGIC
SPECIMENS
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Respiratory Tract Specimens
• Sputum
– Obtain at least 3 consecutive morning
sputum specimens (through deep cough)
– Use wide-mouthed jar with Saccomano’s
fixative

 Alveolar macrophages – sputum from deep

cough
 Absence of alveolar macrophages –
specimen is not sputum but SALIVA
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• Bronchoalveolar lavage/bronchial
washing
– Performed in patients with AIDS to rule
out Pneumocystis carinii
• Bronchial brushings

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 Peritoneal, pleural and pericardial
fluids
• To prevent Jelly-like clots, add 300 u
of heparin per 100 mL of aspirate

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 GIT Specimens
• Types of specimen
– Gastric lavage
– Gastric brush
– FNA (for submucosal lesions)

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 Urine
• At least 50 mL is needed
• Second urine is preferred
• Use of preservatives is NOT recommended
• Use for the diagnosis of urothelial
malignancy
• Types of specimens:
– Males: Voided urine
– Females: Catheterized specimen (to prevent
contamination of vulvar cells)
– Washings from bladder or renal pelvis
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GYNECOLOGIC SPECIMEN

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Staining of Smear Preparations

• Papanicolau Method (Paps Smears)

– Staining method of choice for exfoliative
cytology

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 Advantages of Paps Smear
• Transparent blue staining of cytoplasm is obtained
due to the action of high alcoholic content of the
cytoplasmic counterstain, allowing overlapping cells
to be seen.
• Excellent nuclear detail is produced.
• Color range is predictable and of great value in the
identification and classification of cells producing a
good differential coloring of basophilic and
acidophilic cells.
• It is valuable in comparing cellular appearances in
smears with their counterpart in similarly stained
sections.
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 Vaginal Hormonal Cytology
• Relatively inexpensive
• May be performed regularly even in
pregnant women without undue risk

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 Papanicolaou Staining
• Remove PEG fixativeMethod
in 50% alcohol, 2 min
• Hydrate in 95% alcohol, 2 min, and 70% alcohol, 2 min
• Rinse in water, 1 min
• Stain in Haris’s hematoxylin, 5 min
• Rinse in tap water, 2 min
• Differentiate in 0.5% aq. HCl, 10 s
• Rinse in tap water, 2 min
• ‘Blue’ in Scotts tap water substitute, 2 min
• Rinse in water, 2 min
• Dehydrate in 70%, 95%, 95% alcohol for 2 min in each
solution
• Stain in OG 6
• Rinse in two changes of 95% alcohol for 2 min each
• Stain in EA 50, 3 min
• Rinse in 95% alcohol
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 Sites for the Detection of Genital
Cancer
1. Upper lateral third of vaginal wall
- For vaginal hormonal cytology; more
accesible and less likely to be
contaminated by celular debris or
vaginal discharges.
2. Ectocervix
 Transformation Zone
3. Endocervix

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 Cells found in cervico-vaginal
smears
• Mature superficial cells/superficial
cells
– 45-50 um
– With dark pyknotic nuclei
– With true acidophilia (under estrogen
influence)
• Pseudoacidophilia
– Drying of smears especially before fixation
– Prolapsed and drying of vaginal epithelium
– Infection
– chemicals
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• Intermediate cells
– Medium-sized polyhedral or elongated cells with
basophilic cytoplasm showing vacuoles
 Navicular Cells
 Boat-shaped cells with strong tendency to fold or curl
on edges (presence of progesterone)
 Pregnancy cells
 Round, oval, boat-shaped cells with translucent
basophilic cytoplasm (glycogen accumulation)
 Nucleus pushed aside or towards the cell membrane
 With double walled boundary appearance (deeper blue
stain of cytoplasm at the periphery)

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• Parabasal cells
– 15-30 um
– Thick, round to oval
– Smaller than intermediate cells
– Similar to fried fresh eggs with sunny side up
(presence of large nucleus)
– Normally found:
• From 2 weeks of age to puberty
• After childbirth
• Abortions
• After menopause

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 Other cells
o Endometrial cells
o Similar in appearance to parabasal cells
o Slightly cylindrical with less basophilic
cytoplasm
o Occurring in groups of 3 or more
o Found during and 1-10 days after
menstruation

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o Endocervical glandular cells
o Cytoplasm is usually stained pale
blue/gray, finely vacuolated
o Nuclei with finely granular chromatin
o Having a honeycomb appearance when
viewed on end

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o Basal cells
o Small, round to slightly oval cells with
relatively large nuclei
o Strongly basophilic cytoplasm
o Found before puberty and after
menopause

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o Doderlein bacillus
o Presence means healthy vagina
o Most common organism of the normal
vaginal flora
o Found due to:
o Last trimester of pregnancy
o DM
o Infection
o Estrogen deficiency
o Paps : Lavender/blue
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o Candida albicans
o Commonly seen in:
o Patients with DM
o Patients taking oral contraceptives
o Immunocompromised states
o Leukemia and lymphoma

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o Trichomonas vaginalis
o Pear-shaped
o Causes Strawberry cervix
o Paps: Blue green to blue gray

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o Gardnerella vaginalis
o Clue cells – squamous epithelial cells
which contains cocobacilli in the
cytoplasm

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o Koilocytes
o Squamous epithelial cells that show the
cytopathic effects of HPV
o Cells with atypical nucleus surrounded
by perinuclear halo

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 FERNING
• Cervical mucus exhibiting palm leaf
pattern
– Due to formation of salt crystals
• Signifies a high, persistent estrogen
effect
• One of the basis of the diagnosis of
early pregnancy

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 Quantitative Evaluation of Vaginal
Cytology
 Maturation Index (MI)
- Percentage of cells from the main layers
of vaginal epithelium (superficial,
intermediate, deep (parabasal/basal)
cells)
- Pyknosis – used as a criterion for mature
superficial cells

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 Acidophilic Index (AI)
- Percentage of cells staining pink-orange
to red with Paps smear
- Not a reliable index due to possible
pseuodacidophilia

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 Pyknotic Index (PI)
- Percentage of cells with shrunken, dark,
small structureless nuclei.

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Report for Cytologic Smear
• For the diagnosis of cancer
CLASS DESCRIPTION
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
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Other Cytological Staining Methods

• Polychrome staining method – rapid and

to some extent, differential
• Cresyl violet method – simple and rapid
technique with resulting clarity of
morphologic cellular details
• Supravital staining by Wet Film method
• PAS staining
• Giemsa procedures
• Feulgen reaction
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 Additional Microscopic Exam for
Cytologic Smears
• Acridine Orange Fluorescence Techniue
– RNA in cytoplasm and nucleus stains brick
to orange red
– DNA stains green and yellow
• Phase Contrast Microscopy
– Second best choice for routine cytologic
examination
– Used for hormonal evaluation of
gynecologic specimen and for cancer
detection
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• Interference Microscopy
– Determines the dry weight of individual
cells or cellular constituents, cancer
cells, nucleus and cytoplasmic dry wet
content being less than that of normal.
– Very expensive and complex.

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THANK YOU!

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