LEC 03: CYTOLOGY

Specimens for Examination FIXATION

1. Peritoneal, pericardial and pleural fluids Why FIX?
2. CSF Exfoliated cells decompose rapidly which may destroy
3. Nipple discharge cellular and nuclear details, in turn will give inadequate
4. Brochial brushings/ washings resilts for diagnosis
5. Sputum
6. Gastric washings Common Fixatives
7. Urine sediment 1. Equal parts of 95% Ethanol & Ether
8. Prostatic secretion/ fluid 2. 95% EtOH
9. Cervicovaginal (paps) smear 3. Carnoy’s Fluid
4. Equal partd of tertiary butyl alcohol & 1 part 95% EtOH
Cytologic components of a paps smear 5. SCHAUDINN’S FLUID – Sat. aq. Hg2Cl, absolute EtoH, HAc
1. Endocervical/ Ectocervical smears (Transformation zone) 6. Methanol – for dried films
Where there is an abrupt transition: Stratified
Squamous (Ectocervix) into Simple Columnar Used for practical purposes: 95% EtOH,
Epithelium (Endocervix) Used in OPD: Hairspray - acceptable but not optimal. It
Crucial sample because it is here where most of the may not have the right alcohol percentage (60 - 70%),
dynamic changes actually originates including not getting the right dehydrating effect.
metaplasia, dysplasia & more importantly the
evolution of cancer lesions. STAINING
2. Vaginal smears (for hormonal cytology) PAPANICOULAOU or Pap’s Smear
To evaluate the hormonal status of the patient
Advantage:
Collection of Specimen Transparent blue staining of cytoplasm is observed
Excellent nuclear staining
Chromatin pattern is very important to distinguish
precursor lesions such as dysplasia from invasive cancer.
Color range is predictable and of great value in
identification of cells

Disadvantage:
Procedure is lengthy & complicated
Does not give accurate acidophilic index

Results:
Epithelial cytoplasm:
o Pink–red to Orange (eosinophilic): Superficial
o Greenish Blue to Blue–Gray (basophilic): Intermediate
Routine Cervical/ Endocervical (Pap) Smear Vesicular nucleus Blue
Pyknotic nucleus: Dark blue to black
Bacteria (L. acidophilus): Dark blue
Fungal mycelia: Violet
Trichomonas vaginalis: Pale greenish blue blob of
cytoplasm

Swab is done in gentle rotation motion in order to

mechanically exfoliate the cell or the epithelial surface then
immediately made into a smear.

Cytohormonal assessment (CHMI)
Ideally: Upper lateral third of the vaginal wall
Where there is less contamination & hormone
influence is bet mirrored
Very Important:
FIX IMMEDIATELY
The moment the cells are removed from the bodily
surface, it immediately undergoes drying & autolytic
changes.
Fixative will suspend the cells from autolytic
changes.
Vaginal Cytology
The vagina epithelium is responsive to sex steroid, particularly
E & P, & undergoes predictable changes through the cycle in
response to changes in blood concentrations of ovarian
hormones. Rising levels of E cause the vaginal epithelium to
become “cornified” – the surface cells become large &
flattened, with small (pyknotic) or absent nuclei.
Effect of E: Promote the maturation of vaginal mucosa
Maturation: Changing the cell towards the superficial
cell type becoming more orange in color in Pap’s smear.
In essence, vaginal cytology is a type of endocrine assay. PYKNOTIC INDEX (P.I) – “karyo-pyknotic index”; percentage of
Tracking changes in the morphology of desquamated vaginal cells having shrunken, dark, small (less than 6 u) nuclei
epithelial cells provides a convenient means of assaying
changes in in E & P levels. CHMI
P I (P) S (E)
Vaginal smear may be taken regularly and often. 0 0 100 Shift to the Right
0 10 90 There is small amount of P that influences
Hormonal changes are best mirrored in the upper third of the intermediate differentiation
vagina. 0 80 20 Shift to the Midzone Effect
Interluteal phase of the menstrual cycle
They can also be taken from the lateral walls because their 0 100 0 Absolute Midzone Effect
more accessible and less likely to be contaminated by cellular P level are exaggerated
Perhaps seen in a pregnant state
debris or discharge.
100 0 0 Shift to the Left
Ovarian function is no longer there
Note: UPPER THIRD LATERAL WALL OF VAGINAL MUCOSA Atrophy in menopause (surgical included)
20 40 40 Not a good smear
Represents contamination, inflammation

Correlate CHMI: Age, Menstrual History, Gynecologic History

Vaginal Cells
Superficial Cells
Large (30 – 60u)
Polyhedral flat cells
Cytoplasm: may be acidophilic or basophilic
Presence of small dark pyknotic nuclei (less than 6 u)

Anuclear cells are abnormal which may be derived from:

1. Smear contamination by the cells form the vulva
Histology of Vaginal Wall Mucosa:
2. Epidermization of the vagina or cervix resulting from
In the stratified nature of the epithelium: cells originate prolapse
from the parabasal layer (progenitor cells) & as they 3. Leukoplakia of the cervix
mature upward towards the layer, it transforms into the 4. Ruptured membranes in pregnany women
intermediate type to superficial type. 5. Marked hyper-estrinism
o P: Intermediate type
o E: Superficial type Intermediate Cells
Medium large (20 – 30u)
Polyhedral or elongated
Cytoplasm: basophilic with vacuoles
Vesicular nuclei (6 – 9u)

Pregnant Cells (Navicular)

Upon exfoliation, these are the cells that you would find:
Metaplastic Cell: Actually endocervical cell undergoing
metaplasia
Together with polar affinity aids us in ID the different cell
lines
Round to oval “boat shaped” cells
Has a translucent basophilic cytoplasm (due to glycogen
accumulation)
Cytoplasm stains deep blue or blue gray + cell membrane
Boat – shaped intermediate cells with a strong tendency
to fold and curl their edges
Expression of excess P effect
Found in the latter half of menstrual cycle, during
pregnancy, menopause
May also be found as a result of abnormal androgen
stimulation either endogenous or exogenous
Older Term: Pregnancy Cell (but it does not mean it is
indicative of pregnancy, it simply means that the P level
is quite high)

Parabasal Cells
Quantitation in Vaginal Cytology
CYTOHORMONAL MATURATION INDEX (C.H.M.I) – percentage
proportion of cells from the 3 layers of the vaginal epithelium
CHMI = Parabasal / Intermediate / Superficial
ACIDOPHILIC INDEX (A.I) – percentage of cells that stain pink –
orange to red with Pap’s & red in Shorr method
Round to oval cells
Smaller than intermediate (15 – 25u)
Thick cytoplasmic edge
“Sunny side up” like cells
Have strong basophilic cytoplasm & vesicular nuclei (6 - 9
µ)
Found from 2 weeks of age to puberty, after childbirth,
abortion, or miscarriages and after menopause
Tall columnar epithelial ells with basal nuclei
Occurs in groups and strips of 3 or more cells (“picket
fence configuration)
Cytoplasm: Basophilic with whispy mucin vacuole
Endocervical Cells Image: Adequacy
Tall columnar epithelial Cells with basal nuclei Satisfactory for evaluation
Occurs in groups and strips of 3 or more cells (“picket
fence configuration) Pap smear specimens are considered satisfactory for
Cytoplasm: Basophilic with whispy mucin vacuole interpretation if there are:
Important: since it tells you that you have sampled the Adequate numbers of well – visualized squamous cells
Transformation Zone. present
Adequate numbers of well – visualized endocervical cells
Endometrial cells or squamous metaplastic cells (from the transformation
Found during menstruation period (in groups) seen up to zone)
10 days from LMP Properly labeled specimens
Endometrial stromal cells: seen in tight clusters of small,
oval dark cells; Glandular cells: Slightly larger Image: Negative for Intraepithelial lesion or Malignant cell
Nucleus: small & moderately dark Formerly Class I
Cytoplasm: Basophilic & maybe vacuolated Intraepithelial Lesion: Dysplasia, HPV
Significant: Premenopausal or menopausal period
Image: Adequacy
Specimen Unsatisfactory
Normal Bacterial Flora
Pap smear specimens are considered unsatisfactory for
“Lactobacillus acidophilus” (Doderlein Bacilli)
interpretation if there are:
Gram + slender rod bacteria
Inadequate numbers of well- visualized squamous cells
Predominant organism of the vaginal normal flora:
present
establishes the low pH that inhibits the growth of
Inadequate numbers of well-visualized endocervical cells
pathogens
or squamous metaplastic cells (from the transformation
Stains pale blue to lavender
zone).
Energy is obtained by the fermentation of glycogen
> 75% of the cells obscured by blood or inflammation
derived from disintegrating epithelial cells
Improperly labeled specimens
Numerous in the luteal phase and during pregnancy
Change in the flora is indicative of change in the pH,
Usually, these smears are recommended for repeat sampling.
which makes the milieu vulnerable for infection. Smears
with infection such as Candidiasis or Trichomonas have
Specimens to which the following conditions apply will be
no lactobacillus in the background.
rejected:
1. Specimen is submitted without a requisition
Reporting
2. Specimen is not labeled with the patient name.
CLASS SYSTEM (up to 1988)
3. The patient name (or other identifying information) on
Class I Negative for Malignant Cells
the specimen and requisition do no correspond
Class II Atypical cells present, but Negative for Malignancy
4. The specimen is labeled appropriately but the requisition
Class III Suspicious for Malignant Cells
is not labeled.
Class IV Strongly Suggestive for Malignant Cells
5. The specimen slide(s) is (are) irreparably broken.
Class V Conclusive (Positive) for Malignant Cells
6. Specimen is submitted from an unauthorized source.
Confusing: Class I, III & IV were gray areas which where
EPITHELIAL CELL ABNORMALITIES
subjected to various interpretation depending on how
Squamous
the institution looks at the diagnosis.
Atypical squamous cell of Undetermined Significance
(ASC – US) favor mild dysplasia
The BETHESDA SYSTEM
Low Grade Squamous intraepithelial lesion
Specimen Adequacy:
encompassing HPV cytopathic effects
Satisfactory for Evaluation
Low Grade Squamous Intraepithelial lesion (LSIL)
Satisfactory but limited by ….
(encompassing: HPV/ mild dysplasia/ CIN 1)
Unsatisfactory
General Categorization High Grade Squamous Intraepithelial Lesion (HSIL)
Negative for Intraepithelial lesions or malignant cell (encompassing: moderate & severe dysplasia. CIS, CIN 2
Benign Cellular Changes & CIN 3)
Epithelial cell abnormalities o With features suspicious for invasion (if
Descriptive Diagnosis invasion is suspected)
Infections/ Radiation Effects o Biopsy is warranted
Atypical squamous/ glandular cells of unknown
Significance Image: Squamous Cell CA (Large Cell Keratinizing)
Low grade squamous intraepithelial lesion 95% of malignancy of uterine cervix as most common
High grade squamous intraepithelial lesion Note the nuclear & cytoplasmic changes of the cells
Squamous Cell Carcinoma
Adenocarcinoma
Others