A branch of General Cytology which deals with the microscopic study of cells that have been desquamated naturally

or by mechanical means from the epithelial surfaces.

EXFOLIATIVE CYTOLOGY (Papanicolaou smears)

Recommended for : 1. Detection of malignant cells 2. Detection of precancerous (precursor) lesions 3. Assessment of female hormonal status 4. Determination of genetic (phenotypic) sex 5. Detection of the presence of infx microorganisms

SPECIMENS FOR EXAMINATION 1.  Peritoneal, pericardial and pleural fluids 2.  CSF 3.  Nipple discharge 4.  Bronchial brushings / washings 5.  Sputum 6.  Gastric washings 7.  Urine sediment 8.  Prostatic secretions / fluid 9.  Cervicovaginal (Paps) smear


Collection Procedure: Use an unlubricated speculum (saline or warm water may be used). After visualization of the cervix is accomplished, insert the sampling device (e.g. cytobrush®) into the endocervical canal and rotate one complete turn. Withdraw the cytobrush and spread the collected material quickly and evenly onto the slide opposite the frosted end.


Cytologic components of a Pap smear
The endocervical mucus will prevent air-drying during collection of the subsequent cervical component. Using the extended-tip spatula ( e.g. Ayerst®), scrape material with the spatula from the whole circumference of the cervix. Withdraw the spatula and spread the collected material quickly and evenly onto the slide adjacent to the frosted end. Fix Immediately (drop slide into fixative or spray with fixative, holding the spray bottle approximately 8 to 12 inches from the slide).

1.#Endocervical#/#Ectocervical#smears# (Transforma6on#zone)# 2.#Vaginal#smears#(for#hormonal#cytology)#




• WHY FIX? Exfoliated cells decompose rapidly which may destroy cellular and nuclear details, in turn will give inadequate results for diagnosis. • COMMON FIXATIVES 1. Equal parts of 95% ethanol and ether 2. 95% ethanol 3. Carnoy’s fluids 4. Equal parts of tertiary butyl alcohol and 1 part 95% ETOH 5. Schaudinn’s Fluid – Sat. Aq. Hg2Cl, Abs. ETOH, HAc 6. Methanol – for dried films

PAPANICOULAOU or Pap’s Smear ADVANTAGE: - Transparent blue staining of cytoplasm is observed - Excellent nuclear staining - Color range is predictable and of great value in identification of cells DISADVANTAGE: - Procedure is lengthy and complicated - Does not give accurate acidophilic index

Cytoplasm – either eosinophilic / basophilic

RESULTS : Epithelial cytoplasm – either pink-red to orange to (eosinophilic) greenish blueto blue- gray (basophilic) 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
vesicular nucleus – blue pyknotic nucleus – dark blue to black

Trichomonas vaginalis – pale greenish blue blob of cytoplasm

Vaginal cytology:
Vaginal epithelium •  Responsive to sex steroids, particularly estrogen and progesterone •  Undergoes predictable changes through the cycle •  Responds to changes in blood concentrations of ovarian hormones. •  Rising levels of estrogen cause the vaginal epithelium to become "cornified" •  The surface cells become large and flattened, with small or absent nuclei.

Vaginal cytology:
Vaginal cytology •  A type of endocrine assay. •  Provides a convenient means of assaying changes in estrogen & progesterone levels •  Changes in the morphology of desquamated vaginal epithelial cells

Vaginal cytology:
Vaginal smears may be taken regularly and often. Hormonal changes are best mirrored in the upper third of the vagina. They can also be taken from the lateral walls because their more accessible and less likely to be contaminated by cellular debris or discharge.





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

SUPERFICIAL CELLS Anucleate cells are abnormal which may be derived from: 1.  Smear contamination by the cells from the vulva 2. Epidermization of the vagina or cervix resulting from prolapse 3. Leukoplakia of the cervix 4. Ruptured membranes in pregnant women 5. Marked hyper-estrinism •  Medium large (20-30u) •  Polyhedral or elongated


•  Cytoplasm: basophilic with vacuoles •  Vesicular nuclei (6-9u)



•  Round to oval boat-shaped cells
•  Round to oval cells •  Smaller than intermediate (15-25 u) •  Thick •  “Sunny-side up” like cells •  Have strong basophilc cytoplasm and vesicular nucei (6-9 u ) •  Found from 2 weeks of age to puberty, after childbirth, abortion or miscarriages and after menopause.

•  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 progesterone 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 • Tall columnar epithelial cells with basal nuclei •  Occurs in groups and strips of three or more cells ( picket fence” configuration) •  Cytoplasm : basophilic with whispy mucin vacuole

•  Found during menstruation period ( in groups) seen up to 10 days from LMP •  Endometrial stromal cells : seen in tight clusters of small, oval dark cells •  Glandular cells: slightly larger •  Nucleus: small and moderately dark •  Cytoplasm: basophilic and maybe vacuolated

Normal Bacterial Flora
Lactobacillus acidophilus

•  Gram (+) slender rod bacteria •  Predominant organism of the vaginal normal flora: establishes the low pH that inhibits the growth of pathogens •  Stains pale blue to lavender •  Energy is obtained by the fermentation of glycogen derived from disintegrating epithelial cells •  Numerous in the luteal phase and during pregnancy

CYTOHORMONAL MATURATION INDEX (C.H.M.I.) – percentage proportion of cells from the three layers of the vaginal epithelium. CHMI = P / I / S ACIDOPHILIC INDEX (A.I.) - percentage of cells that stain pinkorange to red with Pap’s and red in Shorr method

CLASS SYSTEM (up to 1988)
Class I Class II Class III Class IV Class V Negative for Malignant cells Atypical cells present, but Negative for Malignancy Suspicious for Malignant Cells Strongly Suggestive for Malignant Cells Conclusive (Positive) for Malignant Cells

PYKNOTIC INDEX (P.I) – “karyo-pyknotic index”; percentage of cells having shrunken, dark, small (less than 6µ) nuclei.


Satisfactory for evaluation

Specimen Adequacy:
Satisfactory for Evaluation Satisfactory but limited by… Unsatisfactory

General Categorization:
Negative for Intraepithelial lesion or malignant cell Benign Cellular Changes Epithelial cell abnormalites

Descriptive Diagnosis:
Infections / Radiation effects Atypical squamous / glandular cells of unknown significance Low grade squamous intraepithelial lesion High grade squamous intraepithelial lesion Squamous Cell Carcinoma Adenocarcinoma Others

Pap smear specimens are considered satisfactory for interpretation if there are: •  Adequate numbers of well-visualized squamous cells present •  Adequate numbers of well-visualized endocervical cells or squamous metaplastic cells (from the transformation zone). •  Properly labeled specimens


Specimen Unsatisfactory


Specimen Rejected/Not Processed

Specimens to which the following conditions apply will be rejected: 1.  Specimen is submitted without a requisition. 2. Specimen is not labeled with the patient name. 3. The patient name (or other identifying information) on the specimen and requisition do not correspond. 4. The specimen is labeled appropriately but the requisition is not labeled. 5. The specimen slide(s) is (are) irreparably broken. 6. Specimen is submitted from an unauthorized source.

Pap smear specimens are considered unsatisfactory for interpretation if there are: •  Inadequate numbers of well-visualized squamous cells present •  Inadequate numbers of well-visualized endocervical cells or squamous metaplastic cells (from the transformation zone). •  More than 75% of the cells obscured by blood or inflammation •  Improperly labeled specimens •  Usually, these smears are recommended for repeat sampling.

Negative for Intraepithelial lesion or Malignant cell


Atypical squamous cell (ASC-US)

Atypical squamous cell (ASC-US), favor mild dysplasia




Low Grade Squamous intraepithelial lesion encompassing HPV cytopathic effects

  Low grade squamous

intraepithelial lesion (LSIL) (encompassing: HPV / mild dysplasia / CIN 1)

High grade squamous intraepithelial lesion (HSIL) (encompassing: moderate and severe dysplasia, CIS, CIN 2 and CIN 3) - with features suspicious for invasion (if invasion is suspected)

Squamous cell carcinoma (Large cell keratinizing)

Squamous cell carcinoma (Large cell keratinizing)


Squamous cell carcinoma

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