Exfoliative and Gynecologic Cytology

Exfoliative cytology

The scientific analysis of cells that are shed, scraped, or extracted from the epithelial and mucosal linings of various body organs

AKA Papanicolaou’s Method (Pap Smear)

Primary Branches: Cytopathology & Cytotechnique

Cytopathology

Diagnostic study of cellular abnormalities, such as identifying cancerous cells

Cytotechnique

Procedural study of processing and preparing cellular samples for microscopic evaluation

Pleural, pericardial, and peritoneal fluids

Specimen for serous effusions

Sputum and bronchial brushings/washings

Specimen for respiratory tract

Cervicovaginal (Pap) smears, breast secretion/nipple discharge

Specimen for female genital tract & breast

Prostatic secretions / fluid and urine sediments

Specimen for male genitourinary tract

Gastric washings

Specimen for GI tract

Cerebrospinal fluid

Specimen for CNS

Urinary sediment

Bronchial lavage specimen

Specimen that utilizes proteolytic enzymes during processing

Specimens requiring adhesive

Pleomorphism

Marked variation in cellular size and shape

Hyperchromatism

Intensely dark-staining nuclei due to increased DNA content

Loss of polarity

Disorganized and chaotic cellular arrangement

Atypical mitosis

Abnormal, irregular, or asymmetric cell division figures

Vesicular cytoplasm

Distinctive, bubbly staining characteristics within the cell

Membrane alterations

Cytoplasmic changes

Nuclear degeneration

Non-malignant cellular abnormalities

Scrapings

Specimen are commonly collected from the epithelial lining and mucosal secretions of the female reproductive tract (vagina, cervix, endometrium)

Rubber bulb & glass pipette

Used to aspirate vaginal pool fluids, primarily for cancer screening

Ayre spatula

A contoured wooden or plastic tool used to scrape mucoid specimens for hormonal studies and cervical cytology

Curette (curved / blunt)

A surgical spoon used for mucosal biopsies to extract tissue pieces from the cervix or endometrial lining

3-Sample Collection Protocol

Collection technique for prostatic secretions

Digital rectal prostate massage using strict aseptic technique

Voided urine collected prior to massage

Sample 1 content of 3-Sample Collection Protocol

Expressed prostatic secretions (EPS) collected during massage

Sample 2 content of 3-Sample Collection Protocol

Voided urine collected after massage

Sample 3 content of 3-Sample Collection Protocol

Captures residual secretions in the urethra

“Deep Cough” Sputum Specimen

Primary Collection of bronchial secretions

The best sample for routine cytological study

Bronchoalveolar Lavage (BAL)

Procedure involving a flexible bronchoscope to clinically extract and obtain bronchial secretions

Saccomanno fluid (50% ethanol + 2% carbowax)

Required fixative for sputum cytology

Properly fixes and preserves cell prior to processing

Gastric contents

Typically a watery, acidic fluid that contains significant amount of food debris

Duodenal fluid

A mucoid, alkaline fluid, characteristically greenish-yellow in color (due to bile)

Lumbar puncture / spinal tap

Needle is carefully inserted into the intervertebral spaces, between the 2nd and 3rd, or 3rd and 4th lumbar vertebrae

Collection method for CSF

Thoracentesis

Collection method for pleural fluid

Collected at the left lateral thoracic wall

Pericardiocentesis

Collection method for pericardial fluid

Collected at the left sternal border

Paracentesis abdominis

Collection method for peritoneal fluid

Collected at the midline of the abdomen, below the navel (at the linea alba)

Red bone marrow (myeloid tissue)

Specimen for cellular analysis of bone marrow substance

Collection site: iliac crest or sternum

Extraction tool: Bone marrow borer (or core biopsy needle)

Slide preparation for bone marrow specimen

Touch preparation: Dabbing tissue onto slide

Squash preparation: Pressing the tissue between two slides

Smear preparation: Spreading the aspirate evenly across the glass

Voided urine

Standard, non-invasive collection method

“Clean catch technique”

Catheterized urine

Obtaining a sample by inserting a sterile tube (catheter) through the urethra directly into the bladder

Suprapubic aspiration

An invasive procedure where a needle is inserted through the abdominal wall directly into the bladder to extract a highly sterile sample

Nipple discharge

Specimen for diagnostic evaluation of abnormal, spontaneous, mammary secretions

Assesses pathological discharge (bloody, purulent) to rule out carcinoma or papilloma

The Pap Smear

Primary GOLD STANDARD screening method worldwide for the early detection of cervical malignancy and its precursor lesions

Lateral vaginal wall

Primarily sampled for hormonal evaluation (cytohormonal index) rather than cancer screening

Ectocervix

The outer portion of the cervix

Covered by a protective layer of squamous epithelium

Endocervix

The inner cervical canal

Lined by mucus-secreting glandular (columnar) epithelium

Transformation Zone / Ectocervix-Endocervix junction

The specific area where there is an abrupt transition from ectocervical (squamous) to endocervical (columnar) epithelium

Most commonly sampled site for cervical cancer screening

Adapts to chronic inflammation and infection

High rate of cellular turnover → prone to metaplasia, dysplasia, carcinoma-in-situ

Cervical brushes (Cytobrush)

Excellent for gathering deep endocervical cells

May induce slight bleeding in the sample

Cervical broom (Cervex-brush, Papette)

Designed with varied bristle lengths to collect cells from both the ectocervix and endocervix simultaneously

Cotton swabs

Not ideal for cervical sample collection

The fibers trap cells and often cause severe drying artifacts

Rotary motion

Smearing technique for vaginal smear

Pull-apart method is not advised as it can damage cells

Soap solution for 20 mins → KOH → dry completely

Post-procedure decontamination for instruments used in vaginal smear preparation

2 weeks after 1st day of LMP

Avoid intercourse for 2 days before testing

Refrain from douching for 1 day

Cease using vaginal medication for 48 hours

Time of collection for Pap smears

Initial screening (Pap smear)

No later than 21 years of age or exactly 3 years after the onset of sexual intercourse

Routine screening (Pap smear)

Conducted annually or biannually following the initial baseline test

Can generally be stopped after age 65 (if previous results were consistently normal)

Ages 30 to 65

Recommended to transition to co-testing (Pap smear + HPV testing) every 5 years

Conventional Smear

The collected sample is smeared directly onto a glass slide in the examination room and fixed immediately

Advantage: Traditional, cost-effective method

Liquid-Based Preparation (LBP)

The collection device (e.g., brush head) is detached and deposited directly into a vial of preservative fluid (like BD SurePath)

Advantage: Reduces obscuring background material (blood/mucus) and prevents drying artifacts.

Wet fixation

Immediate submersion of freshly prepared smears into a liquid fixative to preserve cellular state

Gold standard method for fixing ALL gynecological and non-gynecological smears

95% Ethyl Alcohol (Ethanol)

Ether-Alcohol Mixture

100% Methanol

80% Propanol and Isopropanol

Denatured Alcohol

Common fixing agents (Wet Fixation)

95% Ethyl Alcohol

Ideal fixative; recommended in most laboratories for cytological specimen

Causes enough cell shrinkage to yield optimal chromatin detail characteristics of cytological preparations

100% Methanol

Acceptable substitute for 95% ethanol

Causes less shrinkage

More expensive

Ether Alcohol Mixture

Excellent fixative, but not used in most laboratories

Consists of equal parts of: Ether and Ethyl Alcohol

Carbowax (Polyethylene Glycol)

Substitute when immediate liquid submersion isn’t feasible

Application Methods:

Aerosols: Applied by spraying a fine mist directly over the cellular sample.

Liquid Base: Dropped gently onto the slide.

Soaked overnight in 95% alcohol

Crucial processing step prior to staining Carbowax-coated slides

Dissolves the wax and allows the stains to penetrate the cells

Carnoy’s Fixatice

Specifically utilized for hemorrhagic (bloody) samples

Acts as an excellent nuclear fixative, hemolyzes obscuring RBCs to reveal epithelial cells, and preserves cellular glycogen.

Causes significant cellular shrinkage, and makes the sample prone to over-staining with Hematoxylin

It must be prepared fresh immediately before use and discarded right after.

12 inches away from the slide

Proper spray distance when using an aerosol fixative spray

Prevents physical disruption of the smeared cells

OG-6

Monochrome cytoplasmic stain

Selectively targets specific cellular proteins (e.g., keratin)

Number indicates the concentration of phosphotungstic acid used

EA-36

Polychrome cytoplasmic stain

Highlights diverse cellular conditions and metabolic states

Pap stain

Used to differentiate cells in smear preparations of various bodily secretions

5 dyes in three solutions: Hematoxylin, Orange G-6, Eosin Azure, Light Green SF, Bismarck brown Y

Hematoxylin

First stain

Nuclear stain

Orange G

First OG-6 counterstain

Stains keratin

Used to stain small cells of keratinizing squamous cell carcinoma in sputum

EA-36, EA-50, EA-65

Second EA counterstain → Eosin Y, Light Green SF, Bismarck Brown Y

Number denotes proportion of dyes

Eosin Y

Stains superficial epithelial squamous cells, nucleoli, cilia, and RBCs

Light Green SF

Stains the cytoplasm of other cells, including non-keratinized squamous cells

Fast green FCF → can be used as an alternative stain

Bismarck Brown Y

Stains nothing

Often excluded in contemporary formulations

Nuclear basophilia

A hallmark of optimal fixation and Hematoxylin affinity

Sharp, structurally defined nuclei exhibiting deep blue to black

Yellow / Orange

Keratinized & Glycogen-Rich

Intense affinity for Orange G due to high structural protein and carbohydrate content

Pink / Eosinophilic

Mature Superficial Cells

Eosin Y dominance indicating advanced cellular maturation

Blue / Cyanophilic

Immature Epithelium

Intermediate & Parabasal cells exhibiting Light Green SF affinity

Amphophilic (Green & Pink)

Metaplastic Cells

Simultaneous dual-dye affinity reflecting transitional cytoplasmic chemistry

Mature Superficial Cells

Structure: They present as flat, polygonal squamous cells

Cytoplasm: They exhibit a pale, pink-staining appearance, indicating advanced cellular differentiation

Nucleus: They feature small, dark, pyknotic nuclei—a hallmark of end-stage cellular inactivity.

The presence of these fully mature cells strongly indicates peak estrogenic stimulation. Consequently, they are characteristically seen during the late proliferative and ovulatory phases of the menstrual cycle.

Intermediate Cells

Structural Similarity: They share the same flat, polygonal squamous architecture as mature cells

Nuclear Distinction: The primary diagnostic difference is their nuclei, which are significantly less pyknotic (larger and less dense) compared to superficial cells.

The "open," less condensed nucleus demonstrates that the cell is in a transitional, intermediate state

It has progressed past the basal layers but has not yet reached the end-stage inactivity of a terminal superficial cell

Navicular cells

These specialized intermediate cells exhibit a strong tendency to fold or curl at their edges, creating a characteristic "boat" shape

This unique morphological shift is a direct biomarker suggesting a combined estrogen-progesterone effect

Typical physiologic finding during specific states, specifically the normal menstrual cycle, pregnancy, and menopause

Parabasal Cells

Architecture: They are small, compact, round-to-oval cells that have not yet flattened out.

Cytoplasm: They possess a dense, highly basophilic cytoplasm, lacking the expansion and thinning seen in mature estrogen-stimulated cells.

Their shedding indicates a thin, inactive epithelium primarily driven by low estrogen levels or abrupt hormonal withdrawal.

Pre-puberty: (2 weeks of age to puberty)

Hormonal Shifts/Withdrawal: Postpartum (after childbirth) & post-abortion.

Senescence: Post-menopause

Expected Physiologic States for Parabasal Cells

Basal Cells

Anatomical Context: As the deepest foundational layer of the epithelium, they are heavily shielded by the maturing cells above them

PATHOLOGIC → their presence is a strict diagnostic indicator of severe atrophy

This signifies that the epithelium has thinned completely, typically due to a profound absence of estrogenic stimulation

Endocervical Cells

• Top View (En Face):

  • "Honeycomb" Pattern — Cohesive, flat sheets of uniform, tightly packed polygonal cells with central nuclei.

• Side View (Profile):

  • "Piano Key" / "Picket Fence" Pattern — Neat rows of tall columnar cells with basally located nuclei and apical mucin.

• Clinical Significance:

  • Establishes normal glandular health baseline.

  • Loss of polarity (breakdown of honeycomb/piano key alignment) indicates potential glandular dysplasia.

Endometrial Cells

• Structure & Origin: Small, slightly cylindrical cells originating from the glandular upper uterine lining.

• Cytoplasm: Notably less basophilic (lighter-staining).

• Clinical Timing:

  • Tied to active shedding of the uterine lining.

  • Normal finding ONLY during: Active menstruation and up to 1–10 days post-menses.

Hormonal Cytology

Vaginal epithelium responds to stimulation by steroid hormones, mainly estrogens and progesterone and to a lesser degree, to androgens and adrenal steroids.

Maturation index

The percentage study of the parabasal, intermediate, and superficial squamous cells /100 cells counted from exfoliated epithelial cells of a healthy vaginal smear

PROCEDURE

1. Count 100 cells

2. Classify types of squamous cells seen

3. Reporting: P/I/S

Bethesda System

System used for reporting cervical cytology

Pseudostratification & “Rosettes” feathering cytoplasm

Hallmark cellular characteristics for adenocarcinoma