Cellular Pathology Cytology

2hr exam

Cellular Pathology Fields (2)

Histology / Histopathology

• Microscopic study of diseased tissues

• Tissue obtained during:

  • Minor surgical procedures

  • Major surgical procedure

• Diagnosis Based on:

  • Tissue architecture

  • Cell behaviour

  • Cell morphology within tissue environment

  • is the gold standard

    • Provides more information than cytology

Cytology / Cytopathology

• Microscopic study of individual cells

• Cells are:

  • Shed (exfoliated) from epithelial surfaces

  • Removed by non-invasive methods

• Collection methods include:

  • Scraping

  • Brushing

  • Aspiration

  • Minor surgical procedures

• Diagnosis Based on:

  • Cellular morphological changes due to disease

Both fields:

• These fields complement each other to overcome individual limitations

• Both fields are primarily diagnostic

Diagnostic Approach

• Start with cytology

• Follow with histology if diagnosis is unclear

• Improve cyto-histo correlation for better accuracy

Factors Affecting Diagnostic Accuracy

• Good cellularity / Optimum cellular concentration

• Representative specimens / Representative samples

• Correct sampling / Good collection techniques

• Proper preservation and fixation / Cell preservation/fixation methods

• Correct laboratory preparation / Smear slide preparations

• Precise / Clear staining procedures

• Accurate microscopic interpretation

Why Cytology is Needed (advantages and limitations)

Histology disadvantages:

• More complex, expensive, invasive procedures

• Requires hospitalisation, anaesthesia, surgery

• Causes discomfort, pain, anxiety

• Not suitable for monitoring/follow-up

Advantages of Cytology

• Simple, fast, cost-effective

• Minimally invasive

• Easy to perform

• More acceptable to patients

• Little discomfort, repeatable

• Outpatient procedure

• Helps select patients for surgery

• Avoids unnecessary procedures

• Ideal for monitoring after treatment

Limitations of Cytology

• Only cellular sample

• May lack cellularity

• May not be representative

• Cells may not exfoliate

• Contamination (blood/inflammatory cells)

• Fibrosis or necrosis in lesions

• Laboratory errors:

  • Poor techniques

  • Poor interpretation

Specimen Ethics

• Specimen is often the only patient contact

• Consider:

  • Patient’s physical and physiological needs

  • Patient anxiety

  • Impact on treatment decisions

• Treat all specimens with respect

• Cell pathology specimens may be irreplaceable

• Confidentiality

  • Do not disclose reports to patients or relatives

  • Share reports only with medical professionals

Specimen Identification

• Match request form with specimen container

Required Details:

• Name, surname, date of birth

• ID or hospital number

• Date of specimen

• Type and origin of specimen

• Clinical history/details

Fixation

Purpose

• Preserve cells/tissues in a life-like state

• Prepare specimens for further processing

Importance

• Preserves morphological detail

• Maintains cell structure

• Prevents cell lysis

• Prevents bacterial degradation

Mechanism

• Protein coagulation

• Cross-linking of amino acids

Preserves:

• Cell/tissue shape

• Structural relationships

• Chemical components

Prevents:

• Autolysis

• Bacterial degradation

• Loss of substances

• Shape and volume changes

Limitations

• No fixative is perfect

• All fixatives may introduce artefacts

Common Fixatives

• Alcoholic Fixatives:

  • 95% Ethyl Alcohol – fast-acting, excellent penetration, enhances cell transparency

  • Others: Iso-propanol, Methanol, Methylated spirit

  • Alcoholic spray coatings (e.g., polyethylene glycol, PEG) – cells/tissues degrade after removal

Other Fixative:

• Formaldehyde

Specimen Preparation (2)

Histological Preparation

• Tissue processing steps:

  • Dehydration (alcohol)

  • Clearing (de-alcoholisation)

  • Impregnation and embedding

  • Sectioning

  • Mounting on slides

Cytological Preparation

• Cells smeared or deposited on slides

• Can be done before or after fixation

• Depends on specimen type and texture

Specimen Processing: Planning in Advance

• Specimen identification and nature

• Clinical details

• Investigations required:

  • Direct smears

  • Wet preparations

  • Air-dried or wet-fixed smear preparations

  • Protein estimation

  • Special stains

  • Immunocytochemistry

  • Romanovsky stains

• Type of fixative

• Number of slides

• Cell block method

• Concentration gradient technique

Categories of Cytological Specimens

Category 1:

• Smears prepared and alcohol/spray fixed by hospital staff

• Examples:

  • Cervical smears

  • Skin scrapings

  • Endoscopic brushings

  • Nipple discharge direct smears

  • Fine needle aspirations

Category 2:

• Smears prepared and air-dried by hospital staff

• Examples:

  • Tumor imprints (lymph node)

  • Fine needle aspiration direct slides

• Notes:

  • Suitable for Romanovsky staining only

  • Excellent for demonstrating blood cells and non-epithelial elements

Category 3:

• All unfixed specimens

• Fluids: Serous effusions, CSF, urines, cystic fluids

• Semi-solid: Sputa, washings, lavages, pus, abscess, FNA suspensions

Methods of Preparation of Cytological Specimens

Category 1: Spray Fixed Smears

• 5 mins in 50% alcohol to remove waxy PEG layer

• Down to water

• Papanicolaou staining

Category 2: Air-Dried Smears

• Romanovsky staining

• Rehydration technique → Pap stain

Category 3: Unfixed Specimens

• Treat as biohazards

• Handle in safety cabinet

• Take all necessary safety precautions

Staining Techniques

• Cells/tissues are naturally transparent

Purpose:

• Create contrast between components

Importance:

• Essential for diagnosis

• Requires:

  • Good nuclear staining

  • Good cytoplasmic staining

Examples of Specimen Types and Collection Methods

• Exfoliative: Sputum

• Voided: Urine

• Expectorants:

  • Sputum (non-assisted)

  • Bronchial and broncho-alveolar lavages (assisted)

• Washings: Bronchial and gastric

• Scraping: Skin ulcers and cervical smears

• Direct Smears: Nipple discharge

• Brushing: Bronchial and oesophageal/gastric

• Aspiration: Serous effusions

• Imprints: Tumor touch preparations

• Fine Needle Aspirations

Fluids in Cytology for Macro Examination

Scanty/Clear Fluids

• Centrifugation: 2500 rpm for 10 mins

• Cytocentrifugation: 850 rpm for 5 mins

• Cell button directly on slide

• Spray fixation

Turbid Fluids

• Centrifugation → Remove supernatant

• Cell deposit on slide

• Spray/alcohol fixation

Heavily Blood-Stained Fluids

• Buffy layer technique

• Enzymolysis (Streptolysin)

• Haemolysis (Acetic acid)

• Note: Last two methods may affect cell morphology

Concentration Gradient Floatation Technique

• Centrifuge specimen with polysucrose concentration gradient medium

• Excess erythrocytes and granulocytes aggregate and sediment to the bottom

• Lymphoid cells, epithelial cells, and other large cells remain floating in supernatant-gradient medium

Semi-Solid Fluids: Macro Examination

Thick/Pus or Mucoid

• Pour in Petri dish

• Select blood flecks and white streaks

• Sample with loop or pipette

• Smear & squash method

• Fixation in alcohol

Thick Suspensions

• Pour in centrifuge tubes

• Centrifuge @ 2500 rpm for 10 mins → Remove watery supernatant

• Aspirate thick deposit by loop or wide pipette

• Smear & squash method

• Fixation by spray alcohol

Examples:

• Sputum, pus → Petri dish method

• Washings, lavages, BALs, joint fluids, thick effusions, FNA suspensions → Centrifuge method

Fibrinous Clots and Proteinaceous Coagula

• Gently squeeze coagulum/clot

• Cells released in fluid

• Remaining coagulum/clot → placed in 10% formaldehyde for 24 hrs

• Later processed as histology specimen

Cell Block Technique

• Remove supernatant

• Add a few drops of plasma to cell deposit → mix with vortex

• Add equal amount of thrombin reagent → mix

• Leave mixture on hot plate → coagulum forms in 10–20 secs

• Detach coagulum → place in 10% formaldehyde → fix for 24 hrs

• Processed as tissue section

• Additional histochemical and immunocytochemical stains can be performed on multiple sections

Inflammation

• One of the most common forms of benign processes in tissues

• A form of tissue reaction to injury

Role of Cells in Inflammation

• In all types of inflammation, the injured tissue is assisted in its defences by:

  • Leucocytes

  • Cells of the immune system

Direct Action

• Active participation of macrophages in the removal of the invading agent

Indirect Action

• Production of specific antibodies against the attacking agent

4 MAIN TYPES OF INFLAMMATORY REACTION

Acute

• Necrosis and breakdown of tissues

• Polymorphonuclear neutrophils predominate

• Pus formation

  • Pus = liquefaction of necrotic tissue and dead leucocytes

Subacute

• Duration of inflammation is longer

• Less tissue breakdown

• Less reaction to injury

• Presence of:

  • Polymorphonuclear neutrophils

  • Eosinophils

  • Lymphocytes

Chronic

• Slight and slow tissue breakdown

• Concurrent signs of tissue repair

• Lymphocytes predominate

• Presence of:

  • Macrophages

  • Occasional plasma cells

Granulomatous

• A form of chronic inflammation

• Lymphocytes predominate

• Clusters of:

  • Macrophages

  • Epithelioid cells

• Granuloma formation

• Causes Specific agents such as:

  • Mycobacterium tuberculosis

  • Pathogenic fungi

• Runs a protracted course

The first 3 forms of inflammation are based on:

• Duration of the disease

• Patterns of tissue reaction

TISSUE REPAIR / REGENERATION

• Associated with inflammatory processes

• An attempt of the tissue to:

  • Re-establish its original structure

  • Restore its original function prior to injury

1st Step

• Production of a layer of epithelial cells bridging the defect

2nd Step

• Multiplication of this layer restores the entire thickness of the epithelium

Additional Features

• Accomplished with the help of:

  • Connective tissue

  • Blood vessels

• Accompanied by varying degrees of inflammation

TISSUE MORPHOLOGY

• Marked nuclear activity

• High protein synthesis

• Increased chromatin particles

• Large/multiple nucleoli

NORMAL TISSUE TURNOVER AND CONTROL

how tissues regulate cell loss and replacement under normal conditions

• All tissue types suffer permanent wear and tear

• Especially observed in epithelial tissue where continuous loss of cells occurs

• Under normal conditions:

  • Cell loss is matched by formation of new cells

• Requires stringent control of:

  • Cell proliferation

  • Cell maturation

• Allows perfect feedback between:

  • Cell loss

  • Cell gain

Control of Epidermal Cell Proliferation and Differentiation

• Controlled by a complex pattern of interacting mechanisms

• Involves:

  • Hormones

    • Estrogens

    • Androgens

  • Growth factors

Tissue Self-Regulation

• Tissue acts as a self-regulatory system

• Controls steady state between:

  • Cell loss

  • Cell gain

Mechanism

• Through interplay of:

  • Stimulatory factors

  • Inhibitory factors

• Thought to be produced by the tissue itself

Hormone-like Factors

• Intercellular carriers of information within a feedback circuit

• Act between:

  • Maturing tissue cells

  • Proliferative cell population

They all describe the mechanisms maintaining the balance between:

• Cell loss

• Cell gain

CHALONES

• Tissue-specific substances

• Glycoproteins (oligopeptides)

• Have growth-regulating effects

• Operate through a cell membrane receptor mechanism

Functions

• Inhibit proliferation of immature cells

• Retard cells entering mitosis

Mechanism of Mitotic Inhibition

• Delay in the G1-S phase transition

• Therefore:

  • Does not affect DNA synthesis

Additional Effects

• May retard the G2-M cell transition

During Injury

• Produced by injured tissue itself

• Stimulate:

  • Cellular regeneration

  • Tissue repair

• Via an intercellular information mechanism

SCAR TISSUE

• Repaired tissue may retain morphologic evidence of prior injury

• Example:

  • Scar tissue

• Presence of excessive and collagenized connective tissue

HYPERPLASIA

• Abnormal increase in the number of layers of basal epithelial cells

• Increased maturation rate

• Immature but benign epithelial cells

• Usually precedes tissue repair/regeneration

• Hyperplasia is the proliferation of benign cells that still obey the general laws of cell growth and destruction

• Sometimes equilibrium of cell destruction and regeneration is disrupted

• Some forms of hyperplasia may lead to:

  • Origin of neoplastic lesions

METAPLASIA

• Replacement of one epithelium by another not normally present in a given location

• Genetic rearrangement of basal epithelial cells

• Formation of a more resistant epithelium

Significance

• A protective mechanism

Examples

• Columnar epithelium to squamous metaplastic epithelium in:

  • Bronchial lining

  • Cervix

NEOPLASMS

• Meaning = “New growth”

Definition

• Growth of tissues that have escaped controls governing normal proliferation and regeneration of cells

Tumour Formation

• Non-reversible abnormal proliferation of cells leads to formation of a tumour

Tumours Can Be

• Benign

• Malignant

BENIGN TUMORS

General Characteristics

• Neoplastic cells may show morphological resemblance to normal counterparts regarding tissue of origin

• Amount and arrangement usually abnormal

• Slow growth

• Localized growths of cells

• Good encapsulation

• Lack ability to:

  • Metastasize

  • Invade other tissues

Death is Rare Unless

• Tumor puts pressure on a vital organ in a strategic location

• Massive necrosis followed by:

  • Infection

  • Gross haemorrhage

Types

• Epithelial

• Non-epithelial

Common Forms

• Papillomas

• Polyps

Causes

• Can arise due to infectious agents

• Especially viral infections

• If left untreated, some benign tumors may develop into cancer

MALIGNANT TUMORS (CANCER)

• Progressive autonomous proliferation of tissue

• Not subject to laws governing normal orderly growth

• Results in:

  • Loss of law and order

  • Loss of original tissue function

General Characteristics

• Abnormal proliferation of tissue

• Tumor composed of:

  • Abnormal cells

  • Immortal immature cells

• Loss of original tissue function

• Loss of tissue order and polarity

• Non-capsulated

• Invasive growth:

  • Beyond boundaries of original tissue

• Spread and metastasize through:

  • Lymphatic system

  • Vascular system

• Invariably fatal if left untreated

Classification - According to Organ and Tissue of Origin

• Epithelial → Carcinoma

• Epithelial (glandular) → Adenocarcinoma

• Non-epithelial → Sarcoma

• Specific well-defined forms → Leukemia, Mesothelioma

THEORY OF “FITTER CELLS”

• Around 10¹⁶ cell divisions take place in the body

Genetic Errors

• By chance, some cell divisions may develop genetic errors

• Usually:

  • No consequences

  • Lead to non-viable cells

  • Recognized by body defences

• However Occasional genetic errors or mutations may:

  • Form variant cells

  • Produce unstable genetic material

  • Acquire mitotic capability

• Results in the formation of new mutant cells

• Mutant cells are considered “fitter” cells

• Can form:

  • Colonies

  • Clones

• Leading to malignant tumour growth

CARCINOGENIC AGENTS

May induce initiation or enhance mutagenic events in the cell genome

Examples

• Viruses

• Radiation

• Chemicals

ESTABLISHMENT OF CANCER CELL CLONES

• Time required is highly variable

Depends Mainly On

• Status of body defences

• Extent of mutagenic events occurring in the genome of the “fitter” cells

CELLULAR MORPHOLOGICAL CHARACTERISTICS OF MALIGNANCY

GENERAL PRINCIPLES

• The microscopic diagnosis of malignancy depends on:

  • Individual cellular morphology

  • Overall appearance of smears

• There is NO SINGLE CRITERION OR FEATURE

  • Of a cell which can be attributed to a malignant change

CHARACTERISTICS OF MALIGNANT CELLS

• Malignant cells show characteristics that distinguish them from normal cells

• These characteristics are:

  • Mainly nuclear

  • Also cytoplasmic

Recognition Requires Knowledge Of

• General pathology

• Normal tissue histology

• Morphological characteristics of benign cells:

  • Under normal conditions

  • Under abnormal conditions

GENERAL FEATURES OF MALIGNANT CELLS

• Malignant cells tend to:

  • Proliferate uncontrollably

  • Spread to other parts of the body

Hyperplastic Features

• Loss of uniformity

• Loss of cell-to-cell adherence

• High rate of exfoliation

• Nuclear irregularity

• Nuclear variation in:

  • Size

  • Shape

  • Staining reaction

• Irregular chromatin pattern distribution

NOTE:

• Single features are NOT diagnostic of malignancy

• However, they may:

  • Arouse suspicion

  • Be important in detecting early pre-malignant cellular changes

NUCLEAR CHARACTERISTICS of malignant cells

Enlargement of the Nucleus Features

• Nuclear hypertrophy

• Increased DNA and nucleoprotein content

• Increased nuclear volume

• Some malignant cells may contain small malignant nuclei

Therefore Relationship between nuclear volume and cytoplasm must be investigated

Nuclear to Cytoplasmic Ratio (N/C Ratio) Features

• Distinctive feature of malignancy

• High N/C ratio in malignant cells

• Actual cell size is irrelevant

• Large nuclei of benign cells retain a normal N/C ratio

Hyperchromasia Features

• Increased DNA and nucleoprotein content

• Increased polyploidy and chromosomal size

• Increased nuclear stain intensity

  • Dark staining nuclei

Irregular Chromatin Pattern Features

• Abnormal distribution of chromatin

• Coarse chromatin granulation

• Dense chromatin clumping

• Uneven chromatin condensation at nuclear margin

• Thick, irregularly sharp nuclear borders

Anisonucleosis and Pleomorphism

• Variety of shapes and sizes of nuclei

Increase in Size and Number of Nucleoli Features

• Multiple or prominent nucleoli

• Unusually large nucleoli

• Variation in:

  • Number

  • Size

• Irregularly outlined nucleoli

Multinucleation

• May be present in both:

  • Benign cells

  • Malignant cells

In Malignant Cells, Nuclei Show

• High N/C ratios

• Increased chromatin material

• Abnormal chromatin distribution

• Variation in:

  • Size

  • Shape

Abnormal Mitosis Features

• Increased number of cells in mitosis

• Abnormal mitotic figures

CYTOPLASMIC CHARACTERISTICS of malignant cells

• Cytoplasm and its relationship to the nucleus provide evidence of:

  • Origin of the tumour cells

• Cytoplasmic abnormalities alone are NOT diagnostic of malignancy

• They contribute to interpretation

Why Cytoplasmic Changes Alone Are Not Diagnostic

• Cytoplasmic changes are also seen in many benign processes such as:

  • Inflammation

  • Degeneration

  • Metaplasia

  • Radiation effects

  • Viral infection

Cytoplasmic Changes Features

• Variation in staining reaction

• Variation in shapes and sizes

Examples

• Tadpole cells

• Fibre cells

• Bizarre shapes

Additional Features

• Increased vacuolation

  • Example:

    • Adenocarcinoma

• Cannibalism and phagocytosis

MALIGNANT GROUP CHARACTERISTICS

• Cell clumping, pleomorphism, and anisonucleosis

• Loss of polarity of groupings

• Cannibalism and inclusion of cells

• Abnormal mitosis with incomplete cellular separation

• Lack of cytoplasmic perimeter

GENERAL BACKGROUND APPEARANCES

• Haemorrhage / Erythrocytes

• Inflammation

• Necrosis / Degeneration

PURPOSE OF PAP AND H&E STAINING METHODS

• Aim to promote contrast:

  • Between adjacent cells and tissues

  • Between adjacent organelles

Information Obtained

• Cell morphology

• Cell maturity

• Metabolic state

• Characterised by abundant use of alcohols

• In the PAP stain:

  • Alcohol-based dyes are also used

EFFECTS OF ALCOHOL

• Greater cytoplasmic transparency

• Better resolution of overlapping cells

• Sharp cytoplasmic contrast

• Increased nuclear detail

• Alcoholic fixation:

  • Prevents air-drying effects on cells

How the stain achieves differential colouring and visualization of cells/tissues.

Involves Application Of

1. Nuclear stain

2. Counterstain

Important Requirement

• The two stains should be of contrasting colours

• Purpose:

  • To emphasise morphological features of different cell organelles

DISTINCTIVE FEATURE OF PAPANICOLAU STAINING

• Differential colouration of maturing epithelial cells

4 MAIN STEPS IN STAINING PROCEDURES

1. Fixation / Tissue Processing / Sectioning & Dewax in Xylene

2. Nuclear staining

3. Counterstaining

4. Dehydration / Clearing & Mounting

STEP-BY-STEP PROCEDURE OF PAP AND H&E STAINING METHODS

1. INITIAL PREPARATION - Smears and Tissue Sections

   • Bring down to water through:

     • 80% alcohol

     • 70% alcohol

     • 50% alcohol

     • Water

2. SPECIAL PROCEDURES FOR TISSUE SECTIONS

   1. Removal of Mercury Deposits

      1. From mercury fixatives by immersion in 0.5% I₂ solution in 95% alcohol

      2. Due to health and safety reasons Mercuric fixatives are being phased out

   2. Removal of Formalin Pigments

      • By immersion in mixture of 28% ammonia water in 70% alcohol

3. NUCLEAR STAINING - Smears and Tissue Sections

   • Rinse in distilled water

   • Stain in Harris Haematoxylin

     • Regressive method

4. DIFFERENTIATION STEP Smears

   • Immersed in:

     • 0.5% Hydrochloric Acid (HCl)

   • Used in:

     • Papanicolau method

   Tissue Sections

   • Immersed in:

     • Mixture of 1% HCl in 80% alcohol

   • Used in:

     • H&E technique

5. BLUING STEP Smears and Tissue Sections

   • Bluing carried out in:

     • Running tap water

     • OR Scott’s Water Substitute

   Scott’s Water Substitute Contains

   • MgSO₄

   • NaHCO₃

6. COUNTERSTAINING Tissue Sections

   • in 1% aqueous eosin

7. DEHYDRATION Tissue Sections

   • in 95% alcohol

8. SMEAR DEHYDRATION - Gradual Dehydration Through

   • 50% alcohol

   • 70% alcohol

   • 80% alcohol

   • 95% alcohol

9. ORANGE G6 STAINING Smears

   • Stain in Orange G6

   • Alcohol-based stain for keratin

10. ALCOHOL RINSE Smears

    • Rinse in 2 baths of 95% alcohol

11. EA STAINING Smears

    • Stain in:

      • EA50 for gynaecological slides

      • EA65 for non-gynaecological slides

EOSIN AZURE TRICHROME STAINS

Function

• Produce differential cytoplasmic counterstaining

Cells Are Selectively Coloured By Different Alcohol-Based Acidic Dyes

• Eosin Yellow

• Light Green or Fast Green

• Bismark Brown

KERATINIZATION AND BONDING

As Cells Keratinize

• Increase in:

  • Stronger, tighter protein disulphide bonds (S-S)

• Decrease in:

  • Looser sulphydryl bonds (S-H)

S-H Bonds Predominate In

• Parabasal squamous cells

• Intermediate squamous cells

ROLE OF CELL AND TISSUE PERMEABILITY

• Cell and tissue permeability affect staining process

Biological Staining

• Penetration or diffusion:

  • Is a function of time

  • Is rate controlled

DYE DIFFUSION PRINCIPLES

Small Dye Molecules

• Diffuse easily and quickly

Large Dye Molecules

• Take longer to diffuse

RATE-CONTROLLED STAINING

In Multi-Dye Bath

• Smaller dye molecules:

  • Penetrate rapidly into all cells initially

• With time:

  • Larger dye molecules penetrate

  • Displace smaller dye molecules from cells with less dense structure

REASON FOR DYE DISPLACEMENT

• Larger dyes exert:

  • Stronger van der Waals forces

Due To

• Larger conjugated bond systems

• Presence of many covalent bonds

PAPANICOLAU CYTOPLASMIC STAINING

• Cytoplasmic staining cannot be due to simple Coulombic forces

Reason

• Main dyes used:

  • Eosin

  • Light Green

• Both are acidic dyes

BONDING CHARACTERISTICS OF CELLS

Parabasal & Intermediate Squamous Cells

• Predominantly:

  • S-H bonding

Superficial Squamous Cells

• Predominantly:

  • S-S bonding

CELL STRUCTURE AND DYE DISPLACEMENT

Loose Cell Structure (S-H)

• More dye displacement

Strong Cell Structure (S-S)

• Less dye displacement

STAINING OF DIFFERENT CELL TYPES

Small Eosin Dye Molecules

• Mainly stain:

  • Superficial squamous epithelial cells

Larger Light Green Dye Molecules

• Mainly stain:

  • Intermediate epithelial cells

  • Parabasal epithelial cells

FINAL DEHYDRATION AND MOUNTING Smears and Tissue Sections

Complete Dehydration Through

• 95% alcohol

• Absolute alcohol

Final Steps

• Clear in xylene

• Mount in DPX

Anatomy of the Cervix

Cervix divided into 2 regions

Ectocervix

• Outer part projecting into the vagina

• Covered by:

  • Stratified squamous epithelium

Endocervix

• Cervical canal leading to uterus

• Covered by:

  • Columnar glandular epithelium

Squamo-columnar Junction (SCJ)

• Area where:

  • Squamous epithelium meets columnar epithelium

• Dynamic area that changes position throughout life

Position changes due to:

• Hormonal stimulation

• Puberty

• Pregnancy

• Menopause

• Increase in cervical tissue bulk

During:

• Puberty/pregnancy → SCJ moves outward (ectropion)

• Menopause → SCJ retracts inward into endocervical canal

The position varies due to hormone stimulation and increase in bulk of cervical tissue

Transformation Zone (TZ)

• Area between:

  • Original SCJ

  • New SCJ after metaplastic change

• Site of squamous metaplastic epithelium.

• Highly vulnerable to carcinogens during the early active phase.

Importance of TZ

• Most important area in cervical pathology

• Site where:

  • HPV infection commonly occurs

  • Cervical dysplasia develops

  • Most cervical cancers arise

Why TZ is vulnerable

• Active cell division and replacement

• Presence of immature metaplastic cells

• HPV infects basal immature squamous cells

The position varies due to hormone stimulation and increase in bulk of cervical tissue

Types of Transformation Zones

TZ1

• Completely visible

• Entirely ectocervical

TZ2

• Partly inside endocervical canal

• Still fully visible

TZ3

• Located high in endocervical canal

• Not fully visible

• Common after menopause

• More difficult to assess

Pathogenesis of Cervical Cancer

Site of Origin

• Cervical cancer mainly occurs at the Transformation Zone.

Sequence of Pathogenesis

• Cell genome interferences and possible mutations occur.

• A cell line with neoplastic potential emerges.

• Initial chromosomal changes may remain dormant in the genome for years.

• Additional risk factors may later trigger malignant transformation.

Important Concepts

• Multifactorial process.

• Initiators and promoters are probably involved.

Squamous Metaplasia

Normal physiological replacement of columnar epithelium by squamous epithelium.

Why it occurs

• Columnar cells exposed to:

  • Acidic vaginal environment

  • Hormonal influences

  • Friction/irritation

Growth

Reserve cells → Multipotent reserve cells proliferate → Reserve cell hyperplasia → Immature squamous metaplasia → Mature squamous metaplasia

Important Notes

• Squamous metaplasia is NORMAL

• It is NOT dysplasia or cancer

• Immature metaplastic cells are susceptible to HPV infection

Nabothian Cysts

• Metaplastic squamous epithelium may cover:

  • Endocervical glands

  • Endocervical epithelium

Result

• Mucus secretions become trapped

• Leads to formation of:

  • Nabothian cysts

Characteristics

• Benign mucus-filled cysts

• Common incidental finding

Human Papillomavirus (HPV)

• Over 100 HPV subtypes exist

• Around 25-30 infect ano-genital tract

High-Risk HPV Types

• Main high-risk types : HPV 16, HPV 18

• Responsible for about 70% of cervical cancers

• Mechanism of carcinogenesis:

  • E6 protein - Inactivates p53 tumour suppressor gene

  • E7 protein - Inactivates Rb tumour suppressor gene

• Result:

  • Loss of cell cycle regulation

  • Uncontrolled cell growth

  • Eventual cancer development

  • Dysplasia and malignant transformation

Low-Risk HPV Types

• Types: HPV 6, HPV 11

• Associated with Genital warts (condyloma acuminata)

• No major association with cervical cancer

Associated STDs

• Syphilis

• Gonorrhea

• Herpes

• Chlamydia

• Especially Human Papilloma Virus (HPV) infections

HPV and Cervical Cancer

• Commonly associated with CIN (Cervical Intraepithelial Neoplasia) and cervical cancer.

• Has oncogenic properties.

• Inactivates tumor suppressor genes.

Pre-invasive Stage of Cancer

• Most cancers pass through a stage where transformed cells are still confined to the epithelium.

• This stage precedes:

  • Invasion through the basement membrane

  • Invasion into the stroma

Historical Concept

• Pre-invasive stage concept in cervical cancer dates back to early 20th century (1908).

• In the 1920s, tissue biopsy was standard for diagnosis.

• Common findings:

  • Tumour cells confined to epithelium and basement membrane

  • No access to bloodstream or lymphatic vessels

  • No risk of spread

• These findings strengthened recognition of a pre-invasive stage.

Early Terminology Problems

• Different scientists used different terms:

  • Dysplasia

  • Incipient cancer

  • Carcinoma in situ

• Problems caused:

  • Subjective interpretation

  • Lack of precision in reporting

  • Chaotic descriptions

• Result:

  • Inconsistent patient management

  • Varied treatment strategies for same condition

Terminology Systems

Papanicolaou Class System (1950s)

• Class I: Benign

• Class II: Mild cellular changes considered benign

• Class III: Atypical / mild abnormalities, no malignancy

• Class IV: Abnormal cells suggestive of malignancy

• Class V: Abnormal cells diagnostic of malignancy

• Limitations

  • Many modifications by cytopathologists

  • Not used uniformly

  • Evolved into newer descriptive diagnostic systems

Development of CIN Concept (Late 1960s – Dr. Ralph Richart)

• Need for system describing disease continuum (precursor → invasive cancer)

• CIN Classification introduced:

  • Cervical Intraepithelial Neoplasia (CIN)

  • Communicates degrees of abnormality (not certainty of cancer)

• Features

  • More descriptive terminology

  • 3 grades of abnormality

  • Links cytology and histology:

    • Mild dyskaryosis → CIN 1

    • Moderate dyskaryosis → CIN 2

    • Severe dyskaryosis → CIN 3

• Dysplasia and carcinoma in situ merged into one system

• Improved standardisation of:

  • Patient management

  • Treatment strategies

Modern Terminology Systems

BSCC Terminology (Three-tier system)

• CIN 1

• CIN 2

• CIN 3

2002 BSCC Proposal

• More similarity with NCI Bethesda system

NCI Bethesda Terminology (Two-tier system)

• Low Grade Squamous Intraepithelial Lesion (LSIL)

  • HPV changes

  • CIN 1

• High Grade Squamous Intraepithelial Lesion (HSIL)

  • CIN 2

  • CIN 3

Additional Category

• BSCC “Borderline nuclear changes” corresponds to:

  • Atypical Squamous Cells of Undetermined Significance (ASCUS)

Definition of CIN

• Partial or complete replacement of normal cervical epithelium by neoplastic cells

Key Features

• Basement membrane intact

• No invasive pattern

• Tumour cells separated from stroma

Grading of CIN

Depends on:

• Proportion of epithelial thickness replaced by undifferentiated neoplastic cells

  • High nucleo-cytoplasmic ratio

  • Abnormal chromatin

• Level of mitotic figures within epithelium

• Presence of abnormal mitoses

• Degree of disruption of basal epithelial layer

Grade of CIN correlates with:

• Nuclear and cellular abnormalities in superficial squamous cells

CIN Grades

Precancerous Changes

• Development of Cervical Intraepithelial Neoplasia (CIN).

CIN 1

• Mild dysplasia

• Replacement of the Lower one-third of epithelium

• Often regresses spontaneously

CIN 2

• Moderate dysplasia

• Lower 2/3 affected

CIN 3

• Full thickness epithelial replacement by neoplastic cells

• Severe dysplasia/full thickness abnormality

• Includes carcinoma in situ

Progression

• HPV infection → Persistent infection → CIN → Invasive carcinoma over years

Important

• Many lesions regress spontaneously

• Immune system may eliminate HPV DNA

• CIN may progress to cervical cancer within 6–15 years.

• Depends on the extent of mutagenic changes in the cellular genome.

Aims of CIN System

• Improve communication of scientifically accurate reports

• Minimise inter- and intra-observer variability

• Include specimen adequacy as part of report

Natural History of CIN

• Higher CIN grade → lower regression rate and higher progression rate

Important Points

• CIN does not always progress linearly to cancer

• Many lesions regress spontaneously

Regression / Persistence / Progression

• CIN 1:

  • 50% regress within 2 years

  • 32% persist

  • ~11% progress to higher grade

• CIN 2:

  • 43% regress within 2 years

  • 35% persist

  • 22% progress

• CIN 3:

  • Less likely to regress

  • Higher tendency to persist/progress to cancer

Time to Cancer

• Progression typically takes 12 years (range 3–20 years)

Additional Observations

• Cancer may occur without detectable progression through CIN stages

• High-grade lesions may arise without preceding low-grade lesions

Dysplasia vs Dyskaryosis

• Dysplasia = histological term

• Dyskaryosis = cytological term in gynaecology

Dyskaryosis

• Cells with abnormal nuclear features:

  • Mild

  • Moderate

  • Severe

Correlation

• Dyskaryotic cells arise from CIN 1, CIN 2, or CIN 3 lesions

• Sampling superficial cells helps predict underlying histology

• Basis of cervical screening

• Guides patient management

General Guidelines for Management

• 6-month follow-up (cytology ± HPV DNA testing):

  • Borderline changes

  • HPV changes

  • Mild dyskaryosis

• Colposcopy:

  • Moderate dyskaryosis

  • Severe dyskaryosis

Evidence that CIN is a Precursor of Cervical Cancer

• Microscopic morphological similarities

• Similar aetiological risk factors

• Similar biological behaviour

• Progressive natural history studies:

Studies

• Petersen (1955):

  • CIN → Cervical cancer in 30% within 9 years

• Spriggs (1972):

  • CIN → Cervical cancer in 25% within 5 years

• Green (1955–1976):

  • CIN 3 → Cervical cancer:

    • 18% in 10 years

    • 36% in 20 years

Additional Evidence

• Retrospective studies show CIN commonly associated with cervical cancer

• DNA ploidy studies support progression link

• Same carcinogenic effects seen in CIN and cervical cancer

• Age distribution:

  • CIN: <30 years

  • Cervical cancer: >30–35 years

• Screening programmes:

  • Treatment of CIN reduces incidence of cervical cancer

Types of Cervical Cancer

Squamous Cell Carcinoma

• Most common (~70%)

• Arises from squamous epithelium in TZ

Adenocarcinoma

• Arises from glandular endocervical cells

• Harder to detect on smear tests

Cervical Smear Test / Pap Test

• Screening test for:

  • PRE-CANCEROUS lesions

  • Dysplasia

• NOT mainly for cancer detection

• Detect abnormalities before invasive cancer develops

Adequacy of Cervical Smear

• A satisfactory smear should contain:

  • Squamous epithelial cells

  • Columnar epithelial cells

  • Squamous metaplastic cells

• Why? Indicates that the transformation zone has been sampled adequately.

Abnormal smears may require

• Repeat smear

• HPV testing

• Colposcopy

• Biopsy

Colposcopy

Follow-up investigation after abnormal smear.

Procedure

• Cervix examined under magnification

• Acetic acid applied

• Abnormal areas appear white (“acetowhite”)

Allows

• Better visualisation

• Directed biopsy

• Assessment of lesion severity

Liquid-Based Cytology (LBC)

Modern method of cervical screening

Technique

• Cells collected using brush/spatula

• Sample rinsed into preservative fluid

Advantages

• Cleaner preparation

• Less blood/mucus obscuring cells

• Lower false-negative rate

• Better cell preservation

• HPV testing possible from same sample

HPV DNA Testing

Included in many screening programmes

Advantages

• Detects high-risk HPV infection

• Improves accuracy of cytology

• Helps assess significance of lesions

• Assists patient management

• Guides treatment and follow-up

Cervical Screening Programme

Screening is for:

• Healthy

• Asymptomatic women

Statistics

• Around 90% of smears are normal

• Around 10–12% require follow-up or colposcopy

Screening starts after 25 years because:

• HPV infections very common in younger women

• Most regress spontaneously

• Many low-grade lesions disappear naturally

• Avoids overtreatment and unnecessary anxiety

Recall Intervals Every 3 years

• Routine recall interval

• Especially with LBC due to lower false negatives

Progression rate of cervical cancer

• Usually slow

• Approximately 8–15 years

Women over 65

• Recall may occur every 5 years

• Depends on screening history and guidelines

Cervical Screening Procedure & Patient Care

Environment must be appropriate and professional

Woman should feel:

• Comfortable

• Safe

• Relaxed

• Supported

• Respected

• No fear or embarrassment

• Privacy maintained

Importance

• Positive experience encourages repeat attendance

• Helps avoid defaulters

Clinical Requirements - Facilities should include:

• Adequate examination couches

• Sterile equipment

• Good lighting

• Proper privacy

• Trained healthcare professionals

During the Procedure Procedure should be explained clearly

• Reduces anxiety

• Improves cooperation

Relaxed patient leads to:

• Relaxed pelvic muscles

• Easier speculum insertion

• Better visualisation of cervix

• Better sampling

VERY IMPORTANT

• Cervix MUST be visualised properly

• Necessary for accurate transformation zone sampling

HPV Vaccination

Helps prevent:

• HPV infection

• CIN lesions

• Cervical cancer

Vaccines protect mainly against:

• HPV 16

• HPV 18

Some vaccines also cover:

• HPV 6

• HPV 11

AETIOLOGY & PATHOGENESIS OF CERVICAL CANCER

Cervical Cancer

• One of the commonest causes of death from cancer in developing countries.

• In developed countries, especially where organised national or regional screening programmes are set up, cervical cancer mortality is decreasing.

• However, it is still a major problem.

• Site of Origin

  • Cervical cancer mainly occurs at the Transformation Zone.

In the U.K.

• Approximately 4,500 new cases each year.

• Around 1,800 deaths.

• Mostly occurs in unscreened women.

• Age groups affected are decreasing.

Age Range

• Cervical cancer age range: 20–85 years.

• 50% of deaths occur in women aged 35–55 years.

Cervical Tumor Types

• Squamous cell carcinoma: 70%–90%

• Adenocarcinoma: 10%–15%

• Mixed tumors: 5%–10%

• Small cell carcinoma: 5%–10%

• Undifferentiated carcinoma / Lymphoma / Sarcoma / Metastatic carcinoma

Importance of Cervical Studies

Because of its accessibility, the cervix has been extensively studied, especially regarding:

• Epidemiology

• Life history

• Aetiology

These studies implicated many risk factors, suggesting that the cervix has greater vulnerability for cancer development.

Historical Observation (1842 – Rigoni Stern)

observed that cervical cancer was:

• Much more common in:

  • Married women

  • Widows

  • Prostitutes

• Much less common or rare in:

  • Unmarried women

  • Nuns

  • Virgins

Conclusion

• He suggested cervical cancer results from “marital” or “sexual” events.

• Later studies proved this correct since cervical cancer involves sexually transmitted factors.

Cervical cancer risk factors are associated with increased sexual activity at a young age.

• increasing more to this day

Cervical cancer Risk Factors

Associated with increased sexual activity at a young age.

• increasing more to this day

Sexual Factors

• Coitus (major prerequisite factor)

• Age at first coitus

• Frequency of coitus

• Multiple partners

• Promiscuous sexual partners

• Overlapping sexual factors

Example

• 60% of cervical cancer patients had intercourse before the age of 17 years.

Sequence

• Early coitus → increased sexual partners → STDs → Cervical cancer

Immunodeficiency Factors

Examples:

• HIV

• Toxins

• Smoking

Effects

• Decreased surveillance against mutagenic viruses (especially HPV).

• Cervix becomes more vulnerable.

• Inadequate host immune response.

• Cigarette smoke carcinogens found in cervical secretions further suppress local immune response.

• Cervical cells become more susceptible to malignant change.

Oral Contraceptives (OCs)

• Their impact is difficult to assess because they overlap with other sexual risk factors.

• OCs may reduce the use of barrier contraception by male partners.

• Leads to:

  • Unprotected sexual intercourse

  • Increased exposure to STDs

  • Greater exposure to male-related factors*

Male Factors

Possible Transmission of Chemical Carcinogens

Chemical carcinogens may be transmitted during sexual activity, especially in men exposed to:

• Dust

• Tar

• Farms

• Mines

• Foundries

• Fumes

• Industries

Role of Spermatozoa

• Spermatozoa contain large amounts of DNA.

• Possible interactions with cervical epithelium may:

  • Be mutagenic

  • Trigger cell-bound DNA proliferation

Staining Techniques

• Light microscopy remains the most important method for studying and interpreting cells and tissue components.

• Morphological features are visualised using experience, detection, and interpretative skills.

• Many cell and tissue components are colourless.

• These components can only be distinguished if their refractive indices are sufficiently different to create contrast.

• In cytology and histology, refractive index differences alone are not sufficient.

• To study cells and tissues in detail, components must be highly distinguishable.

• Different constituents must be clearly demonstrated from each other.

• This is achieved using STAINING TECHNIQUES.

Basic Principle of Staining

• Two phases are involved:

  • Solid phase: Tissue sections or cell preparations

  • Liquid phase: Stains and dyes

• Interactions involve a series of bonding reactions, similar to chemical and physical reactions.

• These reactions occur between:

  • Dye molecules

  • Cell/tissue component molecules

• The main aim of staining techniques is to apply specific dyes to specific cell/tissue constituents to produce contrast.

• No single dye can demonstrate all cellular elements selectively.

• Therefore, combination staining sequences are used.

Bonding Reactions in Staining

• Hydrophobic bonding

  • Hydrophobic groups have higher affinity for themselves than for water.

• Van der Waals forces

  • Act between all dyes and tissues over short distances.

• Hydrogen bonding

  • Bonding between hydrogen and two electronegative atoms.

• Electrostatic attraction

  • Attraction between oppositely charged ions (cations and anions).

• Covalent bonding

  • Sharing of electrons between atoms/ions.

Classification of Staining Techniques

• Dyeing methods

• Vital staining

• Lysochrome staining methods

• Histochemistry

• Metallic impregnation

Dyeing Methods

Natural Dyes

• In early histology, dyes were extracted from natural plant sources.

• Examples:

  • Haematoxylin

    • Extracted from logwood of a Mexican tree (Haematoxylon campechianum).

    • On its own, haematoxylin has little staining ability.

    • It must be oxidized to haematin using an oxidizing agent (e.g. mercuric oxide).

    • A mordant is added to enhance and make staining permanent.

  • Carmine

  • Orcein

  • Saffron

Mordants

• Usually metal salts or alums.

• Examples:

  • Chromium

  • Aluminium

  • Potassium

  • Lead

  • Iron

• Function:

  • Form an intermediate layer between tissue and dye.

  • Have multiple binding sites:

    • Some attach to tissue

    • Some attach to dye

Examples of mordant dyes

• Ehrlich’s haematoxylin → potassium alum

• Gower’s haematoxylin → chrome alum

• Best’s carmine → aluminium

Synthetic Dyes (Coal Tar Dyes)

• Originally derived from coal tar substances.

• Now mainly derived from benzene (C₆H₆).

  • Colourless liquid.

  • Absorbs/transmits light in the ultraviolet spectrum.

  • Addition of chemical side groups alters electron cloud.

  • This shifts absorption into visible light range.

• Dyes used today are aromatic compounds (benzene derivatives).

Chromophores

• Groups that shift absorption into visible spectrum and produce colour.

• Examples:

  • Nitro group (–NO₂)

  • Quinonoid group

• Benzene + chromophores = chromogens

Chromogens

• Coloured compounds but NOT true dyes.

• Easily removed from tissue.

• Not permanently effective.

• Not reactive enough with tissue environment.

Auxochromes

• Ionizable side groups added to chromogens.

• Help dye bind permanently to tissue components.

• Promote interaction with cell/tissue molecules.

• Example: Picric acid

  • OH group loses H⁺ → negative charge forms

  • This interacts with positively charged tissue ions

• Usually supplied by mordants.

Types of Dyes

Direct Dyes

• Have affinity for tissue components due to opposite charges.

• Anionic dyes

  • Attract cationic tissue components

• Cationic dyes

  • Attract anionic tissue components

• Examples:

  • Eosin

  • Methylene blue

  • Acid fuchsin

  • Neutral red

Indirect Dyes

• Have little affinity for tissues.

• Require mordants for binding.

Metachromasia

• Some dyes absorb multiple wavelengths.

• This results in different tissue components staining in different shades.

• Some structures stain in a different colour than the dye solution.

• Examples:

  • Methylene blue

  • Brilliant cresyl blue

  • Safranin

• Romanowsky stains show metachromasia.

• Buffer maintains constant pH for staining different components:

  • Acidophilic

  • Basophilic

  • Neutral structures

• Examples:

  • Leishman’s stain

  • May-Grünwald-Giemsa stain