Histology Week 11 - Cytology and Changes in Cell Morphology in Disease

Cytology & Changes in Cell Morphology in Disease

Histology vs Cytology

• Histology: Microscopic study of tissues.
• Cytology: Microscopic study of cells.

Diagnostic Cytology

• Dr. George Papanicolaou made significant contributions to this field.
• The Papanicolaou stain is a universal stain for cytological preparations.
• He is best known for creating the Papanicolaou test, commonly known as the Pap smear, which revolutionized the early detection of cervical cancer.

Cytological Preparation

• Health and safety are of paramount importance due to the risks for the technician handling many of the unfixed specimens.
• Many cytologists request that the specimens be unfixed so that cellular detail is not compromised.

Potentially Hazardous Specimens (unfixed):

• Sputum
• Urine
• Blood
• Less common bodily fluids

Precautions for Unfixed Specimens:

• Use of a safety cabinet (Class I).
• Suitable specimen container that can be disinfected.
• A protocol for disinfection procedures and disposal.
• PPE (Personal Protective Equipment)

Specimen Collection

• Collected specimens should be sent to the cytology laboratory without delay.
• Specimens must be labeled and biohazard stickers used for high-risk specimens.
• Lids must be tight and container placed into sealable plastic bags.

Cell Collection Methods

Exfoliative Cytology

• Cells spontaneously shed by the body ("spontaneous exfoliation") into body fluid such as sputum, urine, CSF, and effusion in body cavities.

Abrasive Cytology

• Cells are obtained directly from the surface of the target of interest.
• Samples are taken by scraping, brushing, or washing. E.g., thin prep.

Intervention Cytology/Aspiration Cytology

• Fine-needle aspiration.

Exfoliative Cytology (Natural Spontaneous Exfoliation)

• Based on spontaneous shedding of cells derived from the lining of an organ into a cavity.
• It is the simplest of the three sampling techniques.
• Contents of the sample are derived from several sources.
• Examples: vaginal smear, sputum, urine, effusion.
• The material is collected spontaneously or by a syringe or a cotton swab.

Abrasive Cytology (Artificial Enhanced Exfoliation)

• Cells are obtained directly from the surface of the target of interest.
• Samples are taken by:
  • Scraping: from cervix (pap smear), vagina, oral cavity (buccal mucosal smear), and skin lesions.
  • Brushing, Washing, and Lavage: bronchi, GIT, and urinary tract.
• Examples: cervical scraper, endoscopy, and gastric lavage.
• Samples can be obtained from superficial or deep lesions.
• The purpose of this procedure is to dislodge cells, enrich the sample with cells obtained directly from the surface of the target of interest.

Cervical Cancer

• Cervical cancer is approximately 10% of the global burden of cancer.
• It is the fourth most common cancer affecting women globally.
• An estimated 604,000 new cases and 342,000 deaths in 2020.

Eliminating Cervical Cancer - Australia's 2030 Targets

• 90% of all eligible people will be vaccinated against HPV.
• 70% of eligible people will be screened every 5 years.
• 95% of eligible people will receive optimal treatment for pre-cancer and cancer.
• Positive, culturally safe, and inclusive experience of prevention and care.
• Eliminating cervical cancer in Australia by the year 2035 could make Australia the first country in the world to actively achieve elimination.

Cervical Sample Collection and Guideline Changes

• A cervical sample is collected into a liquid-based medium.
• The sample is first tested for high-risk HPV DNA.
• If HPV is not detected, no cytology is performed.
• If HPV is detected, the same sample is processed for LBC (ThinPrep).
• A pathologist examines the cells microscopically for any abnormalities.

Cervical Screening Pathway

• HPV not detected: Routine 5-yearly screening.
• HPV detected (not 16/18): LBC - reflex or collect cervical sample.
  • Unsatisfactory LBC: Repeat LBC test in 6 weeks.
  • Negative/pLSIL/LSIL: Follow-up HPV test in 12 months.
  • LBC PHSIL or worse: Refer for colposcopic assessment.
• HPV detected (16/18): LBC (reflex or collect at colposcopy).
  • Any LBC result or unsatisfactory: Refer for colposcopic assessment.
• Unsatisfactory HPV test (if self-collection was used): Repeat as soon as practical, ideally within 6 weeks.
• Direct referral to colposcopy is recommended for screening participants who are:
  • Aged 50+ years
  • Aboriginal and/or Torres Strait Islander
  • Overdue for screening by at least 2 years at initial screen

Legend:

• Low risk of developing cervical cancer precursors within the next five years
• Intermediate risk of developing cervical cancer precursors within the next five years
• Higher risk of developing cervical cancer precursors within the next five years

Definitions:

• DES: diethylstilbestrol
• LSL: low grade squamous intraepithelial lesion
• HSL: high-grade squamous intraepithelial lesion
• PLSIL: possible low-grade squamous intraepithelial lesion
• pHSIL: possible high-grade squamous intraepithelial lesion
• Includes PHS, HSIL, cancer, or glandular abnormality

Cervical Smears

• The Pap smear is used as a screening test for cervical cancer.
• Cells are scraped from the opening of the cervix and can be spread directly onto the slide to create a cervical smear (traditional smearing) or put into a liquid-based vial for analysis.

Cervical Smear Preparation

• Remove material from spatula by scraping both sides against the slide. Gently spread to distribute and spread evenly and quickly wearing gloves in indicated direction.

Fixation of Cytological Specimens

• Alcohol Fixation: 95% ethyl alcohol is used for most routine preparations.
• Conventional pap smear require immediate alcohol fixation on slide before proceeding to Pap stain.
• For Papanicolaou staining, it is important to avoid air-drying the sample. It should be placed into the 95% alcohol whilst it is still wet.

Liquid Based Cytology

• The ThinPrep PAP test is 27% more effective than the original PAP smear.
• It is a more accurate technique.
• Cervical cells are collected by the doctor and rinsed into a container with fixative.
• At the lab, the cells are transferred to a glass slide in a thin even layer.
• Recovers almost all the cells collected and creates a clearer slide making detection of disease easier.
• Abnormal cells are sometimes missed in the conventional pap tests because they are hidden by blood or mucous which are often collected with the cells.

Reflex Cytology

• If HPV detected → Reflex LBC (ThinPrep)
• Same specimen used
• Combined report: HPV + LBC with risk category
• Its importance: It checks for any cell changes caused by HPV, like early signs of cancer. The results help doctors decide how serious the risk is and what to do next

Pap Stain

• Multichromatic staining technique
  • Highlights cellular details
• Stains nucleus and cytoplasm
  • Helps to detect abnormal cells, precancerous changes, and cancer
• Identifies squamous and glandular abnormalities

Characteristics of Papanicolaou Staining

• Individual nuclei:
  • Clearly visible at low power (x10 objective) and high power (x40 objective)
  • Blue/purple to black in color
  • Granular and crisp (not hazy)
• Counterstains (cytoplasm):
  • Superficial squamous cells - pink
  • Less mature cells (intermediate and metaplastic) - blue/green
  • Fully keratinized cells - orange.
• The stains should be equal in intensity.
• There should be cytoplasmic translucency with a sharp contrast to the nuclear stain.

Pap Stain - Traditional Pap Smear vs ThinPrep

• Traditional:
  • Cells are smeared directly onto a glass slide during collection.
  • The slide is then immediately fixed with alcohol spray to preserve the cells.
  • After drying, the slide is manually stained using the Pap stain in the lab.
• ThinPrep:
  • Cells are collected into a liquid vial containing a fixative solution (e.g., PreservCyt).
  • In the lab, the sample is processed to create a thin, even layer of cells on a slide.
  • The slide is then stained using the Pap stain, just like the traditional method.

Papanicolaou Staining Consequences of Air-Dried Samples:

• Increase in eosinophilic staining.
• Apparent nuclear enlargement
• Poor staining

Conventional PAP Smear vs ThinPrep

• Conventional PAP Smear
  • Only a fraction of collected sample is used in slide preparation
  • Sample smearing factors often produce poor cell quality
  • Slide may fail to accurately represent sample and not reflect patient's actual condition
  • Cells on slide may overlap or be obscured by blood, mucus or other material, making visualization of cells difficult
• ThinPrep PAP Test
  • Nearly all of collected sample is retained in the ThinPrep Pap Test vial
  • Immediate fixation maintains cell quality
  • Slides accurately represent sample for increased opportunity to detect abnormality
  • Cells on slide are cleared of obscuring elements and distributed evenly for ease of visualization
  • Produces multiple representative and reproducible samples

Types of Cervical Cancer

• 90% (85–90%) are cervical carcinomas (squamous cell carcinomas)
• 5 to 9 % are adenocarcinoma
• The rests are small cell carcinoma and cervical sarcoma
• 90-95% of squamous cell carcinomas of the cervix contain the human papillomavirus DNA (HPV).

HPV

• Two human papillomaviruses (HPV) types (16 and 18) are responsible for nearly 50% of high-grade cervical pre-cancers
• HPV is mainly transmitted through sexual contact and most people are infected with HPV shortly after the onset of sexual activity.
• More than 90% of them clear the infection eventually.

Cervical Cancer – FIGO Staging System

• Stage 0: Carcinoma in situ. Abnormal cells in the innermost lining of the cervix

Cervical Smears - Results

• Normal result: no abnormal cells presented
• Normal squamous epithelial cells in a premenopausal woman. Intermediate cell (blue cytoplasm). Superficial cell (red cytoplasm).

Normal Cell vs Cancer Cell

Morphology Parameters

• Cell arrangement
  • Papillary configurations with fibrovascular cores (Papillary adenocarcinoma)
  • Glandular or tubular structures (adenocarcinoma)
  • Follicles (Follicular neoplasms of the thyroid)
  • Rosettes (neuroblastoma)
  • Feathering/Rosettes/Strips (Adenocarcinoma in situ)
  • Pearls (squamous cell carcinoma)
• Cell sizes & shapes
  • Help to identify specific neoplasms
  • Cells are generally uniform (monomorphic) in normal tissues and benign neoplasms
  • Malignant tumors frequently exhibit significant variations in cell shape (Pleomorphism).
• Cytoplasmic features
  • May reveal the tissue origin or etiology
  • Colors, texture, presence of vacuoles, pigments, and other products
• Nucleus
  • The critical feature of malignancy resides in the nucleus
  • Size, shape, alterations of the nuclear membrane and chromatin,
  • The prominence of the nucleolus, and mitotic activity
• Cancer cells
  • Nuclei are usually darker (hyperchromatic)
  • Chromatin is coarser and unevenly distributed
• Extracellular material and background surround the cells
  • Presence and type of inflammation, blood, various extracellular substances, cell products, necrotic debris, and microorganisms
  • When present with association with malignant cells, necrosis generally indicates invasive cancer.

Cervical Smears - Results (Abnormal)

• Grouped as follows:
  • Infection
  • Reactive changes
  • Epithelial cell abnormalities
    • Unclear result:
      • ASCUS (Atypical squamous cells of undetermined significance)
      • AGUS (Atypical glandular cells of undetermined significance)
        • This result means there are atypical cells of uncertain significance
        • The changes may be due to HPV
        • They may also mean there are changes that may lead to cancer
  • Unsatisfactory result:
    • If there are not enough endocervical cells in the sample or the cells are clumped together, this is considered unsatisfactory. Your doctor may ask you to come back for another Pap test in a few months.

Descriptive Diagnosis - Infection

• Trichomonas vaginalis.
• Fungal organism morphologically consistent with candida species.
• Predominance of coccobacilli consistent with shift in vaginal flora.
• Bacteria morphologically consistent with actinomyces species.
• Cellular changes associated with Herpes simplex virus.

Descriptive Diagnosis - Reactive Changes

• Inflammation (typical repair)
• Atrophy with inflammation (atrophic vaginitis)
• Radiation
• IUD

Descriptive Diagnosis - Epithelial Cell Abnormalities

• Squamous cells
  • Atypical squamous cells of undetermined significance (ASCUS)
  • Low grade squamous intraepithelial lesion (LSIL)
  • High grade squamous intraepithelial lesion (HSIL)
  • Squamous cell carcinoma
• Glandular cells
  • Endometrial cells, cytologically benign in a post menopausal woman
  • Atypical glandular cells of undetermined significance (AGUS)
  • Endocervical adenocarcinoma
  • Endometrial adenocarcinoma
  • Extrauterine adenocarcinoma
  • Adenocarcinoma not otherwise specified.

Cervical Smears - Unclear Result

• Unclear result is common
• You may see words like ‘equivocal, inconclusive, ASCUS, ASC- cannot exclude HSIL (ASC-H) or AGUS’
• These all mean the same thing—that your cervical cells look like they could be abnormal. However, the cytologic changes suggestive of the squamous intra-epithelial lesions, are insufficient for a definitive interpretation
• It is not clear if it’s related to HPV.
• It could be related to life changes like pregnancy, menopause, or an infection.
• The HPV test can help find out if your cell changes are related to HPV. This could be used to assess the risk of future cervical disease
• E.g., result of ASCUS could be the result of mild infection or inflammation of the cervix

Cervical Smear: ASCUS

• Incomplete koilocytosis including poorly defined cytoplasmic halos or cytoplasmic vacuoles with absent or minimal nuclear changes
• Slightly increased N/C ratio
• Minimal nuclear hyperchromasia/ irregularity in chromatin distribution
• Remember: Nuclei are approximately 2 ½ - 3 times the area of the nucleus of a normal intermediate squamous cell

Cervical Smears - LSIL/HSIL/Carcinoma in situ

• LSIL (low-grade dysplasia) or HSIL (high-grade dysplasia): This means precancerous changes are likely to be present
• The risk of cervical cancer is greater with HSIL
• Carcinoma in situ (CIS): This result usually means the abnormal changes are likely to lead to cervical cancer
• Atypical squamous cells (ASC): Abnormal changes have been found and may be HSIL
• Atypical glandular cells (AGC): Cell changes that may lead to cancer are seen in the upper part of the cervical canal or inside the uterus

The PAP Smear Result

• Normal
• ASCUS: Atypical Squamous Cells of Undetermined Significance
• LSIL: Low Grade Squamous Intra-epithelium Lesion
• HSIL: High Grade Squamous Intra-epithelium Lesion

Cervical Smears - HPV/LSIL On Pap Smear

• Normal squamous cells and HPV-infected cells with mild dysplasia (LSIL)

Cervical Smears - Low-grade squamous intraepithelial lesion (LSIL)

• LSIL with HPV effect

Cervical Smears - High-grade squamous intraepithelial lesion (HSIL)

• High-grade dysplasia
• Cells of HSIL can be infrequent, individual, and bland-appearing-can be easy to miss
• Paradoxically, it's easier to overlook a high-grade lesion in ThinPrep than a low-grade one, and the most serious lesions of all--invasive carcinomas--are the easiest of all to miss.
• HPV 16 is the most common HPV subtype in HSIL

Cervical Smears - Adenocarcinoma

• The malignant cells have prominent mucin filled intracytoplasmic vacuoles. Endocervical cells produce mucin in vacuoles--this is called a signet ring.

Cervical Smears - Unsatisfactory result

• Satisfactory: represents a borderline level for ThinPrep specimen. Slightly over 5000 average number of cells were estimated based on four cells average per field at 40x.
• Unsatisfactory: due to scant squamous cellularity. Endocervical cells, as seen here in the form of honeycomb, are not taken into account in the cellularity estimation.

Respiratory Tract

• Sputum is analysed for detection of disease.
  • Usually collected on 3 consecutive days
  • Early morning is preferred collection time
  • Needs to come from deep cough (not spit/saliva)
  • Collected into a sterile container
• Other respiratory cytology include:
  • Bronchial Washings
  • Bronchial Alveolar Lavage
  • Bronchial Brushings
  • EBUS
• Specimens are usually unfixed
• 50% alcohol solution is used if specimen is hazardous and requires fixation.
• Bronchial brushings can be taken for analysis using a small nylon brush via a bronchoscope.

Respiratory Tract - Infections

• Acid fast bacilli (probably Mycobacterium tuberculosis) as seen in sputum specimen smear stained with Ziehl Neelsen technique

Gastrointestinal Tract

• GI Specimens include:
  • Scrapings from oral and laryngeal lesions
  • Brushings using an endoscope from the oesophagus and stomach
  • Transmucosal FNA (Fine Needle Aspiration)
  • Endoscopic Ultrasound (EUS) guided
    • Better visualisation of lesions
    • Allow lymph node sampling
    • GI tract, liver and pancreas all amenable

Urinary Tract

• Urine samples should be sent to the lab for processing without delay. Cells deteriorate quickly in urine.
• Samples from the bladder and ureteric lesions are received as brushings.
• Urine cytology is one of many tools used to diagnose cancers in the urinary tract, including bladder, kidney, prostate, ureter and urethra cancers.
• However, this test alone can't diagnose cancer.
• Urine cytology does find some cancers, but it's not reliable enough to make a good screening test

Fine Needle Aspirates (FNA) - Aspiration Cytology

• This technique is used for both palpable (can be felt) and non- palpable lesions.
• Fine-needle aspiration biopsies are:
  • Very safe
  • Minor surgical procedures
  • Performed by a thyroid surgeon (usually an ENT) or a radiologist. An ultrasound is often done at the same time
• Consists of: A needle with a syringe is used to remove material from a mass. Smearing it on glass slide. Applying a routine stain. Examine it under the microscope.

Fine Needle Aspirates (FNA) - Common Sites

• Common sites for palpable lesions include:
  • Breast
  • Thyroid
  • Soft tissue
  • Lymph nodes
• Non-palpable lesions:
  • Lesions require the aid of an imaging device eg. MRI or CAT before a needle can be inserted to aspirate a sample.

FNA - Fibroadenoma

• A fibroadenoma is a non-cancerous benign lump that is found in breast tissue. When felt under the skin, the lump may feel rubbery and easily moveable within the breast tissue.
• Fibroadenomas are commonly found in young women during their reproductive years.

FNA - Breast Fibroadenoma

• A sheet of epithelial cells in the typical antler pattern.

Cytocentrifugation

• Specimens with little cell content or small volumes are suitable for this technique
  • Urine
  • Cerebrospinal Fluid
• Concentrate cells within a defined area to enable morphological identification and differential counting.

Changes in Cell Morphology

• Disease causes observable changes in cell morphology. This is the vital function of the job of a histology lab and aids in the diagnosis of disease and development of treatments.

Cell Injury

• Reversible Injury:
  • Swelling of endoplasmic reticulum and mitochondria
  • Membrane blebs
  • Myelin figures
• Irreversible Injury (Necrosis):
  • Breakdown of plasma membrane, organelles, and nucleus; leakage of contents; inflammation
• Apoptosis:
  • Condensation of chromatin
  • Membrane blebs
  • Cellular fragmentation
  • Phagocytosis of apoptotic cells and fragments

Necrosis vs Apoptosis

Definitions:

• KARYOLYSIS: Nuclear fading; chromatin dissolution due to action of DNAases & RNAases

• PYKNOSIS: Nuclear shrinkage; DNA condenses into shrunken basophilic mass

• KARYORRHEXIS: Nuclear dissolution; Nuclear fragmentation; Pyknotic nuclei membrane ruptures & nucleus undergoes fragmentation

• ANUCLEAR NECROTIC CELL

• Morphological characteristics of pyknosis and other forms of nuclear destruction.

Reversible vs Irreversible Cell Injury

• Normal kidney tubules with viable epithelial cells.
• Early (reversible) ischemic injury showing surface blebs, increased eosinophilia of cytoplasm, and swelling of occasional cells.
• Necrotic (irreversible) injury of epithelial cells, with loss of nuclei and fragmentation of cells and leakage of contents.

Squamous Metaplasia

• Columnar cells replaced by squamous cells
• Occurs in the cervix and respiratory tract
• Squamous metaplasia is the ground of the cancer and NOT pre-cancer

Apoptotic Cells

• The nuclei of apoptotic cells show various stages of chromatin condensation and ultimately, karyorrhexis