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 diagnostic cytology.
• The Papanicolaou stain is the 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

• 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 Handling 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 should be used for high-risk specimens.
• Lids must be tight, and containers placed into sealable plastic bags.

Cell Collection Types

• Exfoliative cytology
• Abrasive cytology
• Intervention cytology/ aspiration cytology

Exfoliative Cytology (Natural Spontaneous Exfoliation)

• Based on spontaneous shedding of cells derived from the lining of an organ into a cavity.
• 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, enriching 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 Smears

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

Fixation of Cytological Specimens

• Alcohol Fixation:
  • 95% ethyl alcohol is used for most routine preparations.
  • Conventional pap smear requires 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.
• Cells are rinsed into a container with fixative.
• At the lab, the cells are transferred to a glass slide in a thin even layer

The ThinPrep Pap test:

• A type of liquid-based cytology (LBC).
• 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 to highlight cellular details.
• Stains nucleus and cytoplasm to help 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 PAP Test

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

Abnormal Result: 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.

Reactive Changes

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

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 Results

• 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, CIS, ASC, AGC

• 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.

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 the specimen is hazardous and requires fixation.
• Bronchial brushings can be taken for analysis using a small nylon brush via a bronchoscope.

Gastrointestinal Tract

• Specimens include:
  • Scrapings from oral and laryngeal lesions
  • Brushings using an endoscope from the esophagus and stomach.
  • Transmucosal FNA (Fine Needle Aspiration)
  • Endoscopic Ultrasound (EUS) guided
    • Better visualization 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
  • Apply a routine stain
  • Examine it under the microscope

Common Sites for FNA

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

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.

Necrosis vs. Apoptosis

• Key differences in cell size, nucleus, plasma membrane, cellular contents, and adjacent inflammation.
• Necrosis: Enlarged cell size, disrupted plasma membrane, frequent inflammation, and invariably pathologic.
• Apoptosis: Reduced cell size, intact plasma membrane, no inflammation, and often physiologic.

• Pyknosis: Nuclear shrinkage, DNA condenses into shrunken basophilic mass
• Karyorrhexis: Nuclear dissolution, Pyknotic nuclei membrane ruptures & nucleus undergoes fragmentation
• Karyolysis: Nuclear fading, chromatin dissolution due to action of DNAases & RNAases

Squamous Metaplasia

• Columnar cells are replaced by squamous cells.
• Occurs in the cervix and respiratory tract.
• Squamous metaplasia is the ground of cancer and NOT pre-cancer.
• The nuclei of apoptotic cells show various stages of chromatin condensation and ultimately, karyorrhexis.