8. CYTOLOGY

LECTURE 8: CYTOLOGY

Introduction to Cytology

  • Definition: Cytology is the microscopic examination of a single cell type for the diagnosis of diseases, particularly cancer.

  • Nature of Procedure: It is typically pain-free and non-invasive.

  • Collecting Sites: Specimens can be obtained from various body sites and are categorized as gynecologic or non-gynecological.

  • Collection Responsibility: Collection is not performed by lab staff; the specimen is brought to the lab for cytopreparation.

Gynecological Samples

  • Origins: Specimens are collected from the female genital tract, which includes:

    • Cervix

    • Endocervix

    • Vagina

  • Common Test: Known as the “PAP” (Papanicolaou) test.

  • Additional Testing: Possibility of performing further tests such as:

    • HPV (Human Papillomavirus)

    • Chlamydia

    • Trichomoniasis

  • Purpose: The PAP test is primarily performed for cervical cancer screening.

    • Identifies precancerous cervical cell changes; treatment can prevent the development of cervical cancer.

PAP Tests

  • Cell Types:

    • Normal Cells: Standard features are to be examined.

    • Abnormal Cells: Indicate potential diseases or conditions.

Non-Gynecological Samples

  • Includes various fluids and aspirates such as:

    • Urine

    • Sputum

    • CSF (Cerebrospinal Fluid)

    • Bronchial, esophageal, and gastric washings or brushings

    • Pleural, peritoneal, ascitic, and pericardial fluids

    • Fine needle aspirations (FNA)

Cytology vs. Histology

  • Cytology: Focus on suspended cells, examining:

    • Individual cells.

    • Their nuclei and cytoplasm.

  • Histology: Involves the architecture of entire tissues encompassing multiple types of cells.

Cytopreparation

  • Definition: The act of preparing and staining microscopic slides for diagnostic examination.

  • Critical Role: Essential for the final diagnosis; negligent preparation can lead to false positives or negatives.

  • Involves: Various procedures, including:

    • Cell preparation

    • Fixation

    • Smear preparation

    • Staining

Specimen Collection

  • Nongynecologic Cytology:

    • Collected fresh, without fixative or additive, and transported to the lab quickly.

    • Body fluids (Pleural, peritoneal, pericardial, & ascitic) should send the entire amount collected.

    • Some labs add heparin to prevent clotting.

  • CSF Collection:

    • Cells in CSF deteriorate rapidly, requiring immediate laboratory analysis.

    • Direct scrapings for viral lesions (Tzanck smears) must be prepared on-site.

  • Washings (Bronchial, Esophageal & Gastric):

    • No fixative should be added; refrigeration until lab receipt is required.

  • Brushings:

    • Direct smears prepared at collection must be fixed immediately.

  • Urine:

    • Avoid first morning urine as it leads to degradation of cells in bladder.

  • Fine Needle Aspiration:

    • Slides should be prepared at aspiration time.

Specimen Stability

  • Most specimens require fixation immediately post-collection to prevent deterioration.

  • Pre-fixation:

    • Used when transportation or immediate processing is not feasible.

    • Involves solutions with low alcohol content to stabilize cells temporarily.

Fixation

  • Role: Rapid fixation in alcohol is crucial for cytologic interpretation accuracy.

  • Preferred Fixative: 95% ethyl alcohol (ethanol) is used for all cytology specimens due to its ability to preserve chromatin patterns.

  • Effects of Delay: If fixation is delayed,

    • Cells may air-dry, losing morphological detail.

    • Cells exhibit rounding, becoming smaller and denser.

    • Thin-layer specimens that need pre-fixing while still liquid:

    • Must be performed to maintain cellular integrity.

Fixation Solutions

  • Spray Fixation: Contains a mix of alcohol, acetone for fast drying, and polyethylene glycol for a protective layer.

    • Must eliminate the thin coating before staining.

  • Volume Requirements: Fixative volume should equal specimen volume.

Specimen Preparation

  • Cytology specimens typically arrive as cells suspended in fluids such as pericardial or ascitic fluids.

  • Concentration Step: Cells are concentrated by centrifuging at 2000 RPM for 10 minutes, decanting the supernatant to leave a cell pellet (cell button).

Smear Preparation Methods

  • Direct Smears: Includes methods like:

    • Nickel Method: Spreading specimen in circular patterns.

    • Avoid using cotton swabs for collection.

  • Pull Apart Method: Suitable for non-mucous fluids, utilizing two slides to allow natural spreading of sediment.

  • Crush Method: Used for mucoid specimens (e.g., thick sputum), involves mashing between slides.

Cytocentrifuge Preparation

  • Ideal for sparsely cellular specimens, allowing for direct deposition of cells onto slides during centrifugation.

Cell Block Preparation

  • A method designed to process cytology material for histological sectioning, involving:

    • Centrifuging specimens and adding plasma or thrombin.

    • Eosin added for better cellular visibility.

    • Processed like a histology specimen, including embedding and staining.

Papanicolaou Stain (PAP Stain)

  • Purpose: Primarily for cervical cancer screening; also applicable to non-gynecological specimens to evaluate cellular health and disease.

  • Components of PAP Stain:

    • Hematoxylin: Nuclear stain that provides crisp nuclear detail; it binds to the sulphate groups in DNA.

    • OG-6: Acidic cytoplasmic stain highlighting keratin in orange while being bound by phosphotungstic acid.

    • EA (Eosin & Light Green):

    • Eosin Y: Stains superficial squamous cell cytoplasm and various other cells.

    • Light Green: Stains metabolically active cells with intermediates.

Diff-Quik Romanowsky Stain

  • Function: Enhances cytoplasmic detail, highlighting various cellular and extracellular elements.

  • Components:

    • Diff-Quik Fixative Reagent

    • Diff-Quik Solution I

    • Diff-Quik Solution II

Staining Techniques

  • Manual Staining: Slides are moved manually through stains in a specific order.

  • Automated Staining: Slides are processed via automated machinery programmed for staining sequences.

Cross Contamination Risks

  • Introduction: Due to the loose nature of cytology specimens, cell detachment between slides can lead to false diagnoses.

  • Preventative Measures: High-risk specimens should be handled separately; a blue wet film can be used to assess cross-contamination potential.

Slide Storage and Filing

  • Slides are organized and stored by their cytology number and date for effective tracking.