Laboratory techniques in pathology

Tissue Processing - From Biopsy to Slide

• Goal: To preserve tissue structure and prepare it for microscopic examination.
• Process: A sequential replacement of fluids within the tissue.

• Fixation: 10% neutral buffered formalin is standard; prevents autolysis.
• Dehydration: Ascending grades of alcohol remove water.
• Clearing: Xylene removes alcohol, making tissue translucent.
• Embedding: Infiltration with molten paraffin wax, which then solidifies into a block.
• Sectioning: Microtome cuts sections at 4-5 micrometers.

⭐ High-Yield: Fixation artifacts are a major pitfall. Inadequate or delayed fixation can cause nuclear bubbling and cell shrinkage, mimicking pathological changes and confounding diagnosis.

Histological Stains - The Colour of Disease

• Hematoxylin & Eosin (H&E): The foundational stain.

  • Hematoxylin (base): Stains acidic structures (e.g., DNA, RNA in nucleus) blue (basophilic).
  • Eosin (acid): Stains basic structures (e.g., cytoplasm, collagen) pink (eosinophilic).

• Special Stains:

  • Periodic Acid-Schiff (PAS): Stains glycogen, fungi, and basement membranes magenta.
  • Prussian Blue: Stains iron (hemosiderin) blue.
  • Masson Trichrome: Stains collagen blue/green, distinguishing it from muscle/keratin (red).
  • Congo Red: Stains amyloid salmon-pink.
  • Silver Stains (GMS): Stains fungi and reticular fibers black.

⭐ Under polarized light, Congo Red-stained amyloid exhibits classic apple-green birefringence, a pathognomonic finding for amyloidosis.

Immunohistochemistry (IHC) - Antibody Power‑Plays

• Principle: Utilizes specific antibody-antigen binding to visualize proteins in tissue sections. A primary antibody targets the antigen, and an enzyme-conjugated secondary antibody binds to the primary one. Addition of a chromogen results in a colored precipitate at the antigen's location.

• Key Markers & Associated Tumors:
  • Cytokeratin: Epithelial cells (Carcinomas)
  • Vimentin: Mesenchymal cells (Sarcomas)
  • Desmin: Muscle (Rhabdomyosarcoma)
  • GFAP: Glial cells (Astrocytoma)
  • S-100: Neural crest origin (Melanoma, Schwannoma)
  • CD markers: Hematolymphoid cells (Leukemias, Lymphomas)

⭐ Exam Favorite: In a poorly differentiated malignant neoplasm, IHC is critical. A tumor positive for Cytokeratin but negative for Vimentin is a Carcinoma, not a Sarcoma.

Molecular Techniques - Decoding the Cell

• Polymerase Chain Reaction (PCR): Exponentially amplifies specific DNA segments. Essential for detecting low-titer infections (e.g., HIV), genetic mutations, and in forensics.
• Blotting Techniques: Used to separate and identify specific molecules.
  • 📌 Mnemonic: Southern → DNA, Northern → RNA, Western → Protein (SNoW DRoP).
• Fluorescence in situ Hybridization (FISH): Fluorescent probes bind to specific DNA sequences on chromosomes. Used to identify aneuploidy (e.g., trisomy 21), translocations (e.g., BCR-ABL), or deletions.
• Microarray: Assesses expression levels of thousands of genes simultaneously. Key for analyzing tumor gene expression profiles.

⭐ A positive ELISA screen for HIV is confirmed with a Western blot, which detects specific viral proteins (e.g., p24, gp41).

High‑Yield Points - ⚡ Biggest Takeaways

• H&E is the cornerstone stain, with hematoxylin staining nuclei blue and eosin staining cytoplasm pink.
• Immunohistochemistry (IHC) uses antibodies to identify specific proteins (antigens), crucial for tumor classification.
• Flow cytometry is essential for leukemia and lymphoma analysis, sorting cells by surface markers.
• Electron microscopy reveals ultrastructural details, key for diagnosing glomerular diseases.
• PCR amplifies nucleic acids to detect infections and gene mutations.
• Frozen sections provide a rapid intraoperative diagnosis, guiding surgical decisions.