Diagnostic Tools in Pathology and the Principles of Pathology Notes

Patients typically present with symptoms that suggest a potential diagnosis, such as the presence of a mass.
The pathology laboratory examines specimens or biopsy samples for cancer cells to confirm a diagnosis and monitor the progress of treatment.
A comprehensive diagnosis is achieved by combining several factors:
- Clinical history: For example, a patient's smoking history.
- Physical examination: For example, the presence of swollen lymph nodes.
- Pathology investigations: Including blood counts and molecular tests to identify specific diseases.
Diagnostic tools involve examining tissue samples (biopsies) and performing gross pathology.

Tissue processing is essential for several reasons:
- Prevention of autolysis of the tissue.
- Hardening of the tissue, which facilitates section cutting.
- Acting as a mordant to facilitate staining reactions.
- Removal of water from the tissue (dehydration) to allow for embedding the tissue in paraffin wax.

These techniques are fundamental for histopathologic diagnosis and involve using different stains to color cells, tissues, and cell components.
Hematoxylin and Eosin (H&E) Stain:
- This is the gold standard method for most histopathologic diagnoses.
- It has been the primary diagnostic and investigative technique in pathology for over $100$ years.
- Hematoxylin: A natural blue dye that primarily stains nuclei, revealing general tissue morphology.
- Eosin: A red dye that stains cytoplasm, favored for its ease of use and strong contrast with the blue color of hematoxylin.
- Example application: In a normal human smooth muscle longitudinal section, fibers show central nuclei clearly via H&E staining.

Periodic acid Schiff (PAS Stain):
- Stains structures with high carbohydrate content.
- Includes glycogen, mucin, hyaluronic acid, colloid droplets, and the hyaline found in arteriosclerosis.
Sudan Black / Oil Red O:
- Used for the demonstration of fat.
- Oil Red O Results: Fat appears bright red; nuclei appear blue.
- Sudan Black Results: Fat appears black.
Masson’s Trichrome Stain:
- Used to differentiate between collagen and muscle fibers.
- Results: Nuclei are black; collagen and keratin are blue; muscle is red.
Van Gieson’s Stain:
- Used to differentiate collagen from other tissue elements.
- Results: Collagen is red; muscle and cornified epithelium are yellow; nuclei are blue to black.

Process: Fresh tissue is rapidly frozen, turning internal water into ice. The ice acts as the embedding medium, making the tissue firm enough for sectioning.
Advantages:
- It is a quick diagnostic procedure.
- Essential for demonstrating lipids (which dissolve in routine paraffin processing).
- Minimum amount of shrinkage occurs compared to paraffin wax techniques.
- Practically every staining method can be performed.
Disadvantages:
- Section thickness is typically $7-10$ \, $\mu m$ , and thinner sections are difficult to achieve.
- Correlation of serial sections is practically impossible.
- Interpretation requires a very well-trained pathologist.
Equipment types:
- Freezing Microtome: Utilizes carbon dioxide ( $CO_2$ ) gas; a popular method for many years.
- Refrigerated Microtome (Cryostat): Uses standard rotary microtomes or Standard Cambridge rockers within a refrigerated chamber.

Key Difference: Histopathology is the study of diseased tissue, while cytology is the study of individual cells.
Cytology Types:
- Fine Needle Aspiration (FNA): Can be direct or image-guided.
- Exfoliative Cytology: Used for the cervix, endometrial carcinoma, bronchogenic carcinoma, bladder tumors, prostate tumors, and gastric carcinoma.
Biopsy Types:
- Incisional Biopsy: Removal of a small portion of a lesion or mass. Used for large or complex lesions when complete removal is not feasible (e.g., suspected cancers).
- Excisional Biopsy: Removal of the entire lesion or mass for diagnostic and therapeutic purposes. Used for small, localized lesions where complete removal is possible.

Classical histopathology provides direct visual interpretation and data for diagnosis and management.
Additional tests identify specific markers within samples:
- Simple histochemistry.
- Immunohistochemistry (IHC).
- Molecular testing of nucleic acids (DNA, RNA) and proteins.
Molecular pathology assists in the diagnosis and prognosis of human disease states, evolving from single-gene tests to comprehensive gene panels.

Diagnostics move from general to specific: H&E → Immunohistochemistry → Molecular and cytogenetic diagnosis.
Immunohistochemistry Examples (Breast Cancer):
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Molecular/Genetic Examples:
- $BRAF(V600E)$ mutations found in Melanoma, Colon adenocarcinoma, Papillary thyroid carcinoma, Hairy cell leukemia, and Langerhans cell histiocytosis.
- Targeted treatments like PLX4032 (Vemurafenib) specifically target these mutations.

Testing evolution: From single markers/mutations to single genes, gene panels, exome sequencing, and whole-genome sequencing.
Old vs. Modern Assays:
- Limited Assays (IHC, FISH, Allele-specific PCR): Highly specific but provide limited information and require multiple tests.
- NGS: A massively parallel sequencing technology that offers ultra-high throughput.
NGS Functionality: Determines the order of nucleotides in entire genomes or targeted regions. Bioinformatics is used to map individual reads back to the human reference genome.