Pathology techniques

The primary objective of pathological techniques is the diagnostic classification of pathologically altered tissue (histology) and the assessment of cell morphology (cytology). In addition to post-mortem examination, histological and cytological evaluation of tissue is the main task in pathology. Evaluating tissues and cells with light microscopy requires comprehensive skills in specimen assessment, processing, and preservation. However, an alternative to more traditional macroscopic and microscopic investigation may be found in new procedures that focus on the cellular level.

This article provides an overview of the most common methods of examination and staining in pathology.

Macroscopic examination ^[1]

Processing of every specimen includes:

• Measuring and weighing (e.g., enlarged heart: cardiomegaly; enlarged liver: hepatomegaly)
• Photography (e.g., assessment of the appearance before fixation and staining)
• A description including:
  • Shape (e.g., malignant changes in organ shape)
  • Color (e.g., atypical color in malignancies or necrosis )
  • Structure and consistency (e.g., hard and knotty surfaces in the case of liver cirrhosis)
  • Smell (e.g., discharge of foul-smelling pus is a sign of bacterial infection after the opening of cysts or abscess cavities)

In addition, the degree of penetration, the resection edges, lymph node involvement, and visible metastasis are assessed in the case of tumors.

Microscopic examination

Histopathology

Describes architectural tissue changes; tissue samples are collected using the following techniques:

• Punch biopsy
  • Removal of a cone-shaped core of tissue
  • Clinical applications include:
    • Investigation of palpable breast lumps (breast cancer)
    • Suspicious findings during prostate palpation (prostate biopsy)
    • Space-occupying lesion in liver, kidney, or skin (skin biopsy)
• Intraoperative tissue samples
  • Suspicious tissue areas are biopsied or excised surgically.
  • Clinical application: e.g., assessment of hysterectomy specimens or prostatectomy specimens (prostate cancer)
• Endoscopic removal or puncture
  • Suspicious tissue areas are biopsied or excised during a diagnostic endoscopic procedure (e.g., during colonoscopy or gastroscopy).
  • Clinical applications include:
    • Evaluation of gastric antrum and pylorus specimens (atrophic gastritis)
    • Biopsy specimens of the small intestine (gluten-sensitive enteropathy)
    • Colon polyps
    • Biopsy specimens of the rectal mucosa in cases of amyloidosis

Cytopathology ^[2]

Assesses cells and smaller cell clusters (in particular, cytoplasmic and nuclear changes); cells/specimens are sub-grouped depending on their origin and the collection technique:

• Exfoliative cytology: assessment of desquamated cells (e.g., in body fluids) or mechanically harvested cells (e.g., using a brush or a spatula)
  • Swab: e.g., cervical smear during gynecological medical check-ups
  • Lavage: e.g., bronchoalveolar lavage (BAL)
  • Sputum cytology: e.g., if pneumonia or tuberculosis is suspected
  • Effusion cytology: e.g., for differential diagnosis of pleural effusion or ascites
  • Cytology of cerebrospinal fluid: e.g., for differential diagnosis and pathogen detection if meningitis is suspected
  • Urine cytology: e.g., in cases of suspected transitional cell carcinoma
  • Imprints : e.g., to determine skin diseases
• Fine-needle aspiration cytology
  • A procedure in which a thin, hollow needle is used to collect a sample of cells from a lump or mass for analysis.
  • Examples include thyroid cancer, breast cancer

In cytology, cells are analyzed and sampling is easy and minimally invasive. In histology, tissue is obtained with invasive techniques, but it allows for the assessment of the local spreading of tumor (T stage of TNM score).

Every microscopic examination is preceded by the processing and preservation of cells and tissues (embedding and cutting procedures).

• Histopathology
  • Paraffin sections
    • Used for routine diagnostic testing and to prepare histopathological sections for long-term storage
    • Procedure:
      1. Formaldehyde fixation
      2. Dehydration by exposing the specimens to solutions of increasing alcohol concentration
      3. Alcohol removal (“clearing”) by immersing specimens in xylene or toluene
      4. Embedding of specimen in molten paraffin
      5. Paraffin solidification and section cutting
      6. Section transfer to glass slide
      7. Staining (see below)
  • Frozen sections
    • Used for intraoperative sections and special examinations, e.g., (immuno)histochemistry. ^[3][4]
    • Procedure: quick deep-freezing of specimens, followed by preparation of frozen sections (5–7 μm thick), and staining and microscopic analysis
• Cytopathology: cells are smeared onto a glass slide, followed by alcohol fixation (or fixation spray), and staining ^[2]

• For other staining methods, see: “Gram staining” and ”India ink staining.”

To remember the microorganisms that are visible with Giemsa stain, think of “Ricky’s Little Classmates Tried Playing Boring Helicopter Games” (Rickettsia, Leishmania, Chlamydia, Trypanosoma, Plasmodium, Borrelia, Helicobacter pylori, Giemsa).

See “Laboratory methods” for more information on blotting techniques, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and chromosome testing.

• Flow cytometry: identification, quantification, and sorting of single cells in a sample, based on fluorescent tags
  • Procedure: Fluorophore-conjugated antibodies are used to identify specific elements (cluster of differentiation, interleukins) on the cell membrane and/or intracellularly.
    • The sample containing target cells is mixed with primary or secondary fluorescent-tagged antibodies.
    • A laser beam is projected onto individual cells bound to the fluorescent-tagged antibodies.
    • The intensity and pattern of the reflected light will vary depending on each cell's size, granularity, and protein expression (immunophenotype), and on the fluorophore-conjugated antibody.
    • The collected data is either plotted as a histogram (one measure) or a scatter plot (two measures).
  • Clinical uses
    • Workup of hematologic diseases: leukemia and lymphomas, paroxysmal nocturnal hemoglobinuria, fetomaternal hemorrhage
    • Workup of immunodeficiencies: e.g., quantification of CD4+ T cells in patients with HIV infection
• Electron microscopy
  • Analysis of organelles and accumulated substances within cells with a higher resolution than light microscopy
  • Examples: lesions in the glomerulus (nephrotic syndrome), diagnostic testing for myopathy
• Enzyme histochemistry: enzyme localization in sections and smears
  • Enzyme activity is measured microscopically based on color reactions and may only be performed on freshly isolated tissue or cells
  • Example: detection of acetylcholinesterase activity in Hirschsprung disease
• Immunochemistry methods ^[9][10]
  • Highly specific, antibody-mediated detection of, e.g., proteins and polysaccharides.
  • The antibodies interact with label and visualize differentiation markers, therapeutic target proteins, pathogen proteins, or functional proteins.
  • Immunohistochemistry: performed on a tissue section
  • Immunocytochemistry
    • Performed on cells isolated from the surrounding extracellular matrix
    • Expression profiles are characteristic for particular oncological disorders and provide information about primary tumors, including their potential therapeutic target proteins.
    • Examples
      • Leukocyte markers (e.g., CD20⁺ B lymphocytes) → tumor differentiation, antibody therapy using anti-CD20 (e.g., rituximab)
      • Tumor markers (e.g., PSA, CEA, chromogranin A) → monitoring of various malignancies
  • Additional methods: Western blot and ELISA
    • These methods provide further information about the size and structure of a molecule and may detect smaller amounts of proteins and tissue.
    • Examples of use: HIV testing
• Molecular biological methods: hybridization, DNA amplification, DNA sequencing, and microarray techniques help to detect pathological changes at the genetic level.
  • Hybridization: e.g., fluorescence in situ hybridization (FISH) to detect chromosome aberrations not detectable by karyotyping
  • DNA amplification: e.g., PCR to detect particular viruses
  • Microarray: identification of thousands of genes within a tissue sample; detects differences in the RNA expression profile of various tumors
  • DNA sequencing: analysis of nucleic acid sequences (e.g., Sanger sequencing method)