Methods of Examination of Tissues and Cells

Introduction

Histopathology = study of tissues under the microscope to detect disease.
Cytopathology = study of cells under the microscope for diagnosis.

Both play a crucial role in disease detection, cancer diagnosis, prognosis, and research.

Methods of examination of tissue and cells in a histopathology lab can be divided into two broad categories:

1. Examination of Tissues (Histopathology proper)

2. Examination of Cells (Cytopathology)

 

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Examination of Tissues 

This includes methods where tissue pieces are processed and studied.

A. Biopsy

• Definition: Removal of tissue from a living person for diagnosis.

• Types:

  1. Incisional biopsy – a small part of the lesion is removed.

  2. Excisional biopsy – entire lesion removed (e.g., mole, small tumor).

  3. Needle biopsy – tissue obtained by fine or core needle.

  4. Endoscopic biopsy – sample taken using an endoscope (e.g., gastric, colon).

• Uses: cancer diagnosis, inflammatory diseases, and infections.

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B. Surgical Specimens

• Tissues/organs removed during surgery.

• Example: appendectomy specimen, cholecystectomy, mastectomy.

• Process:

  • Gross examination → size, shape, color, weight, margins.

  • Sampling → representative sections cut.

  • Microscopy after processing and staining.

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C. Autopsy

• Examination of tissues after death.

• Types:

  • Clinical autopsy (cause of death).

  • Medico-legal autopsy (suspicious deaths).

• Provides information on disease progression, response to treatment, genetic disorders.

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D. Tissue Processing

Before microscopic examination, tissue undergoes stepwise preparation:

1. Fixation

   • Preserves tissue, prevents decomposition.

   • Common fixative: 10% formalin.

   • Other fixatives: Bouin’s, Carnoy’s, glutaraldehyde (for EM).

2. Dehydration

   • Tissue passed through increasing grades of alcohol to remove water.

3. Clearing

   • Alcohol replaced with xylene or chloroform.

4. Embedding

   • Tissue infiltrated with molten paraffin wax → solid block formed.

5. Sectioning

   • Thin slices (3–5 µm) cut by microtome.

6. Staining

   • Routine stain: Hematoxylin & Eosin (H&E).

     • Hematoxylin → stains nuclei blue/purple.

     • Eosin → stains cytoplasm pink.

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E. Special Stains

Used when H&E is not sufficient.

• Periodic Acid–Schiff (PAS) → glycogen, mucin.

• Ziehl–Neelsen stain → Mycobacterium tuberculosis.

• Silver stains → fungi, reticulin fibers.

• Congo Red → amyloid (apple-green birefringence in polarized light).

• Masson’s Trichrome → collagen.

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F. Immunohistochemistry 

• Uses antigen–antibody reactions to detect proteins in tissue.

• Example:

  • ER/PR, HER2 in breast cancer (for targeted therapy).

  • Ki-67 (proliferation marker).

• Advantage: provides diagnosis, prognosis, and therapeutic guidance.

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G. Frozen Section 

• Tissue rapidly frozen in cryostat, sectioned, stained, and examined.

• Provides quick diagnosis during surgery.

• Example: checking tumor margins in breast cancer surgery.

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H. Electron Microscopy 

• Provides ultrastructural details at very high magnification.

• Used in:

  • Renal biopsies (glomerular diseases).

  • Muscle biopsies.

  • Viral identification.

 

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Examination of Cells 

Here, individual cells or clusters are studied without intact tissue architecture.

A. Exfoliative Cytology

• Cells naturally shed or mechanically scraped.

• Most famous: Pap smear (cervical cancer screening).

• Also: sputum cytology, urine cytology.

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B. Fine Needle Aspiration Cytology 

• Technique: Fine needle (22–25G) inserted into lump/swelling, cells aspirated, smeared on slide, stained.

• Stains: May–Grünwald Giemsa (MGG), Papanicolaou.

• Advantages: Quick, cheap, outpatient procedure, minimal risk.

• Uses: breast lump, thyroid nodule, lymph node, salivary gland, soft tissue swelling.

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C. Body Fluids Cytology

• Examining cells in:

  • Pleural fluid

  • Peritoneal/ascitic fluid

  • Cerebrospinal fluid (CSF)

  • Synovial fluid

• Detects infections (TB, bacteria), malignancy (metastatic cells).

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D. Imprint Cytology (Touch Prep)

• Fresh tissue touched on slide → cell imprint → stained and studied.

• Rapid diagnosis in breast, thyroid, lymph nodes.

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E. Cytochemistry

• Uses chemical stains to highlight cell contents.

• Example:

  • Sudan Black → lipids (used in leukemias).

  • PAS → glycogen.

  • Peroxidase stain → myeloid cells.

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F. Flow Cytometry

• Cells in suspension are passed through laser beam → fluorescent antibodies used to detect cell markers.

• Provides immunophenotyping.

• Uses:

  • Leukemia & lymphoma classification.

  • Detection of minimal residual disease.

 

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Comparison Table: Tissue vs. Cell Examination