Methods of Examination of Tissues and Cells

Ms. Sangeeta Gourh

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)

 

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.

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.

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.

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.

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.

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.

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.

H. Electron Microscopy 

  • Provides ultrastructural details at very high magnification.

  • Used in:

    • Renal biopsies (glomerular diseases).

    • Muscle biopsies.

    • Viral identification.

 

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.

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.

C. Body Fluids Cytology

  • Examining cells in:

    • Pleural fluid

    • Peritoneal/ascitic fluid

    • Cerebrospinal fluid (CSF)

    • Synovial fluid

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

D. Imprint Cytology (Touch Prep)

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

  • Rapid diagnosis in breast, thyroid, lymph nodes.

E. Cytochemistry

  • Uses chemical stains to highlight cell contents.

  • Example:

    • Sudan Black → lipids (used in leukemias).

    • PAS → glycogen.

    • Peroxidase stain → myeloid cells.

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.

 

Comparison Table: Tissue vs. Cell Examination

Feature Tissue (Histopathology) Cells (Cytopathology)
Sample Biopsy, surgical specimen FNAC, Pap smear, body fluids
Architecture seen? Yes (tissue pattern preserved) No (only single cells)
Processing time Long (fixation, embedding, sectioning) Short (smear, stain, examine)
Common stains H&E, special stains, IHC Pap, MGG, cytochemistry
Use Tumor typing, staging, prognosis Screening, rapid diagnosis
Example Breast carcinoma biopsy Breast lump FNAC