Pathology techniques

Last updated: August 15, 2022

Summarytoggle arrow icon

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.

Specimen typestoggle arrow icon

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


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

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:

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).

Fixationtoggle arrow icon

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]

Staining methodstoggle arrow icon

Most commonly used stains [5][6][7]
Type of stain Staining color Application
Routine staining
Hematoxylin-eosin staining (H&E staining)
  • Routine histopathological staining
Papanicolaou staining (PAP staining)
Giemsa staining
Special staining
Pappenheim staining (MGG staining)
  • Differentiation between blood and bone marrow components
Van Gieson stain
Masson-Goldner staining

Periodic acid–Schiff reaction (PAS reaction)

Prussian blue reaction
Congo red
Von Kossa staining
  • Calcification
Ziehl-Neelsen stain (acid-fast stain)
Auramine-rhodamine stain [8]
Silver stain
  • Black: certain protein functional groups
Mucicarmine stain
Fluorescent antibody stain

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).

Special methods in pathology and molecular biologytoggle arrow icon

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

Referencestoggle arrow icon

  1. Raju K. Evolution of Pap stain. Biomed Res Ther. 2016; 3 (2).doi: 10.7603/s40730-016-0006-8 . | Open in Read by QxMD
  2. Kempf W, Hantschke M, Kutzner H, Burgdorf WHC. Dermatopathology. Springer Science & Business Media ; 2008
  3. Histology Stains. Updated: December 10, 2018. Accessed: December 10, 2018.
  4. Annam V, Kulkarni MH, et al. Comparison of the modified fluorescent method and conventional Ziehl–Neelsen method in the detection of acidfast bacilli in lymphnode aspirates. Cytojournal. 2009; 6 (13).doi: 10.4103/1742-6413.53887 . | Open in Read by QxMD
  5. Geller SA, Horowitz RE. Gross examination. Methods Mol Biol. 2014; 1180: p.3-19.doi: 10.1007/978-1-4939-1050-2_1 . | Open in Read by QxMD
  6. Al-Abbadi MA. Basics of cytology. Avicenna J Med. 2011; 1 (1): p.18-28.doi: 10.4103/2231-0770.83719 . | Open in Read by QxMD
  7. Buchwalow IB, Böcker W. Immunohistochemistry: Basics and Methods. Springer Science & Business Media ; 2010
  8. Jaafar H. Intra-operative frozen section consultation: concepts, applications and limitations. Malays J Med Sci. 2006; 13 (1): p.4-12.
  9. Meier-Ruge WA, Bruder E. Current concepts of enzyme histochemistry in modern pathology. Pathobiology. 2008; 75 (4): p.233-243.doi: 10.1159/000132384 . | Open in Read by QxMD
  10. Matos LL, Trufelli DC, de Matos MG, da Silva Pinhal MA. Immunohistochemistry as an important tool in biomarkers detection and clinical practice. Biomark Insights. 2010; 5: p.9-20.
  11. Kumar V, Abbas AK, Aster JC. Robbins & Cotran Pathologic Basis of Disease. Elsevier Saunders ; 2014
  12. Goljan EF. Rapid Review Pathology. Elsevier Saunders ; 2018

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