• Clinical science

Pathology techniques

Abstract

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 learning card provides an overview of the most common methods of examination and staining in pathology.

Specimen types

Macroscopic examination

Processing of every specimen includes:

  • Measuring and weighing (e.g., enlarged heartcardiomegaly, enlarged liverhepatomegaly)
  • Photography
  • 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

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).

Fixation

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: formaldehyde fixationdehydration by exposing the specimens to solutions of increasing alcohol concentration → alcohol removal (“clearing”) by immersing specimens in xylene or toluene → embedding of specimen in molten paraffinparaffin solidification and section cutting → section transfer to glass slide → staining (see below)
    • Frozen sections: Used for intraoperative sections and special examinations, e.g., (immuno)histochemistry.
      • Procedure: quick deep-freezing of specimens → preparation of frozen sections (5–7μm thick, including staining and microscopic analysis, must be performed in a cooling chamber (= cryostat) at -18°C (-0.4°F)
  • Cytopathology
    • Smears: cells are smeared onto a glass slide → alcohol fixation (or fixation spray) → staining

Staining methods

Routine stainings

Special stainings

Staining Blue Red Black Yellow Green Application
Routine staining Hematoxylin-eosin staining (H&E staining)
  • Routine histopathological staining
Papanicolaou staining (PAP staining)
  • Nucleus
  • Bacteria
  • Cytoplasm and keratin
  • Mucus
  • Mucus in acidic environment
Giemsa staining
  • Intracellular: nucleus
  • Bacteria
  • Other basophilic substrates

Routine cytopathological staining:

  • Differentiation between blood components
  • Parasites (Trypanosoma cruzi), protozoa (malaria, leishmaniasis)
Special staining Pappenheim staining (MGG staining)
  • Nucleus (blue-violet)
  • Other basophilic substrates
  • Basophilic granules
  • Differentiation between blood and bone marrow components
Van Gieson's stain
  • Muscles
  • Cytoplasm
Masson-Goldner staining

Periodic acid–Schiff reaction (PAS reaction)

Nucleus

Prussian blue reaction Iron in mitochondria
  • Nucleus

Iron detection

Congo red Nucleus
  • Amyloid (β-fibrils, green after polarization)
Von Kossa staining
  • Nucleus
Calcium phosphate
  • Calcification
Ziehl-Neelsen stain (acid-fast stain)

Examples

H&E staining

Giemsa staining

Pappenheim staining

Van Gieson's staining

PAS reaction

Prussian blue reaction

Congo red

References:[1][2][3][4]

Special methods in pathology and molecular biology

See the learning card on laboratory methods for more information on blotting techniques, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and chromosome testing.

  • 1. Barone J, Castro MA. Kaplan USMLE Step 1 Lecture Notes 2016. New York, NY, USA: Kaplan Medical; 2016.
  • 2. Raju K. Evolution of Pap stain. Biomed Res Ther. 2016; 3(2). doi: 10.7603/s40730-016-0006-8.
  • 3. Kempf W, Hantschke M, Kutzner H, Burgdorf WHC. Dermatopathology. Springer Science & Business Media; 2008.
  • 4. Hill M. Histology Stains. https://embryology.med.unsw.edu.au/embryology/index.php/Histology_Stains. Updated December 10, 2018. Accessed December 10, 2018.
  • Kasper DL, Fauci AS, Hauser SL, Longo DL, Lameson JL, Loscalzo J. Harrison's Principles of Internal Medicine. New York, NY: McGraw-Hill Education; 2015.
  • Jaafar H. Intra-operative frozen section consultation: concepts, applications and limitations. Malays J Med Sci. 2006; 13(1): pp. 4–12. pmid: 22589584.
  • Meier-Ruge WA, Bruder E. Current concepts of enzyme histochemistry in modern pathology. Pathobiology. 2008; 75(4): pp. 233–243. doi: 10.1159/000132384.
  • Kumar A, Galaev IY, Mattiasson B. Cell Separation. Springer; 2007.
  • Al-Abbadi MA. Basics of cytology. Avicenna J Med. 2011; 1(1): pp. 18–28. doi: 10.4103/2231-0770.83719.
  • Buchwalow IB, Böcker W. Immunohistochemistry: Basics and Methods. Springer Science & Business Media; 2010.
  • Kumar V, Abbas AK, Aster JC. Robbins & Cotran Pathologic Basis of Disease. Philadelphia, PA: Elsevier Saunders; 2014.
  • Geller SA, Horowitz RE. Gross examination. Methods Mol Biol. 2014; 1180: pp. 3–19. doi: 10.1007/978-1-4939-1050-2_1.
  • Howat WJ, Wilson BA. Tissue fixation and the effect of molecular fixatives on downstream staining procedures. Methods. 2014; 70(1): pp. 12–19. doi: 10.1016/j.ymeth.2014.01.022.
  • Goljan EF. Rapid Review Pathology. Philadelphia, PA: Elsevier Saunders; 2018.
  • Datta BN. Textbook of Pathology. Jaypee Brothers Medical Publishers; 2004.
  • Gray F, Duyckaerts C, De Girolami U. Escourolle & Poirier's Manual of Basic Neuropathology. Oxford University Press; 2013.
  • 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: pp. 9–20. pmid: 20212918.
last updated 12/14/2018
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