Pathology techniques

Summary

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.
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Specimen types
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:
o Shape (e.g., malignant changes in organ shape)
o Color (e.g., atypical color in malignancies or necrosis )

o Structure and consistency (e.g., hard and knotty surfaces in the case
of liver cirrhosis)
o 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

o Removal of a cone-shaped core of tissue

o 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
o Suspicious tissue areas are biopsied or excised surgically.
o Clinical application: e.g., assessment of hysterectomy specimens
or prostatectomy specimens (prostate cancer)
 Endoscopic removal or puncture
o Suspicious tissue areas are biopsied or excised during a diagnostic
endoscopic procedure (e.g., during colonoscopy or gastroscopy).
o 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)
o Swab: e.g., cervical smear during gynecological medical check-ups
o Lavage: e.g., bronchoalveolar lavage (BAL)
o Sputum cytology: e.g., if pneumonia or tuberculosis is suspected
 o Effusion cytology: e.g., for differential diagnosis of pleural
effusion or ascites
o Cytology of cerebrospinal fluid: e.g., for differential diagnosis
and pathogen detection if meningitis is suspected
o Urine cytology: e.g., in cases of suspected transitional cell carcinoma
o Imprints : e.g., to determine skin diseases
 Fine-needle aspiration cytology
o A procedure in which a thin, hollow needle is used to collect a sample of
cells from a lump or mass for analysis.
o 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).
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Fixation

Every microscopic examination is preceded by the processing and preservation of

cells and tissues (embedding and cutting procedures).

 Histopathology
o 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)
o 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]

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Staining methods

Most commonly used stains [5][6][7]

Application Staining color Type of

stain

Routine staining

 Routine histopathological  Blue

Hemato
staining Nucleus, en
o
xylin-
doplasmic
eosin
reticulum, ri
bosomes staining
o Calcium pho (H&E
sphate staining)
 Red: Cellular and
extracellular protein
s like cytoplasm, coll
agen fibers

 Routine cytopathological  Blue Papanic

staining: cervical o Nucleus olaou
carcinoma, dysplasia o Bacteria staining
 Red (PAP
o Cytoplasm a staining)
nd keratin
o Mucus
 Yellow: mucus in
acidic environment
 Green: collagen
 Most commonly used stains [5][6][7]

Application Staining color Type of

stain

Routine staining

 Routine cytopathological  Blue Giemsa

staining: o Intracellular: staining
o Differentiation nucleus
between blood o Bacteria
components o Other basop
o Parasites (e.g., Trypan hilic substrat
osoma, Plasmodium, L es
eishmania)  Red
o Bacteria oIntracellular:
(e.g., Chlamydia, Borre eosinophilic
lia, Helicobacter pylori, cytoplasm,
Rickettsia) mast
cell granules
o Collagen
fibers
o Basal
membrane
o Parasites
 Green: melanin

Special staining

 Differentiation between blood  Blue Pappen

and bone marrow components o Nucleus heim
o Other basop staining
hilic substrat (MGG
es staining)
o Basophilic gr
anules
 Red
o Erythrocytes
o Eosinophilic
granules

 Detection of changes  Red Van

in connective tissue o Connective Gieson
tissue stain
o Collagen
 Black
o Elastic fibers
o Nucleus
 Most commonly used stains [5][6][7]

Application Staining color Type of

stain

Routine staining

 Yellow
o Muscles
o Cytoplasm

 Detection of changes  Red Masson-

in connective tissue o Cytoplasm Goldner
o Erythrocytes staining
o Fibrin
o Osteoid
o Muscles
 Green
o Bones
o Connective
tissue
o Mucus

 Fungi (e.g., aspergillosis)  Blue: nucleus

Periodi
 Parasites  Red
c acid–
 Macrophage staining o Neutral glyc
Schiff
in Whipple disease osaminoglyc
Glycogen storage diseases
reactio
 ans
 Alpha 1 antitrypsin deficiency o Mucopolysa
n (PAS
ccharides
reactio
o Carbohydrat
n)
es
o Glycogen

 Iron detection  Blue: iron in mitoch Prussian

o Siderosis (iron ondria blue
overload)  Red: nucleus reaction
o Alveolar macrophages

 Amyloidosis  Blue: nucleus Congo

 Amyloid deposits  Red: amyloid (β- red
fibrils, green after
polarization)

 Calcification  Red: nucleus Von

 Most commonly used stains [5][6][7]

Application Staining color Type of

stain

Routine staining

 Black: Kossa
calcium phosphate staining

 Acid-fast rods (e.g., Mycobact  Red: high mycolic Ziehl-

eria, Nocardia) acid content Neelsen
 Protozoa (e.g., oocysts stain (ac
of Cryptosporidium) id-fast
stain)

 Acid-fast rods (e.g., Mycobact  Reddish-yellow: hig Auramin

eria, Nocardia) h mycolic e-
acid content rhodami
ne
stain [8]

 Various bacteria  Black: certain Silver

(e.g., Pseudomonas, Leptospir protein functional stain
a, Legionella, H. groups
pylori, Treponema)
 Various fungi
(e.g., Pneumocystis, Coccidioid
es, Cryptococcus, Candida)

 Cryptococcus neoformans  Bright red Mucicar

o Thick cell mine
walls contai stain
ning polysac
charides
o Mucin

 Bacteria (e.g., Treponema  Red Fluoresc

pallidum, Pneumocystis o Tubulin ent
jirovecii) o Mitochondri antibod
 Protozoa (e.g., Giardia, Cryptos a y stain
poridium) o Endoplasmic
reticulum
 Green
o Actin
o Endoplasmic
reticulum
 Most commonly used stains [5][6][7]

Application Staining color Type of

stain

Routine staining

 Blue: nucleus

 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, Helicobact
er pylori, Giemsa).
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Special methods in pathology and molecular biology

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
o 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).
o 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
o Analysis of organelles and accumulated substances within cells with a
higher resolution than light microscopy
o Examples: lesions in the glomerulus (nephrotic syndrome), diagnostic
testing for myopathy
 Enzyme histochemistry: enzyme localization in sections and smears
o Enzyme activity is measured microscopically based on color reactions
and may only be performed on freshly isolated tissue or cells
o Example: detection of acetylcholinesterase activity in Hirschsprung
disease
 Immunochemistry methods [9][10]
o Highly specific, antibody-mediated detection of,
e.g., proteins and polysaccharides.

o The antibodies interact with label and visualize differentiation markers,

therapeutic target proteins, pathogen proteins, or functional proteins.
o Immunohistochemistry: performed on a tissue section
o 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

o 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.
o Hybridization: e.g., fluorescence in situ hybridization (FISH) to
detect chromosome aberrations not detectable by karyotyping
o DNA amplification: e.g., PCR to detect particular viruses
o Microarray: identification of thousands of genes within a tissue sample;
detects differences in the RNA expression profile of various tumors

o DNA sequencing: analysis of nucleic acid sequences (e.g.,

Sanger sequencing method)

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last updated 08/20/2021