Special stains-
histochemistry
Introduction and overview of special stains
and histochemical methods
In the histopathology laboratory, the term “routine staining” refers to the hematoxylin and eosin stain (H&E)
that is used “routinely” with all tissue specimens to reveal the underlying tissue structures and conditions.
The term “special stains” has long been used to refer to a large number of alternative staining techniques that
are used when the H&E does not provide all the information the pathologist or researcher needs
Histochemistry and cytochemistry, which is a field to localize chemical components of cells and tissues on
histological sections by using various techniques and analyze the functions based on morphology. They
applied for a single purpose
Normal skin Verhoff’s for elastic fiber Masson’s trichrome for collagen Masson fontana
Histochemistry is a combination of chemistry and histology, in which reactions are carried out on tissue
sections or similar preparations and the results examined under a microscope, with the object of
combining the advantages of chemical or biochemical specificity and histological localization
Routine Special
H& E Histochemistry
Enzyme histochemistry
Immunohistochemistry
In situ hybridization
Genetic testing and cytogenetics
The term “special stains” is of uncertain provenance, but one can be certain that it began to be used after
1876 when H&E was introduced
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The histochemical techniques are based on biochemical reactions which make it possible to reveal in
situ, various constituents .
Lists of target substances to be demonstrated by histochemical
• nucleic acid
• Carbohydrates
• Amyloid
• Lipids
• Connective tissue fibers
• Pigments
• Microorganisms
• Enzyme histochemistry
• Neuroendocrine system
After a tissue specimen has been examined with Haematoxylin and Eosin, a special stain is applied to
a sample for a more in–depth evaluation and allow target substances and foreign elements to be
identified. This includes components in tissue sections, based on their: chemical, biological and
pathological character for example; lipids, calcium, carbohydrates, nerve fibres and fungi to name a
small few.
Requirements of histochemistry
• Specificity
• Sensitivity
• Reproducibility
• Reaction deposits
• Fixation
• Method of preparation
Specificity- selectivity
The ability to discriminate between individual tissue components and to colour one or few of these
leaving the other unstained
Sensitivity
The capacity of the stain to demonstrate a tissue substance in low concentration
A satisfactory staining method is one which combined high specificity and high sensitivity
Diagnostic Specificity
Diagnostic Specificity is the ability of a test to correctly exclude individuals who do not have a given disease or
disorder. For example, a certain test may have proven to be 90% specific. If 100 healthy individuals are tested
with that method, only 90 of those 100 healthy people will be found to be not to have the disease (“normal”).
The other 10 people also do not have the disease, but their test results seem to indicate they do. For that 10%
their “abnormal” findings are a misleading false-positive result.
Diagnostic Sensitivity
Diagnostic Sensitivity is the ability of a test to correctly identify people who have a given disease or disorder. For
example, a certain test may have proven to be 90% sensitive. That is, if 100 people known to have a certain
disease are tested with that method, the test will correctly identify 90 of those 100 cases of disease. The other
10 people who were tested also have the disease but the test will fail to detect it. For that 10%, the finding of a
“normal” result is a misleading false-negative result. A test’s sensitivity becomes particularly important when you
are seeking to exclude a dangerous disease
Reproducibility is the ability of an assay to provide consistent results (when testing the same samples) in
different laboratories
The capacity of an assay to remain unaffected by minor alterations to test protocols that may occur over
time within a single laboratory or when deployed into many laboratories
• Reaction deposits
1Presentation of Normal Chemical Distribution: The substance being analyzed must not diffuse away
from its original site.
2Presentation of Normal Chemical Composition: The procedure must not block or denature the
reactive chemical groups being analyzed, or change normally non reactive groups into reactive groups.
3Detectability of the Reaction Product: The reaction product should be colored or electron scattering,
so that it can be visualized easily with a light or electron microscope.
Fixation
Each materials has suitable fixative so the material under investigation should be well-preserved
Aspect H&E Special stains
Primary interest Nucleus and Nucleus and
cytoplasm cytoplasm
Medical diagnosis Mostly the diagnosis
of infectious agents
Special stains can answer these questions: and cancer based on
• chemical
Is a certain class of molecules present or absent? composition
• Where are the molecules located in the
Bases of Morphology Morphology/colour
preparation? interpretation
• How many of the molecules are Quantitative No As needed
present? Control needed No Yes
Frequency of use Routine As needed
Substrate specific No Yes
Quality control and quality management and accreditation
Generally, when special staining is performed on a section of tissue, a positive control section is also
stained. This involves simultaneous staining of a section of tissue known to contain the substance in
question. This will be given to the pathologist or researcher at the same time as the test tissue section,
and serves various purposes. In particular, it helps to determine whether the special stain technique is
working, and also serves as a reference of what the substance should look like in the test tissue section.
• Method of preparation and technical aspects
biological stain commission
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