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Enzyme Histochemistry Enzyme Preservation

Introduction Fixation
o Fixative should be at 4C for
Enzymes are large tertiary the shortest time.
proteins that acts as catalyst and are Formol Calcium
vital in cellular metabolism while o Recommended for tissue
maintaining hemostasis in man. blocks
Histochemistry is defined as the o Help maintain cell membrane
identification, localization and integrity
qualification in cells and tissue and o Used on smears
by chemical or physical test of o Cryostat Sections
specific substances, reactive groups Buffered Formalin or Formal
and enzyme catalyzed substance. Saline
Enzyme Histochemistry o Satisfactory in most cases
(commonly used)
This technique produces a final Acetone, Formalin Vapour and
coloured reaction product that is Formalin-Alcohol Mixtures
localized to the cells with an intensity
proportional to the enzyme activity.
Applying on enzyme detection Principle of Enzyme Histochemistry
system to tissue section or smears.
Histochemistry procedures are
Classification based on the simple premise that
tissue cells, when placed in a
Classified according to their effect
solution chemically react with the
on the substrate:
solution to produce a colored
o Oxidoreductases
insoluble product.
The most important
groups to the The amount and location of the end-
diagnostic enzyme product can then be evaluated in the
histochemist. context of the cells or tissue.
Known as oxidases Classical histochemical reaction and
and dehydrogenases. generally based on one of the FOUR
o Transferases PRINCIPLES:
o Hydrolases
Oxidative and 1. Simple ionic interactions
hydrolytic enzymes 2. Reactions of aldehydes with
o Lyases schiffs reagent or silver
o Isomerases compounds
o Ligases 3. Coupling of aromatic
diazonium salts with aromatic
Activators residues on protein
4. Conversion acting on a
Ions of Calcium
substrate to form colored ppt.
Magnesium
Manganese
Sodium
Potassium
Types of Histochemical Reactions Self-coulered Substrate

Simultaneous capture Technique:


Post incubation coupling o
Self-coloured substrate
Coloured Solube Substrate
Intramolecular Rearrangement
Simultaneous Capture

Most important technique Enzyme


Principle:
o Gomoris metal ppt.
Solube
technique Hydrolysis Grouping
o Azp dye method
remove
Technique:
o Enzyme + Substrate +
Primary rxn product +
Diazonium salt + Final rxn Coloured Insolube PRP
product (FRP)
Disadvantages: Avantages:
o Diffusion of FRP o Diazonium coupling not
o Rate of hydrolysis of required
substrate
o Diffusion coefficient of the
PRP for the buffer
Intramolecular Rearrangement
o Rate of coupling of the PRP
and diazo salt Technique:
o Enzyme salt and enzyme o
same PH Soluble substrate
Post Incubation Coupling Technique
Hydrolysis
Technique:
o Enzyme + Substrate + PRP +
Insoluble and remain at site Molecular
of production for initial rearrangement in
incubation -> FRP in Spatial structure
substrate medium
Advantages:
o Incubation Stage
o Optimum PH
Coloured insoluble rxn product
Enzyme
Diazonium Salt
PRP
Disadvantages:
o PRP is not completely
insoluble
o Diffusion
Factors Influence the Reaction Diazonium salts or freshly
hexazotized pararosaniline are used
pH
to react with the napthol group to
o Maintained
form a coloured insoluble dye.
Concentration
o Inhibit the enzyme activity Tetrazolium Salt Method
Temperature
Tetrazolium salts are colourless,
o Can alter the enzyme
watersoluble salts that accept
reaction rate
enzymatically-released hydrogen
o Some methods requiring
from the substrate to form highly
incubation at 3C
coloured water-insoluble micro-
o Room temperature
crystalline deposits known as
Demonstration Method Formazans.

1. Metal Precipitation Indigogenic Method


2. Azo-dye
The substrate containing indoxyl
3. Tetrazolium Salt
group, which released as a PRP,
4. Indigogenic Method
which in turn is oxidized by the
5. Diaminobenzidine (DAB)
capture agent potassium
Cytochrome Oxidase or the
ferricyanide to form a turquoise FRP.
peroxidase to form a brown
The incubation solution also includes
pigment
potassium ferricyanide which
The metabolism of glycogen
prevents over-oxidation of the FRP.
by phosphorylase to produce
a polysaccharide that is
colored blue/black with iodine
Diagnostic Application of Enzymes
Metal Precipitation Method Histochemistry
Routinely used to identify 1. Skeletal muscle fiber typing
phosphatases 2. Nerves and ganglia in suspected
o Acid Phosphatase Hirschsprungs disease
o Alkaline Phosphatase 3. Gastrointestinal tissue to assed
o Adenosine Triphosphate malabsorption
(ATPase) 4. Demonstration of lymphoid and
myeloid cells
Azo-dye Method
5. Identification of early liver
The azo-dye methods are used to degeneration
identify acid and alkaline
Skeletal Muscle
phosphatases, non-specific esterase
and chloroacetate esterase. Diagnosis of neuromuscular disease
The different substrates contain a and congenital myopathies
napthol group that can be cleaved
by one of the above enzymes.
Skeletal Muscle Biopsy Two methods:
o Fast Blue RR Method
Application of enzyme histochemical
Which gives a vivid
method to cryostat sections of
reaction product
unfixed skeletal muscle shows:
(Particularly intense in
o The presence of different
mast cell cytoplasm)
fiber types o Pararosaniline Method
o Changes in the number, size,
Which gives a
and relative proportion of
pinkish-red reaction
different fiber which are
product.
valuable in establishing the
diagnosis. Miscellaneous
Demonstration of Nerves and Ganglia ACP Method
Suspected Hirschsprungs Disease o In the identification of
prostate carcinoma
In Hirschsprungs disease in
o Tumor is infiltrating the
children, a variable segmented of
colon/bladder wall or
the rectum and colon is devoid of
o In bone metastases
ganglionic cells.
Application of acid and alkaline
In the effected segment peristalsis is
phosphatases methods to cryostat
impossible and the large bowel
sections of jejunal mucosal biopsy
become obstructed.
specimen.
Demonstration of Specific Lactase or ALP Method
Sucrose deficiency in Jejunal Biopsies o In vascular endothelial
tumors
An ALP method can be applied, ALP
activity resides on the erythrocyte
surface, is a sensitive marker of
structural and functional integrity of
the mucosal absorptive cells.
ACP demonstrate some of the
inflammatory cells in lamina, propria,
also identifies lysososmal activity in
villons enterocytes and glandular
crypt epithelial cells.
Demonstration of Mast Cells and White
Cells of Myeloid Series

Chloroacetate esterase techniques


o Formalin-fixed paraffin
sections
o To assist in the identifications
of tissue mast cells and
myeloid white cells.