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Sargaiyan V et al. Enzyme Histochemistry: A Review.
Review Article
Enzyme Histochemistry: A Review
1 2
Vinod Sargaiyan , Anupam Bansal
1
Departments of Oral and Maxillofacial Pathology and Microbiology, Mansarovar Dental
College and Research Centre, Bhopal, 2Oral and Maxillofacial Surgery, Surendera Dental
College and Research Institute, Sri-Ganganagar, Rajasthan, India
Abstract
Enzyme histochemistry serves as a link between
Corresponding Author: biochemistry and morphology. It is based on
Dr. Vinod Sargaiyan metabolization of a substrate provided to a tissue
Department of Oral and Maxillofacial enzyme in its orthotopic localization. Visualization
Pathology and Microbiology, is accomplished with an insoluble dye product. It is
Mansarovar Dental College and a sensitive dynamic technique that mirrors even
Research Centre, Bhopal, early metabolic imbalance of a pathological tissue
India lesion, combined with the advantage of
E mail: vinodl476@gmail.com histotopographic enzyme localization. With the
advent of immunohistochemistry and DNA-oriented
Received: 14-03-2014 molecular pathology techniques, the potential of
enzyme histochemistry currently tends to be under
Revised: 24-04-2014 recognized.
Accepted: 28-04-2014 Keywords: Enzyme Histochemistry, diazotization,
orthotopic.
This article may be cited as: Sargaiyan V, Bansal A. Enzyme Histochemistry: A Review. J
Adv Med Dent Scie 2014;2(2):191-195.
Introduction:
Enzyme are biocatalysts synthesized by biologic technique when they are combined
living cells, that increase the rate of with histology.[1-4]
reactions without themselves being
changed in the overall process. Principles of enzyme histochemistry :
Histochemistry is defined by Pearse as “the Histochemistry procedures are based on the
identification, localization and simple premise that tissues or cells, when
quantification in cells and tissues and by placed in a solution chemically react with the
chemical or physical tests, of specific solution to produce a colored insoluble end
substances, reactive groups and enzyme- product. The amount and location of the end
catalyzed substances.” Thus, any chemical product can then be evaluated in the context
procedure that localizes a substances with of the cell or tissue.[5,6]
in cells or tissues for subsequent
microscopy is a histochemical technique. In Classical histochemical reactions are
broad sense, Enzyme histochemistry is the generally based on one of the 4 principles:
1.Simple ionic interactions.
science that encompasses immunologic and 2.Reactions of aldehydes with Schiff’s
molecular reagent or silver compounds.
Journal of Advanced Medical and Dental Sciences Research |Vol. 2|Issue 2| April-June 2014 191
SSaarrggaaiiyyaann VV eett aall.. EEnnzzyymmee HHiissttoocchheemmiissttrryy:: AA RReevviieeww.
3.CCoouupplliinngg ooff aarroommaattiicc ddiiaazzoonniiuumm ssaallttss wwiitthh Disadvantage :
aarroommaattiicc rreessiidduueess oonn pprrootteeiinn.. 1.PPRRPP iiss nnoott ccoommpplleetteellyy iinnssoolluubbllee
4.CCoonnvveerrssiioonn aaccttiinngg oonn aa ssuubbssttrraattee ttoo ffoorrmm aa 2.DDiiffffuussiioonn iiss aallwwaayyss tthheerree.
colored ppt.
3.SSeellff ccoolloouurreedd ssuubbssttrraattee :
TTyyppeess ooff hhiissttoocchheemmiiccaall rreeaaccttiioonnss:[1] Techique:
1. Simultaneous capture.
2. PPoosstt iinnccuubbaattiioonn ccoouupplliinngg..
3. Self coloured substrate.
4. IInnttrraammoolleeccuullaarr rreeaarrrraannggeemmeenntt..
1. Simultaneous capture:
-Most imp. Technique.
Principle:
-Gomori’s Metal ppt. TTeecchhnniiqquuee
-Azo dye method.
Technique:
Advantage:
1.DDiiaazzoonniiuumm ccoouupplliinngg nnoott rreeqquuiirreedd.
4. IInnttrraammoolleeccuullaarr rreeaarrrraannggeemmeenntt:
Technique:
Disadvantage :
1.Diffusion of PRP.
2.RRaattee ooff hhyyddrroollyyssiiss ooff ssuubbssttrraattee.
3.DDiiffffuussiioonn ccooeeffffiicciieenntt ooff tthhee PPRRPP ffoorr tthhee
buffer.
4.RRaattee ooff ccoouupplliinngg ooff tthhee PPRRPP aanndd ddiiaazzoo ssaalltt.
5.DDiiaazzoo ssaalltt aanndd EEnnzzyymmee ssaammee ppHH ..
2. PPoosstt iinnccuubbaattiioonn ccoouupplliinngg: DDiiaaggnnoossttiicc AApppplliiccaattiioonnss ooff eennzzyymmee
Technique: histochemistry:
EEnnzzyymmee hhiissttoocchheemmiiccaall tteecchhnniiqquueess aarree nnoott
wwiiddeellyy aapppplliieedd ttoo ssuurrggiiccaall aanndd necropsy
mmaatteerriiaall ffoorr ddiiaaggnnoossttiicc ppuurrppoosseess,, mmaaiinnllyy
bbeeccaauussee ooff tthhee ttoottaall oorr ppaarrttiiaall lloossss ooff eennzzyymmee
aaccttiivviittyy,, wwhhiicchh ooccccuurrss when a tissue is
routtiinneellyy ffiixxeedd aanndd pprroocceesssseedd iinnttoo ppaarraaffffiinn..
TThhee ccuurrrreenntt ccoommmmoonn uusseess ooff eennzzyymmee
hhiissttoocchheemmiissttrryy iinn ssuurrggiiccaall hhiissttooppaatthhoollooggy
Advantages: laboratories
1.CCaassee wwhheerree lloonngg ffiirrsstt iinnccuubbaattiioonn ssttaaggee iiss 1. SSkkeelleettaall mmuussccllee bbiiooppssyy..
necessary 2. RRaappiidd aanndd eeaassyy ddeetteeccttiioonn ooff ggaanngglliiaa aanndd
2.OOppttiimmuumm ppHH ffoorr eennzzyymmee aanndd ffoorr nneerrvveess iinn ccaasseess ooff ssuussppeecctteedd HHiirrsscchhsspprruunngg’’ss
diazonium salt separately disease.
192
Sargaiyan V et al. Enzyme Histochemistry: A Review.
3. Demonstration of specific lactase or which may hamper accurate
sucrase deficiency in jejunal biopsies. diagnosis.Although many of the
4. Demonstration of mast cells & white cells morphological changes in skeletal muscle
of the myeloid series. can be seen on an H & E stain, special
5. Miscellaneous: methods are necessary to demonstrate some
of the structural abnormalities of diagnostic
Skeletal muscle biopsy: importance, and the most imp. Of these are
Application of enzyme histochemical enzyme histochemical techniques.[6]
methods to cryostat sections of unfixed Following methods are used rountinely:
skeletal muscle shows the presence of Adenosine triphosphate.
different fiber types, and changes in the NADH diaphorase.
number, size and relative proportions of Phosphorylase.
different fibers which are valuable in 1. Adenosine triphosphatase:
establishing the diagnosis. ATPase methods are used in combination to
Muscle biopsy samples are of 2 types: distinguish between type1 and type 2 fibers,
and to further subdivide the type 2 fibers into
Open muscle biopsies: 2A,2B and 2C subtypes. This distinction is
These are received in the laboratory as strips diagnostically important since some muscle
of skeletal muscle, preferably tied at each diseases have characteristic patterns of loss,
end to a piece of orange stick.The biopsy atrophy or grouping of specific fiber types or
sample should be received fresh(unfixed) in subtypes. Some types of structural fiber
the lab as soon as possible after surgical abnormality (eg. periodic paralysis) are also
removal.It is transported from operating demonstrated by the ATP-ase methods.
theater to laboratory wrapped in gauze 2. NADH diaphorase:
soaked in normal saline, then squeezed till Demonstrates mitochondria and the fine
just damp, to minimize drying. Long delays detail of the sarcoplasmic reticulum of the
between surgical removal and freezing can fiber. It is used to detect very minor or early
result in unwanted freezing artifact.On structural abnormality in the sarcoplasmic
arrival, muscle biopsy is cut into suitable reticulum network of the fiber, as well as
block sized pieces(0.5 X 0.5 cm) and mitochondrial abnormalities. eg.
oriented so that transverse sections will be Mitochondrial myopathies.
cut.[6]
3. Phosphorylase: Also distinguish between
2.Needle biopsy samples:These are taken by type 1 and 2 fibers but fades very quickly. It
a Bergstrom needle and can be quickly and is used to exclude McArdle’s disease, a
easily obtained from the thigh under primary phosphorylase deficiency.
L.A,after nicking the skin with a sharp 4. Acid phosphatase or non-specific
scalpel blade.The biopsy samples are placed esterase:
on a guaze damped by saline and transferred To identify macrophages in necrotic fibers
to the lab as quickly as possible.Under and abnormal lysosomal activity in muscle
dissecting microscope, biopsies are gently fibers.
manipulated and trimmed so that the fibers in 5. Cholinesterase :
each are running in the same direction, and a To highlight atrophic fibers and to
composite block is made of all the demonstrate intramuscular nerve twigs.
samples.Whether the sample is an open
biopsy or needle biopsy,it is important that
freezing be as rapid as possible,since slow
freezing will produce ice crystal artifact
Journal of Advanced Medical and Dental Sciences Research |Vol. 2|Issue 2| April-June 2014 193
Sargaiyan V et al. Enzyme Histochemistry: A Review.
Detection of nerves & ganglia in suspected integrity of the mucosal absorptive cells.This
Hirschsprung’s disease: is particularly useful in assessing histological
In Hirschsprung’s disease in children, a recovery.
variable segment of the rectum and colon is Acid phosphatase demonstrates some of the
devoid of ganglionic cells.In the effected inflammatory cells in lamina propria, and
segment peristalsis is impossible and the also identifies lysosomal activity in villous
large bowel becomes obstructed.The enterocytes and glandular crypt epithelial
diagnosis may be suspected clinically and cells.
radiologically but requires histological
confirmation,usually by the examination of Demonstation of mast cells & white cells
one or more suction biopsy specimens of of myeloid series :
rectal mucosa and submucosa.The biopsy Chloroacetate esterase techniques have
sample is orientated under dissecting recently been applied to formalin-fixed
microscope control so that sectioning will paraffin sections to assist in the identification
include mucosa and submucosa,then snap- of tissue mast cells and myeloid white cells.
frozen at -170 C in isopentane cooled in Two methods are suitable:
liquid N and sectioned in a 1. Fast blue RR method: which gives a
2 vivid blue reaction product (particularly
cryostat.Preliminary sections are stained with intense in mast
H & E and the submucosa examined for the cell cytoplasm).
presence of ganglia.If sufficient submucosa 2. Pararosanilin methd: which gives a
is present or if no ganglia are seen after pinkish-red reaction product.
examination of a no. of H & E stained
levels,then 2 or 3 sections are stained by Miscellaneous:
cholinesterase method to demonstrate the 1. Use of acid phosphatase in the
fine nerve twings in the mucosal lamina identification of prostate carcinoma.eg.when
propria. the tumor is
infiltrating the colon or bladder wall,or in
Demonstration of specific lactase or bone metastases.
sucrase deficiency in jejunal biopsies: 2. Application of acid and alkaline
For the assessment of jejunal mucosal phosphatase methods to cryostat sections of
biopsies in suspected celiac disease, the jejunal mucosal
specimen can be examined under the biopsy specimens.
dissecting microscope and the presence or 3. Use of alkaline phosphatase methods in
absence of villi noted. Paraffin sections vascular endothelial tumors.
stains with H & E are used to assess villous
height, gland hyperplasia and intensity of Conclusion:
inflammatory cell infiltrate in the lamina Enzyme histochemistry serves to detect early
propria. Alternatively, the biopsy can be metabolic changes in biopsy and autopsy
snap frozen, sectioned in a cryostat, and H & tissue before manifestation on H &E staining
E stained for rapid diagnosis. or immunohistochemistry. As such, it
constitutes a valuable complement to other
Advantages: special techniques, i.e. immune
An alkaline phosphatase method can be histochemistry and molecular pathology. An
applied; Alkaline phosphatase activity exclusive diagnostic domain of enzyme
resides on the enterocyte surface, is a histochemical analysis is the aganglionosis
sensitive marker of structural and functional of the distal rectum mucosa, which cannot
Journal of Advanced Medical and Dental Sciences Research |Vol. 2|Issue 2| April-June 2014 194
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