Diagnostic pathology

1. Enzyme histochemistry
Dr Shikha Pandey
3rd
year resident
CMC-TH

2. Enzyme histochemistry
-Enzyme histochemistry is a morphological technique applied to functional
questions in histopathology.
-It constitutes a link between biochemistry and morphology and provides
important information complementary to conventional histology,
immunohistochemistry and molecular pathology.
-The father of enzyme histochemistry was A.G.P. Pearse.

3. • Enzyme: are biocatalysts synthesized by living cells, that increase the
rate of reactions without themselves being changed in the overall
process.
• Histochemistry is defined by “the identification, localization and
quantification in cells and tissues and by chemical or physical tests, of
specific substances, reactive groups and enzyme catalyzed
substances.”

4. Principles of enzyme histochemistry
• Histochemistry procedures are based on the principle that tissues or
cells, when placed in a solution, chemically react with the solution to
produce a colored insoluble end product.
• The amount and location of the end product can then be evaluated in
the context of the cell or tissue.

5. • Six basic reaction classification: Oxidoreductases, transferases,
hydrolases, lysases , isomerase and ligases.
• Oxidoreductases, hydrolases and transferases are used in
histopathology.

6. Enzymes used in enzyme histochemistry
1. Oxidoreductases
Enzymes: These catalyze oxidation-reduction reactions.
Examples:
• Succinate dehydrogenase (SDH):
Substrate: Succinate.
Application: Demonstrates mitochondrial activity.
• Nicotinamide adenine dinucleotide (NADH) dehydrogenase:
•Substrate: NADH
•Application: Visualizes metabolic activity in muscle biopsies.

7. • Cytochrome oxidase:
Substrate: Cytochrome C.
Application: Detects mitochondrial function.

8. 2. Hydrolases
• Enzymes: These catalyze the hydrolysis of chemical bonds
a. Phosphatases
• Acid Phosphatase (ACP):
• Substrate: Sodium β-glycerophosphate.
• Application: Identifies lysosomal activity, such as in Gaucher’s
disease.
• Alkaline Phosphatase (ALP):
• Substrate: Sodium β-glycerophosphate or naphthol AS-BI phosphate.
• Application: Highlights cellular and tissue differentiation, such as in bone.

9. b. Esterases
• Nonspecific Esterase:
• Substrate: α-naphthyl acetate.
• Application: Differentiates leukemias and histiocytic disorders.
• Cholinesterases (Acetylcholinesterase and Butyrylcholinesterase):
• Substrate: Acetylthiocholine or butyrylthiocholine.
• Application: Identifies cholinergic nerve endings or differentiates nerve fiber
types.

10. c. Glycosidases
• β-Glucuronidase:
• Substrate: Napthol glucuronide.
• Application: Detects lysosomal enzyme activity.
d. Proteases
• Peptidases:
• Substrate: Proteins or peptides.
• Application: Studies protein digestion or degradation processes.

11. 3. Transferases
• Enzymes: These transfer functional groups from one molecule
to another.
• Examples:
• Tyrosine Hydroxylase:
• Substrate: Tyrosine.
• Application: Studies catecholamine synthesis.

12. 4. Lyases
• Enzymes: These catalyze the breaking of chemical bonds without
hydrolysis or oxidation.
• Examples:
• Aldolase:
• Substrate: Fructose-1,6-bisphosphate.
• Application:Demonstrates glycolytic activity.

13. 5. Isomerases
• Enzymes: These catalyze the rearrangement of molecular
structures.
• Examples:
• Phosphoglucoisomerase:
• Substrate: Glucose-6-phosphate.
• Application: Assesses glycolytic pathway function.

14. • Enzymes: These hydrolyze lipids.
• Examples:
• Lipase:
• Substrate: Triglycerides.
• Application: Identifies fat metabolism and lipid storage
disorders.

15. 7. Peroxidases
• Enzymes: These catalyze oxidation using hydrogen peroxide.
• Examples:
Horseradish Peroxidase (HRP):
• Substrate: Hydrogen peroxide and diaminobenzidine (DAB).
• Application: Commonly used in immunohistochemistry to detect
antigens.

16. • Classical histochemical reactions are generally based on one of the 4
principles:
1.Simple ionic interactions.
2.Reactions of aldehydes with Schiff’s reagent or silver compounds.
3.Coupling of aromatic diazonium salts with aromatic residues on
protein.
4.Conversion acting on a substrate to form a colored precipitate.

17. Diazonium salts:
• Are prepared by treating primary aromatic amines with an acid solution of sodium
nitrate.
• The resulting salt, generally a chloride, will react readily with phenols or with aryl
amines to produce corresponding intensely coloured isoluble azo dye, but a few
are dyes.
• The salts themselves are usually colourless.
• Diazonium salts can not be kept indefinitely.
• Must be stored in a cool dark place and renewed at six month intervals.
• The solubility of diazonium salts is limited, especially in the alkaline pH
range.

18. Procedure
1. Tissue Preparation:
- Tissues are typically fresh-frozen to preserve enzymatic activity.
- Cryostat sections are then prepared to maintain the native state of the
enzymes.
- It is recommended to freeze specimens on dry ice (CO2 ) at –80°C or in
isopentane stored in a –25°C freezer.

19. - Liquid nitrogen carries the disadvantage of cracking freezing artifact in large
tissue specimens
- After freezing, the tissue can be stored without loss of enzymatic activity for
longer periods at –25°C in a small Eppendorf tube (biopsies) or plastic bag
(surgical resections).

20. 2. Incubation with Substrate: Sections are incubated with a substrate
specific to the enzyme of interest. The enzyme catalyzes the conversion
of this substrate into a product.
3. Visualization: The product of the enzymatic reaction is often coupled
with a chromogen, resulting in a colored precipitate that can be observed
under a microscope.
4. Controls: Negative controls, such as heat-inactivated sections or
omission of the substrate, are essential to confirm the specificity of the
staining.

21. • Types of histochemical reactions
1. Simultaneous capture.
2. Post incubation coupling.
3. Self coloured substrate.
4. Intramolecular rearrangement.

22. 1. Simultaneous capture: -
• Most imp. Technique.
• Technique:

23. • Incubation mixture containing a substrate and a diazonium salt are
applied to suitable sections in a buffered solution.
• Enzyme present in the section hydrolyses the substrate to form an
invisible primary reaction product (PRP)
• This complex couples with the diazonium salt to produce the final
reaction product (FRP) which is visible, often coloured deposit under
the microscope.

24. • The PRP is colourless where as the FRP is coloured.
• The substrate must be soluble in water or in the bufer medium so that
there is suficient substrate available for the enzyme to hydrolyse.
• The histochemical method must be performed at a pH at which the enzyme
shows maximal activity, usually pH 7.0 for most enzymes.
• The diazonium salt also has an optimal pH for its most efficient rate of
coupling, this should also be considered.
• Eg:Diazo method for alkaline phosphatase

25. • 2. Post incubation coupling Technique:

26. • In this reaction the enzyme hydrolyses the substrate and
produces a reasonably insoluble PRP
• The subsequent coupling is carried out in a separate
solution.
• This type of method relies upon the PRP remaining at
the initial site of hydrolysis.
• It is important that diffusion of the PRP does not occur
during the coupling process.

27. • Advantages:
1. Case where long first incubation stage is necessary.
2. Optimum pH for enzyme and for diazonium salt separately.
3. This process avoids the deterious effect of diazonium salts.
Disadvantage :
1.PRP is not completely insoluble.
2.Diffusion is always there.

28. • 3.Self coloured substrate Techique:

29. • Involves the use of a coloured substrate that is also soluble.
• During hydrolysis the enzyme removes the soluble grouping without
affecting the colour.
• PRP ; coloured and insoluble,and no coupling stage is necessary.
• The insoluble coloured reaction product is precipitated at the site
of enzyme activity.

30. • Advantage:
1.Diazonium coupling not required.

31. • Intramolecular rearrangement:
Produces a coloured insoluble participate at the sites of enzyme activity of an
otherwise colourless substrate.

32. • Current common uses of enzyme histochemistry in surgical
histopathology laboratories
1. Skeletal muscle biopsy.
2. Rapid and easy detection of ganglia and nerves in cases of suspected
Hirschsprung’s disease.

33. 3. Demonstration of specific lactase or sucrase deficiency in jejunal
biopsies.
4. Demonstration of mast cells & white cells of the myeloid series.
5. Increased lysosomal acid phosphatase activity can serve as an early
marker for ischemic damage.

34. 6. Use of acid phosphatase in the identification of prostate
carcinoma.eg. when the tumor is infiltrating the colon or bladder
wall,or in bone metastases.
7. Application of acid and alkaline phosphatase methods to cryostat
sections of jejunal mucosal biopsy specimens.
8. Use of alkaline phosphatase methods in vascular endothelial tumors.

35. 1. Skeletal muscle biopsy:
• Application of enzyme histochemical methods to cryostat sections of
unfixed skeletal muscle shows;
- Presence of different fiber types, and
- Changes in the number, size and relative proportions of different
fibers.
Muscle biopsy samples are of 2 types:

36. • Open biopsy:
- These are received in the laboratory as strips of skeletal muscle,
preferably tied at each end.
- The biopsy sample should be received fresh (unfixed) in the lab as soon
as possible after surgical removal.
- It is transported from operating theater to laboratory wrapped in gauze
soaked in normal saline, then squeezed till just damp, to minimize
drying.
- On arrival, muscle biopsy is cut into suitable block sized pieces(0.5 X 0.5
cm) and oriented so that transverse sections will be cut.

37. • Needle biopsy samples:
-These are taken by a Bergstrom needle .
-The biopsy samples are placed on a guaze damped by saline and
transferred to the lab as quickly as possible.
-Under dissecting microscope, biopsies are gently manipulated and
trimmed so that the fibers in each are running in the same direction, and
a composite block is made of all the samples.
-Whether the sample is an open biopsy or needle biopsy, freezing should
as rapid as possible, since slow freezing will produce ice crystal artifact.
Bergstrom
needle

38. • Following methods are used rountinely:
- Adenosine triphosphate: Differentiate type I and II fibers
- NADH diaphorase: Demonstrates mitochondria and the fine detail of
the sarcoplasmic reticulum of the fiber. eg. Mitochondrial
myopathies.
- Phosphorylase: Also distinguish between type 1 and 2 fibers but fades
very quickly. It is used to exclude McArdle’s disease, a primary
phosphorylase deficiency.

39. Detection of nerves & ganglia in suspected Hirschsprung’s disease:
• The biopsy sample is orientated under dissecting microscope control
so that sectioning will include mucosa and submucosa, then snap
frozen at -170 C in isopentane cooled in liquid N2 and sectioned in a
cryostat.
• Preliminary sections are stained with H & E and the submucosa
examined for the presence of ganglia.
• If sufficient submucosa is present or if no ganglia are seen after
examination of a no. of H & E stained levels, then 2 or 3 sections are
stained by cholinesterase method to demonstrate the fine nerve
twings in the mucosal lamina propria.

40. - Hirschsprung disease with characteristic
increase in acetylcholinesterase activity in
parasympathetic nerve fibers of muscularis
mucosae and lamina propria mucosae (left),
- Normally innervated rectum mucosa (right).
-Increased unmodulated release of
acetylcholine from the sacral roots S2–S5, due
to aplasia of nerve cells of the enteric nervous
system in the rectum, leads to elevation of
acetylcholinesterase activity as a reliable
diagnostic parameter

41. Hypoganglionosis of myenteric
plexus (right). Normal myenteric
plexus (left). Lactic dehydrogenase
reaction

42. Demonstration of specific lactase or sucrase deficiency in jejunal biopsies:
• For the assessment of jejunal mucosal biopsies in suspected celiac
disease, the specimen can be examined under the dissecting
microscope and the presence or absence of villi noted.
• Paraffin sections stains with H & E are used to assess villous height,
gland hyperplasia and intensity of inflammatory cell infiltrate in the
lamina propria.
• Alternatively, the biopsy can be snap frozen, sectioned in a cryostat,
and H & E stained for rapid diagnosis.

43. • Alkaline phosphatase method: Alkaline phosphatase activity resides
on the enterocyte surface.
- Sensitive marker of structural and functional integrity of the mucosal
absorptive cells.
• Acid phosphatase demonstrates; inflammatory cells in lamina propria,
identifies lysosomal activity in villous enterocytes and glandular crypt
epithelial cells.

44. Demonstation of mast cells & white cells of myeloid series;
- Chloroacetate esterase techniques have recently been applied to
formalin-fixed paraffin sections to assist in the identification of tissue
mast cells and myeloid white cells.
Two methods are suitable:
1. Fast blue RR method: which gives a vivid blue reaction product
(particularly intense in mast cell cytoplasm).
2. Pararosanilin method: which gives a pinkish-red reaction product.

45. • Other examples:

46. Cerebellum with contusion
Decrease of lactic
dehydrogenase activity in
Purkinje cells (right) in
comparison to normal
cerebellum (left)

47. Senile plaques stained with acid
phosphatase in the dentate gyrus
of hippocampus (left) higher
magnification (right)

48. References
1. https://jamdsr.com/pdf2b/EnzymeHistochemistryAReview.pdf
2. Current Concepts of Enzyme Histochemistry in Modern Pathology.
William A. Meier-Ruge Elisabeth Bruder.

49. THANK-YOU