CYTOCHEMISTRY by Brian M. Denney, RMT: Phosphatases
CYTOCHEMISTRY by Brian M. Denney, RMT: Phosphatases
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ENZYMATIC TECHNIQUES
PHOSPHATASES
May be Alkaline or Acid
Are groups of isoenzymes that are capable of
hydrolysing monophosphate esters at a particular pH
Substrate: -naphthyl
General Equation:
-naphthyl Phosphatases
- naphthol
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2+
3+
4+
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cytoplasm
Pale, with a moderate amount of blue
staining
Strongly stained granules in 80-100% of
cytoplasm and beginning to coalesce
Strong blue precipitated staining
Strongly stained granules packed into the
cytoplasm (nucleus is the only visible
component)
Deep blue or brilliant staining with no
visible cytoplasm
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General equation:
Esterase
Naphthyl esters
naphthol
H20
Napthol + diazonium salt/ diazo dye (colorless) colored
azodye
Naphthol AS-D Chloroacetate esterase
- substrate: Naphthol AS-D Chloroacetate
- used to demonstrate specific esterases
- to distinguish myeloid leukemias from monocytic
leukemias
- very stable enzyme; can still be visualized in paraffin
embedded sections even after months (i.e. myeloid cells in
granulocytic sarcoma and mast cells in systemic mast cell
disease)
Reagents:
o
o
o
o
o
PEROXIDASE (MYELOPEROXIDASE)
- are enzymes that catalyse the oxidation of substrates in
the presence of hydrogen peroxide
- are present in the primary granules of neutrophils,
eosinophils and to lesser extent monocytes while absent in
lymphocytes and in all eythroid lines
- used in differentiating acute myelogenous leukemia and
acute monocytic leukemia from ALL
- best cytochemical stain for auer rods ( Faggot cell- bundle
of auer rod)
Interpretation:
MPO
3,3- DAB + Hydrogen peroxide (oxidizing
agent)
colored precipitate (made up of quinone rings) + H2O (by
product)
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Reagent:
o
o
o
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Fixative : formalin/ethanol
Periodic acid (1%) 1 g periodic acid
dissolved in 100 mL distilled H2O
Schiff Reagent if becomes pink = expired
- to see if still viable- add several drops of
Schiff reagent + 10 mL of 37% formaldehyde
- Light purple = viable ; Colorless = not viable
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