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Myeloperoxidase staining (MPO Staining)
- Myeloperoxidase is a lysosomal enzyme that can be found in the azurophil granules of neutrophils and monocytes.
- These produce hypohalous acids to carry out their antimicrobial activity, including hypochlorous acid, the sodium salt of which is the chemical in bleach.
- It can serve as a sensitive predictor for myocardial infarction in patients presenting with chest pain.
- It is a member of the XPO subfamily of peroxidases.
- Myeloperoxidase deficiency is a hereditary deficiency of the enzyme, which predisposes to immune deficiency.
- The Myeloperoxidase is encoded by the MPO gene on chromosome 17.
- Azide is used as an inhibitor of MPO. There is also another inhibitor known as 4-aminobenzoic acid hydrazide (4-ABH).
- The MPO protein is a 150-kDa cationic homodimer which is composed of two 15-kDa light chains and two variable-weight glycosylated heavy chains bound to a prosthetic heme group.
Objective
- To differentiate the blasts of acute myeloid leukemia (AML) from those of acute lymphoblastic leukemia (ALL).
- To diagnose congenital deficiency of neutrophil myeloperoxidase.
Myeloperoxidase staining Principle
In presence of hydrogen peroxide, myeloperoxidase present in the leukocyte granules oxidize substrates from colorless form to an insoluble blue/brown derivative at the site of the activity. Benzidine or 3,3′-diaminobenzidine or p-phenylenediamine dihydrochloride are used as substrates.
Requirements for Myeloperoxidase staining (MPO)
- 10% formal ethanol or buffered formal acetone as a Fixative.
- Benzidine or 3,3′-diaminobenzidine or p-phenylenediamine dihydrochloride as a Substrate.
- Sorensen’s phosphate buffer, pH 7.3
- 3% Hydrogen Peroxide
- Add 30 mg DAB in a 60 ml buffer, add 120 ul hydrogen peroxide and mix.
- Hematoxylin as a Counterstain.
Myeloperoxidase staining(MPO) Procedure
- Prepare a smear and air dry it.
- Then fix the smear by using formal ethanol or buffered formal acetone for 60 seconds.
- Rinse the smear with running tap water for 30 seconds.
- Flood the smear with a working substrate.
- Then incubate it for 10 minutes.
- Counterstain the smear with the help of hematoxylin for 3-5 minutes.
- Again rinse the smear with the running tap water and air dry it.
- Now, the slide is ready to observe under the microscope.
Myeloperoxidase staining Result
- Positive MPO Reaction: Neutrophilic granulocytes except blast forms, Eosinophils, Monocytes except blast forms.
- Negative MPO Reaction: Basophils (weakly positive), Lymphocytic cell series, Erythrocyte cell series.
Interpretation
- Myeloperoxidase is present in the primary granules of myeloid cells. Early myeloblasts are negative, with granular positivity appearing progressively as they mature.
- In many cases of AML (without maturation-M1, with maturation-M2 and promyelocytic leukemia-M3), Myeloperoxidase activity has been found in more than 80% blasts. Auer rods are strongly MPO positive.
- Cells of monocytic series display a less intense positive reaction that is characterized by fine granular deposits scattered throughout the cell.
- Lymphoblasts and lymphoid cells are MPO negative.
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Rothera’s test Principle, Procedure, Result. 
