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

1. Prepare a smear and air dry it.
2. Then fix the smear by using formal ethanol or buffered formal acetone for 60 seconds.
3. Rinse the smear with running tap water for 30 seconds.
4. Flood the smear with a working substrate.
5. Then incubate it for 10 minutes.
6. Counterstain the smear with the help of hematoxylin for 3-5 minutes.
7. Again rinse the smear with the running tap water and air dry it.
8. 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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