Myeloperoxidase Reaction (MPO) Stain

Introduction

Myeloperoxidase is a lysosomal enzyme, located in the azurophilic granules of the neutrophils and its precursors, eosinophils and monocytes. This staining is mainly used to differentiate acute myeloid leukaemia from acute lymphoblastic leukaemia.

Principle

Peroxidase in leukocyte granules oxidizes benzidine to form insoluble, stable and non-diffusible reaction product in the presence of hydrogen peroxide (H₂O₂).  This product is colourless to blue or brown derivatives which is localized at the site of the enzyme. Copper sulphate or nitrate can be used to enhance the staining.

Materials

1. Air dried peripheral blood or bone marrow smear (within 24 hours of collection)
2. Fixative – 10% formal ethanol (10 mL of 40% formalin and 90 mL of absolute ethanol)
3. The staining solution:

• 30% Ethanol: 100 mL
• Benzidine dihydrochloride: 0.3 g
• 0.132M (3.8%) ZnSO₄·7H₂O: 1 mL
• Sodium acetate: 1 g
• 3% hydrogen peroxide: 0.7 mL
• 1 N sodium hydroxide: 1.5 mL

4. Giemsa stain

Method

1. Prepare air dried film – preferable within 24 hours of collection (peroxidase is unstable in the light).
2. Fix slide in fixative at room temperature for 60 seconds.
3. Rinse in slow running tap water for 15 – 30 seconds and air dry.
4. Place slide on staining rack and fully cover the air-dried slide with the staining solution and incubate for 1 minute. 
5. Rinse in slow running tap water for 1 minute.
6. Counterstain with Giemsa stain at 1:10 dilution for 10 minutes. 
7. Wash off counter stain. 
8. Wipe the back of the slide and edges with Kim wipes. Be careful not to touch the smear. 
9. Dry the slide using a hair dryer on the lowest speed (not more than 10 seconds at a time) or air dry in a tilted position.
10. Mount the slide with Depex and cover the zone of morphology with a cover slip. 
11. This slide is now ready for viewing. 

Enzyme activity may be preserved for as long as 3 weeks if preparations are stored in the dark.