Myeloperoxidase
Reaction (MPO)

White Blood Cell Resources
Associated Diseases

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

  • Sample: Blood film or bone marrow smear prepared within 12 hours of blood collected in heparin, oxalate or EDTA (MPO activity is not inhibited by these anticoagulant)
  • Fixative – 10% formal ethanol (10 mL of 40% formalin and 90 mL of absolute ethanol)
  • The staining solution ingredients are as follows:
  • 30% Ethanol: 100 mL
  • Benzidine dihydrochloride: 0.3 g
  • 132M (3.8%) ZnSO4·7H2O: 1 mL
  • Sodium acetate: 1 g
  • 3% hydrogen peroxide: 0.7 mL
  • 1 N sodium hydroxide: 1.5 mL

Method

  1. Prepared air dried film – preferable within 24 hours of collection (peroxidase is unstable in the light).
  2. Place the slide in fixative for 60 seconds at room temperature.
  3. Wash for 15-30 seconds with running tap water and air dry.
  4. Place slide on staining rack and pipette the staining solution onto the slide and stain for 1 minute.
  5. After staining wash with tap water for about 1 minute.
  6. Counterstain with Giemsa stain 1:10 dilution for 10 minutes.
  7. Wash off counterstain, clean, dry and examine.

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

Bone marrow aspirate with myeloperoxidase stain x400 magnification