Simplified Staining Method for Dual Esterase Using Single Incubation and Single Mixed Reagent

Choi, Jong Weon; Pai, Soo Hwan

Abstract

BACKGROUND: Dual esterase reaction enables simultaneous visualization of the cells exhibiting different characteristics by combining two esterase stains on a single slide. However, most methods previously reported require the smear slides to be stained twice, using separate coupling reagent with each substrate. Authors attempted to combine chloroacetate esterase (CAE) and alpha-naphthyl butyrate esterase (ANBE) staining methods by using single incubation and single mixed reagent.
METHODS
For dual cytochemical reaction, buffy coat slides of normal subjects and bone marrow smear slides of patients with acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS) were used. Eighty mg of fast blue BB salt was dissolved in 50 mL of 0.1 mol/L phosphate buffer (pH 8.0). In the fast blue BB solution, 2 mL of acetone containing 4 mg of naphthol AS-D chloroacetate (Sigma, USA) and 8 mg of alpha-naphthyl butyrate (Sigma, USA) was added. Air dried slides were fixed with buffered formalin-acetone solution for 1 minutes. The fixed slides were allowed to be incubated with the single mixed reagent solution at room temperature for 20 minutes. Aquous hematoxylin dye was used for counterstaining.
RESULTS
Granulocytes were stained bright blue by single-incubated dual esterase reaction. Monocytes and their precursors showed dark brownish positivity in cytoplasmic granules. Megakaryocytes in bone marrow revealed positive reaction for only ANBE by dual esterase method. The activity of CAE in myeloid lineage was markedly decreased in patients with MDS and some cells revealed dual staining activity with both punctate blue and brownish granules. A few neutrophils in karryorrhexis observed in remission marrow of AML patient showed strong positive reaction for CAE.
CONCLUSIONS
Dual esterase staining method using single incubation is very simple in staining procedure and of use in simply identifying cellular characteristics and differentiation of myeloid and monocytic lineages on a single slide.