α-Naphthyl Acetate Esterase (α-NAE) Stain

α-Naphthyl Acetate Esterase (α-NAE) Stain

α-Naphthyl Acetate Esterase (α-NAE) Stain


Histological staining techniques have revolutionized our understanding of cellular processes, and the α-Naphthyl Acetate Esterase (α-NAE) stain holds a significant place in this field. This staining method, which reveals esterase distribution within cells, plays a crucial role in diagnostics and research. In this article, we will delve into the core principles of α-NAE staining, its essential components, the procedure to conduct the test, expected outcomes, and vital precautions.

Principle of α-Naphthyl Acetate Esterase (α-NAE) Stain

At the heart of α-NAE staining lies a chemical reaction between hydrochloric acid para fuchsin and sodium nitrite, forming a diazonium salt called six-azo-pararosaniline. This diazonium salt reacts with α-naphthol, produced by the breakdown of α-Naphthyl Acetate by esterase. The outcome is a distinctive red-brown precipitate within the cell’s cytoplasm. This technique is referred to as “Non-specific Esterase Stain” due to its lack of specificity toward esterases.

Key Components of the Stain

  • Fixative (solution A): Formaldehyde
  • Pararosaniline solution (solution B): Parafuchsin
  • Sodium nitrite solution (solution C): Sodium nitrite
  • Phosphate buffer (solution D): Phosphate
  • α-Naphthyl acetate solution (solution E): α-Naphthyl acetate
  • Methyl green solution (solution F): Methyl green
  • NaF solution: Sodium fluoride

Test Procedure

A. Preparing the Working Solution:

Dip Staining:

  • Create Diazonium salt solution by combining 1ml of solution B and 1ml of solution C. Allow it to rest for 2 minutes.
  • Introduce 30ml of solution D into the dye vat.
  • Pour the Diazonium salt solution into the tank, ensuring thorough mixing.
  • Gently add 1ml of solution E to the tank. (For the NaF inhibition test, include 1ml of NaF solution.)

Drop Staining:

  • Mix 50μl each of solution B and solution C in a test tube. Wait for 1 minute.
  • Add 1.5ml of solution D and 50μl of solution E. Allow it to stand for 2 minutes. (For the NaF inhibition test, introduce 1 drop of NaF solution.)

B. Conducting the Test:

  • Begin by drying the cell smear. Apply the working solution directly or fix the smear with solution A for 30-60 seconds.
  • Rinse with distilled water and allow it to air dry or use absorbent paper.
  • Apply the working solution to the slide, ensuring comprehensive coverage of the smear. Incubate at 37℃ for 30 minutes.
  • Rinse with distilled water and allow it to air dry or use absorbent paper.
  • Redye with solution F for 1-2 minutes. Rinse with distilled water and let the smear air dry for microscopic examination.

Anticipated Results

Positive reactions manifest as red or red-brown granules in the cytoplasm. Different cell types exhibit unique patterns:

  • Dot Type: Mature T Lymphocytes display 1-4 brown or red-brown circular granules with clear contours.
  • Diffuse Type: Red-brown granules are uniformly spread throughout the cytoplasm.
  • Monocyte Type: Evenly stained red-brown granules are diffusely spread.

Reference Range

The stain’s reactions differ across cell types:

  • Monocytes: Strong positive reaction, inhibited by NaF.
  • Granulocytes: Mostly negative, occasional weak positive reactions.
  • Megakaryocytes and Platelets: Positive.
  • T Lymphocytes: Dot positive.
  • B Lymphocytes and Plasmacytes: Negative.
  • Monocyte-Derived Cells: Strong positive reaction.
  • Gaucher Cells and Sea Blue Histiocytes: Positive.
  • Erythroblasts: Negative.


  • Adhere to recommended test numbers for specific staining methods.
  • Follow the ICSH recommendation method.
  • Seal reagent bottles tightly to prevent evaporation.
  • Ensure solutions are well-mixed and at room temperature.
  • Prepare working solutions fresh and use them within 10 minutes.
  • Limit α-NAE Stain usage to clinical and research labs, with interpretation by trained experts.
  • Respect instruction sheets, labels, and expiration dates.
  • Dispose of waste following biohazard guidelines.


The α-Naphthyl Acetate Esterase (α-NAE) stain offers a fascinating glimpse into cellular esterase activity. By grasping the principles, components, and procedures of this staining technique, researchers and clinicians can unlock its potential to unravel the complexities of cell behavior and function.

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