Principle
Leucocytes esterase are enzymes that hydrolyze acyl or chroacyl esters of alpha-napthol. There are nine isoenzymes. Five isoenzymes are stained specifically with napthol AS-D chloroacetate esterase and correspond to the specific esterase of granulocytes. Four remaining are stained with a napthyl acetate esterase and beta napthyl butyrate esterase and are non-specific esterases.
Chemicals
1) Na2HPO4
2) K2HPO4
3) Acetone
4) 40% formalin
5) Alpha napthyl acetate
5) Ethylene monomethyl ether
7) Pararosaniline
S) NaNO2 (sodium nitrite)
9) Concentrated HCL
10) Methyl green
Reagents
Reagent 1:
Buffered formal acetone (pH 6.6), Na2HPO4 (20 mg), KH2 PO4 (100 mg), and distilled water (30 ml). Dissolve the salts and add acetone (45 ml) and formalin (25 ml).
Adjust pH to 6.6 with 1N HCL or 1N NaOH. The reagent has been stable for 2 months.
Reagent 2:
Phosphate buffer (pH 7.6)
Solution A: dissolves 0.91 grams of K2HPO4 in 100 ml of distilled water.
Solution B: dissolves 4.75 grams of Na2HPO4 in 500 ml of distilled water.
Working buffer: Solution A 65 ml + Solution B 435 ml (adjust pH to 7.6).
Reagent 3:
Pararosaline stock
Dissolve 1 gram of pararosaniline hydrochloride in 20 ml of distilled water + 5 ml of concentrated HCL with gentle warming. Filter after cooling. Store at room temperature.
Reagent 4:
4% sodium nitrite: 200 mg of sodium nitrite in 5 ml of distilled water. Stable for 1 week at 4–10°C.
Reagent 5:
Methyl green
Procedure
1) Fix the air-dried smear in cold buffered formal acetone (4–10°C) for 30 seconds.
2) Rinse well with distilled water and dry completely for 10–30 minutes.
3) Prepare hexazotised pararosaline by mixing equal volume of stock pararosaline and sodium nitrite solution.
4) Make incubation mixture as below:
Phosphate buffer: 8.9 ml
Hexazotized pararosaline: 0.6 ml
Alpha-napthyl acetate: 10 mg + 0.5 ml of ethylene glycol monomethyl ether
Dissolve and adjust the pH to 6.1.
5) Put the slide in the incubation mixture for 45 minutes at room temperature.
6) Wash in distilled water.
7) Counterstain with 2% methyl green for 1-2 minutes.
8) Wash with distilled water and dry.
Results
Enzyme activity is seen as dark red / brown granules.
Interpretation
- Neutrophils and other granulocytes are negative but may show a positive reaction of varying intensity in MDS.
- Most T lymphocytes and lymphoblasts show focal dot-like positivity.
- Leukemic erythroblasts may show focal or diffuse positivity.
- Normal and leukemic Monocytes stain strongly.
- Megakaryocytes stain strongly, and leukemic megakaryoblasts show focal or diffuse positivity. Platelets show positive reaction and its staining is used as a control in peripheral smear.
- As nonspecific esterase is ubiquitous (monocytes, T lymphocytes and megakaryocytes), it is made more specific by fluoride sensitivity. Only Monocytic cells have fluoride sensitivity.
- M7 may be mistaken for ALL but it shows strong positivity, helping in its identification.
Image source: PathologyOutlines