NON SPECIFIC ESTERASE

(NSE)

PRESENTED BY –
ABHISHEK BHARTI
ADHOC-LAB TECHNICIAN
DEPARTMENT OF HAEMATOPATHOLOGY
CONTENT:-

 INTRODUCTION
 PURPOSE
 PRINCIPLE
 REQUIRMENT OF REAGENTS
 PREPARATION OF REAGENTS
 PROCEDURE
 INTERPRETATION
INTRODUCTION:-

 Non-Specific Esterase is used as one of the

diagnostic criteria for Acute Monocytic Leukemia
(FAB M5).
 Here we look for the presence of non-specific
esterase activity and there inhibition by sodium
fluoride (NaF).
 It is special stain.
PURPOSE:-
 NSEis used in diagnosis of acute monocytic
leukemia.
PRINCIPLE:-
 Leucocyte esterases are a group of enzymes that hydrolyse
acyl or chloroacyl esters of α-naphthol or naphthol.
 Enzyme histochemical stain that relies on endogenous esterase activity to
hydrolyze exogenous alpha naphthyl acetate substrate, which yields naphthol,
a reddish brown product visible under light microscopy.
Non Specific Esterase + Alpha Naphthyl Acetate(substrate)
Non Specific Esterase + Alpha
Enzymatic Hydrolysis

Free Naphthol + Diazonium Salt GBC Garnet (coupling dye)

Colored Deposits(at the site of enzymatic activity)

Reddish brown product in cytoplasm

REQUIRMENT OF REAGENTS:-

 Fixative- 40 % Formaldehyde
 Acetone
 Alpha naphthyl acetate
 Fast Garnet GBC sulfate salt (Sigma F 8761)
 Sodium Floride (NaF)
 Poshphate Buffer
 Haematoxylin
PREPARATION OF REAGENTS:-

INCUBATION MIXTURE:-
1) FIRST TUBE- 10 mg Alpha naphthyl acetate + 0.5 ml Acetone.
2) SECOND TUBE – 10 mg Fast Garnet GBC sulfate salt + 10 Poshphate Buffer.

POSHPHATE BUFFER:-
A) 20.0 gms KH2pO4 Dissolve in 1000 ml D/W.
B) 21.3 gms Na2Hpo4 Dissolve in 1000 ml D/W.
Mix 768 ml (A) + 232 ml of (B). Adjust pH 6.3 with meter and make the volume 1
liter. Keep at RT 18-20 C. Used in NSE Buffer.
 PROCEDURE:-
Take 2 slide of the same patient(Labelled on slide case No,Requisition No and Patient Name),mark
one slide as NSE & other as NAF.

Dry slide are fixed in formalin vapour at 4 C for 20 minutes

Wash the slide in running tap water for 10 minutes

Prepare incubation mixture,filter and separate into,One part is used as it on NSE slide and another
one is mixed with 1.5 mg/ml sodium floride and use it on slide labelled NAF.

Cover the staining rack and keep for 20 minutes at room temperature

Wash the slide in running tap water for 10 minutes

NSE and NAF slide are stain with Haemotoxylin for 35 minutes

Wash the slide in running tap water for 10 minutes

Observe the microscope

INTERPRETATION:-
 Negative control- Neutrophil, Eosinophil , Basophil.
 Positve control- Megakaryocyte strong,Lymphocytes,Monocytes.
 Monoblasts positive >03 %.