NEUTROPHIL (LEUCOCYTE) ALKALINE PHOSPHATASE TEST

NEUTROPHIL (LEUCOCYTE) ALKALINE PHOSPHATASE

Leucocyte alkaline phosphatase (LAP) activity reflects intracellular metabolic activity. The enzyme is located in cytoplasmic granules. This enzyme activity is greatly increased in leucocytosis; and is mainly associated with bacterial infections. It is particularly useful in differentiating leukaemias from leukaemoid reactions. 

Principle The alkaline phosphatase activity is demonstrated by the hydrolysis of alpha-naphthol phosphate by the enzyme to liberate naphthol. This, in turn, unites with a diazotised amine to form an insoluble coloured precipitate. The intensity of the colour is proportional to the concentration of the enzyme. 

Reagents

1. Fixative

10 % Formalin-methanol: Add 10 ml of formalin to 90 ml of absolute methanol. The reagent is stable for 2-4 weeks if stored at -20°C. 

2. Buffer

Stock solution 0.2 M Propanediol

2-amino-2-methyl-1,3 propanediol 21 g

 Distilled water 1L

up to 1L

Store at 4°C. 

3.Working buffer  
0.05 M Propanediol, pH 9.4-9.6 

Stock buffer250 ml 

2N Hydrochloric acid 3.5 ml 

Distilled water up to 1L

Store at 4°C. 3. Substrate

Sodium salt of Naphthol AS-B1 phosphate . 0.5 g

 Fast violet B0.04 g

 Working buffer60 ml 

Prepare just before use. Shake well to dissolve and filter 

4. Mayer's haematoxylin 

Haematoxylin 1 g 

Distilled water 500 ml

 Heat to boiling point. 

Add distilled water 500 ml Sodium iodate 0.2 g 

Aluminium potassium sulphate 50g

Shake well to dissolve, filter and store in a brown bottle.50g

Specimen Prepare blood smears from freshly withdrawn blood without anticoagulant. Fix immediately. If the blood sample is collected in EDTA, the blood smears should be made in less than one hour and fixed immediately.

Once fixed, the smear can be stored at 4°C for 24 hours or at -20°C for 3-4 weeks.

Prepare positive control smears from normal healthy individuals or from pregnant women and store as above.

Technique

(i) Fix the test and control smears in the cold fixative at 0 to 5°C for 30 seconds.

(ii) Wash gently in tap water and air dry.

(iii) Place in the freshly prepared substrate for 15 minutes.

(iv) Rinse in tap water. 

(v) Stain with Mayer's haematoxylin for 6-8 minutes.

(vi) Rinse in tap water and air dry. Mount in an aqueous mounting medium.

(vii) Examine under the oil immersion objective.

Alkaline phosphates activity is indicated by the presence of bright blue granules in the cytoplasm of mature neutrophils.

Count at least 100, preferably 200, neutrophils. Each cell is rated 0-4 on the basis of intensity of the stain as shown in Table. [After examination, remove the cover glass and dry the smear for re-examination at a later date, if required). Eosinophils, basophils, lymphocytes and monocytes should not be counted.

	SCORING CRITERIA IN THE LAP REACTION
CELL RATING	AMOUNT(%)	GRANULE SIZE	STAIN INTENSITY	CYTOPLASM BACKGROUND
0	-			Unstained
1+	20-50	small	Faint to moderate	Unstained to pale pin
2+	40-80	Small/medium	Moderate/strong	Unstained to pale pin
3+	80-100	Medium /large	Strong	pink
4+	100	Medium/large	brilliant	Pink Cytoplasm not visible

Results A rating in a total of 100 cells is counted. The sum of the ratings in 100 cells is the smear score. A normal score is 14 to 100 per 100 neutrophils. A score of 200 is considered normal in pregnancy and in children. Elevated alkaline phosphatase activity is observed in infections, in non-leukaemic myeloproliferative disorders such as polycythaemia, in Hodgkin's disease, aplastic anemia, Down's syndrome, cirrhosis and in pregnancy. 

Low scores are found in chronic myeloid leukemia, paroxymal nocturnal haemoglobinuria and infectious mononucleosis.

Either Fya or Fyb. An antibody, anti-Fy3 was first detected in the serum of an individual of the phenotype Fy(a-b-). Anti-Fy3 is directed against the cells other than Fy(a-b-) phenotype.

Anti-Fya and anti-Fyb are present in serum invariably as a result of immune response. They can be detected by the antihuman globulin test. They are inactivated by proteolytic enzymes, and cannot be detected by the enzyme method.