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LEUKOCYTE ALKALINE PHOSPHATASE (LAP)

LAP is useful for differentiating CML from leukemoid

reaction.

PRINCIPLE

LAP is enzyme found in secondary granules of neutrophils.

The substrate naphthol AS-BI phosphate is hydrolyzed by

the enzyme at an alkaline pH.

This hydrolyzed substrate in combination with a dye such as

fast red violet LB or fast blue BB produces a colored

precipitate at site of the enzyme activity.

SCORING

LAP activity is scored in the mature

polymorphonuclear cells and bands only.

The activity scores range from 0 to 4+.

The scores of 100 mature polymorphonuclear cells

and bands are added for the LAP score.

Score No. of Cells Score x No. of Cells

0 20 0

1 45 45

2 25 50

3 5 15

4 5 20

Total 100 130 = LAP score

RESULTS OF LEUKOCYTE ALKALINE PHOSPHATASESTAIN

Finding Score

NormalChronic myelogenous leukemiaLeukemoid reactionPolycythemia veraSecondary polycythemia

20 – 100< 13

> 100100 – 20010 – 100

SUMMARY OF LAP INTERPRETATION

A normal neutrophil alkaline phosphatase reaction

Negative neutrophil alkaline phosphatase (NAP) reaction. A low NAP score is seen in 95% of cases of chronic granulocytic leukaemia. This test is redundant if there is ready availability of karyotypic or molecular genetic analysis to detect t(9;22) and BCR-ABL fusion respectively.

The Leukocyte Alkaline Phosphatase (LAP) test was used years ago to determine if a patient had CGL or an infection.

Normal cells (PMNs) contain the enzyme leukocyte alkaline phosphatase (LAP). In CML the PMNs lack or contain a decreased amount of LAP (Right).(LAP Stain x Oil)Both peripheral blood samples shown above were stained for LAP. The sample on the left is a normal blood sample, and shows positive LAS staining. The sample on the right is from a CML patient. LAP staining is characteristically low or absent in CML.

     ALKALINE  PHOSPHATASE  REACTION

                                                                                                                               

                              

Reaction product: red granulation

Neutrophils:  The more mature the cell, the more positive the reaction; maximum intensity occurs in segmented forms of neutrophilic leucocyte. The reaction intensity is evaluated according to Kaplow's scoring system: 0  points - no reaction  1 point - weak diffuse positive reaction or single granules 2 points - diffuse positive reaction and a few granules  3 points - strong positive reaction with numerous granules  4 points - very strong reaction with a lot of granules, covering the nucleus.  The score from evaluation of the 100 neutrophils shall be between 20 - 100 points. Other blood cells do not contain the enzyme.

Staining: azo-dye method

Comment: Very weak, diffuse only reaction of alkaline phosphatase in granulocytes, which is seen in a form of pink coloured cytoplasm (score 1 according to Kaplow’ scale).

Magnification: x 1000

Comment: Diffuse and granular phosphatase reaction of little intensity in a segmented neutrophil leucocyte (score 2 according to Kaplow’ scale).

Comment: Intense positive phosphatase reaction shown as red granules in a segmented neutrophil leucocyte (score 3 according to Kaplow’ scale).

Comment: Very strong activity of alkaline phosphatase reaction  is seen in a segmented neutrophil leucocytes (score 4 according to Kaplow’ scale) and a weak diffuse reaction (score 1) is seen in  the segmented neutrophil leucocyte above.