Cytochemical Stain

2006200368

2006200366

2006838665

2006838514

2006838393

2006838379

2006838507

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.