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LAP is useful for differentiating CML from leukemoid reaction.

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.

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 0 1 2 3 4 Total

No. of Cells 20 45 25 5 5 100

Score x No. of Cells 0 45 50 15 20 130 = LAP score

RESULTS OF LEUKOCYTE ALKALINE PHOSPHATASE STAIN Finding Score Normal 20 – 100 Chronic myelogenous leukemia < 13 Leukemoid reaction > 100 Polycythemia vera 100 – 200 Secondary polycythemia 10 – 100

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.

A normal neutrophil alkaline phosphatase reaction

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.

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.

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