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 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

SUMMARY OF LAP INTERPRETATION
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.

     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.

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