Cytochemical Stain
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Transcript of 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.