Leukocyte Alkaline Phosphatase Stain

1. Rabab Salama Clinical and Chemical Pathology Consultant,
HCQM Specialist

2. Study of chemical elements in
cells.

4. Substrate + Enzyme >>>> Product
+
Visible product <<<< Chromogen

5. Types
 Myeloperoxidase (MPO)
 Leukocyte Alkaline Phosphates (LAP or NAP)
 Acid Phosphates, TRAP
 Estrase
 Sudan Black B stain (SBB)
 Iron stain
 Reticulin stain
 Periodic Acid Schiff (PAS)

6. Leukocyte Alkaline Phosphatase (LAP) stain
 The leukocyte alkaline phosphatase (LAP) stain is helpful in
determining whether a high peripheral blood leukocytosis is a
reactive process or a leukemia (chronic myelogenous leukemia, or
CML).
 The more differentiated cells in the reactive process will stain
more readily with LAP, while leukemic cells will not.
 The cells on a smear can be assessed and an "LAP score" can be
generated. A high score generally indicates a "leukemoid reaction"
or reactive condition (with an infection or other inflammatory
process) while a low score suggests CML.
Purpose:

7. Leukocyte Alkaline Phosphates (LAP)
Neutrophil Alkaline Phosphates (NAP)
Principle:
Alkaline phosphatase within neutrophils hydrolyzed
naphthol AS phosphate. Hydrolyzed substrate
couples with dye (fast blue BB salt), ppt at site of
enzyme activity. Degree of staining is proportional
to enzymatic activity.
Result:
The reaction product is blue and granular

8. Sampling:
 Peripheral blood
 Fresh sample
 Heparinized or capillary blood sample
(EDTA inhibits LAP)
 If count below 5,000/cmm, use buffy
coat
Leukocyte Alkaline Phosphates
(LAP)

9. Reagents
 Fixatives
 4% formalin methanol.
 Substrate
 Naphthol AS phosphate alkaline solution.
 Fast blue BB salt or fast violet B salt.
 Counterstain
 Neutral red.
Leukocyte Alkaline Phosphates
(LAP)

10. Leukocyte Alkaline Phosphates
(LAP)
Interpretation:
 Count 100 neutrophils and score them (0/+4), then calculate the
final score by adding the total scores.
Grading (LAP scoring):
 (0) No stain
 (+1) Faint stain
 (+2) Moderate stain
 (+3) Strong stain
 (+4) Strong stain without cytoplasmic background
Normal Range: 30-185

11. Positive LAP reaction
Negative LAP reaction
Leukocyte Alkaline Phosphates
(LAP)

13. N.B
 Thin smears or thick smears may falsely elevate
results.
 Only segmented or bands are scored.
 Fresh samples as enzyme activity decreases and
slides should be scored as quick as possible, as the
dye tends to fade.
 Eosinophils are negative but could be mistaken and
counted in the score.
Leukocyte Alkaline Phosphates
(LAP)

14. LAP decreased in:LAP elevated in:
CML.Leukomoid reaction.
Paroxymal Nocturnal
Hemoglobinuria.
Pregnancy
Sickle cell anemia.Polycythemia vera.
Hypophosphatasia.Aplastic anemia.
Multiuple myeloma
Obstructive juindice.
Hodgkins` disease.