Leukocyte Alkaline Phosphatase Stain

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
Reticulinstain
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 "leukemoidreaction"
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:
Alkalinephosphatasewithinneutrophilshydrolyzed
naphtholASphosphate.Hydrolyzedsubstrate
coupleswithdye(fastblueBBsalt),pptatsiteof
enzymeactivity.Degreeofstainingisproportional
toenzymaticactivity.
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)Nostain
(+1)Faintstain
(+2)Moderatestain
(+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.