11. Control
Internal control
Cells in BM known to be positive for the stain
External control
Normal blood smear containing neutrophils,
lymphocytes and monotypes (eg. PAS, LAP)
Slide from patient to have the disease 11
12. Types of specimens
Peripheral blood.
Bone marrow aspirated and imprints.
Paraffin section from bone marrow biopsy.
Aspirated and imprints of LN, spleen.
Fresh samples to ensure optimal enzyme activity
Smears from non-enzymatic stains as PAS and
SBB stains may remain stable for months.
12
13. Control slides do not exhibit proper
staining pattern?
Reagents:
Expiry date
Contamination
Procedures:
Steps not followed correctly
Smear:
Age of the slide.
Storage: some enzymes diminish in activity over time
(fresh samples)
13
15. PEROXIDASE STAIN
Purpose:
To differentiate a myelogenous or monocytic
leukemia from acute lymphocytic leukemia.
Peroxidase is present in the primary azurophilic
granules of neutrophil, eosinophil and monocyte &
activity increased with maturation, no activity is
found in red cells or lymphocytes.
15
19. PEROXIDASE STAIN :
Red – brown peroxidase found in:
neutrophil and eosinophil {promyelocyte – Myelocyte –
Metamyelocyte}
Finely granular staining found in: - Monocyte
Negative stain found in:
( early Myeloblast, lymphblast, basophiles and plasma
cell)
19
20. NOTES:
MPO is sensitive to light, smears
should be stained immediately
and stored in dark.
Positive control from healthy
individuals.
Overincubation: false positive
peroxides in RBCs. 20
23. Leukocyte alkaline phosphatase 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:
23
24. 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
24
25. Sampling:
Leukocyte Alkaline phosphates (LAP)
Peripheral blood
Fresh sample
Heparinized or capillary blood sample
(EDTA inhibits LAP)
If count below 5,000/cmm, use buffy
coat
25
26. 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)
26
27. 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
27
30. N.B
Leukocyte Alkaline phosphatase (LAP)
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. 30
32. Acid phosphatase ( with tartrate resistance)
Like NAP but at pH (5)
Purpose: diagnosis of hairy cell leukemia.
32
33. Acid phosphatase
Principle: ACP enzyme present in white cells hydrolyzed the
substrate naphthol AS-BI phosphoric acid. Hydrolyzed.
Substrates couples with Diazo dye with ppt. at the site of
enzymatic activity.
Result:
The reaction product is red granules 33
34. Acid phosphatase ( with tartrate resistance)
Principle:
ACP enzyme present in white cells hydrolyzed the
substrate naphthol AS-BI phosphoric acid.
Hydrolyzed substrate couples with Diazo dye with
ppt. at the site of enzymatic activity.
Has 7 isoenzymes.
Tartaric acid inhibits all AP isoenzymes except 5
that are present only in HCL. 34
35. Reagents
Fixatives
Methanol + acetone.
Substrate
Naphthol AS-BI phosphate.
Fast blue BB salt or fast violet B salt.
Counterstain
Methyl green or hematoxylin.
Acid phosphatase
35
36. Acid phosphates
(with tartrate resistance)
Hairy cell leukemia, TRAP stain. Acid
phosphatase reaction after incubation
with tartaric acid. Granular staining is
seen in the lymphocytes.
36
39. Specific estrases (1,2,7,8,9) of granulocytes
staining specifically with substrate Naphthol
AS-D chloroacetate, estrase .
NOT inhibited by Sodium fluoride.
Estrases
39
40. Non specific estrases (3,4,5,6) act on many
substrates
α naphthol acetate (ANAE)
α naphthol butyrate (ANBE)
Naphthol AS-D acetate (NASDA)
Naphthol AS acetate (NASA)
NSE of monotypes, megakaryocytes and playelets.
NSE are inhibited by Sodium fluoride.
Estrases
40
44. Principle:
Result:
The reaction product is orange red granules
Non Specific Esterase:
{with fluoride inhibition}
Differentiate myelocytic and monocytic leukemia.
Purpose:
44
45. NSEs α-naphthyl acetate positivity in M5b.
Note the granular positivity in the monoblasts and immature monocytes
Non Specific Esterase
45
46. Interpretation
(+ve) brick – red staining which found in:
Megakaryocyte and platelets, Histocyte,
Macrophage, Monocyte & Lymphoblast of ALL
(-ve) for granulocytes
If fluoride added, only monocyte non specific
esterase will be inhibited.
46