Cytochemistry i

1. Rabab Salama Clinical and Chemical Pathology Consultant,
HCQM Specialist
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2. Diagnostic tools in hematology
1.Routine stain& morphology
2.Cytochemistry
3.Immunophenotyping
4.Cytogenetics
5.Molecular biology
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3. Study of chemical elements in
cells.
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4. 4

5. Substrate + Enzyme >>>> Product
+
Visible product <<<< Chromogen
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6. Components of Cytochemistry
Fixative
Buffer
Substrate
Chromogen (dye)
Counterstain
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7. Fixatives
Types:
Methanol, ethanol, acetone, formation vapor,
formaldehyde, formal acetone, formal
ethanol
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8. Factors Affecting Fixation
1.Timing
Most fixatives 5-7 min
 MPO: 15 sec
 LAP: 30 sec
1.Type of fixative
2.Temperature
At room temperature (LAP, SBB) – at 4°C
(MPO, estate, LAP)
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9. Buffers
pH
Molarity
Time
Temperature
Factors Affecting Buffers:
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10. Counterstains
 MPO
 Mayer's Hematoxylin
 SBB
 nuclear counterstain: 1% Aqueous cresol
violet
 Background stain: 0.2% aqueous light green +
2 drops glacial AA
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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.
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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)
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14. 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) 14

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.
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16. 16

17. PEROXIDASE STAIN
Principle:
 In the presence of myeloperoxidase, H2O2 oxidizes
substrate ( benzedine or DAB) forming black ppt.
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18. Reagents
 Fixatives
Formal ethanol (formaldehyde and absolute
ethanol)
 Substrate
Benzedin (carcinogenic) –diaminobenzedine (DAB)
H2O2
 Counterstain
Mayer’s hematoxylin
PEROXIDASE STAIN
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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)
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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

21. Myeloperoxidase (MPO)
bluish-black granules red brown precipitate
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22. Myeloperoxidase stain, bone marrow
aspirate
The red granular staining
peroxidase activity.
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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:
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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
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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
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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)
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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
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28. Leukocyte Alkaline phosphatase (LAP)
Positive LAP reaction
Negative LAP reaction
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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

31. 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.
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32. Acid phosphatase ( with tartrate resistance)
Like NAP but at pH (5)
Purpose: diagnosis of hairy cell leukemia.
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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
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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.
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37. Specific
estrase
(SE)
• Glucoacetate esterase
• >> granulocytic lineage
Non-specific
esterase
(NSE)
• α naphthol acetate eatrase
• α naphthol butyrate esterase
• naphthol AS-D esterase
• naphthol AS esterase
>> monocytic lineage
Estrases
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38. Group of hydrolases
9 isoenzymes
Estrases
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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
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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
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41. Principle:
Specific esterase or chloroacetate
Result:
The reaction product is blue black granules 41

42. Reagents
 Fixatives
 Buffered formalin acetone.
 Substrate
 Naphthol AS-D chloroacetate.
+
 New Fuchsin (or fast blue BB).
 Counterstain
 Hematoxylin.
Specific esterase or chloroacetate
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43. Specific esterase or chloroacetate
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44. Principle:
Result:
The reaction product is orange red granules
Non Specific Esterase:
{with fluoride inhibition}
 Differentiate myelocytic and monocytic leukemia.
Purpose:
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45. NSEs α-naphthyl acetate positivity in M5b.
Note the granular positivity in the monoblasts and immature monocytes
Non Specific Esterase
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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.
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