Cytochemistry i

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
Reticulinstain
Periodic Acid Schiff (PAS)
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15. PEROXIDASE STAIN
Purpose:
Todifferentiateamyelogenousormonocytic
leukemiafromacutelymphocyticleukemia.
Peroxidaseispresentintheprimaryazurophilic
granulesofneutrophil,eosinophilandmonocyte&
activityincreasedwithmaturation,noactivityis
foundinredcellsorlymphocytes.
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16. 16

17. PEROXIDASE STAIN
Principle:
Inthepresenceofmyeloperoxidase,H2O2oxidizes
substrate(benzedineorDAB)formingblackppt.
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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.
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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 "leukemoidreaction"
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:
Alkalinephosphatasewithinneutrophilshydrolyzed
naphtholASphosphate.Hydrolyzedsubstratecouples
withdye(fastblueBBsalt),pptatsiteofenzyme
activity.Degreeofstainingisproportionalto
enzymaticactivity.
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)Nostain
(+1)Faintstain
(+2)Moderatestain
(+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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29. 29

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.
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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
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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)
•αnaphthol acetate eatrase
•αnaphthol butyrate esterase
•naphthol AS-D esterase
•naphthol AS esterase
>> monocyticlineage
Estrases
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38. Group of hydrolases
9 isoenzymes
Estrases
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39. Specificestrases(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 specificestrases(3,4,5,6) act on many
substrates
αnaphthol acetate (ANAE)
αnaphthol 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
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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 myelocyticand monocyticleukemia.
Purpose:
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45. NSEs α-naphthylacetate positivity in M5b.
Note the granular positivity in the monoblastsand 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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