Cytochemical Stains

SANTOS, Carlo Miguel N.
HEMATOLOGY LECTURE OLA

3D - MT Submitted on 18 February 2019

Hematologic Cytochemical Stains
STAIN USES RESULT
Myeloperoxidase Stain the Peroxidase found in Myeloblast: Strong (+)

granules of cells in Monoblast: Faint (+)
myelocytic and monocytic
cell lines BLACK TO BLUE TO
BROWN PRECIPITATE
Differentiate ALL and AML
Sudan Black B Stains lipids such as sterols, Myeloblast: Strong (+)

neutral fats, and Monoblast: Faint (+)
phospholipids. These lipids
are found primarily in BLACK PRECIPITATE
granules of Neutrophils and
Granulocytes
Differentiate ALL and AML
Used in samples that are not

freshly drawn
Periodic Acid Schiff Periodic acid oxidizes Lymphocytic cells (+)

glycogen, mucoproteins and Erythrocytic cells (+)
other high molecular weight Benign cells (-)
carbohydrates to aldehydes
BRIGHT PINK
Differentiate between AML
(diffuse +) and ALL (block +)
Identifying abnormal
erythroblast and dysplastic
megakaryocytes in AML and
MDS
Acid Phosphatase Reaction Acid phosphatase is present Normal isoenzymes of acid
in all nonerythroid cells and phosphatase (-) : inactivated
hydrolyzes substrate naphthol by tartrate
AS-BI phosphoric acid
RED PRECIPITATE
Specific Esterase Differentiate myelocytic Isoenzymes 1,2,7,8, and 9

series from monocytic series stain with
a.) Naphthol AS-D Chloro
Distinguish normal and Acetate Esterase (CAE):
leukemic cells of both ceries Neutrophilic series and Mast
Cells
Marker of cytoplasmic Myeloblasts especially in
maturation in myeloid mature stages: Strong (+)
leukemia Auer Rods
BRIGHT RED
Non-Specific Esterase Inhibited by sodium fluoride Isoenzymes 3,4,5, and 6 stain

with
Identify monocytic a.) Alpha-Naphthol Acetate
component in AML Esterase (ANAE): Leukemic
and Normal Monocyte:
Strong (+): DIFFUSE RED
OR BROWN COLOR
b.) Alpha-Naphthol Butyrate

Esterase: T-cells (+):
BROWN AND GRANULAR
Combined ANAE and CAE Avoid the need to compare a.) Naphthol AS-D Chloro
results from ANAE and CAE Acetate Esterase (CAE):
Neutrophilic series and Mast
Detect Myelocytic Leukemia Cells
Myeloblasts especially in
Identify Monocytic and mature stages: Strong (+)
Granulocytic component of Auer Rods
AML
BRIGHT RED
a.) Alpha-Naphthol Acetate
Esterase (ANAE): Leukemic
and Normal Monocyte:
Strong (+): DIFFUSE RED
OR BROWN COLOR
Toluidine Blue Basic thiazine metachromatic Basophilic granules and mast

dye with high affinity to cells (+)
acidic tissue components
BRIGHT RED AND
Enumeration of basophils and PRUPLE
mast cells
Perl’s Iron Stain (Prussian Detecting siderotic granules Siderotic granules

Blue Reaction) in nRBCs and some
reticulocytes BLUE
LEUKOCYTIC ALKALINE PHOSPHATASE

- Alkaline Phosphatase activity is found predominantly in mature neutrophil and
metamyelocyte
- It used to differentiate between Leukemoid reaction and CML
- The enzyme activity is associated with a poorly characterized intracytoplasmic membrane
component distinct from primary and secondary granules
Procedure
1. Fix air dried blood films for 30 seconds in cold 4% formalin methanol
2. Rinse with tap water and air dry
3. Incubate slides with working substrate solution for 15 minutes to 40 mL stock
solution. Add 24mg of fast blue BB
4. Wash in tap water and air dry
5. Counterstain for 3 minutes in 0.02% aqueous neutral red. Rinse with water and air
dry
Results
a. Reaction product is blue and granular
b. Intensity of reaction varies from negative to strongly positive
Count 100 NEUTROPHILS and score them 0 to +4 then calculate the final score by adding the
total scores
Grading Interpretation
0 negative / no granules
+1 occasional granules
+2 moderate no. of granules
+3 numerous no. of granules
+4 heavily positive / numerous granules

crowding cytoplasm overlying nucleus
Reference Range
a. LAP Score = 38 to 178
Correlated Diseases
ELEVATED DECREASED
1. Leukemoid reactions 1. CML

2. New born babies 2. Hereditary hypophosphatasia
3. Pregnant women 3. PNH (Paroxysmal Nocturnal
4. Aplastic anemia Hemoglobinuria)
5. Pernicious anemia 4. Sickle Cell Anemia
6. Neutrophilia of infection
7. Polycythemia vera
8. Hodgkin lymphoma
9. Myelofibrosis
10. Essential thrombocytosis
11. Multiple myeloma
12. Obstructive jaundice

Cytochemical Stains

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What are some of the hematologic cytochemical stains discussed and what are they used for?

What are some of the hematologic cytochemical stains discussed and what are they used for?

What are the procedures and results of leukocytic alkaline phosphatase staining?

What are the procedures and results of leukocytic alkaline phosphatase staining?

SANTOS​, Carlo Miguel N.

HEMATOLOGY LECTURE OLA

STAIN USES RESULT

Myeloperoxidase Stain the ​Peroxidase​ found in Myeloblast: Strong (+)

Sudan Black B Stains lipids such as sterols, Myeloblast: Strong (+)

Differentiate ALL and AML

Used in samples that are not

Periodic Acid Schiff Periodic acid oxidizes Lymphocytic cells (+)

Specific Esterase Differentiate myelocytic Isoenzymes 1,2,7,8, and 9

Non-Specific Esterase Inhibited by sodium fluoride Isoenzymes 3,4,5, and 6 stain

b.) Alpha-Naphthol Butyrate

Toluidine Blue Basic thiazine metachromatic Basophilic granules and mast

Perl’s Iron Stain (Prussian Detecting siderotic granules Siderotic granules

LEUKOCYTIC ALKALINE PHOSPHATASE

+2 moderate no. of granules

+3 numerous no. of granules

+4 heavily positive / numerous granules

1. Leukemoid reactions 1. CML

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Myeloperoxidase Stain the Peroxidase found in Myeloblast: Strong (+)