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
in all nonerythroid cells and
hydrolyzes substrate naphthol
AS-BI phosphoric acid
Specific Esterase Differentiate myelocytic
series from monocytic series
Distinguish normal and
leukemic cells of both ceries
Marker of cytoplasmic
maturation in myeloid
leukemia
Non-Specific Esterase Inhibited by sodium fluoride
Identify monocytic
component in AML
Combined ANAE and CAE Avoid the need to compare
results from ANAE and CAE
Detect Myelocytic Leukemia
Identify Monocytic and
Granulocytic component of
AML
Normal isoenzymes of ​acid
phosphatase​ (-) : inactivated
by tartrate
RED PRECIPITATE
Isoenzymes 1,2,7,8, and 9
stain with
a.) Naphthol AS-D Chloro
Acetate Esterase (CAE):
Neutrophilic series and Mast
Cells
Myeloblasts especially in
mature stages: Strong (+)
Auer Rods
BRIGHT RED
Isoenzymes 3,4,5, and 6 stain
with
a.) Alpha-Naphthol Acetate
Esterase (ANAE): Leukemic
and Normal Monocyte:
Strong (+): DIFFUSE RED
OR BROWN COLOR
b.) Alpha-Naphthol Butyrate
Esterase: T-cells (+):
BROWN AND GRANULAR
a.) Naphthol AS-D Chloro
Acetate Esterase (CAE):
Neutrophilic series and Mast
Cells
Myeloblasts especially in
mature stages: Strong (+)
Auer Rods
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