Special Stain
Cytochemical Reactions in Acute Leukemia
Cellular Element Cytochemical
Blasts Identified Stained Reaction
Myeloblasts strong positive; Neutrophil primary Myeloperoxidase
monoblasts faint positive granules (MPO)
Myeloblasts strong positive;
monoblasts faint positive Phospholipids Sudan Black B (SBB)
Myeloblasts strong positive Cellular enzyme Specific esterase
Nonspecific esterase
Monoblasts strong positive Cellular enzyme (NSE)
Variable, coarse or block-like
positivity often seen in
lymphoblasts and
pronormoblasts, myeloblasts Glycogen and
usually related substances Periodic acid-Schiff
negative although faint diffuse
reaction may
occasionally be seen
Sudan Black B:
Sudan Black B (SBB) is a fat soluble dye which has very
high affinity for neutral fats and lipids.
It is similar to that of Myeloperoxidase (MPO) staining
pattern of leukocytes and monocytes.
Purpose: To distinguish acute myelogenous and
monocytic leukemia from lymphocytic leukemia.
:It has few advantages over MPO
SBB can be used for staining smears more than 2 weeks
.old
SBB stains both azurophilic and specific granules in
neutrophils, whereas MPO stains azurophilic granules
.only
.There is only a little fading of the SBB stain over time
:Principle
Sudan black B dye is fat soluble, then it stains fat
particles (Steroles, phospholipids and neutral fats)
These are present in azurophilic and secondary granules
.of myelocytic and lysosomal granules of monocytic cells
During staining, the dye leaves the solvent because of its
high solubility in lipids than solvent. On microscopic
examination, varying degree of black colored pigments
.are seen in the positive reaction
:Requirements
Sample: Fresh anticoagulated whole blood or bone
marrow smear may be used. The slides must be fixed as
.soon as possible
Fixative: 40% formaldehyde solution vapor
Stain: SBB 0.3 g in 100 ml absolute ethanol
Phenol buffer: Dissolve 16 g crystalline phenol in 30 ml
absolute ethanol. Add to 100 ml distilled water in which
.0.3 g Na2HPO4.12H2O has been dissolved
Working SBB stain solution: Add 40 ml buffer to 60 ml
SBB solution (The composition of working stain may
.slightly vary upon different products)
Counterstain: May–Grunwald–Giemsa or Leishman
.stain
:Procedure of Sudan Black B Stain
Fix air dried smears in formalin vapour, formaldehyde or
.formalin-ethanol fixative for 10 minutes
.Wash gently in water for 5-10 minutes
Place the slides in the working stain solution for 1 hour
.in a Coplin jar with a lid on
Remove and flood the slides with 70% alcohol for 30
seconds. Tip the 70% alcohol off and flood again. Repeat
.this three times
.Rinse in running tap water and air dry
Counterstain without further fixation with Leishman
.stain or May–Grunwald–Giemsa stain
.Air dry and examine microscopically
Sudan Black B
Positive sudan black
B (SBB) stain in a
patient with AML ,
Not the black staining
cytoplasmic granules
in the myeloblasts
Production of black and granular pigment indicates
.positive reaction
Lipids are present in azurophilic and secondary granules
.of myelocytic cells. Hence, these are SBB positive
The staining becomes more intense as cells mature from
.myeloblast to mature forms
Basophils are generally not positive but may show bright
.red/purple metachromatic staining of the granules
Lipids are present in lysosomal granules of monocytic
cells. Monocytic cells show variable reactions, from
negative to weakly positive.
Lymphoid cells are SBB negative. However, in ALL, less
than 3% of blast cells show positive reaction.
Myelogenous cells show coarse staining granules with
faint staining pattern for myelobast and increase
staining with maturation.
.Auer rods are +vely stained
.Monocytic cells show finely scattered granules
ve lymphocytic staining except Burkitt`s lymphoma-
.cells, may show +ve staining vacuoles
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