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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