Topic 4: Cytochemistry: Cytochemical Stains

Learning Objectives

At the end of this topic, students will be able to:

1. Describe how cytochemical staining can aid in the diagnosis of leukemias

2. Explain the importance of cytochemistry in differentiating leukemic blast of
acute myelogenous leukemia from leukemia of lymphoid origin.

Presentation of Contents

CYTOCHEMISTRY
- The microscopic study of chemical constituents within cells, useful for the
identification of malignant cell types and abnormal cell constituents
- Helps to differentiate the leukemic blast of acute myelogenous leukemia from
leukemia of lymphoid origin.

CYTOCHEMICAL STAINS
I. Enzymatic Techniques
A. Peroxidases
- catalyze the oxidation of substances by H2O2
- primary neutrophilic granules
- routine stain on all newly diagnosed leukemias
- marker for Auer rods
1. Diamino Benzidine Method
- (+) dark brown granules
2. Cyanide Resistant Peroxidase Stain
- eosinophilic component of leukemia
- (+) brown
Myeloperoxidase (MPO)
- Marker for primary granules and Auer rods
- Peroxidase activity produces dark brown granules in cytoplasm of
granulocytes and monocytes
- done on fresh specimens.

B. Esterases

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 – differentiates monocytes from granulocytes
1. Specific esterase
- Naphthol AS-D Chloroacetate esterase
Chloroacetate esterase
- Marker of mature and immature neutrophils and
mast cells
- Enzyme activity results in bright red granules in
cytoplasm of neutrophils, neutrophil precursors and mast
cells
2. Nonspecific esterase (NSE)
1. ά Naphthyl Acetate Esterase
- Marker for monocytes, megakaryocytes and plasma cells
Monocytes stain red-brown; focal or dotlike for lymphocytes
2. ά Naphthyl Butyrate Esterase
- Useful for monocytes, promonocytes and monoblasts
- Enzyme activity results in dark red precipitates in the
Cytoplasm
- inhibited by fluoride

C. Phosphatases
1. Acid Phosphatase (ACP) Stain
- present in all hematopoietic cells
- located in lysosomes
- (+) purplish to dark red granules
2. Tartrate Resistant Acid Phosphatase (TRAP)
- Marker for Hairy cell leukemia
- Activity is indicated by purple to dark red granules in the
Cytoplasm
- Useful for differentiating subgroups of acute lymphoblastic
leukemia and delineating hairy cell leukemia from other chronic
lymphoid neoplasias
3. Leucocyte Alkaline Phosphatase (LAP)
- Neutrophil is the only leukocyte that contains this activity
- 100 cell count is done; neutrophils are scored from 0 with no
activity to 4 with a large amount of activity
- Used to differentiate CML from leukemoid reaction
- CML has decreased activity

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II. Nonenzymatic Techniques
A. Periodic Acid Schiff Stain (PAS)
- Marker for glycogen, glycoproteins, mucoproteins and high MW
carbohydrates
- Activity results in bright fuchsia pink
- Erythroblasts in M6 leukemia are positive
- Lymphoblastic leukemia shows blocky or chunky pattern
- L1 and L2 block pattern; L3 negative.
B. Sudan Black B (SBB)
- Marker for phospholipids and lipids
- Dark purple-black granules in neutrophil precursors
- Lymphoblasts are negative
C. Toluidine Blue
- binds acid mucopolysaccharides to form metachromatic
complexes
- for mast cell disease, acute and chronic basophilic leukemia
- (+) red violet (purplish red) metachromatic granules of basophils
and mast cells
- Useful for recognition of mast cells and basophils
- Strongly metachromatic
D. Perl’s Prussian Blue Stain
- for ferric iron; sideroblasts & siderocytes
- (+) blue to blue green with red nucleus

III. Immunocytochemical Techniques

A. Immunoperoxidase
B. Terminal Deoxynucleotidyl Transferase (TdT)
- Marker for immature lymphocytes or primitive lymphoid cells
- Used to differentiate AML from ALL
- Present in 90% cases of ALL

C. Megakaryocyte Precursors – can use Gp Iib/IIIa, Platelet Factor 4,

FVIIIR:Ag, GpI for M7 leukemia
D. Lymphocyte Subpopulations – uses respective specific surface marker

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