Cytochemistry

Myeloperoxidase:

Peroxidase is present in primary granules of myeloid cells.
The peroxidase reaction is positive for myeloid cells and negative for lymphoid and erythroid cells. It is thus useful in identifying granulocytic leukemias.

The enzyme peroxidase is present in the primary granules of myeloid cells. These primary granules first appear in the early promyelocyte and persist through subsequent stages. In the presence of hydrogen peroxide, the enzyme peroxidase oxidizes the substrate 3 amino 9 ethylcarbazole to a red brown color.

Sudan Black B:

Phospholipids, neutral fats, and sterols are stained by Sudan Black B. SBB is used to differentiate AML from ALL. A positive result is rare in ALL. Sudan Black is the most sensitive stain for granulocytic precursors. Sudan black stains phospholipids and other lipids. This is believed to be due to physical solubility of the dye in the lipid particles. These lipid particles occur in both primary and secondary granules. They also occur to some extent in monocytic lysozomal granules. They may rarely occur in lymphocytes.

Specific Esterase:

Demonstrates myeloid differentiation in paraffin tissue sections. This is used to aid in differentiation of granulocytes, lymphocytes, and monocytes. The esterase enzyme within the cell hydrolyzes the substrate naphthol AS-D chloroacetate. The hydrolyzed substrate then couples with the diazo salt (hexazotized pararo saniline). The diazo dye precipitates out at the site of enzymatic activity.

Non specific Esterase:

Is used to identify monocytic cells. It is diffusely positive in monocytic cells and negative in granulocytic cells. Aids in the differentiation of granulocytes, lymphocytes, and monocytes. The esterase enzyme within the cell hydrolyzes the substrate alpha-naphthol butyrate. The hydrolyzed substrate then couples with the diazo salt (hexazotized pararosaniline). The diazo dye formed precipitates out at the site of enzymatic activity.

Periodic Acid-Schiff reaction:

Stains for glycogen and related compounds. Lymophocytes, granulocytes, monocytes, and megakaryocytes may be positive. Normal erythroid precursors are negative. This test is not used to distinguish between AML and ALL. It is good however, in distinguishing erythroleukemia from pernicious anemia. It is used in identifying M6 leukemias, and the reaction is negative in Burkitt’s lymphoma. Periodic acid-Schiff stains glycogen. Periodic acid oxidizes glycols to aldehydes. The aldehydes react with Schiff’s reagent.

Terminal Deoxynucleotidyl Transferase Test:

TdT is an enzyme marker for primitive lymphocytic cells. TdT is a DNA polymerase, present in lymphocytic cells. A specific antibody is incubated with the cells. If TdT is present in the nucleus of the cells, the positive cells will fluoresce.