PERIODIC-ACID-SCHIFF (PAS) STAIN

This reaction stains polysaccharides. Periodic acid oxidizes carbohydrates and similar compounds to aldehydes. The aldehydes can then react with the Schiff reagent (leuko-fuchsin) to release fuchsin and stain the cellular components containing oxidizable compounds. A variety of intracellular compounds react with the PAS reagents, but in the blood and marrow cells glycogen is the most abundant compound, as evidenced by amylase digestion that blocks the staining. In the blood from normal people, the cytoplasm of polymorphonuclear leukocytes stains intensely pink or red, with granular appearance in some cells. Monocyte cytoplasm stains faintly pink and may contain fine or coarse granules. Erythrocytes do not stain. Platelets stain intensely. In the normal marrow, the earliest granulocyte precursors do not stain. Cytoplasmic staining increases with increasing maturity of the granulocytic cells. PAS reaction may be helpful in the diagnosis of some cases of acute lymphoblastic leukemia and some subtypes of acute myeloid leukemia.

MYELOPEROXIDASE
Peroxidase is an enzyme from oxydoreductase group. It is found in myeloid cells and is then called myeloperoxidase. Myeloid cells can destroy microorganisms with the help of this enzyme. Peroxidase is present in primary granules of neutrophils, and in the granules of eosinophils and monocytes. The identification of this enzyme in the cytoplasm of leukocytes is useful in distinguishing acute myelogenous leukemia from acute lymphocytic leukemia, and to detect or confirm diminished peroxidase activity in individuals with myeloperoxidase deficiency. In the presence of peroxidase, H2O2 is split liberating O2 that oxidizes benzidine or 4chlor-1-naphthol. Positive reaction goes yellow-brown stained granules in the cytoplasm of a cell. The positive reaction is different in the cells of granulocytopoiesis from promyelocyte to segmented granulocyte. Positive reaction to peroxidase is also possible in sense of monocytes. In myeloblasts, megakaryocytes, lymphocytes, thrombocytes, and erythrocytes the reaction is negative. Positive myeloperoxidase reaction differentiates myelogenous from lymphatic acute leukemia. However, a negative result does not exclude myeloblastic leukemia. Smears of the peripheral blood and bone marrow, in which the activity of peroxidase is to be examined, must not be older than 3 days. Anticoagulants, especially EDTA, inhibit the peroxidase reaction.

Immunohistochemical diagnosis of acute myelogenous leukemia. A, Bone marrow aspirate shows increased blasts from patient with acute myeloid leukemia with inv(16) (Wright-Giemsa stain, 50). B, Bone marrow biopsy from the same patient shows 100% cellularity with sheets of blasts (hematoxylin-eosin stain, 40).