CYTOCHEMICAL DEMONSTRATION OF ACID PHOSPHATASE IN HEMATOPOIETIC CELLS IN HEALTH AND IN VARIOUS HEMATOLOGICAL DISORDERS USING AZO DYE TECHNIQUES

Abstract

Acid phosphatase activity in the formed elements of blood and bone marrow has been demonstrated using unfixed smears and azo dye coupling techniques with naphthol AS-MX or AS-TR phosphates as substrates. A similar staining pattern was also obtained using a new substrate, hydroxycarbazole phosphate. When this compound was used, hydrolysis was more rapid and good staining was obtained using smears fixed at room temperature (25°C) in 60% acetone buffered to pH 4.2-4.5 with citrate. Such preparations showed good cytological detail; however, the azo dye is less chromogenic than that formed with the substituted naphthol phosphates and nonspecific staining due to free hydroxycarbazole was observed. The use of ethylene diamine tetraacetic acid (EDTA) as an anticoagulant did not interfere with the reaction. Moderate reduction in staining intensity was observed using smears stored at room temperature longer than 24 hours prior to incubation. Dye precipitation was observed in most cells and was strongest in bone marrow osteoclasts and phagocytic reticulum cells. Immature myeloid cells showed moderately strong activity and this was particularly evident in peripheral blood smears of some patients with untreated chronic myelogenous leukemia. Increased staining was also observed in the plasma cells of 2 patients with untreated multiple myeloma. No consistent deviation from the normal was observed in the remaining disorders studied.