Cytochemical Demonstration of "Acid" Phosphatase in Bone Marrow Smears

Abstract

Using Gomori"s method (1941), "acid" phosphatase was demonstrated in human and animal bone marrow smears. These were made on coverslips, dried in air, fixed in cold acetone for 30 sec., rinsed in distilled water, and incubated at 37[degree]C for 6-10 hrs. in the following medium: 5 parts distilled water, 3 parts acetate buffer at pH 4.0, 1 part 2% Na glycerophosphate, and 1 part 2% Pb nitrate. Incubation mixtures at pH 3.5 and 4.5 gave identical results. At pH 4.0, very little cloudiness was seen. Control coverslips were incubated in the same mixture, to which 2 mg. of NaF were added. The coverslips were washed in water for 2 min., immersed in yellow ammonium sulfide for 2 min., rinsed, and mounted in balsam. The areas of phosphatase activity were brown-black and the controls negative. Ethyl alcohol, formol, Na molybdate, and NaCN did not interfere with the phosphatase activity. Colchicine, urethane, and pteroyl- glutamic acid did not affect the reaction in normal and pernicious anemia marrow. Bone marrow smears from subjects without blood disorders and from a case of Addisonian pernicious anemia, one of chronic myeloid leucemia, and two cases of lymphoid leucemia were studied. The results were constant. In normal marrows, eosinophiles gave a strong reaction on the granules. Nonspecific granules of the myelo-blasts stained less intensely. Neutrophile granules were negative. In rat"s mesentery, myeloblast nuclei were nearly negative. They had a filamentous aspect in more mature white cells. The red cell cytoplasm stained poorly, but the nuclear structure was more granulous than filamentous. More mature elements showed a condensation of the nucleus. Red blood cells were entirely negative. Megakaryocytic nuclei stained heavily. The cytoplasm gave a weak reaction. Platelets were negative, as were nucleoli. White and red cell series, in chronic myeloid leucemia, gave reactions comparable to those of normal marrow. Lymphoid leucemia cells gave a very faint reaction in nucleus and cytoplasm. Megaloblastic series in pernicious anemia marrow did not present quantitative differences when compared with the normoblastic series. Chicken and rat marrow smears showed similar reactions even after 16-18 hrs. of incubation. The reaction was localized in the nucleus of chicken red blood cells.