Histochemical double staining method for simultaneous demonstration of acid and alkaline phosphatases on bone marrow smears.

Abstract

A double staining method was devised for simultaneous histochemical demonstration of acid and alkaline phosphatase activities on the same bone marrow smears. The fixation was done by neutral formaldehyde vapor for 5 minutes at room temperature. The incubation for demonstration of acid phosphatase was firstly carried out by immersing the smears in a medium containing naphthol-ASBI-phosphate as the substrate and Fast red violet LB salt as the coupler at 37°C for 2 hours. Next, the smears were incubated in a medium for alkaline phos-phatase using naphthol-ASMX-phosphate as the substrate and Fast blue RR as the coupler at 37°C for 3 hours. Interesting results were obtained concerning the cells of neutrophilic series and the reticuloendothelial cells. Immature neutrophils disclosed red staining indicating acid phosphatase activity and gradually lost red color with increasing blue staining due to increasing alkaline phosphatase activity along with cell maturation. Reticuloendothelial cells showed intense acid and alkaline phos-phatase activity, and they could be classified into four groups according to the behavior to the enzyme staining: 1) The group of cells possessing only acid phosphatase activity. 2) The group of cells showing more intense reaction to acid phosphatase than to alkaline phosphatase. 3) The group of cells showing more intense reaction to alkaline phosphatase than to acid phosphatase. 4) The group of cells having only alkaline phosphatase activity. The average rate of appearance was 49% for the 1st group, 3% for the 2nd group, 40% for the 3rd group and 8% for the 4th group. Reticuloendothelial cells belonging to the 1st group were found increased in the bone marrow of the patients with malignant reticulosis.