Kinetic determination of granulocyte alkaline phosphatase by the GEMSAEC

Granulocyte alkaline phosphatase (ALP) activity is measured kinetically using the GEMSAEC centrifugal analyzer and p-nitrophenylphosphate as substrate. Measurements of enzyme activity are made at pH 9.8 utilizing 1.0 M diethanolamine as a buffer containing 0.5 mM magnesium chloride. Granulocytes are separated from heparinized blood using dextran sedimentation, followed with ficoll-hypaque separation. The separated cells are suspended in saline, and the red cells are lysed. Manual counting of stained smears of the cells isolated showed that 95 percent of these cells were granulocytes. After lysing the red cells, the granulocyte suspension is rinsed in 12.5 percent heat inactivated serum in saline and separated by centrifugation. After 200 microliters of the diluent solution (12.5 percent heat inactivated serum in saline) is added to the cells, they are sonicated for seven sec at 0 degrees in an ice bath. The extracts are then assayed kinetically for ALP activity. Granulocyte ALP activity varies from day to day in normal individuals. The daily variation of ALP activity observed correlates closely with and is confirmed by the Sigma Histochemical Procedure based on Kaplow' Scoring Method. The coefficient of variation of the ALP method within assay performed on two consecutive days on a pooled sample is 0.75 percent and 1.38 percent, respectively, and between assays 4.82 percent.