FLOW-CYTOMETRY OF LEUKOCYTE ALKALINE-PHOSPHATASE IN NORMAL AND PATHOLOGICAL LEUKOCYTES

Leucocyte alkaline phosphatase (LAP) is an enzyme expressed on the ext ernal aspect of the neutrophilic granulocyte plasma membrane, and repr esents a specific marker for the fully differentiated granulocyte. In this report we characterize 1B12.1, a monoclonal antibody raised again st human bone alkaline phosphatase, by its ability to recognize the LA P protein, As assessed by Western blot analysis, following electrophor esis under non-reducing conditions, the antibody specifically reacts w ith LAP upon forced expression of the protein in simian COS-7 fibrobla sts. In addition, the 1B12.1 antibody recognizes partially purified LA P isolated from peripheral blood granulocytes. With this antibody we d eveloped a quantitative flow-cytometry-based method for the determinat ion of LAP. Double fluorescence flow cytometry demonstrated that the L AP protein was present in relatively high amounts in neutrophilic gran ulocytes, but not in monocytes, natural killer cells, or B and T lymph ocytes of normal individuals. The protein was completely absent in gra nulocytes obtained from chronic myeloid leukaemia and paroxysmal noctu rnal haemoglobinuria patients. Higher than normal levels of LAP protei n were evident in neutrophilic granulocytes of patients suffering from -polycythaemia vera, essential thrombocythaemia and severe aplastic an aemia. However, the highest amounts of LAP protein were present in the granulocytes of normal individuals treated with G-CSF for the isolati on of peripheral blood stem cells.