Assessment of alkaline phosphatase on the surface membrane of neutrophils by immunofluorescence

Expression of alkaline phosphatase (ALP) on the surface membrane of neutrop hils (mNAP) was studied by immunofluorescence using an anti-ALP monoclonal antibody. Fluorescent intensity distribution of mNAP was analyzed using FAG S (fluorescence-activated cell sorter). The mean fluorescent intensity (MFI ) of the mNAP in this assay was well correlated with the neutrophil ALP (NA P) score demonstrated cytochemically (r = 0.832). mNAP levels in Various he matological disorders were evaluated by % mNAP(+) cells and MFI. The levels in aplastic anemia and polycythemia vera were significantly higher, and in chronic myelocytic leukemia and paroxysmal nocturnal hemoglobinuria (PNH), the revels were significantly lower compared with the levels in healthy vo lunteers. Two-color immunofluorescence with anti-ALP and anti-CD16 showed t hat the PNH clone was essentially negative for mNAP, whereas residual norma l neutrophils (CD16(+)) had levels slightly higher than those in normal ind ividuals. Highly reproducible results were obtained in the blood samples wh ich were stored at 4 degrees C for at least 24 hr without any treatment pri or to immunofluorescent staining. No degradation of fluorescent intensity w as seen 4 days after staining and fixation. The mNAP assay is simple, witho ut subjective evaluation for quantification, and is useful far differential diagnosis of hematological disorders. (C) 1999 Wiley-Liss, Inc.