Flow cytometry of leucocyte alkaline phosphatase in normal and pathologic leucocytes

Abstract

Summary. Leucocyte alkaline phosphatase (LAP) is an enzyme expressed on the external aspect of the neutrophilic granulocyte plasma membrane, and represents a specific marker for the fully differentiated granulocyte. In this report we characterize 1B12.1, a monoclonal antibody raised against human bone alkaline phosphatase, by its ability to recognize the LAP protein. As assessed by Western blot analysis, following electrophoresis under non-reducing conditions, the antibody specifically reacts with LAP upon forced expression of the protein in simian COS-7 fibroblasts. In addition, the 1B12.1 antibody recognizes partially purified LAP isolated from peripheral blood granulocytes. With this antibody we developed a quantitative flowcytometry-based method for the determination of LAP. Double fluorescence flow cytometry demonstrated that the LAP protein was present in relatively high amounts in neutrophilic granulocytes, but not in monocytes, natural killer cells, or B and T lymphocytes of normal individuals. The protein was completely absent in granulocytes obtained from chronic myeloid leukaemia and paroxysmal nocturnal haemoglobinuria patients. Higher than normal levels of LAP protein were evident in neutrophilic granulocytes of patients suffering from polycythaemia vera, essential thrombocythaemia and severe aplastic anaemia. However, the highest amounts of LAP protein were present in the granulocytes of normal individuals treated with G-CSF for the isolation of peripheral blood stem cells.

Pericytes resident in postnatal skeletal muscle differentiate into muscle fibres and generate satellite cells

“…AP is broadly expressed in many pre-and postnatal tissues, and in different cell types such as osteoblasts 37 , embryonic stem cells 38 , bone marrow stroma cells 39 , liver and kidney cells 40 , granulocytes 41 and B cells 42 ; however, in postnatal mouse striated muscles, AP is expressed only by vessel-associated cells, in both endothelial cells and pericytes, as previously reported 9,[14][15][16] , and exhaustively confirmed in this work through a variety of histochemical and immunochemical methods. Most importantly, by using virtually all possible experimental approaches, we demonstrated that both AP and Cre expression are invariably absent in quiescent satellite cells, as well as in proliferating and differentiating myogenic cells in vitro or in vivo, even after cardiotoxin-induced damage.…”

Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders

“…To investigate granulocyte activation in myeloproliferative disorders, we measured LAP, 24,25 CD16, 25 and CD157 26 expression levels on circulating granulocytes.…”

Bis-indols: a novel class of molecules enhancing the cytodifferentiating properties of retinoids in myeloid leukemia cells

“…27 CD11b, CD38, CD14, ICAM-1, and integrin ␣-5 were measured by flow cytometry using specific monoclonal antibodies (Becton Dickinson, Franklin Lakes, NJ). 28 The nitrobluetetrazolium (NBT) reduction assay was performed on extracts of phorbol myristate acetate-stimulated cells, 20 and the percentage of NBT ϩ cells was determined as described. 20 Western blot analysis, JNK kinase activity, and determination of cytokine secretion and gel retardation assays Extracts of NB4 cells (from 3 ϫ 10 6 cells) were subjected to Western blot analysis as reported.…”