Flow cytometry of leucocyte alkaline phosphatase in normal and pathologic leucocytes

Alessandro Rambaldi, Kensaku Masuhara, Gian Maria Borleri, Ricardo Amaru, Maurizio Giannì, Mineko Terao, Tiziano Barbui, Enrico Garattini

Research output: Contribution to journalArticle

Abstract

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 flow cytometry-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.

Original languageEnglish
Pages (from-to)815-822
Number of pages8
JournalBritish Journal of Haematology
Volume96
Issue number4
Publication statusPublished - 1997

Keywords

  • alkaline phosphatase
  • diagnosis
  • flow cytometry
  • leucocyte
  • retinoic acid

ASJC Scopus subject areas

  • Hematology

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  • Cite this

    Rambaldi, A., Masuhara, K., Borleri, G. M., Amaru, R., Giannì, M., Terao, M., Barbui, T., & Garattini, E. (1997). Flow cytometry of leucocyte alkaline phosphatase in normal and pathologic leucocytes. British Journal of Haematology, 96(4), 815-822.