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The diagnosis of systemic mastocytosis (SM) is based primarily on the histologic and immunohistochemical evaluation of a bone marrow trephine biopsy specimen. Although mast cell (MC) specific antigens like tryptase and chymase are detectable in routinely processed tissue, no immunohistochemical markers that can be used to discriminate between normal and neoplastic MCs are yet available. We have investigated the diagnostic value of an antibody against CD25 for the immunohistochemical detection of MCs in bone marrow sections in 73 patients with SM and 75 control cases (reactive marrow, n = 54; myelogenous neoplasms, n = 21) and correlated the results with the presence of c-kit mutations. While MCs in almost all patients with SM (72 of 73) expressed CD25, none of the control samples contained CD25-positive MCs. Irrespective of the SM subtype, most of neoplastic MCs expressed CD25. In 3 patients with advanced MC disease, pure populations of neoplastic MCs were obtained and found to express CD25 mRNA by RT-PCR analysis. In addition, all patients with CD25-positive MCs contained c-kit mutations, while all control cases exhibited wild type c-kit. CD25 therefore appears to be a reliable immunohistochemical marker for the discrimination of neoplastic from normal/reactive MCs, with potential as a diagnostic tool in SM.