Immunophenotypic analysis using multicolor flow cytometry was performed on bone marrow (BM) aspirate specimens collected in EDTA anticoagulant tubes and processed within 12 hours of collection using a standard lyse/wash technique (PharmLyseTM, BD Biosciences, San Diego, CA, USA). For each analysis, a minimum of 200,000 events were acquired on FACSCanto II instruments (BD Biosciences). As described previously, the panel of monoclonal antibodies used included reagents specific for CD2, CD3 (surface and cytoplasmic), CD4, CD5, CD7, CD10, CD13, CD14, CD15, CD19, CD20, CD22, CD25, CD33, CD34, CD38, CD43, CD45, CD52, CD56, CD64, CD117, CD123, CD303, myeloperoxidase, HLA-DR, and TdT (BD Biosciences) [5 (link)]. An isotype control was used for each antibody.
Immunohistochemical studies were performed using formalin-fixed, paraffin-embedded tissue specimens using the avidin–biotin–peroxidase complex method on automated Leica Bond stainers (Leica Biosystems, Buffalo Grove, IL, USA). The antibodies utilized included TCL1, TCF4/CD123 multiplex stain, MYC, p53, and Ki-67 as described previously [6 (link),7 (link),8 (link)].
Immunohistochemical studies were performed using formalin-fixed, paraffin-embedded tissue specimens using the avidin–biotin–peroxidase complex method on automated Leica Bond stainers (Leica Biosystems, Buffalo Grove, IL, USA). The antibodies utilized included TCL1, TCF4/CD123 multiplex stain, MYC, p53, and Ki-67 as described previously [6 (link),7 (link),8 (link)].
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