title: Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL.
abstract: BACKGROUND
  The development of multiparameter flow cytometry (FCM) and increasingly sophisticated analysis software has considerably improved the exploration of hematological disorders. These tools have been widely applied in leukaemias, lymphomas, and myelodysplasias, yet with very heterogeneous approaches. Consequently, there is no extensive reference document reporting on the characteristics of normal human bone marrow (BM) in multiparameter FCM. Here, we report a reference analysis procedure using relevant antibody combinations in normal human BM.
  
  
  METHODS
  A first panel of 23 antibodies, constructed after literature review, was tested in four-color combinations (including CD45 in each) on 30 samples of BM. After evaluation of the data, a second set of 22 antibodies was further applied to another 35 BM samples. All list-modes from the 65 bone marrow samples were reviewed collectively. A systematised protocol for data analysis was established including biparametric representations and color codes for the three major lineages and undifferentiated cells.
  
  
  RESULTS
  This strategy has allowed to obtain a reference atlas of relevant patterns of differentiation antigens expression in normal human BM that is available within the European LeukemiaNet. This manuscript describes how this atlas was constructed.
  
  
  CONCLUSIONS
  Both the strategy and atlas could prove very useful as a reference of normality, for the determination of leukemia-associated immunophenotypic patterns, analysis of myelodysplasia and, ultimately, investigation of minimal residual disease in the BM.
authors.affiliation: Biopathology, Institut Paoli Calmettes, Marseilles, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Arnoulet
authors.forename: Marie C
authors.initials: MC
authors.lastname: Béné
authors.forename: Françoise
authors.initials: F
authors.lastname: Durrieu
authors.forename: Jean
authors.initials: J
authors.lastname: Feuillard
authors.forename: Chantal
authors.initials: C
authors.lastname: Fossat
authors.forename: Bernard
authors.initials: B
authors.lastname: Husson
authors.forename: Hélène
authors.initials: H
authors.lastname: Jouault
authors.forename: Marc
authors.initials: M
authors.lastname: Maynadié
authors.forename: Francis
authors.initials: F
authors.lastname: Lacombe
mesh_terms: D053301:B-Cell Maturation Antigen / Q000032:analysis; D001853:Bone Marrow; D002454:Cell Differentiation; D003116:Color; D030562:Databases, Protein; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007962:Leukocytes / Q000166:cytology*; D012015:Reference Standards; D015398:Signal Transduction
chemical_list: D053301:B-Cell Maturation Antigen