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Flow Cytometric Analysis of Normal B Cell Differentiation

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Flow Cytometric Analysis of Normal B Cell Differentiation Leukemia (1999) 13, 419–427 1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu Flow cytometric analysis of normal B cell differentiation: a frame of reference for the detection of minimal residual disease in precursor-B-ALL PLu´cio1, A Parreira1, MWM van den Beemd2, EG van Lochem2, ER van Wering3, E Baars3, A Porwit-MacDonald4, E Bjorklund4, G Gaipa5, A Biondi5, A Orfao6, G Janossy7, JJM van Dongen2 and JF San Miguel6 BIOMED-1 Concerted Action: 1Department of Hematology, Portuguese Institute of Oncology, Lisbon, Portugal; 2Department of Immunology, University Hospital Dijkzigt/Erasmus University Rotterdam, The Netherlands; 3Dutch Childhood Leukemia Study Group, The Hague, The Netherlands; 4Department of Pathology, Karolinska Hospital, Stockholm, Sweden; 5Clinica Pediatrica, University of Milan, Ospedale San Genaro, Monza, Italy; 6Department of Hematology, University of Salamanca, Salamanca, Spain; and 7Department of Immunology, Royal Free Hospital, London, UK During the last two decades, major progress has been made in Introduction the technology of flow cytometry and in the availability of a large series of monoclonal antibodies against surface mem- brane and intracellular antigens. Flow cytometric immunophen- Differentiation of B cells from early committed progenitors to otyping has become a diagnostic tool for the analysis of normal mature B-lymphocytes is a multistep maturation process that and malignant leukocytes and it has proven to be a reliable can be monitored by the coordinated acquisition and loss of approach for the investigation of minimal residual disease leukocyte differentiation antigens. Several hypothetical mod- (MRD) in leukemia patients during and after treatment. In order els of B cell differentiation have been proposed in which the to standardize the flow cytometric detection of MRD in acute consecutive sequence of antigen expression is described, thus leukemia, a BIOMED-1 Concerted Action was initiated with the enabling the definition of different stages of B cell participation of six laboratories in five different European coun- 1–3 tries. This European co-operative study included the immuno- maturation. phenotypic characterization and enumeration of different pre- Initially, most concepts of human early B cell development cursor and mature B cell subpopulations in normal bone were derived from studies on precursor-B-acute lymphoblastic marrow (BM). The phenotypic profiles in normal B cell differen- leukemia (precursor-B-ALL).4–6 More recently, studies have tiation may form a frame of reference for the identification of focused on the investigation of normal bone marrow (BM)7–9 aberrant phenotypes of precursor-B cell acute lymphoblastic leukemias (precursor-B-ALL) and may therefore be helpful thanks to technical improvements and refinements of multi- in MRD detection. Thirty-eight normal BM samples were anal- parameter flow cytometry, which allows the analysis of large yzed with five different pre-selected monoclonal antibody numbers of normal B cells, even when present at very low combinations: CD10/CD20/CD19, CD34/CD38/CD19, CD34/ frequencies.10 In addition, extensive panels of monoclonal ؊ CD22/CD19, CD19/CD34/CD45 and TdT/CD10/CD19. Two CD19 antibodies against B cell-associated leukocyte differentiation immature subpopulations which coexpressed B cell-associated antigens have been generated, whole blood/BM staining antigens were identified: CD34؉/CD22؉/CD19؊ and ؉ ؉ ؊ ؎ methods have been developed, new fluorochromes with high TdT /CD10 /CD19 , which represented 0.11 0.09% and 11,12 of the total BM nucleated cells, respectively. sensitivity emission signals have been discovered and 0.05% ؎ 0.04 These immunophenotypes may correspond to the earliest methods for flow cytometric intracellular stainings have been stages of B cell differentiation. In addition to these minor improved substantially.13 These combined technical advances ؉ subpopulations, three major CD19 B cell subpop- have contributed to a better insight in the discrete maturation ulations were identified, representing three consecutive stages of normal B cell development. Several controversies /maturation stages; CD19dim/CD34؉/TdT؉/CD10bright/CD22dim CD45dim/CD38bright/CD20؊ (subpopulation 1), CD19؉/CD34؊/ concerning the consecutive expression of antigens throughout TdT؊/CD10؉/CD22dim/CD45؉/CD38bright/CD20dim (subpopulation B cell differentiation have been clarified, and minor B cell and CD19؉/CD34؊/TdT؊/CD10؊/CD22bright/CD45bright/CD38dim/ subpopulations have been identified in normal BM, such as (2 CD20bright (subpopulation 3). The relative sizes of subpopula- those coexpressing CD34 and CD20 or CD34 and CD22.14–16 tions 1 and 2 were found to be age related: at the age of 15 Nevertheless, some controversies remain: classical models3,5,6 years, the phenotypic precursor-B cell profile in BM changed have been challenged by the possible existence of parallel from the childhood ‘immature’ profile (large subpopulations 1 17 and 2/small subpopulation 3) to the adult ‘mature’ profile (small pathways of B cell development; furthermore, differences subpopulation 1 and 2/large subpopulation 3). When the immu- between fetal and postnatal BM B cell precursors have been nophenotypically defined precursor-B cell subpopulations reported5,17,18 indicating also that postnatal age-related differ- from normal BM samples are projected in fluorescence dot- ences which mainly involve the relative distribution of the dif- plots, templates for the normal B cell differentiation pathways ferent BM B cell subsets, exist.9,19,20 However, in these studies can be defined and so-called ‘empty spaces’ where no cell procedures have been used in which pre-enrichment steps for populations are located become evident. This allows discrimi- nation between normal and malignant precursor-B cells and mononuclear cells are included. This limits the potential value can therefore be used for MRD detection. of the enumeration of the different BM cell subsets due to the Keywords: normal B cell differentiation; minimal residual disease; existence of important levels of uncontrolled loss of cells of precursor-B-ALL; flow cytometry; lymphopoiesis interest.21 Accordingly, to the best of our knowledge, no sys- tematic study of B cell differentiation at different ages has been performed in a large series of healthy individuals using a whole bone marrow sample preparation technique. In the present study, we performed a standardized flow cytometric analysis at six different sites of a total of 38 normal human BM samples from healthy individuals whose age Correspondence: A Orfao, Servicio General de Citometria, Laborato- rio de Hematologia, Hospital Clinico Universitario, Paseo de San ranged from 2 to 75 years. Our major goal was to investigate Vicente, 58–82, 37007 Salamanca, Spain; Fax: 34 923 29 46 24 the BM B cell compartment in order to generate a frame of Received 29 December 1997; accepted 21 October 1998 reference for the identification of leukemia-associated pheno- Flow cytometric analysis of B cell differentiation PLu´cio et al 420 types in precursor B-ALL cases. The definition of the normal Immunophenotypic analysis patterns of B cell differentiation was based on: (1) the objec- tive identification of clearly defined subsets of B cells corre- For standardization purposes, all groups used the same set of sponding to discrete stages of B cell maturation; (2) the rela- monoclonal antibodies conjugated with fluorescein isothiocy- tive distribution of each of these subsets from the total BM anate (FITC), phycoerythrin (PE) and either peridin chlorophyll cellularity; and (3) the definition of flow cytometry dot-plot protein (PerCP) or the PE/cyanine 5 (PE/Cy5) fluorochrome templates corresponding to the normal B cell differentiation tandem. The antibodies’ clones were carefully selected on the pathways. basis of their reactivity patterns and the absence of back- ground staining. Their source and specificity were as follows: CD10-FITC (W8E7), CD19-FITC (leu12), CD20-PE (leu16), CD22-PE (leu14), CD34-FITC (HPCA2), CD34-PE (HPCA2), Materials and methods CD38-PE (leu17) and CD45-PerCP (Hle1) were purchased from BD, CD10-PE (J5) and CD19-PE/Cy5 (J4.119) were from Specimen collection Coulter/Immunotech (Miami, FL, USA), CD19-PE/Cy5 (SJ25- C1) was from CALTAG Laboratories (San Francisco, CA, USA) BM samples were collected from 38 healthy donors by six and TdT-FITC (HT6) from DAKO (Glostrup, Denmark). participating European immunophenotyping laboratories Subsequently, the triple fluorochrome-conjugated mono- involved in the BIOMED-1 Concerted Action: Investigation of clonal antibody combinations were extensively tested in the Minimal Residual Disease in Acute Leukemia: International six participating laboratories according to predefined guide- Standardization and Clinical Evaluation (Department of Hem- lines. Based on the combined results, the following five triple atology, Portuguese Institute of Oncology, Lisbon, Portugal; immunofluorescence stainings were established: CD10-FITC/ Department of Immunology, University Hospital CD20-PE/CD19-PE-Cy5, CD34-FITC/CD38-PE/CD19-PE-Cy5, Dijkzigt/Erasmus University Rotterdam, Rotterdam, The CD34-FITC/CD22-PE/CD19-PE-Cy5, CD19-FITC/CD34-PE/ Netherlands; Dutch Childhood Leukemia Study Group, The CD45-PerCP, and TdT-FITC/CD10-PE/CD19-PE-Cy5. Irrel- Hague, The Netherlands; Department of Pathology, Karolin- evant monoclonal antibody reagents of the same isotypes as ska Hospital, Stockholm, Sweden; Clinical Pediatrica, Univer- in the triple combinations,
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