Leukemia (2000) 14, 816–825 2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu BIOMED-1 Concerted Action report: Flow cytometric characterization of CD71 cell subsets in normal bone marrow as a basis for the diagnosis and follow-up of T cell acute lymphoblastic leukemia (T-ALL) A Porwit-MacDonald1, E Bjo¨rklund1, P Lucio2, EG van Lochem3, J Mazur1, A Parreira2, MWM van den Beemd3, ER van Wering4, E Baars4, G Gaipa5, A Biondi5, J Ciudad6, JJM van Dongen3, JF San Miguel6 and A Orfao6 1Department of Pathology, Karolinska Hospital, Stockholm, Sweden; 2Instituto Portugues de Oncologia, Lisboa, Portugal; 3Department of Immunology, Erasmus University, Rotterdam, and University Hospital, Rotterdam, The Netherlands; 4Dutch Childhood Leukemia Study Group, The Hague, The Netherlands; 5Centro di Ricerca M Tettamanti, Monza, Italy; and 6Department of Hematology, University Hospital, Salamanca, Spain The European BIOMED-1 Concerted Action was initiated in multiparameter flow cytometry for the investigation of MRD 1994 to improve and standardize the flow cytometric detection can reach a high sensitivity and specificity for the identifi- of minimal residual disease (MRD) in acute leukemia (AL). 8–11 Three different protocols were defined to identify the normal cation of residual leukemic T lymphoblasts. A cooperative subsets of B, T and myeloid cells in bone marrow (BM), and study conducted by six European centers involved in the were applied to the different types of AL in order to study BIOMED-1 Concerted Action ‘Investigation of Minimal aberrant immunophenotypes. Using sensitive acquisition Residual Disease in Acute Leukemia: International Standardiz- methods (‘live gate’) T cell subsets in normal BM could be ation and Clinical Evaluation’ applied multiparameter flow identified with five triple-stains: CD7/CD5/CD3, CD7/CD4/CD8, cytometry in which triple antibody combinations were used CD7/CD2/CD3, CD7/CD38/CD34 and TdT/CD7/surface or cyto- plasmic (cy)CD3 (antibodies conjugated with FITC/PE/PECy5 or together with sensitive data acquisition methods (such as ‘live PerCP, respectively). The identification of T cell subsets in BM gate’ acquisition). Using this approach we obtained detailed allowed definition of ‘empty spaces’ (ie areas of flow cytometric information on cell subsets that are present in normal BM at plots where normally no cells are found). All studied T-ALL very low frequencies and that may remain undetected using cases (n = 65) were located in ‘empty spaces’ and could be conventional methods of analysis. discriminated from normal T cells. The most informative triple The immunophenotyping methods (staining protocols and staining was TdT/CD7/cyCD3, which was aberrant in 91% of T- ALL cases. In most cases, two or more aberrant patterns were flow cytometry) were carefully standardized in order to found. Apparently the immunophenotypes of T-ALL differ sig- develop a highly reproducible and sensitive approach to nificantly from normal BM T cells. This is mostly caused by identify both major and minor subsets of T cells present in BM their thymocytic origin, but also the neoplastic transformation and their relative distribution according to age. Once normal might have affected antigen expression patterns. Application T cell subsets were identified, we focused our attention on of the five proposed marker combinations in T-ALL contributes the ‘empty spaces’ left in multidimensional flow cytometry dot to standardized detection of MRD, since cells persistent or plots of each studied antigen combination, as has previously reappearing in the ‘empty spaces’ can be easily identified in 12–14 follow-up BM samples during and after treatment. Leukemia been reported for precursor B cells. Subsequently, we (2000) 14, 816–825. used the same triple antibody combinations and flow cytome- Keywords: T cells; bone marrow; acute lymphoblastic leukemia; try protocols to analyze 65 consecutive T-ALL cases diag- immunophenotype; flow cytometry; minimal residual disease nosed in the six participating laboratories. Our aim was to assess the incidence of aberrant phenotypes, which can be used for the follow-up of MRD in T-ALL patients. Introduction It has long been assumed that blasts from T cell acute lym- Materials and methods phoblastic leukemia (T-ALL) reflect the immunophenotypes of early stages of T cell differentiation.1–3 Most T-ALL cases dis- Specimen collection play phenotypes corresponding to various stages of thymocyte differentiation, which normally are not found in bone marrow (BM) or peripheral blood (PB).4 Normal BM samples: Normal BM samples (n = 35) were The aberrant immunophenotypic characteristics of leu- obtained from healthy donors (n = 29) and patients with idio- kemic T lymphoblasts allow discrimination from T cell subsets pathic thrombocytopenia (ITP) (n = 6), recruited in the six Eur- in BM and thus can be used for detection of minimal residual opean centers involved in the BIOMED-1 Concerted Action disease (MRD).5–8 A comprehensive analysis of the immuno- on MRD. The following centers participated: Portuguese Insti- phenotypic characteristics of T cells and the subsequent tute of Oncology, Lisbon, Portugal; Karolinska Hospital, identification of all T cell subsets in normal BM are important Stockholm, Sweden; University of Salamanca, Salamanca, for the reliable distinction between normal and leukemic T Spain; Department of Immunology, Erasmus University, Rot- cells. terdam and University Hospital, Rotterdam, The Netherlands; Previous studies in T-ALL strongly suggest that the use of Dutch Childhood Leukemia Study Group, The Hague, The Netherlands; and M Tettamanti Research Center, Monza, Italy. Informed consent was obtained from all donors or parents Correspondence: A Porwit-MacDonald, Department of Pathology, in cases of children less than 15 years old in accordance with Karolinska Hospital, 171 76 Stockholm, Sweden; Fax: 46 851775843 J Mazur was on leave from Department of Epidemiology and Health local Medical Ethics Committees. The samples from patients Care Planning, Institute Mother and Child, Warsaw, Poland with ITP were obtained for diagnostic purposes. The median Received 14 June 1999; accepted 15 December 1999 age of the donors was 15 years, ranging from 2 to 75 years BIOMED-1 Concerted Action report A Porwit-MacDonald et al 817 old (mean age 24), the male/female ratio was 0.8. Moreover, studies showed that both intracellular staining procedures 16 follow-up BM samples from children receiving chemo- yielded similar results.14,15 therapy for B-precursor ALL were included to investigate if T For standardization purposes, we used triple combinations cell content and distribution changed during cytostatic of the same Mab clones labeled with identical fluorochromes treatment. (FITC/PE/PECy5 or PerCP) in all immunostainings of normal BM in all participating centers: CD7/CD5/CD3, CD7/CD4/CD8, CD7/CD2/CD3, CD7/CD38/CD34 and Leukemic samples: T-ALL samples (n = 65) were obtained TdT/CD7/cyCD3 (Table 1). Isotype-matched immunoglobulins in the participating centers for diagnostic purposes. The mor- and a sample stained for CD3-FITC/CD4-PE/CD8-PE/Cy5 were phological diagnosis of ALL was made on the basis of micro- used as negative and positive controls, respectively. Addition- scopic investigation of May–Gru¨nwald-Giemsa stained BM ally, expression of CD13, CD33 and CD19 within the CD7+ smears. The immunologic diagnosis of T-ALL was made cell population was studied in BM samples from a total of 24 according to well-established criteria.4 The median age of the normal donors in Salamanca. The immunostaining of leu- patients was 13 years, ranging from 1 to 52 years (mean 16), kemic samples was performed using the same triple antibody and the male/female ratio was 1.8. combinations and sample preparation procedures as for normal BM. Sample preparation Data acquisition and analysis Bone marrow aspirates were collected in either heparin or EDTA anticoagulant and maintained at room temperature (RT) Data acquisition was performed in all centers in a stan- until processed (not longer than 24 h). The nucleated cell dardized manner, using FACScan flow-cytometers (BD), count was adjusted to 1–2 × 107 cells/ml. For sample prep- equipped with either LysisII or CellQuest (BD) software pro- aration, a stain and then lyse/wash technique was used. grams. Instrument set-up and calibration was performed in a Briefly, 100 µl of BM cell suspension (at least 1 × 106 standardized way as previously described.12 For the analysis nucleated cells) were incubated for 10–15 min (RT, in the of normal BM samples, a two-step acquisition procedure was dark) with saturating amounts of five different triple combi- used. First, 15 × 103 non-gated events were acquired from the nations of monoclonal antibodies (Mab, Table 1). After incu- BM samples. Then a total of 105 to 2 × 106 cells were meas- bation, 2 ml of FACS lysing solution (Becton Dickinson (BD), ured with a live gate set on the basis of CD7 expression and San Jose, CA, USA) diluted 1/10 in distilled water was added low/intermediate side scatter (SSC) characteristics. In this and samples were incubated for another 10 min at RT in the second step, data acquisition exclusively concerned CD7+ dark. Subsequently, cells were washed and re-suspended in 1 cells with lymphoid scatter generally representing approxi- ml of PBS. mately 10% of BM cells (Table 2). In practice, this meant that Cytoplasmic (cy)CD3 and nuclear terminal deoxynucleoti- between 5 × 104 and 2 × 105 CD7+ lymphoid BM cells were dyl transferase (TdT) were detected after staining for surface acquired for further evaluation. The expression of the other T markers (as above), and fixation/permeabilization with Perme- cell markers was analyzed within this CD7+ lymphocyte gate afix (OPM; Ortho, Raritan, NY, USA) or Fix & Perm (F/P; An in order to characterize the different CD7+ cell subsets. der Grub, Vienna, Austria).15 The reagents were used follow- Additional live gates were based on CD34+ and TdT+ events ing the manufacturer’s recommendations.