CD34+ cell subpopulations detected by 8-color flow cytometry in bone marrow and in peripheral blood stem cell collections: application for MRD detection in leukemia patients
Fast development in polychromatic flow cytometry (PFC) makes it possible to study CD34+ cells with two scatter and eight fluorescence parameters. Minimal residual disease (MRD) is determined as persistence of leukemic cells at submicroscopic levels in bone marrow (BM) of patients in complete remission. MRD can be present in collections of hematopoietic stem cell from blood (HSC-B). Using PFC, we have defined patterns of antigen expression in CD34+ cell subpopulations in BM and applied them as templates in MRD analysis. Twelve BM samples from hospital control (HC) patients with no signs of hematological malignancy were studied using five 8-color monoclonal antibody combinations detecting subsets of CD34+ cells. These patterns have been used as templates to determine levels of MRD in HSC-B collections from six AML patients. Several subsets of CD34+ precursor cells were found to be present at very low frequencies (<10−4) in BM and/or HSC-B collections. All six HSC-B collections from AML patients showed MRD by 8-color technique and only three by previously applied 3-color method. The 8-color technique showed promising results in efficient detection of different CD34+ subpopulations of HSC-B and in MRD quantification. Monitoring of MRD should become a part of quality control of HSC-B collections.
KeywordsHematopoietic stem cell transplantation Minimal residual disease Acute myeloid leukemia CD34+ cells Flow cytometry
The excellent technical assistance of Agnieszka Dul, Britt-Marie Johansson, Ana Lodoli, and Kia Heimersson is gratefully acknowledged. We thank Lewis Edgel for linguistic consultation.
- 10.Menendez P, Perez-Simon JA, Mateos MV, Caballero MD, Gonzalez M, San Miguel JF, et al. Influence of the different CD34+ and CD34− subsets infused on clinical outcome after non-myeloblative allogeneic peripheral blood transplantation from human leukocyte antigen-identical sibling donors. Br J Haematol. 2002;119:135–43.CrossRefPubMedGoogle Scholar
- 13.Matarraz S, Lopez A, Barrena S, Fernandez C, Jensen E, Flores J, et al. The immunophenotype of different immature, myeloid and B-cell lineage-committed CD34(+) hematopoietic cells allows discrimination between normal/reactive and myelodysplastic syndrome precursors. Leukemia. 2008;22:1175–83.CrossRefPubMedGoogle Scholar
- 18.Porwit-MacDonald A, Bjorklund E, Lucio P, van Lochem EG, Mazur J, Parreira A, et al. BIOMED-1 concerted action report: flow cytometric characterization of CD7+ cell subsets in normal bone marrow as a basis for the diagnosis and follow-up of T cell acute lymphoblastic leukemia (T-ALL). Leukemia. 2000;14:816–25.CrossRefPubMedGoogle Scholar
- 19.Lucio P, Gaipa G, van Lochem EG, van Wering ER, Porwit-MacDonald A, Faria T, et al. BIOMED-I concerted action report: flow cytometric immunophenotyping of precursor B-ALL with standardized triple-stainings. BIOMED-1 concerted action investigation of minimal residual disease in acute leukemia: international standardization and clinical evaluation. Leukemia. 2001;15:1185–92.CrossRefPubMedGoogle Scholar
- 20.Kern W, Danhauser-Riedl S, Ratei R, Schnittger S, Schoch C, Kolb HJ, et al. Detection of minimal residual disease in unselected patients with acute myeloid leukemia using multiparameter flow cytometry for definition of leukemia-associated immunophenotypes and determination of their frequencies in normal bone marrow. Haematologica. 2003;88:646–53.PubMedGoogle Scholar
- 21.San Miguel JF, Vidriales MB, Lopez-Berges C, Diaz-Mediavilla J, Gutierrez N, Canizo C, et al. Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukemia identifies different patient risk groups and may contribute to postinduction treatment stratification. Blood. 2001;98:1746–51.CrossRefPubMedGoogle Scholar
- 26.Laane E, Derolf AR, Bjorklund E, Mazur J, Everaus H, Soderhall S, et al. The effect of allogeneic stem cell transplantation on outcome in younger acute myeloid leukemia patients with minimal residual disease detected by flow cytometry at the end of post-remission chemotherapy. Haematologica. 2006;91:833–6.PubMedGoogle Scholar
- 29.Brunning R, Matutes E, Harris N, Flandrin G, Vardiman J, Bennett J. Acute myeloid leukemias. In: Jaffe ES, Harris NL, et al., editors. World Health organization classification of tumours. Patholoy & genetics. Tumours of haematopoietic and lymphoid tissues. Lyon: IARC Press; 2001. p. 75–107.Google Scholar
- 32.Altman DG. Practical statisitcs for medical research. London: Chapman & Hall; 1991. p. 403–9.Google Scholar
- 35.Goussetis E, Theodosaki M, Paterakis G, Peristeri J, Petropoulos D, Kitra V, et al. A functional hierarchy among the CD34 + hematopoietic cells based on in vitro proliferative and differentiative potential of AC133+ CD34(bright) and AC133(dim/)-CD34+ human cord blood cells. J Hematother Stem Cell Res. 2000;9:827–40.CrossRefPubMedGoogle Scholar
- 43.Seriu T, Yokota S, Nakao M, Misawa S, Takaue Y, Koizumi S, et al. Prospective monitoring of minimal residual disease during the course of chemotherapy in patients with acute lymphoblastic leukemia, and detection of contaminating tumor cells in peripheral blood stem cells for autotransplantation. Leukemia. 1995;9:615–23.PubMedGoogle Scholar
- 45.Venditti A, Buccisano F, Tamburini A, Del Poeta G, Maurillo L, Del Moro B, et al. The amount of minimal residual disease after consolidation therapy predicts outcome in acute myeloid leukemia. Blood. 1999;94:695A.Google Scholar