Abstract
Immunophenotyping by multiparameter flow cytometry is a rapid and efficient technique to simultaneously assess and correlate multiple individual cell properties like size and internal complexity along with antigen expression in a population of cells. This method is utilized for rapid characterization of the blasts and classification of acute myeloid leukemia (AML), in both the peripheral blood (PB) and bone marrow (BM). This technique is not only useful in the initial diagnosis but also in monitoring and determining prognosis of the disease through minimal residual disease (MRD) testing. This chapter provides an overview of procedures for specimen processing, staining, and immunophenotyping of AML and describes the principles of data analysis for AML classification and MRD testing.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Swerdlow SH, Campo E, Harris NL et al (eds) (2017) WHO classification of tumours of haematopoietic and lymphoid tissues, 4th edn. IARC, Lyon
Craig FE, Foon KA (2008) Flow cytometric immunophenotyping for hematologic neoplasms. Blood 111:3941–3967
Chen X, Cherian S (2017) Acute myeloid leukemia immunophenotyping by flow cytometric analysis. Clin Lab Med 37:753–769
van Lochem EG, van der Velden VH, Wind HK et al (2004) Immunophenotypic differentiation patterns of normal hematopoiesis in human bone marrow: reference patterns for age-related changes and disease-induced shifts. Cytometry B Clin Cytom 60(1):1–13
Gorczyca W, Sun ZY, Cronin W et al (2011) Immunophenotypic pattern of myeloid populations by flow cytometry analysis. Methods Cell Biol 103:221–266
Loken MR, Chu SC, Fritschle W (2009) Normalization of bone marrow aspirates for hemodilution in flow cytometric analyses. Cytometry B Clin Cytom 76(1):27–36
Wood BL (2007) Myeloid malignancies: myelodysplastic syndromes, myeloproliferative disorders, and acute myeloid leukemia. Clin Lab Med 27(3):551–575. vii
Wood BL, Arroz M, Barnett D et al (2007) 2006 Bethesda international consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia. Cytometry B Clin Cytom 72(Suppl 1):S14–S22
Wood BL (2016) Principles of minimal residual disease detection for hematopoietic neoplasms by flow cytometry. Cytometry B Clin Cytom 90(1):47–53
Xu J, Jorgensen JL, Wang SA (2017) How do we use multicolor flow cytometry to detect minimal residual disease in acute myeloid leukemia? Clin Lab Med 37:787–802
Zhou Y, Wood BL (2017) Methods of detection of measurable residual disease in AML. Curr Hematol Malig Rep 12:557–567
Dohner H, Estey E, Grimwade D et al (2017) Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood 129(4):424–447
Terwijn M, van Putten WL, Kelder A et al (2013) High prognostic impact of flow cytometric minimal residual disease detection in acute myeloid leukemia: data from the HOVON/SAKK AML 42A study. J Clin Oncol 31(31):3889–3897
Venditti A, Buccisano F, Del Poeta G et al (2000) Level of minimal residual disease after consolidation therapy predicts outcome in acute myeloid leukemia. Blood 96(12):3948–3952
Zhu HH, Zhang XH, Qin YZ et al (2013) MRD-directed risk stratification treatment may improve outcomes of t(8;21) AML in the first complete remission: results from the AML05 multicenter trial. Blood 121(20):4056–4062
Freeman SD, Virgo P, Couzens S et al (2013) Prognostic relevance of treatment response measured by flow cytometric residual disease detection in older patients with acute myeloid leukemia. J Clin Oncol 31(32):4123–4131
Walter RB, Gooley TA, Wood BL et al (2011) Impact of pretransplantation minimal residual disease, as detected by multiparametric flow cytometry, on outcome of myeloablative hematopoietic cell transplantation for acute myeloid leukemia. J Clin Oncol 29(9):1190–1197
Getta BM, Devlin SM, Levine RL et al (2017) Multicolor flow cytometry and multigene next-generation sequencing are complementary and highly predictive for relapse in acute myeloid leukemia after allogeneic transplantation. Biol Blood Marrow Transplant 23(7):1064–1071
van Dongen JJ, Lhermitte L, Böttcher S, EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708) et al (2012) EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia 26(9):1908–1975
Schuurhuis GJ, Heuser M, Freeman S et al (2018) Minimal/measurable residual disease in AML: a consensus document from the European LeukemiaNet MRD working party. Blood 131(12):1275–1291
Reilly JT (1996) Use and evaluation of leukocyte monoclonal antibodies in the diagnostic laboratory: a review. Clin Lab Haematol 18:1–5
Owens MA, Loken MR (1995) Quality control of flow cytometer and reagents. In: Owens MA, Loken MR (eds) Flow cytometry: principles for clinical laboratory practice. Quality assurance for quantitative immunophenotyping. Wiley, New York, pp 45–72
Wood BL (2013) Flow cytometric monitoring of residual disease in acute leukemia. Methods Mol Biol 999:123–136
Chen W, Luu HS (2017) Immunophenotyping by multiparameter flow cytometry. Methods Mol Biol 1633:51–73
Stelzer GT, Shults KE, Loken MR (1993) CD45 gating for routine flow cytometric analysis of human bone marrow specimens. Ann N Y Acad Sci 677:265–280
Borowitz MJ, Guenther KL, Shults KE et al (1993) Immunophenotyping of acute leukemia by flow cytometric analysis. Use of CD45 and right-angle light scatter to gate on leukemic blasts in three-color analysis. Am J Clin Pathol 100:534–540
Harrington AM, Olteanu H, Kroft SH (2012) A dissection of the CD45/side scatter “blast gate”. Am J Clin Pathol 137:800–804
Orfao A, Chillón MC, Bortoluci AM et al (1999) The flow cytometric pattern of CD34, CD15 and CD13 expression in acute myeloblastic leukemia is highly characteristic of the presence of PML-RARalpha gene rearrangements. Haematologica 84(5):405–412
Kussick SJ, Wood BL (2003) Using 4-color flow cytometry to identify abnormal myeloid populations. Arch Pathol Lab Med 127:1140–1147
van de Loosdrecht AA, Alhan C, Béné MC et al (2009) Standardization of flow cytometry in myelodysplastic syndromes: report from the first European LeukemiaNet working conference on flow cytometry in myelodysplastic syndromes. Haematologica 94(8):1124–1134
Hurwitz CA, Raimondi SC, Head D et al (1992) Distinctive immunophenotypic features of t(8;21)(q22;q22) acute myeloblastic leukemia in children. Blood 80:3182–3188
Kita K, Nakase K, Miwa H et al (1992) Phenotypical characteristics of acute myelocytic leukemia associated with the t(8;21)(q22;q22) chromosomal abnormality: frequent expression of immature B-cell antigen CD19 together with stem cell antigen CD34. Blood 80:470–477
Baer MR, Stewart CC, Lawrence D et al (1997) Expression of the neural cell adhesion molecule CD56 is associated with short remission duration and survival in acute myeloid leukemia with t(8;21)(q22;q22). Blood 90:1643–1648
Iriyama N, Hatta Y, Takeuchi J et al (2013) CD56 expression is an independent prognostic factor for relapse in acute myeloid leukemia with t(8;21). Leuk Res 37:1021–1026
De J, Zanjani R, Hibbard M et al (2007) Immunophenotypic profile predictive of KIT activating mutations in AML1-ETO leukemia. Am J Clin Pathol 128:550–557
Ouyang J, Goswami M, Peng J et al (2016) Comparison of multiparameter flow cytometry immunophenotypic analysis and quantitative RT-PCR for the detection of minimal residual disease of core binding factor acute myeloid leukemia. Am J Clin Pathol 145(6):769–777
Exner M, Thalhammer R, Kapiotis S et al (2000) The “typical” immunophenotype of acute promyelocytic leukemia (APL-M3): does it prove true for the M3-variant? Cytometry 42:106–109
Takenokuchi M, Kawano S, Nakamachi Y et al (2012) FLT3/ITD associated with an immature immunophenotype in PML-RARα leukemia. Hematol Rep 4(4):e22
Montesinos P, Rayón C, Vellenga E et al (2011) Clinical significance of CD56 expression in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens. Blood 117:1799–1805
Muñoz L, Nomdedéu JF, Villamor N et al (2003) Acute myeloid leukemia with MLL rearrangements: clinicobiological features, prognostic impact and value of flow cytometry in the detection of residual leukemic cells. Leukemia 17:76–82
Creutzig U, Harbott J, Sperling C et al (1995) Clinical significance of surface antigen expression in children with acute myeloid leukemia: results of study AML-BFM-87. Blood 86:3097–3108
Alsabeh R, Brynes RK, Slovak ML et al (1997) Acute myeloid leukemia with t(6;9) (p23;q34): association with myelodysplasia, basophilia, and initial CD34 negative immunophenotype. Am J Clin Pathol 107:430–437
Lai YY, Li Y, Shi Y et al (2012) Characteristics of 11 patients with acute myeloid leukemia accompanied with karyotype aberration t(6;9). Zhongguo Shi Yan Xue Ye Xue Za Zhi 20:1293–1296
Oyarzo MP, Lin P, Glassman A et al (2004) Acute myeloid leukemia with t(6;9)(p23;q34) is associated with dysplasia and a high frequency of flt3 gene mutations. Am J Clin Pathol 122:348–358
Slovak ML, Gundacker H, Bloomfield CD et al (2006) A retrospective study of 69 patients with t(6;9)(p23;q34) AML emphasizes the need for a prospective, multicenter initiative for rare ‘poor prognosis’ myeloid malignancies. Leukemia 20:1295–1297
Medeiros BC, Kohrt HE, Arber DA et al (2010) Immunophenotypic features of acute myeloid leukemia with inv(3)(q21q26.2)/t(3;3)(q21;q26.2). Leuk Res 34:594–597
Raya JM, Martín-Santos T, Luño E et al (2015) Acute myeloid leukemia with inv(3)(q21q26.2) or t(3;3)(q21;q26.2): clinical and biological features and comparison with other acute myeloid leukemias with cytogenetic aberrations involving long arm of chromosome 3. Hematology 13:1607845415Y0000000003
Bernstein J, Dastugue N, Haas OA et al (2000) Nineteen cases of the t(1;22)(p13;q13) acute megakaryblastic leukaemia of infants/ children and a review of 39 cases: report from a t(1;22) study group. Leukemia 14:216–218
Carroll A, Civin C, Schneider N et al (1991) The t(1;22) (p13;q13) is nonrandom and restricted to infants with acute megakaryoblastic leukemia: a pediatric oncology group study. Blood 78:748–752
Inaba H, Zhou Y, Abla O et al (2015) Heterogeneous cytogenetic subgroups and outcomes in childhood acute megakaryoblastic leukemia: a retrospective international study. Blood 126:1575–1584
Nomdedéu J, Bussaglia E, Villamor N et al (2011) Immunophenotype of acute myeloid leukemia with NPM mutations: prognostic impact of the leukemic compartment size. Leuk Res 35:163–168
Liu YR, Zhu HH, Ruan GR et al (2013) NPM1-mutated acute myeloid leukemia of monocytic or myeloid origin exhibit distinct immunophenotypes. Leuk Res 37:737–741
Angelini DF, Ottone T, Guerrera G et al (2015) A leukemia-associated CD34/ CD123/CD25/CD99+ immunophenotype identifies FLT3-mutated clones in acute myeloid leukemia. Clin Cancer Res 21:3977–3985
Lin LI, Chen CY, Lin DT et al (2005) Characterization of CEBPA mutations in acute myeloid leukemia: most patients with CEBPA mutations have biallelic mutations and show a distinct immunophenotype of the leukemic cells. Clin Cancer Res 11:1372–1379
Hou HA, Lin LI, Chen CY et al (2009) Reply to ‘heterogeneity within AML with CEBPA mutations; only CEBPA double mutations, but not single CEBPA mutations are associated with favorable prognosis’. Br J Cancer 101:738–740
Tang JL, Hou HA, Chen CY et al (2009) AML1/RUNX1 mutations in 470 adult patients with de novo acute myeloid leukemia: prognostic implication and interaction with other gene alterations. Blood 114:5352–5361
Schnittger S, Dicker F, Kern W et al (2011) RUNX1 mutations are frequent in de novo AML with noncomplex karyotype and confer an unfavorable prognosis. Blood 117:2348–2357
Haferlach T, Kohlmann A, Klein HU et al (2009) AML with translocation t(8;16)(p11;p13) demonstrates unique cytomorphological, cytogenetic, molecular and prognostic features. Leukemia 23:934–943
Dang H, Chen Y, Kamel-Reid S et al (2013) CD34 expression predicts an adverse outcome in patients with NPM1-positive acute myeloid leukemia. Hum Pathol 44:2038–2046
Chen CY, Chou WC, Tsay W et al (2013) Hierarchical cluster analysis of immunophenotyped classify AML patients with NPM1 gene mutation into two groups with distinct prognosis. BMC Cancer 13:107
Acknowledgments
This research was supported by the Intramural Research Program of the National Institutes of Health.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Galera, P.K., Jiang, C., Braylan, R. (2019). Immunophenotyping of Acute Myeloid Leukemia. In: McCoy, Jr, J. (eds) Immunophenotyping. Methods in Molecular Biology, vol 2032. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9650-6_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9650-6_15
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9649-0
Online ISBN: 978-1-4939-9650-6
eBook Packages: Springer Protocols