One tube with eight antibodies for 14-part bone marrow leukocyte differential using flow cytometry
Open Access
- 14 March 2016
- journal article
- research article
- Published by Wiley in Cytometry Part B: Clinical Cytometry
- Vol. 92 (4), 299-309
- https://doi.org/10.1002/cyto.b.21369
Abstract
Background Bone marrow analysis by flow cytometry is part of the routine diagnosis of hematological disorders in medical laboratories. Differential leukocyte count and identification of abnormal cell subsets is currently performed through morphological examination on bone marrow smears by skilled cytologists. In this work, we propose a single 8‐color tube for providing equivalent information, using flow cytometry. Methods 99 bone marrow samples were classified into 2 groups, (i) 51 samples, obtained from either healthy donors (n = 4) or patients with various diseases at diagnosis or during remission that did not present a hematological malignancy (n = 47), and (ii) 48 pathological samples with quantitative and/or qualitative abnormalities. A panel of eight antibodies—CD3‐FITC/CD10‐PE/CD38‐PerCP‐Cy5.5/CD19‐PECy7/CD36‐APC/CD16‐APC‐H7/CD34‐BV421/CD45‐V500—was tested to identify the main cell subsets at different stages of maturation using a FACSCanto‐II analyzer. Results We first proposed a strategy of sequential gating leading to the identification of 14 leukocyte subsets, that is, erythroblasts, monocytes, B‐lymphoid cells from hematogones to plasma‐cells (5 subsets), T‐ and NK‐cells, polymorphonuclear cells (neutrophils, eosinophils, and basophils), myeloblasts and other immature granular cells. This approach was validated by comparing flow cytometry and microscopic morphological examination, both in cases of normal and abnormal samples. Interestingly, cell identification, and numeration by flow cytometry was easy to perform and highly reproducible. Conclusion A very simple, rapid, and reproducible flow cytometric approach, using a combination of eight antibodies allows determination of the cellular composition of bone marrow with high precision. © 2016 International Clinical Cytometry SocietyKeywords
This publication has 26 references indexed in Scilit:
- Transitional B cell subsets in human bone marrowClinical and Experimental Immunology, 2013
- EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocolsLeukemia, 2012
- Flow cytometry and IG/TCR quantitative PCR for minimal residual disease quantitation in acute lymphoblastic leukemia: a French multicenter prospective study on behalf of the FRALLE, EORTC and GRAALLLeukemia, 2012
- Evaluation of an 8‐color flow cytometric reference method for white blood cell differential enumerationCytometry Part B: Clinical Cytometry, 2010
- Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation studyHaematologica, 2009
- Standardization of flow cytometry in myelodysplastic syndromes: report from the first European LeukemiaNet working conference on flow cytometry in myelodysplastic syndromesHaematologica, 2009
- Flow cytometric differential of leukocyte populations in normal bone marrow: Influence of peripheral blood contamination1Cytometry Part B: Clinical Cytometry, 2008
- Total nucleated cell differential for blood and bone marrow using a single tube in a five‐color flow cytometerCytometry Part B: Clinical Cytometry, 2007
- “6 markers/5 colors” extended white blood cell differential by flow cytometryCytometry Part A, 2007
- The CD14+ CD16+ blood monocytes: their role in infection and inflammationJournal of Leukocyte Biology, 2006