title: Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes.
abstract: Immunophenotyping of blood lymphocyte subpopulations in neonates and young infants is hampered by the limited amount of blood that can be collected. Contamination of the flow cytometric "lympho-gate" by normoblasts and analysed erythrocytes, and therefore the underestimation of the relative frequencies of lymphocyte subpopulations, interferes with the precise calculation of absolute counts. A microassay was developed by adapting the lysed whole blood technique. Triple immunostaining in a single antibody staining step was used to reduce washing steps and cell loss. Introduction of a triple staining for CD71 (expressed by erythroid precursors), glycophorin A (GpA, expressed by all erythroid cells), and CD45 (expressed by all leucocytes) permitted the relative frequencies of normoblasts (CD71(+)/GpA+/CD45(-) population) and unlysed erythrocytes (CD71(-)/GpA+/CD45(-) population)to be identified and measured within the "lympho-gate" of neonatal cord blood samples. Particularly high frequencies were found (median: 31%) in cord blood samples from preterm neonates. These erythroid cells disappear rapidly by 1 week of age The relative frequencies of erythroid cells can be used to calculate correct lymphocyte subpopulation values. Using only 0.5-0.8 ml of blood, this micro- assay would also be suitable for rapid prenatal immunodiagnosis of congenital immunodeficiencies.
authors.affiliation: Department of Immunology Erasmus University Rotterdam The Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: de Vries
authors.forename: S
authors.initials: S
authors.lastname: de Bruin-Versteeg
authors.forename: W M
authors.initials: WM
authors.lastname: Comans-Bitter
authors.forename: R
authors.initials: R
authors.lastname: de Groot
authors.forename: G J
authors.initials: GJ
authors.lastname: Boerma
authors.forename: F K
authors.initials: FK
authors.lastname: Lotgering
authors.forename: J J
authors.initials: JJ
authors.lastname: van Dongen
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D000944:Antigens, Differentiation, B-Lymphocyte / Q000032:analysis; D001327:Autoimmune Diseases / Q000276:immunology; D004912:Erythrocytes / Q000276:immunology; D015672:Erythroid Precursor Cells / Q000276:immunology; D005312:Fetal Blood / Q000276:immunology*; D005434:Flow Cytometry; D006021:Glycophorins / Q000032:analysis; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D007231:Infant, Newborn / Q000276:immunology*; D007234:Infant, Premature / Q000276:immunology; D007235:Infant, Premature, Diseases / Q000276:immunology; D017493:Leukocyte Common Antigens / Q000032:analysis; D018655:Lymphocyte Count; D016131:Lymphocyte Subsets / Q000276:immunology*; D011990:Receptors, Transferrin; D012680:Sensitivity and Specificity; D018709:Statistics, Nonparametric
chemical_list: D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C068126:CD71 antigen; D006021:Glycophorins; D011990:Receptors, Transferrin; D017493:Leukocyte Common Antigens