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.

Additional Metadata
Keywords Antigens, CD/analysis, Antigens, CD45/analysis, Antigens, Differentiation, B-Lymphocyte/analysis, Autoimmune Diseases/immunology, Erythrocytes/immunology, Erythroid Progenitor Cells/immunology, Fetal Blood/*immunology, Flow Cytometry, Glycophorin/analysis, Humans, Immunophenotyping/*methods, Infant, Newborn/*immunology, Infant, Premature, Diseases/immunology, Infant, Premature/immunology, Lymphocyte Count, Lymphocyte Subsets/*immunology, Receptors, Transferrin, Sensitivity and Specificity, Statistics, Nonparametric
Persistent URL hdl.handle.net/1765/9083
Journal Archives of Disease in Childhood: an international peer-reviewed journal for health professionals and researchers covering conception to adolescence
Citation
de Vries, E, de Bruin-Versteeg, S, Comans-Bitter, W.M, de Groot, R, Boerma, G.J.M, Lotgering, F.K, & van Dongen, J.J.M. (1999). Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes. Archives of Disease in Childhood: an international peer-reviewed journal for health professionals and researchers covering conception to adolescence. Retrieved from http://hdl.handle.net/1765/9083