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Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes
  1. E de Vriesa,b,
  2. S de Bruin-Versteega,
  3. W M Comans-Bittera,c,
  4. R de Grootc,
  5. G J M Boermad,
  6. F K Lotgeringe,
  7. J J M van Dongena
  1. aDepartment of Immunology Erasmus University Rotterdam The Netherlands, bDepartment of Paediatrics Bosch Medicentrum Hertogenbosch, cDepartment of Paediatrics University Hospital/Sophia Children’s Hospital Rotterdam, dDepartment of Clinical Chemistry University Hospital Rotterdam, eDepartment of Obstetrics and Gynaecology
  1. Dr E de Vries Department of Immunology Erasmus University Rotterdam PO Box 1738 3000 DR Rotterdam The Netherlands. Email:vandongen{at}


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.

  • erythroid cell contamination
  • lymphocyte subpopulations
  • microassay
  • normoblasts

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