http://hdl.handle.net/1765/9083

Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes


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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.



Keywords


Automatically Extracted Terms
  • blood
  • unlysed erythrocytes
  • normoblast
  • lympho-gate
  • erythrocyte
  • erythroid cell contamination
  • unlysed
  • lymphocyte subpopulations
  • lymphocyte
  • erythroid
  • sample
  • frequency
  • subpopulation
  • lymphocytes
  • cord blood samples
  • contamination
  • neonate
  • microassay
  • immunostaining
  • immunophenotyping


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