Abstract
The increased susceptibility to infections in neonates is probably based on immaturity of the immune system. The reported immunophenotypic differences in neonatal, infant, and adult lymphocyte subpopulations may reflect age-related maturational changes. Detailed immunophenotyping is needed for further investigation of these maturational processes.
Flowcytometric immunophenotyping of neonatal and infant blood lymphocyte subpopulations poses several problems. Firstly, the blood volume needed for detailed characterization of lymphocyte subpopulations is too large for studies in young infants, especially in small prematures. Secondly, erythroid cell contamination (e.g. normoblasts) of the flowcytometric “lympho-gate” may lead to considerable miscalculations of the relative and absolute cell numbers.
We developed a whole blood microassay for flowcytometric analysis of lymphocyte subpopulations using triple immunological staining with fluorochrome conjugated antibodies (FITC, PE, and the duochrome PE-Cyanine 5). This microassay only needs 20μl per test tube, which is five times less than in conventional test systems. Due to the single-step incubation our microassay is fast and cell loss is minimal.
The triple labelings allow detailed analysis of subpopulations of lymphocytes, as well as identification and quantification of the erythroid cell contamination in the flowcytometric “lympho-gate”. This contamination is measured with a triple labeling for CD71. GpA and CD45, which are expressed by erythroid precursors, all erythroid cells, and all leukocytes respectively. We detected 17.9% (range 1.3-41.9) CD71+GpA+CD45- erythroid cells within the “lympho-gate” in 7 cord blood samples (gestational ages 27-42 weeks). If the “lympho-gate” is not adjusted for this erythroid contamination, the relative numbers of lymphocyte subpopulations will be underestimated down to 50% of their real values.
In conclusion, we developed a fast, reliable microassay for immunophenotyping of infant lymphocyte subpopulations. The erythroid cell contamination of the flowcytometric “lympho-gate” is much larger than previously assumed, but can easily be identified with the CD71/GpA/CD45 triple staining. This method is an important tool for future studies of the immune system in neonates and young infants.
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De Vries, E., Versteeg, S., Cornans-Bitter, W. et al. 245 MICROASSAY FOR IMMUNOPHENOTYPING OF INFANT LYMPHOCYTE SUBPOPULATIONS. Pediatr Res 36, 43 (1994). https://doi.org/10.1203/00006450-199407000-00245
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DOI: https://doi.org/10.1203/00006450-199407000-00245
