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Lymphocyte phenotypes in infants are altered by separation of blood on density gradients
S G Kutvirt1, S L Lewis, T L Simon
1Department of Pathology, University of New Mexico School of Medicine, Biomedical Research Facility, Albuquerque 87131.
Insights
Density gradient separation alters lymphocyte subsets in paediatric and adult immunophenotyping. Whole blood analysis is recommended for accurate paediatric immunological disorder diagnosis and monitoring.
Area of Science:
- Immunology
- Cell Biology
- Paediatric Medicine
Background:
- Flow cytometry is crucial for diagnosing and monitoring paediatric immunological disorders.
- Current practices sometimes use density gradients for cell preparation, despite adult studies advising against it.
Purpose of the Study:
- To evaluate the impact of density gradient separation on lymphocyte phenotypes in neonates, infants, and adults.
- To compare these results with whole blood determinations.
Main Methods:
- Comparison of lymphocyte subset distributions using density gradient separation versus whole blood preparation.
- Analysis of immunophenotypes in neonates, infants, and adults.
Main Results:
- Density gradient separation significantly altered lymphocyte subset percentages across all age groups.
- CD19+ (B cell) and CD8+ (suppressor/cytotoxic T cell) percentages decreased, while CD3-CD56+ (NK cell) percentages increased.
- Selective loss of CD8+CD11b- (cytotoxic T) cells was observed, but CD8+CD11b+ (suppressor T) cells remained unaffected.
Conclusions:
- Density gradient separation introduces bias in lymphocyte subset analysis.
- Whole blood techniques are recommended for accurate immunophenotyping in paediatric subjects.
Abstract:
Flow cytometry techniques for immunophenotyping have revolutionised the diagnosis and monitoring of paediatric immunological disorders. Although recent studies in adult subjects discourage the use of density gradients for cell preparation prior to phenotyping, these procedures continue to be used. The purpose of this study was to determine the effect of density gradient separation on lymphocyte phenotypes from neonates, infants, and adults as compared to whole blood determinations. Subset distributions were different with the two procedures. In all three groups, CD19+ (B cell) and CD8+ (suppressor/cytotoxic T cell) percentages were significantly lower and CD3-CD56+ (NK cell) percentages were significantly higher in the density gradient separated cells. The loss of CD8+ cells in density gradient separation was shown to be a selective event. The CD8+CD11b- (cytotoxic T) subset percentages were lower in the density gradient separated cells, while the percentages of CD8+CD11b+ (suppressor T) cells were not affected by separation procedure. Because of the selective loss of lymphocytes on density gradients, the use of a whole blood technique for immunophenotyping in paediatric subjects is recommended.