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Epigenetic immune cell counting is a DNA (de)methylation-based technique which can be used to quantify lymphocyte subsets on dried blood spots (DBS). The foregoing techniques allow for a retrospective investigation of immune cell profiles in newborns. In this study, we used this technique for determining lymphocyte subcounts as a potential biomarker for necrotizing enterocolitis (NEC). We investigated whether this technique can be implemented in the field of neonatology, by testing whether regulatory T cell (Treg) levels are pre-existently low in preterms with NEC. Newborn screening (NBS) cards from 32 preterms with NEC and 32 age- and weight-matched preterm controls, and 60 healthy term newborns, were analyzed. Relative and absolute cell counts were determined for CD3+, CD4+, CD8+, Th17, and Treg T cells. For both relative and absolute cell counts of CD3+, CD4+, CD8+, and Th17 T cells, significant differences were found between healthy term controls and both preterm groups, but not between preterm groups. For Tregs, no significant differences were found in either relative or absolute counts between any of the newborn groups. This study demonstrates the principle of epigenetic immune cell counting to analyze lymphocyte subsets in preterm neonates. [ABSTRACT FROM AUTHOR]

Epigenetic immune cell counting provides a potentially more robust platform to diagnose immune defects in adults and newborns. Counting by chromatin: Peripheral immune cell counts can be wielded to diagnose a variety of disorders. There are limitations to traditional methods such as flow cytometry, including the type of sample needed for analysis. Baron et al. took advantage of distinct immune cell epigenetic signatures and devised a real-time quantitative polymerase chain reaction (qPCR) method to perform immune cell counting without the requirement of viable cells. They examined different types of samples from healthy adults or those that were infected with HIV (and consequently had fewer CD4+ T cells). The epigenetic qPCR method correlated well with flow cytometry and could also be applied to dried blood spots to identify newborns with primary immunodeficiencies. Although it is not ready to be deployed clinically, the advantages of using epigenetic qPCR make this an intriguing approach to develop further. Immune cell profiles provide valuable diagnostic information for hematologic and immunologic diseases. Although it is the most widely applied analytical approach, flow cytometry is limited to liquid blood. Moreover, either analysis must be performed with fresh samples or cell integrity needs to be guaranteed during storage and transport. We developed epigenetic real-time quantitative polymerase chain reaction (qPCR) assays for analysis of human leukocyte subpopulations. After method establishment, whole blood from 25 healthy donors and 97 HIV+ patients as well as dried spots from 250 healthy newborns and 24 newborns with primary immunodeficiencies were analyzed. Concordance between flow cytometric and epigenetic data for neutrophils and B, natural killer, CD3+ T, CD8+ T, CD4+ T, and FOXP3+ regulatory T cells was evaluated, demonstrating substantial equivalence between epigenetic qPCR analysis and flow cytometry. Epigenetic qPCR achieves both relative and absolute quantifications. Applied to dried blood spots, epigenetic immune cell quantification was shown to identify newborns suffering from various primary immunodeficiencies. Using epigenetic qPCR not only provides a precise means for immune cell counting in fresh-frozen blood but also extends applicability to dried blood spots. This method could expand the ability for screening immune defects and facilitates diagnostics of unobservantly collected samples, for example, in underdeveloped areas, where logistics are major barriers to screening. [ABSTRACT FROM AUTHOR]