One, two, three—how many cells for me?

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Science Immunology  07 Sep 2018:
Vol. 3, Issue 27, eaav0446
DOI: 10.1126/sciimmunol.aav0446


An alternate strategy is described for enumerating peripheral immune cells in health and disease by epigenetic quantitative PCR.

Whereas most clinical specialties have a single core test or battery of evaluations to assess the health of the relevant body system, the immune system remains more enigmatic both in technical complexity and terminology. As a starting point toward quantifying immune health, flow cytometry can be used to enumerate the various subsets of lymphocytes in peripheral blood, but there are challenges in the broad use of this technology. First, it requires access to expensive and complex technology, a skilled operator, and sufficient optimization to generate robust healthy control reference ranges. Second, it requires the ability to collect and rapidly assess fresh blood samples or carefully cryopreserve samples for future study. So, is it possible to measure cell subset frequencies without counting intact cells? Baron et al. provide a possible epigenetic strategy. They began by defining immune cell subset-specific unmethylated DNA regions that are identified by using bisulfite sequencing to transform unmethylated cytosine phosphate guanosine (CpG) to TpGs, thus marking them. For T cell subsets they identified appropriate unmethylated elements in CD3+, CD4+, and CD8+. For B cells, natural killer cells, and neutrophils, they used genome wide profiling to detect unmethylated regions that uniquely associated with each leukocyte subset of interest (for example, methylated in control subsets). Next, they established quantitative polymerase chain reaction (qPCR) assays for those regions (also known as epigenetic qPCR) and used various control, calibration, and quantification strategies to allow calculation of cell concentrations using the epigenetic qPCR technology. The authors validated this epigenetic counting strategy by measuring the correlation of traditional flow cytometry in healthy donors and HIV+ patients—with CD4+ T cell lymphopenia). Finally, they overcame one of the limitations of flow cytometry by demonstrating that this strategy could be applied to dried blood spots, such as those used in newborn screenings, from both healthy and immunodeficient patients. This strategy was able to identify many types of immune deficiencies, including those missed on the widely used T cell receptor excision circles (TRECs) assay. This novel strategy remains to be developed further and tested with additional samples, but it is fascinating and holds promise for altering our clinical and research laboratory strategies.

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