Research ArticleIMMUNODIAGNOSTICS

Epigenetic immune cell counting in human blood samples for immunodiagnostics

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Science Translational Medicine  01 Aug 2018:
Vol. 10, Issue 452, eaan3508
DOI: 10.1126/scitranslmed.aan3508
  • Fig. 1 Bisulfite sequencing–derived DNA methylation profiles of cell-specific marker genes in purified immune cells.

    Immune cell types, isolated from adult healthy donors, are arranged in columns with AMPs, and the associated gene names are arranged in rows. Different gene loci are separated by red dashed lines, and AMPs within the same locus are separated by black dashed lines. Each individual line represents a single CpG site. Methylation rates are color-coded ranging from yellow (0%) to blue (100%).

  • Fig. 2 Comparison of immune cell quantification by flow cytometry and epigenetic qPCR.

    Immune cells from blood samples of 25 independent donors were measured with flow cytometry (y axis) and epigenetic qPCR (x axis). (A) Relative immune cell counts are shown as percentage of total leukocytes. (B) Absolute immune cell counts are shown as cell number per microliter of whole blood. The regression line is depicted in red as computed from all data points, and the black line indicates the bisectrix.

  • Fig. 3 Method comparison of T cell subsets in an HIV+ cohort.

    Samples were analyzed from 97 HIV+ subjects. Relative counts of CD3+, CD4+, and CD8+ T cells in percentage of total nucleated cells determined by (A) flow cytometry and epigenetic qPCR in liquid whole blood, (B) flow cytometry as in liquid blood and epigenetic qPCR from DBS, and (C) comparison of epigenetic qPCR from liquid blood and DBS. On the left hand side, data are presented as scatterplots. The regression line is depicted in red as computed from all data points, and the black line indicates the bisectrix. On the right hand side, Bland-Altman plots show average cell counting of the respective analyses (x axis) plotted over their relative difference (y axis). Gray lines reflect limits of agreement. Central red lines illustrate the systematic bias. The respective 95% CIs are shown as dotted gray lines. Top, total CD3+ T cells; middle, CD4+ T cells; bottom, CD8+ T cells.

  • Fig. 4 Epigenetic qPCR on DBS from newborns.

    Copies from cell type–specific qPCRs (y axis) plotted against GAPDH (glyceraldehyde-3-phosphate dehydrogenase) copies (x axis). DBSs from healthy neonates (n = 250; gray circles) estimate reference ranges for each assay, as defined by 99% confidence region (red ellipse) and 99.9% confidence region (dashed and gray ellipse). Twenty-four DBSs from PID-diagnosed newborns are shown as colored circles, each referencing disease characteristics shown in Table 2. (A) Unmethylated CD3G/D, indicating T cells. (B) MVD, indicating NK cells. (C) LRP5, indicating B cells.

  • Fig. 5 Epigenetic qPCR on DBS from newborns with IPEX or SCN.

    DBS from healthy controls (gray-shaded boxes) and newborns with (A) confirmed IPEX and (B) SCN were subjected to epigenetic qPCR for quantification of the percentage of Treg cells within CD3+ T cells depicted in (A) or the percentage of CD15+ neutrophils of all nucleated cells depicted in (B). Healthy cohorts [n = 13 in (A) and n = 26 in (B)] are represented in the boxplot, and results from diseased patients are depicted in red. Each box represents the interquartile range, and the central line shows the median.

  • Table 1 Cell type–specific epigenetic qPCR systems.

    RDls, relative determination of unmethylated DNA (locus specific) in %; RDu, relative determination of unmethylated DNA (universal) in %; EF, efficiency factor; DDu, definitive determination of unmethylated DNA (universal) in %.


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  • Table 2 Genetic defects and diagnostic classification by TREC/KREC and epigenetic qPCR for PID patients.
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Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/452/eaan3508/DC1

    Fig. S1. Schematic illustration of the different quantification approaches for epigenetic cell counting.

    Fig. S2. Correlation analysis of absolute quantification for T cell subsets in an HIV cohort.

    Fig. S3. Epigenetic immune cell quantification on DBS from newborns.

    Table S1A. Selected candidate regions for neutrophils and NK and B cells from genome-wide discovery on Illumina’s Infinium methylation-specific array.

    Table S1B. Calculated “β values” from genome-wide discovery on Illumina’s Infinium methylation-specific array.

    Table S2. Data from method comparison analysis.

    Table S3. Stability testing of DBS.

    Table S4. Epigenetic qPCR from DBS spotted with diluted blood.

    Table S5. Oligonucleotides for bisulfite sequencing and epigenetic qPCR.

  • The PDF file includes:

    • Fig. S1. Schematic illustration of the different quantification approaches for epigenetic cell counting.
    • Fig. S2. Correlation analysis of absolute quantification for T cell subsets in an HIV cohort.
    • Fig. S3. Epigenetic immune cell quantification on DBS from newborns.
    • Table S1A. Selected candidate regions for neutrophils and NK and B cells from genome-wide discovery on Illumina’s Infinium methylation-specific array.
    • Table S2. Data from method comparison analysis.
    • Table S3. Stability testing of DBS.
    • Table S4. Epigenetic qPCR from DBS spotted with diluted blood.
    • Table S5. Oligonucleotides for bisulfite sequencing and epigenetic qPCR.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1B (Microsoft Excel format). Calculated “β values” from genome-wide discovery on Illumina’s Infinium methylation-specific array.

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