期刊
CLINICAL CHEMISTRY
卷 64, 期 3, 页码 566-575出版社
AMER ASSOC CLINICAL CHEMISTRY
DOI: 10.1373/clinchem.2017.279935
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资金
- Else Kroner-Fresenius-Stiftung [2014_A193]
- German Research Foundation [WA 1706/8-1]
- German Ministry of Education and Research [01KU1402B]
- Ministry for Innovation, Science and Research of German Federal State of North Rhine-Westphalia, Germany
- Dutch Province of Limburg, the Netherlands
BACKGROUND: White blood cell counts are routinely measured with automated hematology analyzers, by flow cytometry, or by manual counting. Here, we introduce an alternative approach based on DNA methylation (DNAm) at individual CG dinucleotides (CpGs). METHODS: We identified candidate CpGs that were non-methylated in specific leukocyte subsets. DNAm levels (ranging from 0% to 100%) were analyzed by pyrosequencing and implemented into deconvolution algorithms to determine the relative composition of leukocytes. For absolute quantification of cell numbers, samples were supplemented with a nonmethylated reference DNA. RESULTS: Conventional blood counts correlated with DNAm at individual CpGs for granulocytes (r = -0.91), lymphocytes (r = -0.91), monocytes (r = -0.74), natural killer (NK) cells (r = -0.30), T cells (r = -0.73), CD4+ T cells (r = -0.41), CD8+ T cells (r = -0.88), and B cells (r = -0.66). Combination of these DNAm measurements into the Epi-Blood-Count provided similar precision as conventional methods in various independent validation sets. The method was also applicable to blood samples that were stored at 4 degrees C for 7 days or at -20 degrees C for 3 months. Furthermore, absolute cell numbers could be determined in frozen blood samples upon addition of a reference DNA, and the results correlated with measurements of automated analyzers in fresh aliquots (r = 0.84). CONCLUSIONS: White blood cell counts can be reliably determined by site-specific DNAm analysis. This approach is applicable to very small blood volumes and frozen samples, and it allows for more standardized and cost-effective analysis in clinical application. (c) 2017 American Association for Clinical Chemistry
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