• Media type: E-Article
  • Title: Toward Clinical Application of Leukocyte Counts Based on Targeted DNA Methylation Analysis
  • Contributor: Sontag, Stephanie; Bocova, Ledio; Hubens, Wouter H G; Nüchtern, Selina; Schnitker, Matthis; Look, Thomas; Schröder, Kema M; Plümäkers, Birgit; Tharmapalan, Vithurithra; Wessiepe, Martina; Kraus, Thomas; Kramer, Jan; Rink, Lothar; Koschmieder, Steffen; Wagner, Wolfgang
  • Published: Oxford University Press (OUP), 2022
  • Published in: Clinical Chemistry
  • Extent: 646-656
  • Language: English
  • DOI: 10.1093/clinchem/hvac006
  • ISSN: 1530-8561; 0009-9147
  • Keywords: Biochemistry (medical) ; Clinical Biochemistry
  • Abstract: <jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Differential leukocyte counts are usually measured based on cellular morphology or surface marker expression. It has recently been shown that leukocyte counts can also be determined by cell-type–specific DNA methylation (DNAm). Such epigenetic leukocyte counting is applicable to small blood volumes and even frozen material, but for clinical translation, the method needs to be further refined and validated.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>We further optimized and validated targeted DNAm assays for leukocyte deconvolution using 332 venous and 122 capillary blood samples from healthy donors. In addition, we tested 36 samples from ring trials and venous blood from 266 patients diagnosed with different hematological diseases. Deconvolution of cell types was determined with various models using DNAm values obtained by pyrosequencing or digital droplet PCR (ddPCR).</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Relative leukocyte quantification correlated with conventional blood counts for granulocytes, lymphocytes, B cells, T cells (CD4 or CD8), natural killer cells, and monocytes with pyrosequencing (r = 0.84; r = 0.82; r = 0.58; r = 0.50; r = 0.70; r = 0.61; and r = 0.59, respectively) and ddPCR measurements (r = 0.65; r = 0.79; r = 0.56; r = 0.57; r = 0.75; r = 0.49; and r = 0.46, respectively). In some patients, particularly with hematopoietic malignancies, we observed outliers in epigenetic leukocyte counts, which could be discerned if relative proportions of leukocyte subsets did not sum up to 100%. Furthermore, absolute quantification was obtained by spiking blood samples with a reference plasmid of known copy number.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Targeted DNAm analysis by pyrosequencing or ddPCR is a valid alternative to quantify leukocyte subsets, but some assays require further optimization.</jats:p></jats:sec>
  • Description: <jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Differential leukocyte counts are usually measured based on cellular morphology or surface marker expression. It has recently been shown that leukocyte counts can also be determined by cell-type–specific DNA methylation (DNAm). Such epigenetic leukocyte counting is applicable to small blood volumes and even frozen material, but for clinical translation, the method needs to be further refined and validated.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>We further optimized and validated targeted DNAm assays for leukocyte deconvolution using 332 venous and 122 capillary blood samples from healthy donors. In addition, we tested 36 samples from ring trials and venous blood from 266 patients diagnosed with different hematological diseases. Deconvolution of cell types was determined with various models using DNAm values obtained by pyrosequencing or digital droplet PCR (ddPCR).</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Relative leukocyte quantification correlated with conventional blood counts for granulocytes, lymphocytes, B cells, T cells (CD4 or CD8), natural killer cells, and monocytes with pyrosequencing (r = 0.84; r = 0.82; r = 0.58; r = 0.50; r = 0.70; r = 0.61; and r = 0.59, respectively) and ddPCR measurements (r = 0.65; r = 0.79; r = 0.56; r = 0.57; r = 0.75; r = 0.49; and r = 0.46, respectively). In some patients, particularly with hematopoietic malignancies, we observed outliers in epigenetic leukocyte counts, which could be discerned if relative proportions of leukocyte subsets did not sum up to 100%. Furthermore, absolute quantification was obtained by spiking blood samples with a reference plasmid of known copy number.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Targeted DNAm analysis by pyrosequencing or ddPCR is a valid alternative to quantify leukocyte subsets, but some assays require further optimization.</jats:p></jats:sec>
  • Footnote: