Clinical Application of Leukocyte Counts Based on Targeted DNA Methylation Analysis

Leukocyte subsets are usually quantified with automated cell counting devices and particularly for stratification of lymphocyte subsets with flow cytometry. Despite broad application, conventional methods have several limitations.

DNA methylation (DNAm) is a covalent modification of cytosine residues, mostly at CG dinucleotides (CpG sites). By measuring the methylation level at CpG sites with cell-type–specific hypo- or hypermethylation, it is possible to quantify the composition of leukocyte subsets using statistical algorithms (deconvolution models).

Biomedical Engineers at the RWTH Aachen University (Aachen, Germany) and colleagues optimized and validated targeted DNAm assays for leukocyte deconvolution using 332 venous and 122 capillary blood samples from healthy donors. In addition, they 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).

DNA was isolated from 150 µL venous or 50 µL capillary blood and bisulfite-treated with the EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA). PCR amplicons were sequenced on a PyroMark Q96 ID (Qiagen, Hilden, Germany) and analyzed with Qiagen’s PyroMark Q96 CpG 1.0.9. Digital Droplet Polymerase Chain Reaction (ddPCR) was carried out with the QX200 Droplet Digital reader (Bio-Rad, Hercules, CA, USA). Absolute leukocyte quantification by pyrosequencing was also performed.

The investigators reported that 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 and ddPCR measurements. In some patients, particularly with hematopoietic malignancies, they 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. The results confirmed cell-type–specific hypomethylation in the genes WDR20, CD4, CD8A, WIPI2, SLC15A4, and CENPA, which was more pronounced compared to the Illumina BeadChip data or reverse nonnegative least square (NNLS) estimation.

The authors concluded that targeted DNAm analysis by pyrosequencing or ddPCR is a valid alternative to quantify leukocyte subsets, but some assays require further optimization. The study was published on February 14, 2022 in the journal Clinical Chemistry.

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