Institute for Stem Cell Biology, Faculty of Medicine, RWTH Aachen University, Aachen, Germany.
Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Aachen, Germany.
Clin Chem. 2023 Nov 2;69(11):1283-1294. doi: 10.1093/clinchem/hvad143.
Cell-type specific DNA methylation (DNAm) can be employed to determine the numbers of leukocyte subsets in blood. In contrast to conventional methods for leukocyte counts, which are based on cellular morphology or surface marker protein expression, the cellular deconvolution based on DNAm levels is applicable for frozen or dried blood. Here, we further enhanced targeted DNAm assays for leukocyte counts in clinical application.
DNAm profiles of 40 different studies were compiled to identify CG dinucleotides (CpGs) with cell-type specific DNAm using a computational framework, CimpleG. DNAm levels at these CpGs were then measured with digital droplet PCR in venous blood from 160 healthy donors and 150 patients with various hematological disorders. Deconvolution was further validated with venous blood (n = 75) and capillary blood (n = 31) that was dried on Whatman paper or on Mitra microsampling devices.
In venous blood, automated cell counting or flow cytometry correlated well with epigenetic estimates of relative leukocyte counts for granulocytes (r = 0.95), lymphocytes (r = 0.97), monocytes (r = 0.82), CD4 T cells (r = 0.84), CD8 T cells (r = 0.94), B cells (r = 0.96), and NK cells (r = 0.72). Similar correlations and precisions were achieved for dried blood samples. Spike-in with a reference plasmid enabled accurate epigenetic estimation of absolute leukocyte counts from dried blood samples, correlating with conventional venous (r = 0.86) and capillary (r = 0.80) blood measurements.
The advanced selection of cell-type specific CpGs and utilization of digital droplet PCR analysis provided accurate epigenetic blood counts. Analysis of dried blood facilitates self-sampling with a finger prick, thereby enabling easier accessibility to testing.
基于细胞类型特异性 DNA 甲基化（DNAm）的方法可用于确定血液中白细胞亚群的数量。与基于细胞形态或表面标记蛋白表达的传统白细胞计数方法不同，基于 DNAm 水平的细胞去卷积适用于冷冻或干燥的血液。在这里，我们进一步增强了基于 DNAm 水平的白细胞计数在临床应用中的靶向 DNAm 检测方法。
我们汇编了 40 项不同研究的 DNAm 图谱，使用计算框架 CimpleG 来识别具有细胞类型特异性 DNAm 的 CG 二核苷酸（CpG）。然后，在 160 名健康供体和 150 名患有各种血液系统疾病的患者的静脉血中使用数字液滴 PCR 测量这些 CpG 的 DNAm 水平。进一步使用静脉血（n = 75）和干燥在 Whatman 纸上或 Mitra 微采样装置上的毛细血管血（n = 31）进行去卷积验证。
在静脉血中，自动细胞计数或流式细胞术与粒细胞（r = 0.95）、淋巴细胞（r = 0.97）、单核细胞（r = 0.82）、CD4 T 细胞（r = 0.84）、CD8 T 细胞（r = 0.94）、B 细胞（r = 0.96）和 NK 细胞（r = 0.72）的相对白细胞计数的表观遗传估计相关性良好。干燥血样也获得了类似的相关性和精度。通过参考质粒的添加，能够从干燥血样中准确地进行绝对白细胞计数的表观遗传估计，与传统的静脉（r = 0.86）和毛细血管（r = 0.80）血样测量相关。
先进的细胞类型特异性 CpG 选择和数字液滴 PCR 分析的利用提供了准确的表观遗传血液计数。干燥血样分析便于通过手指穿刺进行自我采样，从而更容易获得检测。
