Abstract
BackgroundGenome-wide DNA methylation (DNAm) studies have proven extremely useful to understand human hematopoiesis. Due to their active DNA content, nucleated red blood cells (nRBCs) contribute to epigenetic and transcriptomic studies derived from whole cord blood. Genomic studies of cord blood hematopoietic cells isolated by fluorescence-activated cell sorting (FACS) may be significantly altered by heterotopic interactions with nRBCs during conventional cell sorting.ResultsWe report that cord blood T cells, and to a lesser extent monocytes and B cells, physically engage with nRBCs during FACS. These heterotopic interactions resulted in significant cross-contamination of genome-wide epigenetic and transcriptomic data. Formal exclusion of erythroid lineage-specific markers yielded DNAm profiles (measured by the Illumina 450K array) of cord blood CD4 and CD8 T lymphocytes, B lymphocytes, natural killer (NK) cells, granulocytes, monocytes, and nRBCs that were more consistent with expected hematopoietic lineage relationships. Additionally, we identified eight highly differentially methylated CpG sites in nRBCs (false detection rate <5 %, |Δβ| >0.50) that can be used to detect nRBC contamination of purified hematopoietic cells or to assess the impact of nRBCs on whole cord blood DNAm profiles. Several of these erythroid markers are located in or near genes involved in erythropoiesis (ZFPM1, HDAC4) or immune function (MAP3K14, IFIT1B), reinforcing a possible immune regulatory role for nRBCs in early life.ConclusionsHeterotopic interactions between erythroid cells and white blood cells can result in contaminated cell populations if not properly excluded during cell sorting. Cord blood nRBCs have a distinct DNAm profile that can significantly skew epigenetic studies. Our findings have major implications for the design and interpretation of genome-wide epigenetic and transcriptomic studies using human cord blood.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0129-6) contains supplementary material, which is available to authorized users.
Highlights
Genome-wide DNA methylation (DNAm) studies have proven extremely useful to understand human hematopoiesis
Higher nucleated red blood cell (nRBC) counts have been observed in response to prenatal exposure to infection, preeclampsia, maternal obesity, diabetes, and smoking [17,18,19,20,21,22]. nRBCs are generally resistant to lysis protocols and tend to sediment in the mononuclear cell fraction during purification by density gradient centrifugation, further complicating the isolation of pure hematopoietic cell populations [23]
We detected events positive for expression of both erythroid (CD235) and monocyte (CD14) or B cell (CD19) markers. These findings indicate that heterotopic red blood cell (RBC)-to-white blood cell (WBC) doublets can be undetected by fluorescence-activated cell sorting (FACS) using conventional singlet gating
Summary
Genome-wide DNA methylation (DNAm) studies have proven extremely useful to understand human hematopoiesis Due to their active DNA content, nucleated red blood cells (nRBCs) contribute to epigenetic and transcriptomic studies derived from whole cord blood. Genome-wide profiling of isolated blood cells can provide a large amount of information, data interpretation is notoriously difficult in mixed cell populations [5,6,7] To address this issue, The proportion of nRBCs in cord blood varies considerably between individuals. NRBCs are generally resistant to lysis protocols and tend to sediment in the mononuclear cell fraction during purification by density gradient centrifugation, further complicating the isolation of pure hematopoietic cell populations [23] Depending on their proportion, the presence of nRBCs could complicate both epigenetic and gene expression studies
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