title: Nucleated red blood cells impact DNA methylation and expression analyses of cord blood hematopoietic cells.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  Heterotopic 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.
authors.affiliation: Child & Family Research Institute, 950 W 28th Avenue, Vancouver, BC V5Z 4H4 Canada ; Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z3 Canada.
authors.forename: Olivia M
authors.initials: OM
authors.lastname: de Goede
authors.affiliation: Child & Family Research Institute, 950 W 28th Avenue, Vancouver, BC V5Z 4H4 Canada ; Department of Pediatrics, University of British Columbia, Vancouver, BC V6T 1Z3 Canada.
authors.forename: Hamid R
authors.initials: HR
authors.lastname: Razzaghian
authors.affiliation: Child & Family Research Institute, 950 W 28th Avenue, Vancouver, BC V5Z 4H4 Canada ; Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z3 Canada ; Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, BC V6T 1Z3 Canada.
authors.forename: E Magda
authors.initials: EM
authors.lastname: Price
authors.affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z3 Canada ; Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Vancouver, BC V5Z 4H4 Canada.
authors.forename: Meaghan J
authors.initials: MJ
authors.lastname: Jones
authors.affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z3 Canada ; Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Vancouver, BC V5Z 4H4 Canada.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Kobor
authors.affiliation: Child & Family Research Institute, 950 W 28th Avenue, Vancouver, BC V5Z 4H4 Canada ; Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z3 Canada.
authors.forename: Wendy P
authors.initials: WP
authors.lastname: Robinson
authors.affiliation: Child & Family Research Institute, 950 W 28th Avenue, Vancouver, BC V5Z 4H4 Canada ; Department of Pediatrics, University of British Columbia, Vancouver, BC V6T 1Z3 Canada.
authors.forename: Pascal M
authors.initials: PM
authors.lastname: Lavoie