title: Accurate Quantification of T Cells by Measuring Loss of Germline T-Cell Receptor Loci with Generic Single Duplex Droplet Digital PCR Assays.
abstract: Quantifying T cells accurately in a variety of tissues of benign, inflammatory, or malignant origin can be of great importance in a variety of clinical applications. Flow cytometry and immunohistochemistry are considered to be gold-standard methods for T-cell quantification. However, these methods require fresh, frozen, or fixated cells and tissue of a certain quality. In addition, conventional and droplet digital PCR (ddPCR), whether followed by deep sequencing techniques, have been used to elucidate T-cell content by focusing on rearranged T-cell receptor (TCR) genes. These approaches typically target the whole TCR repertoire, thereby supplying additional information about TCR use. We alternatively developed and validated two novel generic single duplex ddPCR assays to quantify T cells accurately by measuring loss of specific germline TCR loci and compared them with flow cytometry-based quantification. These assays target sequences between the Dδ2 and Dδ3 genes (TRD locus) and Dβ1 and Jβ1.1 genes (TRB locus) that become deleted systematically early during lymphoid differentiation. Because these ddPCR assays require small amounts of DNA instead of freshly isolated, frozen, or fixated material, initially unanalyzable (scarce) specimens can be assayed from now on, supplying valuable information about T-cell content. Our ddPCR method provides a novel and sensitive way for quantifying T cells relatively fast, accurate, and independent of the cellular context.
authors.affiliation: Department of Dermatology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Willem H
authors.initials: WH
authors.lastname: Zoutman
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Rogier J
authors.initials: RJ
authors.lastname: Nell
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Mieke
authors.initials: M
authors.lastname: Versluis
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Debby
authors.initials: D
authors.lastname: van Steenderen
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Rajshri N
authors.initials: RN
authors.lastname: Lalai
authors.affiliation: Department of Dermatology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Jacoba J
authors.initials: JJ
authors.lastname: Out-Luiting
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: de Lange
authors.affiliation: Department of Dermatology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Maarten H
authors.initials: MH
authors.lastname: Vermeer
authors.affiliation: Laboratory for Medical Immunology, Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands.
authors.forename: Anton W
authors.initials: AW
authors.lastname: Langerak
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands. Electronic address: velden@lumc.nl.
authors.forename: Pieter A
authors.initials: PA
authors.lastname: van der Velden
mesh_terms: D000483:Alleles; D018095:Germ-Line Mutation; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D012015:Reference Standards; D015203:Reproducibility of Results; D017384:Sequence Deletion; D013601:T-Lymphocytes / Q000378:metabolism*
chemical_list: D011948:Receptors, Antigen, T-Cell