title: A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood.
abstract: Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.
authors.affiliation: a Department of Neurological Surgery ; University of California, San Francisco ; San Francisco , CA USA.
authors.forename: John K
authors.initials: JK
authors.lastname: Wiencke
authors.forename: Paige M
authors.initials: PM
authors.lastname: Bracci
authors.forename: George
authors.initials: G
authors.lastname: Hsuang
authors.forename: Shichun
authors.initials: S
authors.lastname: Zheng
authors.forename: Helen
authors.initials: H
authors.lastname: Hansen
authors.forename: Margaret R
authors.initials: MR
authors.lastname: Wrensch
authors.forename: Terri
authors.initials: T
authors.lastname: Rice
authors.forename: Melissa
authors.initials: M
authors.lastname: Eliot
authors.forename: Karl T
authors.initials: KT
authors.lastname: Kelsey
mesh_terms: D017252:CD3 Complex / Q000378:metabolism; D016022:Case-Control Studies; D019175:DNA Methylation; D005434:Flow Cytometry; D005910:Glioma; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013601:T-Lymphocytes / Q000378:metabolism*
chemical_list: D017252:CD3 Complex
keywords: CV, correlation coefficient; DMR, differentially methylated regions; DNA methylation; FACS; FACS fluorescence activated cell sorting; MS, methylation specific; T cells; ddPCR, droplet digital polymerase chain reaction; digital PCR; qPCR, quantitative polymerase chain reaction; real time PCR