10.6084/m9.figshare.1255023.v3
John K Wiencke
John
K Wiencke
Paige M Bracci
Paige
M Bracci
George Hsuang
George
Hsuang
Shichun Zheng
Shichun
Zheng
Helen Hansen
Helen
Hansen
Margaret R Wrensch
Margaret
R Wrensch
Terri Rice
Terri
Rice
Melissa Eliot
Melissa
Eliot
Karl T Kelsey
Karl
T Kelsey
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
Taylor & Francis Group
2015
ddpcr
pcr
T cell proportions
CD 3Z gene
dna methylation
demethylated CpG promoter sites
flow cytometry
qpcr
157 blood specimens
blood CD 3 counts
t cells
FACS
2015-10-27 04:04:54
Dataset
https://tandf.figshare.com/articles/dataset/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/1255023
<div><p>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.</p></div>