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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Journal of Translational Medicine 1/2017

Detection of newly produced T and B lymphocytes by digital PCR in blood stored dry on nylon flocked swabs

Zeitschrift:
Journal of Translational Medicine > Ausgabe 1/2017
Autoren:
Marion Vaglio Tessitore, Alessandra Sottini, Aldo M. Roccaro, Claudia Ghidini, Simona Bernardi, Giovanni Martellosio, Federico Serana, Luisa Imberti
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12967-017-1169-9) contains supplementary material, which is available to authorized users.

Abstract

Background

A normal number of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) is considered a biomarker for adequate new T- and B-cell production. In newborns, detection of TRECs and KRECs by real time PCR from dried blood spotted on filter paper is used for the screening of severe immunodeficiency. In adults, elderly and during diseases, where the number of TRECs is lower than in newborns and children, a large amount of DNA and a sensitive method of amplification are necessary to identify newly produced lymphocytes.

Methods

DNA was prepared from blood of 203 healthy adults (range: 18–91 years old) absorbed for 10 s on flocked swabs and let to dry, or from peripheral blood mononuclear cells. DNA was subjected to digital PCR and to well established conventional real time PCR-based method using TREC- and KREC-specific primers and probes. The number of TRECs and KRECs was expressed per mL of blood. Statistical analysis was performed by nested ANOVA, Pearson coefficient of determination, and by linear regression tests.

Results

The novel method for the storage of dried blood on nylon flocked swabs and the use of digital PCR allow quantification of TRECs and KRECs with high degree of sensitivity, specificity, accuracy, and precision. TRECs and KRECs were amplified by digital PCR in all tested blood samples, including those obtained from elderly individuals (>70 years old) and that were negative by real time PCR. Furthermore, values of TRECs and KRECs obtained by digital PCR were in the range of those acquired by real time PCR.

Conclusions

Our findings demonstrate that DNA isolation from dried blood on flocked swabs followed by digital PCR-based analysis represents a useful tool for studying new lymphocyte production in adults and elderly individuals. This suggests the potential use of the methodology when monitoring of clinical variables is limited by the number of molecules that can be amplified and detected, such as in patients with immunodeficiency or under immunosuppressive therapies.
Zusatzmaterial
Additional file 1: Figure S1. dPCR plots for TREC and KREC quantification. A) Representative dPCR plots of TREC and KREC quantification in HeLa cells. The cell line is used to establish the threshold values, which were set up at 5000 for FAM (TRECs) fluorescence and 4000 for VIC (KRECs) fluorescence. B) Representative dPCR plots of TREC and KREC quantification in a positive sample showing that the threshold values allow a precise separation between positive and negative dot plots. The data points in the plots are color-coded: FAM (blue), VIC (red), FAM plus VIC (green), undetermined (grey) and not amplified (yellow).
Additional file 2: Table S1. Intra-assay and inter-assay variation for dPCR.
Additional file 3: Table S2. TREC and KREC values obtained in different experimental procedures of qRT-PCR and dPCR.
Additional file 4: Figure S2. Levels of TRECs and KRECs in adults divided by age. Mean levels of TRECs and KRECs obtained by dPCR starting from DNA isolated from dried blood adsorbed on FS (red bars) and by qRT-PCR starting from DNA prepared from PBMC (blue bars) in the indicated age groups of adults. Error bars represent standard deviations.
Literatur
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