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

Journal of Translational Medicine

Research

Luisa Imberti¹  Marion Vaglio Tessitore¹  Alessandra Sottini¹  Simona Bernardi¹  Aldo M. Roccaro¹  Giovanni Martellosio¹  Federico Serana¹  Claudia Ghidini¹ 

[1] Centro di Ricerca Emato-oncologica AIL (CREA), ASST Spedali Civili, P.le Spedali Civili, 1, 25123, Brescia, Italy;

关键词: Digital PCR;    Flocked swabs;    KRECs;    TRECs;   

DOI  :  10.1186/s12967-017-1169-9

 received in 2017-01-12, accepted in 2017-03-23,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundA 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.MethodsDNA 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.ResultsThe 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.ConclusionsOur 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.

【 授权许可】

CC BY   
© The Author(s) 2017