Abstract
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.
Highlights
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
TRECs and KRECs were initially measured in separate quantitative real time PCR assays [1, 2]; successively, we modified these assays to allow the simultaneous measure of TRECs and KRECs in peripheral blood mononuclear cells (PBMC) (“reference” method) [6, 7], while others used the method to measure the target molecules on dried blood spotted on filter paper cards [8, 9]
We found that the extent of age-related TREC decrease determined by Digital PCR (dPCR) in DNA obtained from dried blood adsorbed on flocked swabs (FS) was comparable to that reported by Lorenzi et al [3], stating a 1.5-Log change of TRECs over the age range they have measured in liquid whole blood (WB)
Summary
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. Detection of TRECs and KRECs by real time PCR from dried blood spotted on filter paper is used for the screening of severe immunodeficiency. The protocol performed using dried blood spots is currently used for newborn screening of immunodeficiency [8, 9, 15], considering the high number of TRECs in newborns, the low cost of the test, and the easy transportation and storage of spotted cards. The use of dried blood samples on filter paper prepared from adults, elderly people, and patients undergoing immunosuppressive therapies may be challenging due to the low quantity of TRECs and KRECs that can be isolated and the low quantity of DNA that can be extracted by punches of cards. The more DNA is recovered, the better is the chance for obtaining a robust and reliable result; on the contrary, a small amount of DNA would result either in stochastically variable amplification or in the loss of signal due to insufficient template
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