Detection of newly produced T and B lymphocytes by digital PCR in blood stored dry on nylon flocked swabs.
J Transl Med
; 15(1): 70, 2017 04 05.
Article
en En
| MEDLINE
| ID: mdl-28381232
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.Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Linfocitos B
/
Linfocitos T
/
Recolección de Muestras de Sangre
/
Reacción en Cadena en Tiempo Real de la Polimerasa
/
Nylons
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Adolescent
/
Adult
/
Aged
/
Aged80
/
Humans
/
Middle aged
Idioma:
En
Revista:
J Transl Med
Año:
2017
Tipo del documento:
Article
País de afiliación:
Italia