Kanegae Marilia Pyles P, Barreiros Lucila Akune, Sousa Jusley Lira, Brito Marco Antônio S, Oliveira Edgar Borges de, Soares Lara Pereira, Mazzucchelli Juliana Themudo L, Fernandes Débora Quiorato, Hadachi Sonia Marchezi, Holanda Silvia Maia, Guimarães Flavia Alice T M, Boacnin Maura Aparecida P V V, Pereira Marley Aparecida L, Bueno Joaquina Maria C, Grumach Anete Sevciovic, Gesu Regina Sumiko W Di, Santos Amélia Miyashiro N Dos, Bellesi Newton, Costa-Carvalho Beatriz T, Condino-Neto Antonio
Departamento de Imunologia, Universidade de São Paulo (USP), São Paulo, SP, Brasil.
Hospital Municipal Dr. José de Carvalho Florence, São José dos Campos, SP, Brasil.
Rev Paul Pediatr. 2017 Jan-Mar;35(1):25-32. doi: 10.1590/1984-0462/;2017;35;1;00013.
To validate the quantification of T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) by real-time polymerase chain reaction (qRT-PCR) for newborn screening of primary immunodeficiencies with defects in T and/or B cells in Brazil.
Blood samples from newborns and controls were collected on filter paper. DNA was extracted and TRECs, and KRECs were quantified by a duplex real-time PCR. The cutoff values were determined by receiver operating characteristic curve analysis using SPSS software (IBM®, Armonk, NY, USA).
Around 6,881 samples from newborns were collected and TRECs and KRECs were quantified. The TRECs values ranged between 1 and 1,006 TRECs/µL, with mean and median of 160 and 139 TRECs/µL, respectively. Three samples from patients with severe combined immunodeficiency (SCID) showed TRECs below 4/µL and a patient with DiGeorge syndrome showed undetectable TRECs. KRECs values ranged from 10 to 1,097 KRECs/µL, with mean and median of 130 and 108 KRECs/µL. Four patients with agammaglobulinemia had results below 4 KRECs/µL. The cutoff values were 15 TRECs/µL and 14 KRECs/µL and were established according to the receiver operating characteristic curve analysis, with 100% sensitivity for SCID and agammaglobulinemia detection, respectively.
Quantification of TRECs and KRECs was able to diagnose children with T- and/or B-cell lymphopenia in our study, which validated the technique in Brazil and enabled us to implement the newborn screening program for SCID and agammaglobulinemia.
通过实时聚合酶链反应（qRT-PCR）验证对T细胞受体切除环（TREC）和κ链缺失重组切除环（KREC）的定量检测，用于巴西原发性免疫缺陷病（T和/或B细胞存在缺陷）的新生儿筛查。
从新生儿和对照者的滤纸血样中提取DNA，通过双重实时PCR对TREC和KREC进行定量检测。使用SPSS软件（美国纽约州阿蒙克市IBM公司）通过受试者工作特征曲线分析确定临界值。
收集了约6881例新生儿样本并对TREC和KREC进行了定量检测。TREC值在1至1006个TREC/μL之间，平均为160个TREC/μL，中位数为139个TREC/μL。3例重症联合免疫缺陷（SCID）患者的样本显示TREC低于4/μL，1例迪乔治综合征患者未检测到TREC。KREC值在10至1097个KREC/μL之间，平均为130个KREC/μL，中位数为108个KREC/μL。4例无丙种球蛋白血症患者的检测结果低于4个KREC/μL。根据受试者工作特征曲线分析确定的临界值分别为15个TREC/μL和14个KREC/μL，对SCID和无丙种球蛋白血症检测的灵敏度分别为100%。
在我们的研究中，对TREC和KREC的定量检测能够诊断T和/或B细胞淋巴细胞减少症患儿，这验证了该技术在巴西的有效性，并使我们能够实施SCID和无丙种球蛋白血症的新生儿筛查项目。
