RESUMO
OBJECTIVES: Droplet digital PCR (ddPCR) has been reported to have a superior validity over PCR with amplification-refractory mutation system (ARMS-PCR) for detecting the BRAF V600E mutation in thyroid nodule fine-needle aspiration (FNA) samples using cytological diagnosis as the reference. However, the added value of ddPCR on surgical decision-making remains to be illustrated when the technique is combined with FNA cytology. METHODS: A total of 277 consecutive patients with thyroid nodules were subjected to FNA cytology and BRAF V600E testing with ARMS-PCR. Within this patient cohort, 90 patients underwent surgical intervention with pathological diagnosis available. BRAF V600E testing with ddPCR was performed retrospectively using FNA frozen DNA specimens. The clinical validity and utility of ddPCR in comparison with ARMS-PCR were compared using surgical pathology as the reference. RESULTS: Overall, 101 BRAF V600E mutations were detected by ddPCR, including five ARMS negative patients, four of whom were confirmed to have papillary thyroid cancer (PTC) by surgical pathology. Of the 90 patients with surgical pathology, which is considered the gold standard, ddPCR BRAF V600E testing yielded a sensitivity of 91.3% and specificity of 100% for PTC diagnosis, higher than that of ARMS (sensitivity 83.1%, specificity 100%). However, ddPCR only identified one more candidate patient for surgical intervention than ARMS when the techniques were combined with cytology. CONCLUSIONS: This study highlighted the superior performance of ddPCR over ARMS in BRAF V600E detection from thyroid nodule FNA samples. Further studies are needed to evaluate the cost-effectiveness of replacing ARMS-PCR with ddPCR for surgical decision-making.
Assuntos
Análise Mutacional de DNA , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas B-raf/genética , Nódulo da Glândula Tireoide/genética , Nódulo da Glândula Tireoide/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha Fina , Análise Mutacional de DNA/métodos , Análise Mutacional de DNA/normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Estudos Retrospectivos , Glândula Tireoide/patologia , Nódulo da Glândula Tireoide/diagnóstico , Nódulo da Glândula Tireoide/terapia , Adulto JovemRESUMO
OBJECTIVE: To establish a method for the expression of glycogen synthase kinase 3ß with high purity and biological activity. METHODS: E.coli expression system and baculovirus-insect cell expression system were used to produce the kinase, followed by purification using His-tag and GST-tag and determination of its purity and activity by SDS-PAGE and kinase reaction, respectively. RESULTS: Glycogen synthase kinase 3ß produced from E.coli represented 54% of the total bacterial protein, as compared with 96% of the total protein from the insect cell system .Glycogen synthase kinase 3ß produced from insect cell exhibited an one-fold higher biological activity than the protein obtained from E.coli. CONCLUSIONS: Compared with the protein from E.coli system, glycogen synthase kinase 3ß from the insect cell expression system is endowed with a higher purity and bioactivity.
