Original Article
Development and applications of external quality control materials for MTHFR 677C/T genotypes
Yun Bao, Yanqun Xiao, Lingli Jiang, Xueliang Wang, Yixiao Yang, Hualiang Wang
Published 2018-10-11
Cite as Chin J Lab Med, 2018, 41(10): 749-754. DOI: 10.3760/cma.j.issn.1009-9158.2018.10.009
Abstract
ObjectiveTo evaluate the performance of MTHFR 677 genotyping external quality assessment (EQA) program using plasmid DNA constructed in vitro as quality control samples and discuss the problems in clinical laboratories enrolled in the program.
MethodsRecombinant plasmid carrying MTHFR 677C locus sequence was constructed as wild type sample and plasmid with MTHFR 677T mutation was generated with site-directed mutagenesis as mutant type sample. Heterozygous mutant samples were obtained after equal proportion of the two plasmids. EQA scheme were held twice a year in 2016 and 2017, and sample panels contained 5 different samples using recombinant plasmid DNA containing all types of MTHFR 677 locus genotypes. Participating laboratories were asked to test samples using their routine methods and report the results before deadlines. 26, 28, 52 and 56 effective reports were received respectively in the four EQA schemes. The scores of each lab were calculated based on their results and the overall compliance of different samples as well as the sensitivity and specificity of different methods were calculated using Microsoft Excel.
ResultsMTHFR 677 locus genotypes of the constructed plasmid were verified by Sanger sequencing and there was no failure of sample detection in the four EQA schemes, which suggest that the plasmid has good clinical applicability. About 96.15%(25/26), 100%(28/28), 96.15%(50/52)and 98.21%(55/56)of the laboratories submitted correct results for all samples in the four EQA schemes. The overall compliance rate were 99.23% (129/130), 100%(140/140), 96.92% (252/260) and 98.93% (277/280) respectively. All laboratories using digital FISH and microarrays got full marks in four EQA schemes. The compliance rates for fluorescent PCR were 97.5% (39/40), 100% (45/45), 94.29% (66/70) and 100% (95/95) respectively, while the rates were 100% (20/20), 100% (15/15), 90% (36/40) and 92.5% (37/40) for Sanger sequencing.
ConclusionsThe recombinant plasmid DNA constructed in this study can effectively detect the performance of reagents with good clinical applicability. The results of EQA programs suggested that the overall accuracy rate of laboratories enrolled was high enough, while some laboratories′ performance still needs to be improved. Quality controls in clinical laboratories were essential to assure the accuracy of results.(Chin J Lab Med, 2018, 41: 749-754)
Key words:
Methylenetetrahy drofolate reductase(NADPH); Plasmids; Genetic testing; Quality control
Contributor Information
Yun Bao
Department of Clinical Cytogenetics, Shanghai Center for Clinical Laboratory, Shanghai 200126, China
Yanqun Xiao
Lingli Jiang
Xueliang Wang
Yixiao Yang
Hualiang Wang
