Analysis Of The Causes Of False Negative And False Positive In Non-invasive Prenatal Testing And Establishment Of Laboratory Investigation Procedures

Non-invasive prenatal testing(NIPT)is a method which uses massive parallel sequencing technology to sequence free fetal DNA in maternal peripheral blood by separating free DNA(cellfree DNA,cf DNA)from maternal peripheral blood to determine the presence of aneuploidy abnormalities in the fetal chromosomes by analyzing additional chromosomal differences introduced by aneuploid fetal DNA.Whereas for the accuracy of NIPT detection,the result can be affected by the changed biological factors such as the source and content of the free DNA in maternal peripheral blood,so for the target disease detected,the fetal chromosome results detected by NIPT are inconsistent with the invasive diagnosis results,that is,the NIPT false negative and false positive results.In view of this kind of situation,most of the current literature reports are only based on the data from single or several medical centers investigating the reasons of these several cases,but the multi-sample reason investigation and summary have not found.In addition,after receiving feedback from false negative or false positive results,the laboratory can investigate the known influencing factors of NIPT false negative or false positives and analyze the causes,which may relieve the contradiction between doctors and patients,also the accumulated data can further improve technical indicators.At present,the investigation procession of laboratory has not yet been set in the industry.So according to the overall results of this study,the set of laboratory NIPT false negative and false positive investigation procedure can be concluded.This study aimed at 104 subjects who reported NIPT results from 1.46 million subjects who had undergone NIPT testing during the four years from January 1,2013 to December 31,2016,which were inconsistent with the clinical diagnosis and requested a cause investigation.There were 30 NIPT results false negative and 74 NIPT results false positive.Investigate and analyze the laboratory and biological reasons.At the same time,the single nucleotide polymorphism mass spectrometry detection method(SNP-MS)was used to compare 34 SNP sites of maternal leukocyte DNA and NIPT library DNA of the same subject,and the results of false errors and false positives in the laboratory due to operational errors in the laboratory Impact.Chromosome microdeletion and microduplication detection using high-throughput sequencing technology was used to sequence the maternal leukocytes and placental tissues of the subjects,and the effects of these two factors on the NIPT false negative and false positive results were analyzed.The results are as follows:In 104 cases,the maternal leukocyte DNA and NIPT library DNA 34 SNP sites were all 100% identical.None of the 30 false-negative subjects were found to be affected by maternal copy number variation.Of the 74 false-positive subjects,20(27%)detected maternal chromosome microduplications.NIPT false positive chromosomal micro-repetitive fragments are generally larger than maternal leukocyte micro-repetitive fragments.12 of 14 cases(85.7%)NIPT detected chromosomal micro-repetitive fragments with a larger size,with a higher proportion of 3.4% ? 316.7%.In placenta tissue testing,8 cases(80%)of 10 false negative subjects detected the placenta effect,and 4 cases(44.4%)of 9 false positive subjects detected restrictive placental mosaicism.the result shows:1.NIPT false-negative and false-positive laboratory operations affect the survey results: mass spectrometry analysis of 34 SNP sites can be used to identify mixed samples.In this study,the 104 samples of the surveyed subjects were consistent in 34 sites during the laboratory operation,which proved that the laboratory did not mix samples,and the research and analysis can be directly conducted from biological reasons.2.NIPT false negative and false positive biological reasons-the effect of maternal copy number variation: On the one hand,the impact of maternal copy number variation on NIPT mainly occurs in false positive results,and the highest incidence of XO is in the sex chromosome false positive.On the other hand,the NIPT method can detect a certain percentage of maternal copy number variations at the same time.3.NIPT False Negative and False Positive Biological Reasons-Effect of Placental Chimerism: Placental chimerism has a large proportion of effects on both NIPT false negative and false positive,but there is no direct correlation between the placental chimera ratio and the detection of NIPT This may depend on the sampling method of the survey sample,such as sampling location,sampling volume,etc.4.NIPT false-negative and false-positive laboratory investigation process is established: through this study,from the survey object,sample sampling,sample processing,sample transportation to testing methods,five aspects are summarized to establish a set of NIPT false negative or false-positive laboratory cause investigation The process,including investigation of sampling objects,sampling methods,sample processing,sample transportation and testing methods,can be used for reference by similar research.