Views: 0 Author: Site Editor Publish Time: 2024-11-13 Origin: Site
-Facilitates in the true fast implementation of digital PCR technology in NIPT field
In mid-October 2024, the Chinese Journal of Medical Genetics, Volume 41, Issue 10, published an article titled "Expert Consensus on the Application of Digital PCR Noninvasive Prenatal Screening Technology for the Initial Implementation of Fetal Chromosomal Disorder Screening" (https://rs.yiigle.com/cmaid/1517121, DOI:10.3760/cma.j.cn511374- 20240318-00176). The consensus elaborates on the relevant aspects of dPCR-NIPT, including: basic requirements, scope of application, pre-testing service process, testing process, report interpretation, genetic counseling, and limitations of the technology.
The picture of the digital PCR equipment published in the expert consensus is the original picture of the RainSure Scientific DropDx series instrument, and the details can be found in the equipment and product display section of the RainSure Scientific website (https://www.rainsurebio.com).
Fetal chromosome aneuploidy is a major component of prenatal screening tests, which is beneficial to promoting eugenics and improving the quality of childbirth through early prenatal screening and diagnosis. Aneuploidy prenatal testing is a non-invasive, minimally invasive or invasive technological approach that examines pregnant women during a certain time to assess the risk of fetal chromosomal aneuploidy disorders or to further confirm the diagnosis. Conventional prenatal diagnostic methods are invasive, with amniocentesis, chorionic villus sampling, and umbilical vein puncture sampling for karyotyping analysis, which carries a potential risk of fetal miscarriage or infection. Early and mid-trimester routine prenatal screening combines ultrasound examination with serum biomarkers analysis, along with clinical information such as maternal age, gestational age, weight, and the presence of diabetes, to calculate risk values using software for assessment. However, the sensitivity and specificity of these methods are limited. In 1997, Lo et al. discovered cell-free fetal DNA (cff DNA) in the peripheral blood of pregnant women, laying the foundation for non invasive prenatal testing (NIPT) for fetal chromosome aneuploidy. In 2008, Lo et al. further demonstrated the feasibility of fetal chromosome aneuploidy detection based on cff DNA in maternal plasma and large-scale genome sequencing.
At this stage, non-invasive prenatal testing based on NGS (Next Generation Sequencing) has been used in clinical screening for fetal trisomy 21, trisomy 18, and trisomy 13 syndromes, and has demonstrated high sensitivity, specificity, and detection rate in high-risk pregnancies. However, NGS-NIPT is affected by a variety of factors originating from the fetus, the mother, the sequencing process, and data analysis, which may result in false-positive or false-negative results. In addition, the cost of NGS-NIPT testing is expensive, making it difficult to rapidly and effectively implement on a large scale.
With the introduction of microfluidic technology, digital PCR (dPCR), which represents the third generation of PCR technology, has demonstrated higher detection efficiency and sensitivity. It has been shown that dPCR has the potential for more direct, rapid and cost-effective development in chromosomal aneuploidy NIPT applications. Moreover, the combination of dPCR-NIPT and NGS-NIPT technologies for screening can further enhance the accuracy, safety, and timeliness of prenatal screening tests for fetal chromosomal aneuploidies.
In addition, consensus-related recommendations are summarized below:
Summary of recommendations.
[Recommendation 1] The cost of dPCR-NIPT testing is comparable to that of serological screening, while its positive detection rate is similar to that of NIPT, making it a promising alternative to prenatal screening. This could lead to the formation of a sequential prenatal screening and diagnostic strategy of "dPCR-NIPT for initial screening, NIPT for secondary screening, and invasive prenatal diagnosis.".
[Recommendation 2] It is recommended that dPCR-NIPT be performed in accordance with the requirements for personnel, organization, reagents and equipment.
[Recommendation 3] Referring to serologic screening and NIPT, dPCR-NIPT can be used for simultaneous screening of trisomy 21, trisomy 18 and trisomy 13. In addition, given that the incidence and burden of Turner syndrome (45, X) and DiGeorge syndrome (22q11.2Deletion Syndrome) are not less than those of trisomy 18 and 13, simultaneous screening of trisomy 21, 45, X and 22q11.2DS combinations can also be performed using dPCR-NIPT.
[Recommendation 4] It is recommended that pregnant women undergoing dPCR-NIPT screening be categorized by appropriate, cautionary, and inappropriate populations, and to ensure informed consent be obtained.
[Recommendation 5] Different laboratories can establish localized standard dPCR-NIPT procedures and quality control systems according to their own conditions and pre-test and intra-test procedures and quality control.
[Recommendation 6] The criteria for determining the risk of fetal chromosomal diseases should be based on the dPCR-NIPT results.
[Recommendation 7] Genetic counseling and treatment strategies should be provided for different risks suggested by the test.
[Recommendation 8] Given the characteristics of cffDNA and dPCR-NIPT, it is recommended that medical institutions establish a "counseling~follow-up" retrospective mechanism and also ensure proper follow-up work is conducted.
Regarding the technical challenges of dPCR-NIPT
Instrument: There are stringent requirements for the performance of the equipment. Since digital PCR devices used for NIPT need to differentiate between normal and abnormal cff DNA copy number differences (usually less than 4%), they require excellent precision and reproducibility. Given that the copy number difference between normal and abnormal cff DNA is within 5%, increasing the number of droplets (i.e., partition number) to amplify the total amount of cfDNA in the sample can enlarge the difference in normal and abnormal cff DNA copy numbers, thereby enhancing detection sensitivity.
In a study by Dai [16], the minimum number of droplets required under specific conditions was calculated. At a cffDNA of 5% (0.05) and a PDR of 30% (0.3), approximately 18,000 droplets or PCR reactions were required to distinguish an aneuploid pregnancy from an aneuploid pregnancy with a 95% (1.96-fold standard deviation) confidence level.
Reagents: Used for NIPT require the realization of multiple tests, the more targets the more accurate the test. The principle of the reagent is to amplify the copy number difference between normal and abnormal fetal samples by detecting multiple targets. For example, assuming a 1% copy number difference between a normal and an abnormal fetus, the difference between 100 copies of one target is 1 copy. If 10 targets are tested, the difference will be put to 10 copies, which will increase the sensitivity of the test.
In Lašáková’s study [15], the authors used 16 primer pairs and two fluorescently labeled lock nucleic acid probes for chromosome 21 and chromosome 18, respectively.This test was validated in a blinded assay on plasma samples from 30 pregnant women at risk for Down syndrome trisomy 21, with a range of risk from 1:4 to 1:801. The results of the test and the invasive diagnostic procedures were in perfect agreement (sensitivity, specificity, positive predictive value and negative predictive value were all 100%). In a study by Dai [16], 10 sets of primer probes were used for multiplex testing of chromosomes 21, 18 and 13. In a study by Tan [14], 10 ng of broken gDNA and different numbers of primer pairs (1, 5, 10, and 20 pairs for chr21 and chr18, respectively) were used to verify the relationship between the number of primer pairs and the number of target DNA molecules detected by ddPCR. The results showed that the amount of target DNA molecules detected was highly dependent on the number of primer pairs, and that increasing the number of primer pairs can increase the number of target DNA molecules detected, given a certain amount of DNA.
Selection of more sensitive digital PCR equipment and accurately designed and validated multiplex digital PCR reagents are key to improving the sensitivity and specificity of the dPCR-NIPT assay.
About RainSure Scientific Ltd. Co.
RainSure Scientific is an innovative company dedicated to precision diagnostics. Adhering to the mission of "Innovation, Intelligent Manufacturing, Health Promotion", Rainsure Scientific is mainly engaged in the development of innovative diagnostic platforms and IVD diagnostic solutions. The company has developed world-leading scientific instruments and molecular diagnostic products, including microfluidic research platforms, digital PCR systems and supporting reagent kits, based on the company's continuous technological advancements in the fields of microfluidics and intelligent manufacturing. The company's founding team members all graduated from Ivy League universities and worked for a number of internationally recognized life science manufacturers, with rich industry experience and cutting-edge perspectives. RainSure Scientific is committed to empowering customers with innovative technology and intelligent manufacturing to foster the development of the healthcare industry.
