Technical Review Guideline On Fetal Chromosome Aneuploidy Test Kit (High-throughput Sequencing Method)
2021-06-22
Appendix Review and Guiding Principle for Registration Technology of Fetal Chromosome Aneuploidy (T21, T18, T13) Test Kit (High-throughput Sequencing Method) This guiding principle aims to not only guide the registration applicant to prepare and write the registered application data of fetal chromosomal aneuploidy (T21, T18, T13) detection kit (high throughput sequencing method), but also provide references for the technical evaluation department to conduct technical evaluation on registered application data. This guiding principle is based on the general requirements of the reagent, and the applicant shall determine which content is applicable according to the specific characteristics of the product. If the content is not applicable, the reasons and corresponding scientific basis are required to be narrated in details, and according to the specific characteristics of the product, the registered application information content shall be enriched and refined. This guiding principle is the guidance document for the —2—— applicant and the reviewers, but excludes the administrative matters involved in registration approval, nor as law for enforcement. The methods able to meet the requirements of relevant laws and regulations also can be used, but need detail reasons. Meanwhile, its scientific rationality shall be validated to provide detailed research and validation data. Relevant personnel shall use this guiding principle under the premise of following relevant laws and regulations. This guiding principle is formulated under the current laws, regulations and standard systems as well as current cognitive levels. With the constant improvement of laws, regulations and standards as well as the continuous development of science and technology, the related content of this guiding principle will be timely adjusted. I. Scope Trisomies 21, Trisomies 18 and Trisomies 13 (T21, T18 and T13) of fetal chromosome aneuploidies are the most common chromosome aneuploidy clinical diseases, corresponding to T21 syndrome (also known as Down’s syndrome, mongolian or Down syndrome), T18 syndrome (also known as Edwards syndrome) and T13 syndrome (also known as Patau syndrome) with the morbidity of about 1/700, 1/6000 and 1/10000 respectively. A vast majority of —3—— child patients have serious mental retardation and organ deformity. Prenatal screening and prenatal diagnosis are the means to avoid the children with genetic defects. Routine prenatal screening methods include early pregnancy ultrasonography combined with serological screening and second trimester serological screening. The pregnant women with high-risk screening results are recommended to be finalized by prenatal diagnosis and informed to select. The prenatal diagnostic gold standard of fetal chromosomal abnormalities is the pre-intervention prenatal diagnosis surgery, referring to use the chorionic villus sampling / amniocentesis / percutaneous umbilical blood vessel puncture for acquiring corresponding cells in combination with cell biology method for karyotype analysis of fetal chromosome. The principle of high-throughput sequencing method to detect fetal chromosome aneuploidy is that fetal cell-free DNA (cfDNA) exists in maternal plasma of pregnant women with a length of about 75 bp – 250 bp, almost all from placental trophoblastic cells, of which the concentration is closely related to gestational week with a certain proportion (5%-30%) stabilizing in maternal peripheral blood plasma. High-throughput sequencing method is to extract the normal maternal and fetal plasma cell-free DNA from maternal plasma, and —4—— carry out the library building and fragment amplification for final implementation of machine sequencing, so as to obtain chromosome number evaluation results through the software to analyze data. Taking the detection of T21 syndrome as example, when analyzing the cell-free DNA data of T21 fetal maternal plasma, the total cell-free DNA number of chromosome 21 will rise by a small proportion. The statistical algorithm is used to distinguish such tiny difference, so as to achieve the application of cfDNA to prenatal screening of fetal chromosomal aneuploidy. However, the high-throughput sequencing method is unable to detect the abnormality of chromosome structure, nor replace the open neural tube defect screening in traditional screenings. The fetal chromosome aneuploidy (T21, T18, T13) detection kit (high-throughput sequencing method) described in this guiding principle refers to use the high-throughput sequencing method for detecting the fetal cell-free deoxyribonucleic acid (DNA) of peripheral blood plasma for pregnant women, through the analysis of the 21 of cell-free fetal DNA in the samples, analyze the differences between the numbers of chromosome 21, 18 and 13 in fetal chromosome aneuploidy of samples, and expect the prenatal screening detection kit for fetal chromosome aneuploidy diseases, —5—— i.e., T21 syndrome, T18 syndrome and T13 syndrome. The high-throughput sequencing method described in this guiding principle refers to the second-generation sequencing method through library building and fragment amplification for machine sequencing, and the third-generation and fourth-generation single molecule sequencing methods with no need of fragment amplification are not discussed herein. High-throughput sequencing technology has developed rapidly, but if the third-generation and fourth-generation single molecule sequencing methods are applicable to some aspects, such as enterprise internal reference set, performance evaluation and clinical evaluation, this guiding principle can be a reference. The detection kit mentioned in this guiding principle is applicable to the pregnant women with gestational week of 12+0 and above for prenatal screening. But the result is not representative to confirm the pregnant women with trisomy fetus, and the result of prenatal screening shall be confirmed by prenatal diagnosis. The principles of ethics shall comply with relevant national laws, regulations and industrial rules. II. Registered information requirement (1) Survey material —6—— Survey materials mainly include product expected use, product description, biosafety description, the summary and evaluation of major research results related to the product, and other contents. Among them, the other contents include the approval situation of similar products at home and abroad, which shall focus on the methodology, detection limit and other aspects to specify the major differences between the to-be-registered product and the present approved similar products. Survey materials shall comply with the relevant requirements of IVD Reagent Registration Management Measure (CFDA Decree No. 5, hereinafter referred to as the “Measure”) and Announcement on Registration Material Requirement and Approval Certification Document Format for IVD Reagent (CFDA Announcement No. 44 in 2014, hereinafter referred to as the “Announcement”). Survey materials shall expound the test principle in details, which can be added with graphs to specify all reagent and consumable material names, manufacturer, product code or certificate code and detailed compositions for combined use in the entire test. (2) Research data of main raw materials Research data of main raw materials include two parts, i.e., the —7—— internal reference products of the enterprise and the necessary reagents to complete the test.
Internal reference products of the enterprise shall at least include positive reference, negative reference, test limit reference and chimera reference, and specific requirements are shown as follows. 1.1 Positive reference: the simulated samples of T21, T18 and T13 fetal DNA fragments mixed with normal female plasma in a certain proportion. 1.2 Negative reference: including the simulated samples of fetal DNA fragments in normal chromosome number mixed with normal female plasma in a certain proportion, and the simulated samples of fetal DNA fragments with anomaly in other chromosomes mixed with normal female plasma in a certain proportion. The negative reference with other chromosomal abnormalities is selected independently by the applicant according to the common chromosomal abnormality project and the quality control requirement of the product. 1.3 Detection limit reference: the simulated samples of T21, T18, T13 fetal DNA fragments mixed with normal female plasma in a certain proportion such as 5%, 3.5% and 2.5%, 95%. The positive —8—— detection rate of 95% (n≥20) is recommended to use as the determination standard for minimum detection limit. 1.4 Chimera reference product: the proportion of simulated T21, T18 and T13 chimeras is set up independently by the applicant according to the quality control requirements of the product, such as 70% abnormal and 30% normal, 30% abnormal and 70% normal, etc. Fetal DNA fragments with T21, T18, T13 and other chromosomal abnormality can be prepared from abortion tissue or amniotic cells, cell lines and other sources. The applicant shall specify the composition of internal reference product of the enterprise, the preparation and verification methods, the determination method of the concentration of fetal DNA fragments and the selection basis of the proportions. The samples of various internal reference products shall have different sources.
The reagents needed to complete the whole testing process generally shall at least include the extraction and purification reagent of plasma cell-free DNA, library building reagent, library quantitative reagent, sequencing reagent, negative quality control product, positive quality control product and the co-used sequencing —9—— chip and data analysis software. Specific requirements are shown as follows: 2.1 Extraction and purification reagent of plasma cell-free DNA: whether it is included in the registered products or not, the applicant shall submit the principle of co-used extraction and purification reagent, detailed composition, manufacturer, produce code, Class-I medical device registration certificate code and relevant information. In terms of performance, the length range accuracy and purity requirements of DNA fragments extracted by the reagent, the extraction efficiency, repeatability and anti-interference ability shall be evaluated. 2.2 Library building reagent, library quantitative reagent, sequencing reagent: the library building reagent is used for fragmented DNA gap-filling and corresponding partial 5’ terminal phosphorylation (if necessary) and 3’ terminal dephosphorylation (if necessary), then the ligase is applied in combination with the tags and linkers for sequencing and analysis to build the standard library for machine sequencing. Library quantitative reagent is used to quantitatively measure the concentration of library by PCR amplification and the contrast with standard curve, preparing for the subsequent machine sequencing of normalized library and assessing —10—— the quality of library building. Library quantification shall clarify the requirements of library quality, fragment size and concentration, provide analysis graphs, and interpret the parameters (such as whether there is an impure peak or not, the causes and whether the impact on final result exists or not). Library quantification is not limited to the above methods and can be conducted by other methods, such as fluorescence dye method and Qubit quantification. The sequencing reagent is used to expand the fragment of normalized library, and the gene sequencer is applied to capture the signal changes caused by base variation in the amplification process, so as to achieve the purpose of reading sequence. The above three reagents are generally composed of the enzyme with corresponding functions (terminal repair enzyme, DNA ligase, gap repair enzyme DNA polymerase, etc.), nucleotide sequence (primer, linker sequence, tag sequence, library quantitative standard), buffer and dNTP. Specific requirements are shown as follows: 2.2.1 Enzyme: purity, activity and functional test data shall be submitted. For example, the library building process is including but not limited to terminal repair enzyme, DNA ligase, gap repair enzyme, etc., and the selected enzyme shall have terminal blunting, —11—— gap-filling, tag or linker connection and DNA polymerase activity.
