Department of Hematological and Transfusion Immunology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland.
Department of Genetics, Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland.
Transfusion. 2022 Feb;62(2):458-468. doi: 10.1111/trf.16780. Epub 2022 Jan 8.
Fetal blood group (BG) and platelet (HPA) antigens may trigger maternal immunization, causing a fetal disease. Noninvasive prenatal diagnostics (NIPT) predicts fetal genotype, identifying pregnancies with no risk. All current techniques detect fetal antigen alleles with unspecific background and without estimation of fetal fraction, thus new protocols for detection of fetal BG/HPA alleles with ultrahigh sensitivity still need to be tested to improve NIPT.
To design NIPT of clinically important antigens using Ion AmpliSeq HD technology.
Plasma DNA from 36 pregnant women (9-33 week of gestation, 24 immunized with anti-HPA-1a,-3b,-15a, -K, or -D+C+S), with known BG/HPA genotypes of their neonates/partners, was tested on Ion S5 System using the Ion AmpliSeq HD designer custom gene panel. NGS contained 25 rs-targets encoding relevant BG/HPA antigens and 10 markers.
Using the NGS protocol, 76 out of 85 differences in fetal/maternal BG/HPA genotypes were determined in concentration above 2% fetal paternally inherited allele chimerism. The level of unspecific reads for BG/HPA alleles was below 0.87%. In 24 immunized women NGS revealed feto-maternal incompatibility in 11 cases (from 2.44% to 7.41%) and excluded in 10 (<0.05%), three cases had inconclusive results (1.79%, 0.19%, 0.11%). The presence of fetal DNA was confirmed in each case by detecting markers with at least 2% chimerism.
The use of Ion AmpliSeq HD technology improves the prediction of feto-maternal incompatibility, increasing the sensitivity of BG/HPA NIPT and serving confirmation of the fetal DNA at the same workflow.
胎儿血型（BG）和血小板（HPA）抗原可能引发母体免疫，导致胎儿疾病。非侵入性产前诊断（NIPT）预测胎儿基因型，识别无风险妊娠。所有当前技术均使用非特异性背景检测胎儿抗原等位基因，且不估计胎儿分数，因此需要测试新的超敏检测胎儿 BG/HPA 等位基因的方案来改进 NIPT。
使用 Ion AmpliSeq HD 技术设计具有临床重要性的抗原的 NIPT。
从 36 名孕妇（9-33 孕周，24 名孕妇因抗 HPA-1a、-3b、-15a、-K 或-D+C+S 而免疫）的血浆 DNA 中，使用 Ion S5 系统和 Ion AmpliSeq HD 设计器定制基因面板对 Ion S5 系统进行测试，该系统包含 25 个编码相关 BG/HPA 抗原的 rs 靶标和 10 个标记物。
使用 NGS 方案，在 2%以上的胎儿父系嵌合率的浓度下，确定了 85 个胎儿/母体 BG/HPA 基因型差异中的 76 个。BG/HPA 等位基因的非特异性读数水平低于 0.87%。在 24 名免疫孕妇中，NGS 在 11 例（2.44%-7.41%）中发现胎儿-母体不相容，在 10 例（<0.05%）中排除，3 例结果不确定（1.79%、0.19%、0.11%）。通过检测至少 2%嵌合率的标记物，在每个病例中均确认存在胎儿 DNA。
Ion AmpliSeq HD 技术的使用提高了胎儿-母体不相容的预测能力，提高了 BG/HPA NIPT 的灵敏度，并在同一工作流程中确认胎儿 DNA 的存在。
