Ahmadi Mohammad Hossein, Pourfathollah Ali Akbar, Rabiee Maryam, Amirizadeh Naser
Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad, Iran.
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
J Reprod Infertil. 2022 Apr-Jun;23(2):128-134. doi: 10.18502/jri.v23i2.8998.
The main cause of hemolytic disease of the fetus and newborn (HDFN) is the incompatibility of the RHD antigen between mother and fetus. Following the discovery of cell-free fetal DNA (cffDNA), noninvasive fetal RHD genotyping also became possible, which will help in the better management of immunized RHD negative mothers and in the targeted prenatal injection of Rho(D) immune globulin (RhIG). The objective of this study was to establish a reliable method with high accuracy to determine the fetal RHD genotype.
The project was a prospective observational cohort study. After cell-free DNA (cfDNA) extraction from maternal plasma, fetal RHD genotyping was performed by duplex real-time polymerase chain reaction (PCR) and exons 5, 7, and 10 of the RHD gene were examined. SRY and RASSF1A genes were used as internal controls to confirm the presence of cffDNA in maternal plasma.
Out of 40 samples, 33 were RhD positive heterozygous mothers and 7 cases were RHD negative. In three cases where both the fetal RHD and SRY genotypes were negative, RASSF1A was amplified in cell-free DNA sample treated with the BstUI enzyme, and the presence of cffDNA was confirmed.
The findings reveal that the strategy used in this study is reliable and it is possible to determine the fetal RHD status with high accuracy. The strategy can help targeted injection of RhIG and prevent unnecessary injection in RhD negative mothers who carry an RhD negative fetus.
胎儿和新生儿溶血病（HDFN）的主要原因是母亲与胎儿之间RHD抗原不相容。随着游离胎儿DNA（cffDNA）的发现，无创胎儿RHD基因分型也成为可能，这将有助于更好地管理已免疫的RHD阴性母亲，并有助于有针对性地进行产前注射Rho（D）免疫球蛋白（RhIG）。本研究的目的是建立一种可靠且准确性高的方法来确定胎儿RHD基因型。
该项目是一项前瞻性观察队列研究。从母体血浆中提取游离DNA（cfDNA）后，通过双重实时聚合酶链反应（PCR）进行胎儿RHD基因分型，并检测RHD基因的外显子5、7和10。使用SRY和RASSF1A基因作为内部对照，以确认母体血浆中cffDNA的存在。
在40个样本中，33例为RhD阳性杂合子母亲，7例为RHD阴性。在3例胎儿RHD和SRY基因型均为阴性的情况下，在用BstUI酶处理的游离DNA样本中扩增出RASSF1A，证实了cffDNA的存在。
研究结果表明，本研究中使用的策略是可靠的，并且有可能高精度地确定胎儿的RHD状态。该策略有助于有针对性地注射RhIG，并防止对怀有RHD阴性胎儿的RHD阴性母亲进行不必要的注射。
