Yu Yang, Fen Qian, Lin Zi-Lin, Pan Ji-Chun, Zhang Ting, Ma Chun-Ya, Zhang Xiao-Juan, Ge Guo-Feng, Chen Xin, Guan Xiao-Zhen, Ren Le, Sun Dan, Fu Li-Hui, Luo Qun, Wang De-Qing
Department of Blood Transfusion, Center for Clinical Transfusion Medicine, PLA General Hospital, Beijing 100853, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2009 Oct;17(5):1363-7.
This study was aimed to establish a genotyping method to detect ABO group gene of fetus from peripheral blood of pregnant women for prenatal diagnosis of hemolytic disease of newborn (HDN) resulting from ABO blood group incompatibility. 4 pairs of primers were designed according to ABO blood group gene DNA and mRNA sequences. 20 plasma DNA samples from healthy donors were extracted and amplified to explore the best conditions for plasma DNA extraction and PCR amplification. The O group plasma DNA was mixed with A group or B group plasmas by the ratios of 1:1, 2:1, 4:1, 8:1, 10:1, 20:1, 40:1, 100:1 to simulate the status of mixed ABO gene from pregnant maternal blood and to establish the mixed blood group ABO genotyping technology. The pregnant maternal blood samples with more than 30 weeks of gestation were selected for detecting the fetal ABO blood group genotype. The blood samples should be taken as possible as after birth for identification of ABO blood group and evaluation of sensitivity and accuracy of fetal ABO blood group genotyping technology through peripheral blood of pregnant women. The results indicated that the minimal amount of template DNA from single blood plasma for accuracy identification was at least about 0.625 ng, the DNA amount extracted from 500 microl of plasma could meet the requirement for PCR amplification. When the proportion of O group plasma DNA in mixed plasma DNA was <or=10, the non-O group gene could be accurately detected. Among 14 peripheral blood samples from O-group pregnant women, the non-O group gene was amplified in 9 samples; the non-O group gene was not amplified in 5 samples. The identification of peripheral blood ABO group for 5 newborns using serologic method showed that the A group 3 cases, B group 2 cases, O group 1 case, which consisted with genotyping results with consistent rate 100%. It is concluded that in middle and late pregnancy the fetal ABO group gene can be detected accurately by means of established fetal ABO group gene extraction and typing technology, that provides some guidances for the prenatal diagnosis and prevention of HDN.
本研究旨在建立一种从孕妇外周血中检测胎儿ABO血型基因的基因分型方法，用于产前诊断ABO血型不合所致新生儿溶血病（HDN）。根据ABO血型基因的DNA和mRNA序列设计了4对引物。提取并扩增20份健康供者的血浆DNA样本，以探索血浆DNA提取和PCR扩增的最佳条件。将O型血浆DNA与A型或B型血浆按1:1、2:1、4:1、8:1、10:1、20:1、40:1、100:1的比例混合，模拟孕妇血液中混合ABO基因的状态，建立混合血型ABO基因分型技术。选取妊娠30周以上的孕妇血样检测胎儿ABO血型基因型。出生后应尽快采集血样进行ABO血型鉴定，并通过孕妇外周血评估胎儿ABO血型基因分型技术的敏感性和准确性。结果表明，单次血浆模板DNA准确鉴定所需的最小量至少约为0.625 ng，从500 μl血浆中提取的DNA量可满足PCR扩增要求。当混合血浆DNA中O型血浆DNA的比例≤10时，可准确检测非O型基因。在14份O型孕妇外周血样本中，9份样本扩增出非O型基因；5份样本未扩增出非O型基因。采用血清学方法对5例新生儿外周血ABO血型进行鉴定，结果显示A型3例，B型2例，O型1例，与基因分型结果一致，符合率为100%。结论：在妊娠中晚期，通过建立的胎儿ABO血型基因提取和分型技术可准确检测胎儿ABO血型基因，为HDN的产前诊断和预防提供了一定指导。
