孕妇血样采集后体外存放对血浆中胎儿游离DNA水平的影响

Influence of delay in blood-processing on the level of cell-free fetal DNA in maternal plasma sample

目的:探讨孕妇血样采集后体外存放过程中血浆游离DNA水平的影响因素。方法:采集孕妇血样,体外4℃存放6、36h后,实时定量PCR检测孕妇外周游离DNA水平,Annexin Ⅴ/PI双染色法流式分析血样白细胞死亡情况,速率法检测血浆乳酸脱氢酶(LDH)活性,单相酶扩散法测定血浆DNA酶活性。结果:血样体外存放36h后,血浆中β-globin基因的拷贝数较存放6h的样品显著增加,而SRY基因拷贝数明显减少;4℃存放条件下,妊娠晚期孕妇外周血中胎儿DNA占总DNA百分比从6h的(10.3±5.6)%下降到36h的(3.0±2.1)%,差异有统计学意义(P<0.05)。体外存放6h的血样中基本未检出死亡细胞,而存放36h的血样中分别检出了坏死和凋亡的白细胞;36h样品血浆中LDH活性较6h样品显著升高(P<0.05)。单相酶扩散法结果表明,血浆DNA酶在4℃时虽然活性显著减弱,但仍然保持降解DNA的能力。结论:血样采集后体外存放时间过长会导致血浆总游离DNA增多,而胎儿DNA减少,这种变化可能与血样体外存放过程中白细胞死亡和血浆DNA酶对胎儿DNA的降解作用有关;胎儿DNA抽提应在血样采集后尽早(6h内)进行。

Objective：To investigate the influencing factors of cell-free fetal DNA level in the maternal plasma during bloodprocessing. Methods： Aliquots of blood samples from pregnant women with male fetus were processed at 6 h and 36 h after sampling. The SRY and β-globin genes and the total DNA level were quantified by real time quantitative PCR. Death of white blood cells was assayed by flow cytometry after stained with Annexin V/PI. The plasma DNase activity was assayed by radial enzyme-diffusion method and plasma lactate dehydrogenases （LDH） by rate method. Results： A 36 hour delay in bloodprocessing led to a significant increase in the total DNA and decrease in the fetal DNA （SRY gene） in the maternal plasma. The ratio of fetal DNA decreased from （10.3±5.6） % at 6 h after sampling to （3.0±2.1） % at 36 h after sampling under 4℃ （P〈 0.05）. No dead cells were identified in the blood sample 6 h after sampling; however,apoptosis and necrosis of white blood cells were identified 36 h after sampling. The activity of LDH at 36 h was significantly higher than that at 6 h （P〈0.05）. Radial enzyme-diffusion result showed that, though greatly decreased at 4℃, the DNase was still able to degrade DNA. Conclusion： Delay in blood-processing can lead to increase of the total free DNA in maternal plasma but decrease of fetal DNA,which might be related to the death of white blood cells and degradation of fetal DNA by plasma DNase, so the extraction of fetal DNA should be done as early as possible after sampling （within 6 h）.