Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma
Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma
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DOI：
10.1046/j.1537-2995.2001.41020276.x
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发表时间：
2001-02-01
期刊：
影响因子：
2.9
通讯作者：
Busch, MP
中科院分区：
文献类型：
--
作者：
Lee, TH;Montalvo, L;Busch, MP
背景：血浆和血清样本已被用于检测某些病理条件下血清或血浆中的无细胞基因组DNA，如系统性红斑狼疮、肺栓塞和恶性肿瘤，以及母体血清和/或血浆中的胎儿细胞嵌合。在这项研究中，血清和血浆样本中无细胞DNA的基线浓度被评估用于研究输血后嵌合。研究设计和方法：使用HIV检测试剂盒中的试剂从新鲜或储存（4度1-6天）正常供体血清或血浆样本（ACD； EDTA）中提取DNA。样品孵育和洗涤后，用HLA DQ-a引物（GH26和27）或人y染色体引物（SA和SD）扩增纯化DNA，定量测定基因组DNA浓度。结果：新鲜血清样品的游离DNA浓度比新鲜血浆样品高约20倍。血清样品中无细胞基因组DNA的浓度每天都在增加，凝血管在4℃下保存4 - 5天后，其浓度达到基线的100多倍。在储存的ACD全血样本中，无细胞血浆DNA略有增加。将男性白细胞加入新鲜的非抗凝女性血液中，在凝血过程中进行裂解，男性DNA释放到血清样本中。结论：血清样品中游离DNA大部分是在原采集管凝血过程中产生的。新鲜血浆中游离细胞基因组DNA的浓度可能与血液循环中的浓度相同。因此，虽然血清样本不应用于监测患者循环中无细胞DNA的浓度，但在静脉切断术后3至5天，从含有血块（即不含抗凝剂）的样本管中收集的血清可作为筛查输血后微嵌合的DNA来源。
BACKGROUND: Plasma and serum samples have been used to detect cell-free genomic DNA in serum or plasma in certain pathologic conditions such as systemic lupus erythematosus, pulmonary embolism, and malignancies, as well as in fetal cell chimerisms in maternal serum and/or plasma. In this study, baseline concentrations of cell-free DNA in serum and plasma samples were evaluated for the study of posttransfusion chimerism.STUDY DESIGN AND METHODS: DNA was extracted from fresh or stored (4 degreesC for 1-6 days) normal donor serum or plasma samples (ACD; EDTA) by using reagents from an HIV assay kit. After incubation and washing of samples, purified DNA was amplified with HLA DQ-a primers (GH26 and 27) or human Y-chromosome primers (SA and SD) to quantitate the concentration of genomic DNA.RESULTS: Fresh serum samples had concentrations of cell-free DNA that were about 20-fold higher than the concentrations in fresh plasma samples. The concentration of cell-free genomic DNA in serum samples increased daily, to a level more than 100 times baseline after clotted blood tubes were stored at 4 degreesC for 4 to 5 days. There was a small increase in cell-free plasma DNA in stored ACD whole blood samples. Male WBCs, spiked into fresh nonanticoagulated female blood, were lysed during the process of clotting, with male DNA liberated into the serum samples.CONCLUSION: Most cell-free DNA in serum samples is generated during the process of clotting in the original collection tube. The concentration of cell-free genomic DNA in fresh plasma is probably the same as that in circulation. Consequently, while serum samples should not be used to monitor the concentration of cell-free DNA in a patient's circulation, serum collected from sample tubes containing clots (i.e., without anticoagulant), 3 to 5 days after the date of phlebotomy, could be useful as a source of DNA with which to screen for posttransfusion microchimerism.