Quantitative analysis of circulating mitochondrial DNA in plasma

BACKGROUND: Recent studies have demonstrated the existence of circulating mitochondrial DNA in plasma and serum, but the concentrations and physical characteristics of circulating mitochondrial DNA are unknown. The aim of this study was to develop an assay to quantify mitochondrial DNA in the plasma of healthy individuals. METHODS: We adopted a real-time quantitative PCR approach and evaluated the specificity of the assay for detecting mitochondrial DNA with a cell line (rho(0)) devoid of mitochondria. The concentrations and physical characteristics of circulating mitochondrial DNA were investigated by experiments conducted in three modules. In module 1, we evaluated the concentrations of mitochondrial DNA in plasma aliquots derived from four blood-processing protocols. In module 2, we investigated the existence of both particle-associated and free forms of mitochondrial DNA in plasma by subjecting plasma to filtration and ultracentrifugation. In module 3, we used filters with different pore sizes to investigate the size characteristics of the particle-associated fraction of circulating mitochondrial DNA. RESULTS: The mitochondrial DNA-specific, real-time quantitative PCR had a dynamic range of five orders of magnitude and a sensitivity that enabled detection of one copy of mitochondrial DNA in plasma. In module 1, we found significant differences in the amounts of circulating mitochondrial DNA among plasma aliquots processed by different methods. Data from module 2 revealed that a significant fraction of mitochondrial DNA in plasma was filterable or pelletable by ultracentrifugation. Module 3 demonstrated that filters with different pore sizes removed mitochondrial DNA from plasma to different degrees. CONCLUSIONS: Both particle-associated and free mitochondrial DNA are present in plasma, and their respective concentrations are affected by the process used to harvest plasma from whole blood. These results may have implications in the design of future studies on circulating mitochondrial DNA measured in different disease conditions.     

胎儿细胞及游离DNA穿越胎盘的实验研究

目的 从胎盘组织中寻找胎儿细胞及其游离DNA穿越胎盘屏障的实验室证据,为利用孕妇外周血进行无创性产前基因诊断提供实验依据。方法 分娩胎盘组织22份(男婴的12份,女婴的10份),切片,平行分为2组。一组HE染色,光镜观察绒毛中胎儿细胞的分布;另一组用Dig-碱性磷酸酶标记男性性别特异性标记物SRY(sex determining region)基因扩增片断制成探针,通过原位杂交鉴定胎盘绒毛内特别是绒毛间隙是否存在相应的游离DNA。阳性对照由男性外周血有核细胞涂片经原位杂交提供。结果 HE染色光镜观察,22例胎盘组织切片可见胎儿细胞有穿越绒毛毛细血管内皮和滋养层基膜的现象;胎盘组织原位杂交结果显示：分娩男婴的12份胎盘切片的绒毛毛细血管腔内、绒毛滋养层基膜边缘、以及绒毛间隙,可见阳性信号;分娩女婴的10份胎盘切片均无阳性信号出现;阳性对照涂片杂交后可见清晰阳性信号。结论 胎盘组织切片HE染色、镜检观察胎儿细胞分布,结合原位杂交技术鉴定绒毛内、绒毛问隙游离核酸的性质,可以初步反映胎儿微量遗传物质穿越胎盘进入母亲血液循环的过程。     

荧光定量PCR检测Rh阴性孕妇外周血浆游离DNA中胎儿RhD血型

目的 探讨荧光定量PCR(fluorescence quantitative polymerase chain reaction,FQ-PCR)技术对Rh阴性孕妇血浆中游离胎儿DNA进行非创性产前诊断胎儿RhD血型的可行性.方法 选取78份妊娠11～40周、B超确诊为单胎的Rh阴性孕妇血浆.采用9个短串联重复序列(short tandem repeat,STR)多态性位点及Y染色体性别决定区基因(sex-determining region Y chromosome,SRY)确定胎儿DNA的存在;运用FQ-PCR技术对血浆中游离胎儿DNA进行RHD基因外显子5、7、10和内含子4定量分析,以确定胎儿RhD血型的基因型;其基因型结果与产后新生儿脐血血清学检测结果进行对比分析,回顾性评价胎儿基因定型结果的准确性.结果 78份标本中,41份检测到SRY基因,平均浓度为(214.7±120.9)拷贝/ml,产后证实皆为男性.70份FQ-PCR基因定型结果与血清学结果相符,另有5份确定为假阳性,3份基因定型结果不可确定,检测结果总符合率为90%(70/78).5份假阳性标本通过检测RHD1227A等位基因鉴定了4份RhD放散型,FQ-PCR最终结果准确率达到95%(74/78).结论应用FQ-PCR方法进行非创性胎儿RhD血型检测可用于新生儿溶血病的预防和诊断.     

Quantitative analysis of circulating plasma DNA as a tumor marker in thoracic malignancies

BACKGROUND: Increased plasma DNA has been found in cancer patients and may have potential as a tumor marker. The objectives of this study were to develop a controlled, quantitative PCR (QPCR) assay to measure plasma DNA and then evaluate plasma DNA concentrations as a tumor marker in patients with thoracic malignancies. METHODS: We developed a QPCR assay for DNA, using the human beta-actin gene. Plasma samples were analyzed from 58 patients with esophageal cancer (EC; 20 banked samples and 38 prospectively collected samples) and 25 patients with lung cancer (LC; all prospectively collected). Control groups consisting of 51 patients with gastroesophageal reflux disease (GERD; 23 banked samples and 28 prospectively collected) and 11 healthy volunteers were also analyzed. RESULTS: The assay had an experimental variability <4%. In our banked samples, the mean concentration of plasma DNA in EC was 819.0 microg/L (range, 46.2-4738.0 microg/L) vs 432.0 microg/L (6.0-2888.0 microg/L) in GERD (P = 0.02). However, the prospectively collected samples had lower DNA concentrations, and there was no difference between cancer patients and controls. The mean DNA concentration was 10.6 microg/L (range, 7.0-14.0 microg/L) in healthy volunteers and 10.5 microg/L (range, 4.0-23.5 microg/L) in GERD controls vs 13.0 microg/L (range, 4.5-46.5 microg/L) in EC and 14.6 microg/L (range, 3.0-30.0 microg/L) in LC. CONCLUSIONS: Our data indicate that plasma DNA concentrations are of limited diagnostic value when samples are prospectively collected and uniformly handled. This is in contrast to previously published results. Qualitative analysis of DNA may be needed if plasma nucleic acids are to be used as a diagnostic tool in cancer screening.     

Modified salting-out method for DNA isolation from newborn cord blood nucleated cells

The present work describes modification of a widely used salting-out procedure to rapidly extract DNA suitable for PCR, using the ARMS method to amplify a target sequence in the beta-globin gene. The salting-out DNA extraction procedure did not completely remove or decrease the presence of inhibitors to PCR in a considerable number of cord blood samples. By introducing a simple phenol/chloroform step, before ethanol precipitation of the nucleic acid, to certain samples, we were able to eliminate or substantially reduce the presence of inhibitors to PCR without having to re-extract the samples.     

孕DOWN综合征胎儿妇女血浆游离胎儿DNA浓度定量研究

Objective To investigate the application of real-time quantitative PCR in quantification of cell-free fetal DNA maternal plasma in patients bearing fetuses affected with DOWN syndrome. Methods Cell-free fetal DNA in maternal serum was isolated from 30 samples(7 male DOWN syndrome fetal ,3 female DOWN syndrome fe-tal,14 male euploid fetal,6 female euploid fetal). Cell-free fetal DNA levels in maternal serum were measured using real-time quantitative PCR using SRY as marker. Results The median cell-free fetal DNA levels in pregnant carry-ing male fetuses(n=7) and the controls (pregnant carrying male euploid fetuses,n=14)were 318.03±96.74 ge-nome-equivalents/ml and 154.40±39.43 genome-equivalents/ml of maternal serum,respectively (t=3.33,P=0.004 ),which was o in women with female fetuses. Conclusion The cell-free fetal DNA levels in pregnant women with DOWN syndrome fetuses are higher than that in pregnant women with normal fetuses.     

孕DOWN综合征胎儿妇女血浆游离胎儿DNA浓度定量研究

Objective To investigate the application of real-time quantitative PCR in quantification of cell-free fetal DNA maternal plasma in patients bearing fetuses affected with DOWN syndrome. Methods Cell-free fetal DNA in maternal serum was isolated from 30 samples(7 male DOWN syndrome fetal ,3 female DOWN syndrome fe-tal,14 male euploid fetal,6 female euploid fetal). Cell-free fetal DNA levels in maternal serum were measured using real-time quantitative PCR using SRY as marker. Results The median cell-free fetal DNA levels in pregnant carry-ing male fetuses(n=7) and the controls (pregnant carrying male euploid fetuses,n=14)were 318.03±96.74 ge-nome-equivalents/ml and 154.40±39.43 genome-equivalents/ml of maternal serum,respectively (t=3.33,P=0.004 ),which was o in women with female fetuses. Conclusion The cell-free fetal DNA levels in pregnant women with DOWN syndrome fetuses are higher than that in pregnant women with normal fetuses.     

孕DOWN综合征胎儿妇女血浆游离胎儿DNA浓度定量研究

Objective To investigate the application of real-time quantitative PCR in quantification of cell-free fetal DNA maternal plasma in patients bearing fetuses affected with DOWN syndrome. Methods Cell-free fetal DNA in maternal serum was isolated from 30 samples(7 male DOWN syndrome fetal ,3 female DOWN syndrome fe-tal,14 male euploid fetal,6 female euploid fetal). Cell-free fetal DNA levels in maternal serum were measured using real-time quantitative PCR using SRY as marker. Results The median cell-free fetal DNA levels in pregnant carry-ing male fetuses(n=7) and the controls (pregnant carrying male euploid fetuses,n=14)were 318.03±96.74 ge-nome-equivalents/ml and 154.40±39.43 genome-equivalents/ml of maternal serum,respectively (t=3.33,P=0.004 ),which was o in women with female fetuses. Conclusion The cell-free fetal DNA levels in pregnant women with DOWN syndrome fetuses are higher than that in pregnant women with normal fetuses.     

孕DOWN综合征胎儿妇女血浆游离胎儿DNA浓度定量研究

Objective To investigate the application of real-time quantitative PCR in quantification of cell-free fetal DNA maternal plasma in patients bearing fetuses affected with DOWN syndrome. Methods Cell-free fetal DNA in maternal serum was isolated from 30 samples(7 male DOWN syndrome fetal ,3 female DOWN syndrome fe-tal,14 male euploid fetal,6 female euploid fetal). Cell-free fetal DNA levels in maternal serum were measured using real-time quantitative PCR using SRY as marker. Results The median cell-free fetal DNA levels in pregnant carry-ing male fetuses(n=7) and the controls (pregnant carrying male euploid fetuses,n=14)were 318.03±96.74 ge-nome-equivalents/ml and 154.40±39.43 genome-equivalents/ml of maternal serum,respectively (t=3.33,P=0.004 ),which was o in women with female fetuses. Conclusion The cell-free fetal DNA levels in pregnant women with DOWN syndrome fetuses are higher than that in pregnant women with normal fetuses.     

孕DOWN综合征胎儿妇女血浆游离胎儿DNA浓度定量研究

Objective To investigate the application of real-time quantitative PCR in quantification of cell-free fetal DNA maternal plasma in patients bearing fetuses affected with DOWN syndrome. Methods Cell-free fetal DNA in maternal serum was isolated from 30 samples(7 male DOWN syndrome fetal ,3 female DOWN syndrome fe-tal,14 male euploid fetal,6 female euploid fetal). Cell-free fetal DNA levels in maternal serum were measured using real-time quantitative PCR using SRY as marker. Results The median cell-free fetal DNA levels in pregnant carry-ing male fetuses(n=7) and the controls (pregnant carrying male euploid fetuses,n=14)were 318.03±96.74 ge-nome-equivalents/ml and 154.40±39.43 genome-equivalents/ml of maternal serum,respectively (t=3.33,P=0.004 ),which was o in women with female fetuses. Conclusion The cell-free fetal DNA levels in pregnant women with DOWN syndrome fetuses are higher than that in pregnant women with normal fetuses.     

孕DOWN综合征胎儿妇女血浆游离胎儿DNA浓度定量研究

Objective To investigate the application of real-time quantitative PCR in quantification of cell-free fetal DNA maternal plasma in patients bearing fetuses affected with DOWN syndrome. Methods Cell-free fetal DNA in maternal serum was isolated from 30 samples(7 male DOWN syndrome fetal ,3 female DOWN syndrome fe-tal,14 male euploid fetal,6 female euploid fetal). Cell-free fetal DNA levels in maternal serum were measured using real-time quantitative PCR using SRY as marker. Results The median cell-free fetal DNA levels in pregnant carry-ing male fetuses(n=7) and the controls (pregnant carrying male euploid fetuses,n=14)were 318.03±96.74 ge-nome-equivalents/ml and 154.40±39.43 genome-equivalents/ml of maternal serum,respectively (t=3.33,P=0.004 ),which was o in women with female fetuses. Conclusion The cell-free fetal DNA levels in pregnant women with DOWN syndrome fetuses are higher than that in pregnant women with normal fetuses.     

孕DOWN综合征胎儿妇女血浆游离胎儿DNA浓度定量研究

Objective To investigate the application of real-time quantitative PCR in quantification of cell-free fetal DNA maternal plasma in patients bearing fetuses affected with DOWN syndrome. Methods Cell-free fetal DNA in maternal serum was isolated from 30 samples(7 male DOWN syndrome fetal ,3 female DOWN syndrome fe-tal,14 male euploid fetal,6 female euploid fetal). Cell-free fetal DNA levels in maternal serum were measured using real-time quantitative PCR using SRY as marker. Results The median cell-free fetal DNA levels in pregnant carry-ing male fetuses(n=7) and the controls (pregnant carrying male euploid fetuses,n=14)were 318.03±96.74 ge-nome-equivalents/ml and 154.40±39.43 genome-equivalents/ml of maternal serum,respectively (t=3.33,P=0.004 ),which was o in women with female fetuses. Conclusion The cell-free fetal DNA levels in pregnant women with DOWN syndrome fetuses are higher than that in pregnant women with normal fetuses.     

孕DOWN综合征胎儿妇女血浆游离胎儿DNA浓度定量研究

Objective To investigate the application of real-time quantitative PCR in quantification of cell-free fetal DNA maternal plasma in patients bearing fetuses affected with DOWN syndrome. Methods Cell-free fetal DNA in maternal serum was isolated from 30 samples(7 male DOWN syndrome fetal ,3 female DOWN syndrome fe-tal,14 male euploid fetal,6 female euploid fetal). Cell-free fetal DNA levels in maternal serum were measured using real-time quantitative PCR using SRY as marker. Results The median cell-free fetal DNA levels in pregnant carry-ing male fetuses(n=7) and the controls (pregnant carrying male euploid fetuses,n=14)were 318.03±96.74 ge-nome-equivalents/ml and 154.40±39.43 genome-equivalents/ml of maternal serum,respectively (t=3.33,P=0.004 ),which was o in women with female fetuses. Conclusion The cell-free fetal DNA levels in pregnant women with DOWN syndrome fetuses are higher than that in pregnant women with normal fetuses.     

孕DOWN综合征胎儿妇女血浆游离胎儿DNA浓度定量研究

Objective To investigate the application of real-time quantitative PCR in quantification of cell-free fetal DNA maternal plasma in patients bearing fetuses affected with DOWN syndrome. Methods Cell-free fetal DNA in maternal serum was isolated from 30 samples(7 male DOWN syndrome fetal ,3 female DOWN syndrome fe-tal,14 male euploid fetal,6 female euploid fetal). Cell-free fetal DNA levels in maternal serum were measured using real-time quantitative PCR using SRY as marker. Results The median cell-free fetal DNA levels in pregnant carry-ing male fetuses(n=7) and the controls (pregnant carrying male euploid fetuses,n=14)were 318.03±96.74 ge-nome-equivalents/ml and 154.40±39.43 genome-equivalents/ml of maternal serum,respectively (t=3.33,P=0.004 ),which was o in women with female fetuses. Conclusion The cell-free fetal DNA levels in pregnant women with DOWN syndrome fetuses are higher than that in pregnant women with normal fetuses.     

孕DOWN综合征胎儿妇女血浆游离胎儿DNA浓度定量研究

Objective To investigate the application of real-time quantitative PCR in quantification of cell-free fetal DNA maternal plasma in patients bearing fetuses affected with DOWN syndrome. Methods Cell-free fetal DNA in maternal serum was isolated from 30 samples(7 male DOWN syndrome fetal ,3 female DOWN syndrome fe-tal,14 male euploid fetal,6 female euploid fetal). Cell-free fetal DNA levels in maternal serum were measured using real-time quantitative PCR using SRY as marker. Results The median cell-free fetal DNA levels in pregnant carry-ing male fetuses(n=7) and the controls (pregnant carrying male euploid fetuses,n=14)were 318.03±96.74 ge-nome-equivalents/ml and 154.40±39.43 genome-equivalents/ml of maternal serum,respectively (t=3.33,P=0.004 ),which was o in women with female fetuses. Conclusion The cell-free fetal DNA levels in pregnant women with DOWN syndrome fetuses are higher than that in pregnant women with normal fetuses.     

孕DOWN综合征胎儿妇女血浆游离胎儿DNA浓度定量研究

目的 探讨实时定量PCR方法检测妊娠DOWN综合征胎儿妇女血浆游离胎儿DNA的可行性.方法 SRY基因为胎儿游离DNA的标志,应用实时定量PCR分别检测10例妊娠DOWN综合征胎儿(男胎7例,女胎3例)妇女与20例妊娠正常胎儿(男胎14例,女胎6例)妇女血浆标本中游离胎儿DNA含量.结果 7例妊娠DOWN综合征男胎妇女SRY基因当量为(318.03±96.74)拷贝/ml(95%可信区间228.26～407.50拷贝/ml),妊娠正常男胎组SRY基因当量为(154.40±39.43)拷贝/ml(95%可信区间131.63～177.16拷贝/ml);2组差异有统计学意义(t=3.33,P=0.004).妊娠女胎组妇女血浆中SRY基因当量均为0拷贝/ml.结论 妊娠DOWN综合征男胎儿妇女血浆游离胎儿DNA的量较妊娠正常男胎儿妇女高.     

Quantitative real-time detection of parvovirus B19 DNA in plasma

BACKGROUND: As of 2004, the European Pharmacopoeia demands that plasma pools for production of anti-D immunoglobulin should not contain more than 104 IU per mL of parvovirus B19 (B19V) DNA. Hence, before pooling, highly viremic donations have to be identified, and after pooling the level of B19V DNA must be determined. The performance of a new real-time B19V DNA PCR test (Roche, Mannheim, Germany) was studied, using a DNA extractor (NucliSens, bioMerieux, Boxtel, the Netherlands) for isolation of nucleic acid, and using a DNA quantification test (LightCycler apparatus, Roche, Mannheim, Germany) for amplification and detection. STUDY DESIGN AND METHODS: Dilutions of the international B19V DNA standard and reference preparations were tested to determine the precision, linear range, and accuracy of the assay and to calculate the factor for conversion of B19V DNA copies to IUs. The internal control signals, invalid test results, and the effect of cryo-poor plasma were studied as a measure for robustness. Routine performance was assessed by testing 164 manufacturing pools (not screened for B19V) and 1048 test pools of 480 donations each. RESULTS: The copies-to-IU conversion factor was calculated to be 3.34 (95% CI, 3.07-3.63). The assay appears linear between 10(3) and 10(7) IU per mL. Between 10(3) and 10(5) IU per mL, the test can discriminate samples differing a factor two in B19V DNA content. Overall, 0.78 percent of the test results were invalid. Of 127 B19V DNA negative control plasma samples, 7 were contaminated with low levels of B19V DNA. Of 164 nonscreened manufacturing plasma pools, 92 contained B19V DNA (56%); 13 contained more than 10(4) IU per mL. Of 503,040 donations, 29 contained more than 5 x 10(6) IU per mL B19V DNA (1:17,346). CONCLUSION: The B19V DNA quantification test (LightCycler, Roche ) is suitable for quantitative, routine, in-process measurement of B19V DNA levels in plasma pools, using the DNA extractor (NucliSens, bioMerieux) for nucleic acid isolation.     

Pharmacokinetic modeling of bidirectional transfer during peritoneal dialysis

A pharmacokinetic model and distributional clearance terms to describe bidirectional peritoneal transfer were used to examine cefamandole pharmacokinetics in five uninfected patients with end-stage renal disease who were receiving continuous ambulatory peritoneal dialysis. Each patient received intravenous and intraperitoneal 1 gm doses of drug, and serum and dialysate samples were collected over three dialysis dwell periods. The mean systemic availability of cefamandole after intraperitoneal dosing was 0.71 +/- 0.1. No significant differences in the serum-to-peritoneal fluid and peritoneal fluid-to-serum distributional clearances were observed. The time dependence of peritoneal dialysis clearance was examined. The amount of drug found in the dialysate divided by the corresponding serum AUC was empirically found to estimate the time-averaged peritoneal dialysis clearance. A mass balance-area method to calculate distributional clearance was developed that obviates more complicated computer fitting of the data. We present a comprehensive modeling approach that should be useful in the examination of the kinetics of drugs during continuous ambulatory peritoneal dialysis.