Free Plasma DNA Research Articles

Can plasma-free DNA concentration be a diagnostic tool in critically ill septic patients?

Recent evidence suggests that the plasma-free DNA concentration has potential use as a prognostic marker in many clinical situations including sepsis, trauma, and acute stroke [1]. However, its predictive value is arguable. We hypothesized that plasma DNA is increased in septic patients admitted to the ICU compared with nonseptic ICU patients, and it is correlated with disease severity and clinical outcome. Forty-two consecutive patients (11 septic, 31 nonseptic) admitted to a mixed ICU and mechanically ventilated were recruited. Plasma-free DNA concentration was measured by real-time PCR assay for the β-globin gene, and the APACHE II score, SOFA score, serum C-reactive protein (CRP) concentrations, procalcitonin (PCT) concentrations, serum lipid concentrations, and clinical outcome (ICU/hospital days and mortality) were assessed on admission to the ICU. Assessments and samplings were repeated as the diagnosis of the patients changed (sepsis, severe sepsis and septic shock). Finally, 86 plasma samples were collected. Descriptive statistics, Mann–Whitney U, Kruskall–Wallis and Spearman's tests, and receiver operating characteristic analysis were used when appropriate. Demographic data were similar. ICU and hospital mortalities were 26.2% and 33.3%, respectively. The mean DNA concentrations on admission were significantly higher in ICU patients compared with healthy subjects (n = 11) (13,405 GE/ml versus 390 GE/ml, P < 0.05) and septic patients compared with nonseptic patients (33,170 GE/ml versus 1,171 GE/ml, P < 0.001). Furthermore, during the overall ICU stay, increased DNA concentration associated with the increase of severity of illness was noted; however, this increase was statistically significant only between septic and septic shock samples (26,624 GE/ml versus 42,861 GE/ml, P < 0.05). The area under the curve obtained for the plasma-free DNA concentration in distinguishing between septic and nonseptic patients on admission was 0.9 (sensitivity 84%, specificity, 95%; cutoff 4,083 GE/ml). Also, the plasma-free DNA concentration was found to be higher in patients who died in the ICU compared with patients who survived, although not statistically significant. The DNA concentration demonstrated a significant correlation with CRP (P = 0.037, r = 0.365), PCT (P = 0.007, r = 0.457) and high-density lipoprotein (P = 0.015, r = -0.415) concentrations. In conclusion, plasma DNA may be a potentially valuable tool to confirm the diagnosis of sepsis on admission to the ICU and to monitor disease severity.

Monocyte Chemoattractant Protein-1 and Interleukin-8 Levels in Acute Inflammation Induced by Prolonged Brisk Exercise.

Monocyte chemoattractant protein-1 (MCP-1), also known as CCL2, a chemokine that regulates migration and infiltration by monocytes/macrophages, belongs to the CC chemokine subfamily. Interleukin-8 (IL-8), also known as CXCL8, a proinflammatory chemokine with angiogenesis-promoting properties belongs to the CXC chemokine superfamily. Both MCP-1 and IL-8 have important roles in the pathogenesis of many chronic inflammatory disorders, including atherosclerosis and obesity. Their proinflammatory effects are mediated mainly by the CC chemokine receptor 2 (CCR2) and CXCR1/R2, respectively. MCP-1 and IL-8 cause chronic vascular inflammation and induce thrombosis, proliferation and migration of vascular smooth muscle cells, angiogenesis, and oxidative stress. Previous studies indicate that:MCP-1 production from endothelial cells, smooth muscle cells, and regional leukocytes increases in the presence of endothelial dysfunction and atherosclerosis risk factors;MCP-1 and IL-8 expression is increased in atherosclerotic lesions and injured arteries; andeliminating MCP-1 function decreases neointimal hyperplasia after injury and atheroma formation in mice.We studied the association between MCP-1 and IL-8 levels and the degree of inflammation in 15 athletes that participated in the ultra-distance foot race of the 246 Km “Sparthathlon”. This race consists of continuous, prolonged, brisk exercise. We reported earlier that Interleukin-6 (IL-6), C-reactive protein (CRP), Serum amyloid A protein (SAA) and free plasma DNA levels markedly increased (by 8000–, 152– 108– and 10-fold, respectively) over the baseline at the end of the race1. However, IL-6 levels returned to normal by 48h, while CRP, SAA and free plasma DNA remained elevated. Circulated levels of MCP-1 and IL-8 were measured by means of a multi-analyte Biochip Array Technology, using the Evidence analyzer (Randox Laboratories, UK). The measurements were performed before (phase I), at the end (phase II) and 48h post-race (phase III). MCP-1 levels at phase I (216.9±48.5 ng/L) (mean±SE), increased significantly at phase II (592.9±115.7 ng/L) and subsequently decreased at phase III (278.1±62.7 ng/L). At the same time period, IL-8 followed a similar pattern (phase I: 9.4±4.5 ng/L, phase II: 28.5±8.8 and phase III: 8.9±4.3 ng/L). A significant positive correlation between MCP-1 and IL-8 was found at phase III (r=0.845, p<0.01), while this correlation was absent in the other two phases, indicating an independent response of each chemokine to inflammatory stimuli in each athlete.In conclusion, prolonged exercise induces an inflammatory response that is expressed by an increase in circulating MCP-1 and IL-8 levels. Whether these changes have long-term negative effects on the vasculature remains unknown.

Cell-Free Plasma DNA as a Novel Marker of Aseptic Inflammation Severity Related to Exercise Overtraining

Circulating free plasma DNA is implicated in conditions associated with tissue injury, including exercise-induced inflammation, and thus is a potential marker for athletic overtraining. We measured free plasma DNA along with C-reactive protein (CRP), creatine kinase (CK), and uric acid (UA) in 17 recreationally trained men participating in a 12-week resistance training regimen (8 resistance multi-joint exercises selected to stress the entire musculature: bench press, squat, leg press, snatch, hang clean, dead lifts, barbell arm curls, and rowing), consisting of 4 training periods (t1, t2, t3, and t4). Plasma DNA concentrations increased markedly after t1, t2, and t3 and returned to baseline after t4. There were substantial differences between t2 and t1 and between t3 and t2 plasma DNA concentrations. CRP increased by 300% after t2 and by 400% after t3 (there was no difference between t2 and t3 CRP values) compared with baseline (t0). CK increased only after t3. UA increased after t2 and t3, with a greater increase after t3. This study demonstrates that, after chronic excessive resistance exercise, plasma DNA concentrations increase in proportion to training load, suggesting that plasma DNA may be a sensitive marker for overtraining-induced inflammation.

Non‐invasive diagnosis of fetal sex; utilisation of free fetal DNA in maternal plasma and ultrasound

Non-invasive prenatal diagnosis is now a clinical reality, using both early ultrasound and molecular DNA methods. Technical advances in the sensitivity of the polymerase chain reaction (PCR), coupled with the finding that significant levels of fetal DNA (ffDNA) are found in maternal plasma and serum, has enabled the ready detection of paternally inherited genes or polymorphisms. Routine maternal plasma-based genotyping is now available for the determination of fetal sex and RHD blood group status (Van der Schoot et al., 2003). This review touches briefly on the ultrasound diagnoses and then focuses on the application of free ffDNA for fetal sex determination, indicating the Y-chromosome targets exploited in this strategy and the merits of their utilisation.

Detection of maternal deoxyribonucleic acid in peripheral blood of premature and mature newborn infants

Over the past decade, a lot of attention has been directed towards the fetomaternal and maternofetal transfer of nucleated cells and plasma DNA. In some autoimmune diseases, the fetal DNA is suspected to play an important role in the etiology of the disease. In the same way, the presence of maternal cells and free plasma DNA in fetal/newborn circulation gives rise to interesting questions. The aim of our study was to detect maternal deoxyribonucleic acid in the peripheral blood of premature and mature newborn infants. In the case of eight RhD-positive mothers-RhD-negative newborn pairs, peripheral blood samples were collected from the newborn infants within 35-120 min after birth. The maternal origin DNA was determined by real-time PCR amplification of the exon 7 of the RhD-positive allele. In all eight cases, the RhD exon 7 was amplified during the PCR reaction. The result of our study demonstrates that maternal DNA is present in the fetal peripheral circulation. The presence of maternally derived cells/DNA in the blood of newborn infants might have a role in the immunization of the newborn infants and also could be a possible explanation for 'grandmother effect' in the case of Rh-negative nulligravida patients.

Non‐invasive first trimester determination of fetal gender: a new approach for prenatal diagnosis of haemophilia

Ten carriers of haemophilia referred for prenatal diagnosis were offered first trimester non-invasive fetal gender determination by ultrasound and analysis of free fetal DNA (ffDNA) in maternal plasma in an attempt to reduce the need for an invasive diagnostic procedure in female pregnancies. Although repeat testing was required in three cases, fetal gender was determined correctly in all cases (four females, six males) at a median gestation of 12(+3) (11(+2) to 14(+1)) using both methods. In all cases of a female fetus, the mothers opted not to have invasive testing. Both methods provide a reliable option of avoiding invasive testing in female pregnancies.

Possible Neuroprotective Effect of Erythropoietin in Acute Inflammation Induced by Exhaustive Exercise.

Erythropoietin is a glycoprotein hormone produced mainly by interstitial fibroblasts in the kidneys of the adult and by hepatocytes of the foetus. Released into the circulation, erythropoietin makes its way to the bone marrow, where it regulates red cell production by preventing apoptosis of erythroid progenitor cells. Recently, erythropoietin has emerged as a multifunctional growth factor that plays a significant role in the central nervous system (CNS). Both erythropoietin and its receptor are expressed throughout the brain in glial cells, neurons and endothelial cells. Hypoxia and ischaemia have been recognised as important stimuli of erythropoietin expression in the brain. Erythropoietin has potent neuroprotective properties in vivo and in vitro and appears to act in a dual way by directly protecting neurons from ischaemic damage and by stimulating endothelial cells. S-100b, a glial-derived protein is a well-established biomarker for severity of neurological injury and prognosis for recovery. Cell-based and clinical studies have implicated S-100b protein in the initiation and maintenance of a pathological, glial-mediated pro-inflammatory state in the CNS. We studied the possible association between erythropoietin and S-100b in 18 athletes participated in the ultra-distance foot race of the 246Km “Sparthathlon”. This race consists of continuous, prolonged, brisk exercise. We have reported that IL-6, CRP SAA and free plasma DNA levels markedly increased (by 8000-, 152- 108- and 10-fold, respectively) over the baseline at the end of the race1. However, IL-6 levels returned to normal by 48h, while CRP, SAA and free plasma DNA remained elevated. Erythropoietin and S-100b protein levels were determined by means of chemiluminescence (Nichols Institute Diagnostics, CA, USA and Sangtec Medical AB, Bromma, Sweden respectively). The measurements were performed before (phase I), at the end (phase II) and 48h post-race (phase III). Erythropoietin levels at phase I (13.5±6.3 IU/L), were increased significantly at phase II (52.0±52.0 IU/L) and subsequently decreased at phase III (21.0±14.6 IU/L). At the same time period S-100b protein followed the same pattern (phase I: 0.13±0.01 mg/L, phase II: 0.29±0.01 and phase III: 0.13±0.01mg/L). A significantly positive correlation between erythropoietin and S-100b protein was found at phase II (r=0.521, p<0.03), while this correlation was absent in the other two phases (p>0.40 and p>0.08 respectively). Simultaneous measurements of haemoglobin levels showed only a significant drop of haemoglobin at phase III (143.4±12.4, 140.4±13.0 and 124.3±9.5 g/L respectively). Exhaustive exercise induces inflammation and CNS injury, promoting an erythropoietin anti-inflammatory response. We suggest that in this particular inflammatory process, erythropoietin secretion is associated with brain lesions, as they are expressed by the S-100b protein release in the circulation. This is strongly supported by the lack of any association between erythropoietin values and haemoglobin levels.

Dramatic Elevations of Interleukin-6 and Acute-Phase Reactants in Athletes Participating in the Ultradistance Foot Race Spartathlon: Severe Systemic Inflammation and Lipid and Lipoprotein Changes in Protracted Exercise

Plasma IL-6, the serum inflammatory markers C-reactive protein (CRP) and serum amyloid A (SAA), and the tissue destruction marker-free plasma DNA, as well as the circulating lipid profile, were examined in athletes participating in the ultradistance foot race of the 246-km Spartathlon. SETTING, DESIGN, AND PARTICIPANTS: This race consists of continuous, prolonged, brisk exercise. Blood samples were obtained from 15 male athletes, who finished the race in less than 36 h, taken before, at the end of, and 48 h after the end of the race. IL-6, CRP, SAA, and free plasma DNA levels markedly increased (by 8000-, 152- 108-, and 10-fold, respectively) over the baseline at the end of the race. However, IL-6 levels returned to normal by 48 h, whereas CRP, SAA, and free plasma DNA remained elevated. The mean values of cholesterol, triglycerides, low-density lipoprotein, and apolipoprotein B decreased to a minimum value at the end of the race and remained low 48 h after the race. High-density lipoprotein levels, on the other hand, were mildly increased at the end of the race (P < 0.015) and decreased to normal 48 h after the race. Apolipoprotein AI levels decreased significantly during the time course of the exercise and remained low 48 h after the race (P < 0.001). These observations suggest that continuous, prolonged, moderate-intensity exercise is associated with markedly elevated IL-6 and acute-phase reactant concentrations, peripheral tissue damage, and significant changes in serum lipid levels. The biochemical changes observed during the Spartathlon amount to a potent systemic inflammatory response, which might explain severe cardiovascular events that occur during prolonged exercise in compromised individuals.

Reduction in diagnostic and therapeutic interventions by non-invasive determination of fetal sex in early pregnancy

This study reviews our clinical experience of non-invasive techniques for early sex determination. It assesses the effectiveness of these techniques at reducing invasive prenatal testing for X-linked genetic disease or for ambiguous development of the external genitalia. A prospective cohort study of 30 pregnancies was referred to a tertiary unit for prenatal diagnosis. Fetal gender was determined using two non-invasive techniques: analysis of free fetal DNA (ffDNA) in maternal plasma and ultrasound visualisation. The results were compared to fetal gender determined by invasive testing or at birth. Fetal gender was accurately determined by analysis of ffDNA at a mean of 10 + 1 (7 + 6 to 14 + 1) weeks' gestation in all cases. Ultrasound assessment was accurate in 20 of the 23 cases where this was attempted at 12 + 0 (10 + 4 to 14 + 1) weeks' gestation, but could not be determined in the remaining 3 cases. Thirteen of 28 (46%) women chose not to have invasive testing on the basis of these findings. Both the techniques appear to offer an accurate means of assessing fetal gender, giving parents the option of avoiding invasive testing in the 50% of cases where the fetus would not be affected. The molecular technique is performed at an earlier gestation, but female fetal status is predicted by a negative test result. Ultrasound cannot be applied until 11 weeks' gestation but diagnostic signs are sought in both sexes. Combining these approaches offers a highly sensitive method of non-invasive determination of gender in high-risk pregnancies. Health professionals, clinical geneticists and genetics associates, in particular, who refer women at high risk should be aware of these non-invasive options for prenatal sex determination.

Apoptosis as an early event in the development of multiple organ failure?

We have recently developed a simple method of plasma free DNA detection, which enables us to distinguish between apoptotic and genomic (necrotic) DNA. After applying this method to the critically ill, we revealed apoptotic DNA on the day of admission to be higher than later when multiple-organ failure developed. Moreover, apoptotic DNA contributed to total plasma DNA much more than DNA from necrotic cells and its increase predicted future development of multiple-organ failure and death.

Use of maternal plasma for noninvasive determination of fetal RhD status

Determination of the fetal RhD typing using free fetal DNA in maternal plasma is beginning to enjoy widespread acceptance in Europe. Case 1, the partner of an RhD-sensitized patient, was identified with a heterozygous paternal phenotype by serologic testing. Maternal plasma was drawn at 18 weeks' gestation to determine the fetal RhD status. The result was unable to be reported as RhD negative; the patient subsequently underwent amniocentesis to confirm an RhD-negative fetus. Case 2, a partner of another RhD-sensitized patient, was similarly identified with a heterozygous paternal phenotype by serologic testing. Maternal plasma was also drawn at 18 weeks' gestation to determine the fetal RhD status. It returned RhD negative and allowed for the avoidance of invasive testing for the remainder of the pregnancy. Therefore, maternal plasma testing for fetal RhD status represents a new tool in the management of the cases of RhD alloimmunization in pregnancy.

Methylated tumor-specific DNA as a plasma biomarker in patients with glioma

1527 Background: While patients without malignancy have <100μg/ml free DNA in plasma (Chang, J NC I 2002), patients with malignancy demonstrate substantial quantities of tumor-specific DNA in their plasma. Measurements of this DNA are being studied to develop plasma markers to correlate with tumor burden. This approach has not been explored in gliomas. Methods: This study assesses the presence of plasma tumor-specific DNA in gliomas using methylation specific polymerase chain reaction (MSP). With IRB approval, 30 ml venous blood was collected prior to craniotomy from 10 patients with presumed high grade glioma. After surgery, DNA was extracted and quantified from the tumor and plasma samples. The methylation status of p16, p73, RARβ, and MGMT gene promoters was assayed with MSP. The presence of tumor-specific DNA in the plasma was defined as identification of the same methylated promoter (MP) in both plasma and tumor. Results: Histology was: glioblastoma (N=6), anaplastic astrocytoma (2), anaplastic oligo...

A clinical service in the UK to predict fetal Rh (Rhesus) D blood group using free fetal DNA in maternal plasma.

Antenatal determination of fetal blood group is important in pregnancies with a significant risk of hemolytic anemia due to maternal alloimmunization. The International Blood Group Reference Laboratory (part of the National Blood Service) in Bristol, UK, provides a fetal blood group genotyping service to obstetricians caring for immunized pregnant women with heterozygous partners. Since 2001, fetal D typing has been offered using free fetal DNA in maternal plasma. Real-time polymerase chain reaction (PCR) assays are performed to detect the RHD gene. To confirm the presence of fetal DNA when RHD is not detected, Y-chromosome sequences are targeted. When a D-negative female fetus is predicted, maternal buffy coat DNA is tested for eight insertion/deletion polymorphisms. Sequences that are absent from the maternal genome are then targeted in maternal plasma and are used to confirm the presence of free fetal DNA in the blood sample. Currently, 283 pregnancies have been tested, of which 50 are awaiting confirmatory results. Fetal D status was correctly predicted in 223 cases, and no result was obtainable in 7 cases. In three cases, serology on cord blood was discrepant with reported results, but all fetuses had received multiple intrauterine transfusions. The new test has significantly reduced the number of invasive procedures carried out in the UK for fetal D grouping. Antenatal anti-D prophylaxis is currently being introduced in the UK to all D-negative women; in the future, detection of fetal RHD sequences in maternal plasma may allow anti-D to be restricted to pregnancies involving a D-positive fetus.

1669: Plasma Free DNA Offers a Sensitive and Specific Test for Prostate Cancer

1669: Plasma Free DNA Offers a Sensitive and Specific Test for Prostate Cancer

Application of fetal DNA in maternal plasma in noninvasive prenatal diagnosis.

To explore the application of fetal DNA in maternal plasma for noninvasive prenatal diagnosis, the DNA template was extracted by hydroxybenzene-chloroform from 44 maternal (7-41 weeks) plasma. The Fetus-derived Y sequence DYZ-1 gene (149bp) was chosen to be amplified by PCR. The fragment was identified in all the plasma of male bearing pregnant women with the diagnostic accordance rate being 100.00%. Two of the 22 female bearing pregnant women had false positive results. Among the 44 pregnant women, the diagnostic accordance rate was 88.89% at early pregnant stage, 100.00% at medium pregnant stage, and 96.55% at late stage respectively. The final accuracy of 95.45% was obtained in all cases. It was concluded that by means of hydroxybenzene-chloroform extraction the authors of this article promoted the concentration and purity of the DNA template, and diagnosed more accurately. The results showed that free fetal DNA in the maternal plasma could be regarded as the gene resource for noninvasive prenatal diagnosis.

87 Plasma free DNA levels offer a sensitive and specific test to distinguish between benign and malignant prostate disease

87 Plasma free DNA levels offer a sensitive and specific test to distinguish between benign and malignant prostate disease

Free fetal DNA concentration in maternal plasma during normal labour at term.

Our aim was to investigate the effect of uterine contractions on free fetal DNA concentration in maternal plasma at term. Nine pregnant women were admitted for elective induction of labour between 38 and 40 weeks of gestation. All patients carrying male fetuses and without history of pre-term labour and membrane rupture were selected. Maternal venous blood samples were serially collected every hour during labour, one hour after delivery and 24 h after delivery. In order to amplify fetal and total free DNA, primers for SRY and GAPDH genes were respectively chosen. Real- time PCR analysis was performed with an GeneAmp 5700 Sequence Detection System (Applied Biosystems Foster City, USA). Statistical significance was determined using the Wilcoxon test. Median concentration of fetal DNA in plasma before labour was 3081 copies/mL (Range 812-15 864 copies/mL). No significant difference between the number of copies per millilitre before any contractions and during labour was demonstrated. One hour after delivery, significant decrease in the fetal DNA rate was observed with a median concentration of 293 copies/mL (Range 0-2037 copies/mL) (p-value: 0.04). This drop was more significant 24 h after delivery with a median concentration of 0 copies/mL (Range 0-95 copies/mL) (p-value: 0.02). During labour, no changes in free fetal DNA in maternal plasma were demonstrated. This suggests that labour does not have effect on free fetal DNA release in maternal circulation at term.

Determination of RHD zygosity using real-time quantitative PCR.

At present RHD incompatibility is still an obstetric problem despite prophylactic treatment. A very welcome recent technical advance has now made it possible to determine the foetal RHD status in a non-invasive risk-free manner using cell free foetal DNA in maternal plasma. In some cases, however, where there is a high risk that the foetus may be affected by HDN (haemolytic disease of the newborn), it may be of interest to determine whether the father is hetero- or homozygous for the RHD gene, since in the former instance there is only a 50% chance that the pregnancy is affected. It has recently been shown that quantitative PCR assays, in particular real-time Taqman PCR, can be used to determine the RHD gene dosage, and also to determine foetal aneuploidies. We demonstrate that the same real-time Taqman PCR assay we had previously developed for non-invasive analysis of the foetal RHD gene and the foetal Y chromosome from maternal plasma can be used to determine the paternal RHD genotype.

Determination of RHD zygosity using real-time quantitative PCR.

At present RHD incompatibility is still an obstetric problem despite prophylactic treatment. A very welcome recent technical advance has now made it possible to determine the foetal RHD status in a non-invasive risk-free manner using cell free foetal DNA in maternal plasma. In some cases, however, where there is a high risk that the foetus may be affected by HDN (haemolytic disease of the newborn), it may be of interest to determine whether the father is hetero- or homozygous for the RHD gene, since in the former instance there is only a 50% chance that the pregnancy is affected. It has recently been shown that quantitative PCR assays, in particular real-time Taqman PCR, can be used to determine the RHD gene dosage, and also to determine foetal aneuploidies. We demonstrate that the same real-time Taqman PCR assay we had previously developed for non-invasive analysis of the foetal RHD gene and the foetal Y chromosome from maternal plasma can be used to determine the paternal RHD genotype.

Replicate real-time PCR testing of DNA in maternal plasma increases the sensitivity of non-invasive fetal sex determination.

We determined fetal sex in pregnancies referred for invasive prenatal diagnosis procedures by analysis of DNA in maternal plasma. Twelve pregnancies at risk of X-linked haemophilia and 32 pregnancies at risk of chromosomal aneuploidies at a gestational age ranging from 10 to 18 weeks recruited before chorionic villus sampling or amniocentesis were involved in the study. Male fetal DNA in maternal plasma was detected by using real-time polymerase chain reaction with the SRY gene as a marker. The specificity of the system reached 100% (no Y signal was detected in 17 women pregnant with a female fetus) and the sensitivity reached 100% (SRY amplification in 27 examined samples). Amplification of free fetal DNA in maternal plasma is a valid and rapid technique for predicting fetal sex in first- and second-trimester pregnancies and could allow the restriction of invasive sampling procedures to male fetuses at risk of X-linked disorders.