DNA In Human Plasma Research Articles

Facile Preconcentration of Cell-Free DNA in Human Plasma by Ion-Specific Poly Ionic Sorbents Featuring an Anion Exchange Mechanism.

The expanding horizon of diagnostic and therapeutic applications involving nucleic acids (NA) requires novel tools for purification, including minimal sample preparation. In this work, thin-film microextraction devices featuring five poly ionic sorbents were examined as anion exchange extraction phases for the rapid purification of NAs. Each sorbent is composed of a nonionic cross-linker and a methacrylate monomer containing a core tetra-alkyl ammonium moiety with an alkyl, anionic, or cationic residue. Extraction devices were produced through the application of the prepolymer sorbent mixture onto a functionalized nitinol metal support followed by photoinduced free-radical polymerization. The miniaturized extraction devices (10 mm × 3.5 mm) were directly immersed into aqueous samples to isolate NAs via electrostatic interactions with the polycation. The ammonium methacrylate (AMA) monomer containing a propyl trimethylammonium group (AMA-C3N(CH3)3) exhibited the highest affinity for DNA, with 80 ± 10% of DNA being isolated. Recovery of DNA from the sorbents required the introduction of ions in an aqueous solution to exchange the anionic biopolymer from the polycationic moiety. An investigation of three anion species revealed that the AMA-C3N(CH3)3 sorbent showed the highest recoveries, with the perchlorate anion producing a preconcentration factor of 4.36 ± 0.86 while requiring only 250 mM NaClO4. A directly compatible quantitative polymerase chain reaction assay was developed to quantify the recovery of spiked DNA with lengths of 830, 204, and 98 base pairs in heat-treated human plasma. The AMA-C3N(CH3)3 sorbent was uninhibited by the complex human plasma matrix and enabled high preconcentration factors for the spiked DNA at a biologically relevant concentration of 10 pg/mL. While Qiagen's circulating cell-free DNA MinElute extraction kit enabled higher preconcentration of all analytes, the methodology described in this work requires fewer steps, less user intervention, and minimal equipment requirements to isolate DNA, making it more amenable for high-throughput and low resource applications.

Quantitation of oxidized nuclear and mitochondrial DNA in plasma samples of patients with abdominal aortic aneurysm.

There is accumulating evidence that pro-inflammatory features are inherent to mitochondrial DNA and oxidized DNA species. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is the most frequently studied oxidatively generated lesion. Modified DNA reaches the circulation upon cell apoptosis, necrosis or neutrophil extracellular trap (NET) formation. Standard chromatography-based techniques for the assessment of 8-oxodGuo imply degradation of DNA to a single base level, thus precluding the attribution to a nuclear or mitochondrial origin. We therefore aimed to establish a protocol for the concomitant assessment of oxidized mitochondrial and nuclear DNA from human plasma samples. We applied immunoprecipitation (IP) for 8-oxodGuo to separate oxidized from non-oxidized DNA species and subsequent quantitative polymerase chain reaction (qPCR) to assign them to their subcellular source. The IP procedure failed when applied directly to plasma samples, i.e. isotype control precipitated similar amounts of DNA as the specific 8-oxodGuo antibody. In contrast, DNA isolation from plasma prior to the IP process provided assay specificity with little impact on DNA oxidation status. We further optimized sensitivity and efficiency of qPCR analysis by reducing amplicon length and targeting repetitive nuclear DNA elements. When the established protocol was applied to plasma samples of abdominal aortic aneurysm (AAA) patients and control subjects, the AAA cohort displayed significantly elevated circulating non-oxidized and total nuclear DNA and a trend for increased levels of oxidized mitochondrial DNA. An enrichment of mitochondrial versus nuclear DNA within the oxidized DNA fraction was seen for AAA patients. Regarding the potential source of circulating DNA, we observed a significant correlation of markers of neutrophil activation and NET formation with nuclear DNA, independent of oxidation status. Thus, the established method provides a tool to detect and distinguish the release of oxidized nuclear and mitochondrial DNA in human plasma and offers a refined biomarker to monitor disease conditions of pro-inflammatory cell and tissue destruction.

Methods for the purification and detection of single nucleotide KRAS mutations on extrachromosomal circular DNA in human plasma.

Despite recent advances, many cancers are still detected too late for curative treatment. There is, therefore, a need for the development of new diagnostic methods and biomarkers. One approach may arise from the detection of extrachromosomal circular DNA (eccDNA), which is part of cell-free DNA in human plasma. First, we assessed and compared two methods for the purification of eccDNA from plasma. Second, we tested for an easy diagnostic application of eccDNA liquid biopsy-based assays. For the comparison we tested a solid-phase silica purification method and a phenol/chloroform method with salt precipitation. For the diagnostic application of eccDNA we developed and tested a qPCR primer-based SNP detection system, for the detection of two well-established cancer-causing KRAS mutations (G12V and G12R) on circular DNA. This investigation was supported by purifying, sequencing, and analysing clinical plasma samples for eccDNAs containing KRAS mutant alleles in 0.5 mL plasma from 16 pancreatic ductal adenocarcinoma patients and 19 healthy controls. In our method comparison we observed, that following exonuclease treatment a lower eccDNA yield was found for the phenol/chloroform method (15.7%-26.7%) compared with the solid-phase purification approach (47.8%-65.9%). For the diagnostic application of eccDNA tests, the sensitivity of the tested qPCR assay only reached ~10-3 in a background of 105 wild type (wt) KRAS circular entities, which was not improved by general amplification or primer-based inhibition of wt KRAS amplification. Furthermore, we did not detect eccDNA containing KRAS in any of the clinical samples. A potential explanation for our inability to detect any KRAS mutations in the clinical samples may be related to the general low abundance of eccDNA in plasma. Taken together our results provide a benchmark for eccDNA purification methods while raising the question of what is required for the optimal fast and sensitive detection of SNP mutations on eccDNA with greater sensitivity than primer-based qPCR detection.

Plasma contains ultrashort single-stranded DNA in addition to nucleosomal cell-free DNA

SummaryPlasma cell-free DNA is being widely explored as a biomarker for clinical screening. Currently, methods are optimized for the extraction and detection of double-stranded mononucleosomal cell-free DNA of ∼160bp in length. We introduce uscfDNA-seq, a single-stranded cell-free DNA next-generation sequencing pipeline, which bypasses previous limitations to reveal a population of ultrashort single-stranded cell-free DNA in human plasma. This species has a modal size of 50nt and is distinctly separate from mononucleosomal cell-free DNA. Treatment with single-stranded and double-stranded specific nucleases suggests that ultrashort cell-free DNA is primarily single-stranded. It is distributed evenly across chromosomes and has a similar distribution profile over functional elements as the genome, albeit with an enrichment over promoters, exons, and introns, which may be suggestive of a terminal state of genome degradation. The examination of this cfDNA species could reveal new features of cell death pathways or it can be used for cell-free DNA biomarker discovery.

Detection of Tumor DNA in Human Plasma with a Functional PLL-Based Surface Layer and Plasmonic Biosensing.

Standard protocols for the analysis of circulating tumor DNA (ctDNA) include the isolation of DNA from the patient’s plasma and its amplification and analysis in buffered solutions. The application of such protocols is hampered by several factors, including the complexity and time-constrained preanalytical procedures, risks for sample contamination, extended analysis time, and assay costs. A recently introduced nanoparticle-enhanced surface plasmon resonance imaging-based assay has been shown to simplify procedures for the direct detection of tumor DNA in the patient’s plasma, greatly simplifying the cumbersome preanalytical phase. To further simplify the protocol, a new dual-functional low-fouling poly-l-lysine (PLL)-based surface layer has been introduced that is described herein. The new PLL-based layer includes a densely immobilized CEEEEE oligopeptide to create a charge-balanced system preventing the nonspecific adsorption of plasma components on the sensor surface. The layer also comprises sparsely attached peptide nucleic acid probes complementary to the sequence of circulating DNA, e.g., the analyte that has to be captured in the plasma from cancer patients. We thoroughly investigated the contribution of each component of the dual-functional polymer to the antifouling properties of the surface layer. The low-fouling property of the new surface layer allowed us to detect wild-type and KRAS p.G12D-mutated DNA in human plasma at the attomolar level (∼2.5 aM) and KRAS p.G13D-mutated tumor DNA in liquid biopsy from a cancer patient with almost no preanalytical treatment of the patient’s plasma, no need to isolate DNA from plasma, and without PCR amplification of the target sequence.

ChIP-seq of plasma cell-free nucleosomes identifies gene expression programs of the cells of origin.

Cell-free DNA in human plasma provides access to molecular information about the pathological processes in the organs or tumors from which it originates. These DNA fragments are derived from fragmented chromatin in dying cells, and retain some of the cell of origin histone modifications. Here, we apply chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) to 268 human samples. In healthy donors, we identified bone marrow megakaryocytes, but not erythroblasts, as major contributors to the cfDNA pool. In patients with a range of liver diseases, we show that we can identify pathology- related changes in hepatocyte transcriptional programs. In metastatic colorectal carcinoma patients, we detected clinically relevant, and patient-specific information, including transcriptionally active HER2 amplifications. Altogether, cfChIP-seq using low sequencing depth, provides systemic and genome-wide information, can inform diagnosis, and facilitate interrogation of physiological and pathological processes using blood samples.

Extracellular DNA in blood products and its potential effects on transfusion.

Blood transfusions are sometimes necessary after a high loss of blood due to injury or surgery. Some people need regular transfusions due to medical conditions such as haemophilia or cancer. Studies have suggested that extracellular DNA including mitochondrial DNA present in the extracellular milieu of transfused blood products has biological actions that are capable of activating the innate immune systems and potentially contribute to some adverse reactions in transfusion. From the present work, it becomes increasingly clear that extracellular DNA encompassed mitochondrial DNA is far from being biologically inert in blood products. It has been demonstrated to be present in eligible blood products and thus can be transfused to blood recipients. Although the presence of extracellular DNA in human plasma was initially detected in 1948, some aspects have not been fully elucidated. In this review, we summarize the potential origins, clearance mechanisms, relevant structures, and potential role of extracellular DNA in the innate immune responses and its relationship with individual adverse reactions in transfusion.

High-resolution profiling of human cytomegalovirus cell-free DNA in human plasma highlights its exceptionally fragmented nature

Human cytomegalovirus (CMV) infections comprise a leading cause of newborn impairments worldwide and are pervasive concerns among the immunocompromised. Quantification of CMV viral loads is increasingly used to guide definitions of CMV disease but standardization of CMV quantitation remains problematic, mostly due to differences in qPCR amplicon sizes between clinical laboratories. Here, we used plasma cfDNA sequencing data from 2,208 samples sent for non-invasive prenatal aneuploidy screening to detect CMV and precisely measure the length of CMV fragments in human plasma. CMV reads were identified in 120 (5.4%) samples. Median cfDNA fragment size derived from CMV was significantly shorter than cfDNA derived from human chromosomes (103 vs 172 bp, p < 0.0001), corresponding to the 3rd percentile of human cfDNA. Sequencing of cfDNA from seven plasma samples from transplant patients positive for CMV confirmed the extraordinarily short nature of CMV cfDNA fragment size with a median length of 149 bp. We further show that these high-resolution measurements of CMV DNA fragment size accurately predict measured discrepancies in serum viral load measurements by different qPCR assays. These results highlight the exceptionally fragmented nature of CMV cfDNA and illustrate the promise of plasma cfDNA sequencing for quantitating viral loads through detection of fragments that would be unrecoverable by qPCR.

Identification and characterization of extrachromosomal circular DNA in maternal plasma

We explored the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. Through sequencing following either restriction enzyme or Tn5 transposase treatment, we identified eccDNA molecules in the plasma of pregnant women. These eccDNA molecules showed bimodal size distributions peaking at ∼202 and ∼338 bp with distinct 10-bp periodicity observed throughout the size ranges within both peaks, suggestive of their nucleosomal origin. Also, the predominance of the 338-bp peak of eccDNA indicated that eccDNA had a larger size distribution than linear DNA in human plasma. Moreover, eccDNA of fetal origin were shorter than the maternal eccDNA. Genomic annotation of the overall population of eccDNA molecules revealed a preference of these molecules to be generated from 5'-untranslated regions (5'-UTRs), exonic regions, and CpG island regions. Two sets of trinucleotide repeat motifs flanking the junctional sites of eccDNA supported multiple possible models for eccDNA generation. This work highlights the topologic analysis of plasma DNA, which is an emerging direction for circulating nucleic acid research and applications.

Size-tagged preferred ends in maternal plasma DNA shed light on the production mechanism and show utility in noninvasive prenatal testing

Cell-free DNA in human plasma is nonrandomly fragmented and reflects genomewide nucleosomal organization. Previous studies had demonstrated tissue-specific preferred end sites in plasma DNA of pregnant women. In this study, we performed integrative analysis of preferred end sites with the size characteristics of plasma DNA fragments. We mined the preferred end sites in short and long plasma DNA molecules separately and found that these "size-tagged" ends showed improved accuracy in fetal DNA fraction estimation and enhanced noninvasive fetal trisomy 21 testing. Further analysis revealed that the fetal and maternal preferred ends were generated from different locations within the nucleosomal structure. Hence, fetal DNA was frequently cut within the nucleosome core while maternal DNA was mostly cut within the linker region. We further demonstrated that the nucleosome accessibility in placental cells was higher than that for white blood cells, which might explain the difference in the cutting positions and the shortness of fetal DNA in maternal plasma. Interestingly, short and long size-tagged ends were also observable in the plasma of nonpregnant healthy subjects and demonstrated size differences similar to those in the pregnant samples. Because the nonpregnant samples did not contain fetal DNA, the data suggested that the interrelationship of preferred DNA ends, chromatin accessibility, and plasma DNA size profile is likely a general one, extending beyond the context of pregnancy. Plasma DNA fragment end patterns have thus shed light on production mechanisms and show utility in future developments in plasma DNA-based noninvasive molecular diagnostics.

Abstract 4244: Detection and characterization of extrachromosomal circular DNA in human plasma

Abstract Background: eccDNA (extrachromosomal circular DNA) has been reported in most eukaryotes, including in human cells. However, little is known about the eccDNA profiles in human circulating system such as blood. Method: We extracted plasma cell free DNA (cfDNA) from three patients with advanced stage cancers. For each patient, we tested 10ng, 1ng and 0.1ng cfDNA, each with a technical replicate. The cfDNA samples were subjected to ATP-dependent DNase digestion and whole genome amplification (WGA). An additional 2ng cfDNA from each patient skipped the DNase digestion and went directly to WGA step. We sequenced the amplified DNAs using the Illumina 100bp PE read protocol. We analyzed sequencing data using a custom split reads-based strategy to retrieve circular DNA molecules. We further analyzed eccDNAs for their biofeatures using functional annotation tools. Results: We received sequence reads at ~14.8 million (11.4~22.4) for each sample. When read depth≥ 3 as a cutoff, we detected a total number of 2542, 1578, and 57 unique eccDNAs in three patients while negative controls showed &amp;lt;=5 eccDNAs. For non-DNase digested cfDNAs, we detected only 3 eccDNAs. By comparing read numbers mapped to mitochondria and human genome, we found that mitochondrial sequence reads increased significantly from 0.34-1.1% before digestion to 24.1-50.1% after digestion. Numbers of detected eccDNAs were similar between technical replicates and proportional to initial input cfDNA. However, eccDNAs detected also showed few overlaps between technical replicates. Size distribution ranged from 31bp to 19740bp (median 1801-1962bp). To test if eccDNAs were generated in specific genomic regions and if eccDNA-involved genes were enriched in specific pathways, we performed enrichment analysis and observed higher GC content in smaller eccDNAs (&amp;lt;500 bp, 48%) than larger ones (&amp;gt;500bp, 41%) (p&amp;lt;0.001). When compared to random distribution, eccDNAs were more likely located at CpG islands (1.36-1.53 fold enrichment). The eccDNAs were also enriched in exons, 5’UTR, 3’UTR and DNase hypersensitive sites (enrichment folds from 1.39 to 2.3). Using DAVID annotation tool, we observed significant enrichment of the eccDNA-related genes in several categories including the Coiled coil, phosphoprotein, fibronectin, suggesting a potential involvement in gene expression regulation and cell adhesion events. Using GREAT (Genomic Regions Enrichment of Annotations Tool), we found that the eccDNAs were involved in genes associated with invasive cancer cell gene expression regulation (p&amp;lt;0.001). Conclusion: Human plasma contains an abundant number of eccDNAs. Distribution of these eccDNAs is nonrandom event and is likely enriched in genomic regions with known functional consequences and genes that play a role in gene regulations. Further characterization of circulating eccDNAs in peripheral blood will facilitate understanding of their molecular mechanisms and potential clinical utilities. Citation Format: Jing Zhu, Meijun Du, Peng Zhang, Liang Wang. Detection and characterization of extrachromosomal circular DNA in human plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4244. doi:10.1158/1538-7445.AM2017-4244

Sandwich ELISA for Circulating Myeloperoxidase- and Neutrophil Elastase-DNA Complexes Released from Neutrophil Extracellular Traps

Neutrophil extracellular traps (NETs) are DNA scaffolds released by activated neutrophils that contain enzymes from neutrophil granules, such as myeloperoxidase (MPO), neutrophil elastase (NE) and cathepsin-G. NETs are produced by neutrophils in response to various stimuli, including invasion of pathogenic microorganisms. Here we describe a new ELISA method for quantifying the circulating levels of MPO- and NE-associated DNA in human plasma.

Validation of a real-time PCR-based qualitative assay for the detection of methylated SEPT9 DNA in human plasma.

Epi proColon® is a new blood-based colorectal cancer (CRC) screening test designed to determine the methylation status of a promoter region of the SEPT9 (septin 9) gene in cell-free DNA isolated from plasma. We describe the analytical and clinical performance of the test. Analytical performance at 4 testing laboratories included determination of limit of detection, precision, and reproducibility of the SEPT9 test. Clinical performance was evaluated in a prospective study by use of samples (n = 1544) from subjects enrolled in the PRESEPT clinical trial. Results were analyzed by comparison with colonoscopy, the reference standard. The limit of detection for methylated SEPT9 DNA was 7.8 pg/mL (95% CI 6-11 pg/mL) corresponding to <2 genome copies of methylated SEPT9 per milliliter of plasma. In the prospective clinical trial, sensitivity for all stages of CRC was 68% (95% CI 53%-80%) and for stage I-III CRC, 64% (48%-77%). Adjusted specificity, on the basis of negative colonoscopy findings, was 80.0% (78%-82%). The Epi proColon test is a simple, real-time PCR-based assay for the detection of methylated SEPT9 DNA in blood that may provide a noninvasive CRC screening alternative for people noncompliant with current CRC screening guidelines.

Abstract 4701: Cancer-specific genomic rearrangements used to quantify disease burden in plasma from patients with colorectal cancer.

Abstract Tumor-specific chromosomal rearrangements have the potential to serve as highly sensitive biomarkers for tumor detection. Such alterations are not present in normal cells and should be exquisitely specific. It is well recognized that some colorectal cancers (CRC) release small amounts of DNA into plasma. Accordingly, sensitive and specific assays targeting these tumor-specific rearrangements should enable quantification of changes in disease burden if applied to serial plasma samples. Here we report the establishment of efficient procedures for 1) identification of tumor-specific genomic rearrangements, 2) development of sensitive and specific assays to these, 3) quantification of these rearrangements in plasma, and 4) results from applying these procedures to quantify disease burden in serial plasma samples from CRC patients. The tumor-specific genomic rearrangements were identified using a combination of next generation paired-end sequencing of large-insert libraries and SNP array based DNA copy number analysis. Nested real-time PCR assays targeting the rearrangements were applied to plasma samples in order to quantify disease burden. Initially the approach was tested using the CRC cell line SW620 as a tumor model, and serial dilutions of SW620 DNA and normal human plasma DNA as models of patient plasma. Multiple cell line specific rearrangements were identified and nested real-time PCR analysis of these in the serial dilutions revealed an excellent linearity, recovery across three log scales. Also the sensitivity was very good. As little as 6.4 pg cell line DNA could be detected in a background 20 ng of normal plasma DNA, corresponding to DNA from 1-2 tumor cells circulating in 2 ml plasma. We are currently in the process of analyzing 25 CRC patients from which we have more than three years of follow-up and serial plasma samples collected every third month, starting the day before surgery and until month 36. The results from these analyses will be presented. In conclusion: We have shown that the combination of large insert paired end sequencing and copy number analysis readily identifies multiple tumor-specific rearrangements that can be targeted by sensitive and specific nested real-time PCR assays to quantify disease burden in plasma samples. Citation Format: Lone V. Schøler, Rune Thomsen, Heidi Tobiasen, Søren Vang, Hans Jørgen Nielsen, Søren Laurberg, Torben F. Ørntoft, Claus L. Andersen. Cancer-specific genomic rearrangements used to quantify disease burden in plasma from patients with colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4701. doi:10.1158/1538-7445.AM2013-4701

Prospective Clinical Validation of an Assay for Methylated SEPT9 DNA in Human Plasma as a Colorectal Cancer Screening Tool in Average Risk Men and Women ≥50 Years

Prospective Clinical Validation of an Assay for Methylated SEPT9 DNA in Human Plasma as a Colorectal Cancer Screening Tool in Average Risk Men and Women ≥50 Years

Diagnosing Schistosomiasis by Detection of Cell-Free Parasite DNA in Human Plasma

IntroductionSchistosomiasis (bilharzia), one of the most relevant parasitoses of humans, is confirmed by microscopic detection of eggs in stool, urine, or organ biopsies. The sensitivity of these procedures is variable due to fluctuation of egg shedding. Serological tests on the other hand do not distinguish between active and past disease. In patients with acute disease (Katayama syndrome), both serology and direct detection may produce false negative results. To overcome these obstacles, we developed a novel diagnostic strategy, following the rationale that Schistosoma DNA may be liberated as a result of parasite turnover and reach the blood. Cell-free parasite DNA (CFPD) was detected in plasma by PCR.Methodology/Principal FindingsReal-time PCR with internal control was developed and optimized for detection of CFPD in human plasma. Distribution was studied in a mouse model for Schistosoma replication and elimination, as well as in human patients seen before and after treatment. CFPD was detectable in mouse plasma, and its concentration correlated with the course of anti-Schistosoma treatment. Humans with chronic disease and eggs in stool or urine (n = 14) showed a 100% rate of CFPD detection. CFPD was also detected in all (n = 8) patients with Katayama syndrome. Patients in whom no viable eggs could be detected and who had been treated for schistomiasis in the past (n = 30) showed lower detection rates (33.3%) and significantly lower CFPD concentrations. The duration from treatment to total elimination of CFPD from plasma was projected to exceed one year.Conclusions/SignificancePCR for detection of CFPD in human plasma may provide a new laboratory tool for diagnosing schistosomiasis in all phases of clinical disease, including the capacity to rule out Katayama syndrome and active disease. Further studies are needed to confirm the clinical usefulness of CFPD quantification in therapy monitoring.

Is the quantity of circulatory cell-free DNA in human plasma and serum samples associated with gender, age and frequency of blood donations?

Circulatory cell-free DNA (cf-DNA) is increased in a variety of clinical pathologic conditions; therefore, these markers could be widely used as markers for detecting and monitoring several disorders. To better understand the biology of this molecule, we analysed the relationship between the level of circulatory cf-DNA and physiological parameters such as gender, age and frequency of blood donations. Paired plasma and serum samples were obtained from 87 blood donors and 50 healthy adults who had never donated blood. Cf-DNA was extracted from plasma and serum samples using the MagNA Pure LC Instrument. Quantity determination of circulatory cf-DNA was performed by TaqMan real-time PCR for the ubiquitous GAPDH gene. Our data showed that the concentration of cf-DNA in serum was about eightfold higher than that in plasma. Regarding the level of these circulatory species, no significant differences were observed between the age-matched men and women and gender-matched, different-age cohorts, except in women who were older than 60 years of age. Frequent blood donations did not increase the circulatory species. Circulatory cf-DNA in plasma and serum samples is not correlated with human gender and human age except in women who are older than 60 years of age. Frequent blood donation did not affect the quantity of circulatory cf-DNA. The explanation for the latter most likely is the short half-life time of free fetal DNA in maternal circulation.

Evaluation of different assays for the detection of parvovirus B19 DNA in human plasma

Human parvovirus B19 is a frequent contaminant of blood and plasma-derived medicinal products and transmission of this virus has been shown to occur through the administration of contaminated products. Inactivation of the virus has proved difficult and as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus B19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (NAT). We present data comparing the performance of two commercially available kits for the detection and quantitation of parvovirus B19 DNA using the LightCycler, and determine their applicability for the detection of recently discovered variants of the virus. Parvovirus B19 DNA was readily quantified using both commercial assays. However, one kit failed to detect any of the variant viruses. The second kit detected the variant viruses although there was a marked difference in the sensitivity of detection of the different virus genotypes. To improve the detection of these variant viruses a novel assay has been developed and data are presented to show its use for screening pooled plasma for the presence of parvovirus B19 DNA and the variants identified recently.

Detection of Cytomegalovirus DNA in Human Specimens by LightCycler PCR: Melting Point Analysis Is Mandatory To Detect Virus Strains with Point Mutations in the Target Sequence of the Hybridization Probes

In our recently published paper we described the application of the LightCycler technology for the detection of cytomegalovirus (CMV) DNA in human plasma and urine (1). We are now able to give further information gained since September 2000, when this PCR procedure was introduced as the standard assay for the detection of CMV DNA in our diagnostic laboratory. As of June 2001, we had tested 912 specimens using this assay. According to the described test protocol, 185 specimens (20.3%) were positive for CMV DNA. A melting point (Tm )o f 59.2°C was observed for the hybridization probes with PCR amplicons of all positive specimens. Another 12 specimens (1.3%) were negative in quantitative analysis but generated a significant peak, with a Tm of 53.1°C, in the melting point analysis. Gel electrophoresis revealed a distinct PCR product of about 250 bp with these specimens that corresponds to the targeted 254-bp amplicon. DNA sequencing of the PCR products with the decreased melting point confirmed the specific amplification of CMV DNA using these specimens as a template. The decline of the melting point is caused by a point mutation in position 630 of the CMV glycoprotein B gene (GenBank accession no. A13758), resulting in a shift from cytosine to thymidine. This strain variant is not included in the databases and causes a mismatch with position 5 of the LCRed 640-labeled acceptor fluorophore probe. However, the existence of further strain variations in the amplified region cannot be excluded. The melting point analysis allows the reliable detection of CMV strain variants harboring this point mutation. Specificity of the signal should be further confirmed by gel electrophoresis. Melting point analysis is mandatory to detect virus strain variants in the target sequence of the hybridization probes with the described LightCycler CMV PCR assay.

Plasma DNA as a marker of cancerous cell death. Investigations in patients suffering from lung cancer and in nude mice bearing human tumours.

Plasma DNA that circulates mainly as mononucleosomes is a cell death marker. Its significance and prognostic value in cancer as compared to other tumour markers was investigated in 68 patients hospitalised for lung cancers. Prognostic values of the various studied parameters were evaluated using the Cox's model. The cellular origin of plasma DNA was further investigated in nude mice transplanted with human lung adenocarcinoma. Plasma DNA concentrations were increased in cancer patients as compared to normal subjects (P < 0.01). They were higher in patients with extended (Stage 4) disease than in patients with limited stage disease (P < 0.05). Plasma DNA concentrations, serum lactate dehydrogenase activities and neuron-specific enolase concentrations were correlated all together in small cell lung carcinoma (SCLC) and in non-SCLC. Similar relationships were found between survival and each of these three cell death/tumour markers (P < 0.02-0.005). Plasma DNA from mice bearing human tumour hybridised with both mouse and human plasma DNA, while plasma DNA from endotoxin-injected mice hybridised only with mouse plasma DNA. In conclusion, in patients suffering from lung cancer, plasma DNA as well as LDH and NSE represent cell death markers that are correlated with survival. At a time when apoptosis pathways appear to be potential targets for cancer therapy, plasma DNA is a cell death/tumour marker that should be taken into account in studying the cancerous process in human diseases.