Clinical use of donor-derived cell-free DNA in kidney transplantation

Traditional monitoring of kidney transplant recipients for allograft dysfunction caused by rejection involves serial checks of serum creatinine with biopsy of the renal allograft if dysfunction is suspected.This approach is labor-intensive,invasive and costly.In addition,because this approach relies on a rise in serum creatinine above historical baselines,injury to the allograft can be extensive before this rise occurs.In an effort to address this,donor-derived cell-free DNA(dd-cf DNA)is being used with increasing frequency in the clinical setting as a means of diagnosing a rejection of the renal allograft early in the course.This can poten-tially allow for early intervention to minimize not only injury,but the intensity of antirejection therapy needed and the avoidance of side effects.Here,we will review the available methodology for the determination and quantification of dd-cf DNA,the data supporting its use in clinical practice and the limitations of this technology.

Cell-free DNA in the management of prostate cancer:Current status and future prospective

Objective:With the escalating prevalence of prostate cancer(PCa)in China,there is an urgent demand for novel diagnostic and therapeutic approaches.Extensive investigations have been conducted on the clinical implementation of circulating free DNA(cfDNA)in PCa.This review aims to provide a comprehensive overview of the present state of cfDNA as a biomarker for PCa and to examine its merits and obstacles for future clinical utilization.Methods:Relevant peer-reviewed manuscripts on cfDNA as a PCa marker were evaluated by PubMed search(2010-2022)to evaluate the roles of cfDNA in PCa diagnosis,prognosis,and prediction,respectively.Results:cfDNA is primarily released from cells undergoing necrosis and apoptosis,allowing for non-invasive insight into the genomic,transcriptomic,and epigenomic alterations within various PCa disease states.Next-generation sequencing,among other detection methods,enables the assessment of cfDNA abundance,mutation status,fragment characteristics,and epigenetic modifications.Multidimensional analysis based on cfDNA can facilitate early detection of PCa,risk stratification,and treatment monitoring.However,standardization of cfDNA detection methods is still required to expedite its clinical application.Conclusion:cfDNA provides a non-invasive,rapid,and repeatable means of acquiring multidimensional information from PCa patients,which can aid in guiding clinical decisions and enhancing patient management.Overcoming the application barriers of cfDNA necessitates increased data sharing and international collaboration.

Revolutionizing Non-Invasive Biomarker Discoveries: The Power of Methylation Screening Analysis in Cell-Free DNA Liquid Biopsy

Epigenetic changes of DNA, including methylation, have long been recognized as key indicators of various diseases, including aging, cancer, and neurological disorders. Biomarker discoveries based on distinct methylation patterns for both hypermethylation and hypomethylation lead the way in discovery of novel diagnosis and treatment targets. Many different approaches are present to detect the level of methylation in whole genome (whole genome bisulfite sequencing, microarray) as well as at specific loci (methylation specific PCR). Cell-free DNA (cf-DNA) found in body fluids like blood provides information about DNA methylation and serves as a less invasive approach for genetic screening. Cell-free DNA and methylation screening technologies, when combined, have the potential to transform the way we approach genetic screening and personalized therapy. These technologies can help enhance disease diagnostic accuracy and inform the development of targeted therapeutics by providing a non-invasive way for acquiring genomic information and identifying disease-associated methylation patterns. We highlight the clinical benefits of using cell-free DNA (cf-DNA) liquid biopsy analysis and available methylation screening technologies that have been crucial in identifying biomarkers for disease from patients using a non-invasive way. Powering such biomarker discoveries are various methods of cf-DNA methylation analysis such as Bisulfite Sequencing and most recently, Methylation-Specific Restriction Enzyme (MSRE-seq) Analysis, paving the way for novel epigenetic biomarker discoveries for more robust diagnosis such as early disease detection, prognosis, monitoring of disease progression and treatment response as well as discovery of novel drug targets.

Quick recovery and characterization of cell-free DNA in seminal plasma of normozoospermia and azoospermia： implications for non-invasive genetic utilities

We established a quick and reliable method for recovering cell-free seminal DNA （cfsDNA）, by using the binding-washing-elution procedure on the DNA purification column. Low variations （below 15%） among the triplicate values of cfsDNA quantity verified the reproducibility of our cfsDNA recovery method. Similar cfsDNA yield and size distribution between seminal plasma acquired by filtration and centrifugation confirmed the presence of cfsDNA. To investigate the general characterization of cfsDNA, the quantitation and size distribution of cfsDNA from normozoospermic and azoospermic semen were analyzed by real-time PCR and electrophoresis, respectively. CfsDNA concentration in semen with normozoospermia （n = 11） was 1.34 ± 0.65 μg ·mL^-1, whereas a higher cfsDNA concentration was observed in azoospermia （2.56 ± 1.43 μg ·mL^-1, n = 9）. The continuous distribution of DNA fragments ranging from -1 kb to 15 kb and a spectrum of multiples of 180-bp fragments were observed in each normozoospermic and azoospermic sample. Distinct characteristic DNA ladder fragmentations in some azoospermic samples implicated that cfsDNA originate partly from apoptotic cells. CfsDNAs of 36 selected azoospermic patients with known information of Y chromosome microdeletion were subjected to the same microdeletion analysis by multiplex PCR and PCR amplification of sY114 （1 450 bp）. All multiplex PCR reactions with cfsDNA amplified successfully and provided the same result as leukocyte DNA. PCR amplification of sY114 gave a 1 450-bp amplicon as expected. Our data suggested the potential use of cfsDNA in search of biomarker or diagnostic procedures.

Value of dynamic plasma cell-free DNA monitoring in septic shock syndrome: A case report

BACKGROUND Mortality due to septic shock is relatively high.The dynamic monitoring of plasma cell-free DNA(cfDNA)can guide the treatment of septic shock.CASE SUMMARY Herein,we present a typical case of septic shock syndrome caused by the bacilli Acinetobacter baumannii and Pantoea.The patient complained of abdominal pain,fever and chills upon admission to the Emergency Department.Marked decreases in white blood cells and procalcitonin(PCT)were observed after the patient received continuous renal replacement and extracorporeal membrane oxygenation.Plasma cfDNA levels were consistently high,peaking at 1366.40 ng/mL,as measured by a duplex real-time PCR assay with an internal control,which was developed as a novel method for the accurate quantification of cfDNA.The patient died of septic shock on HD 8,suggesting that cfDNA could be used to monitor disease progression more effectively than PCT and the other inflammatory factors measured in this case.CONCLUSION CfDNA may be a promising marker that complements other inflammatory factors to monitor disease progression in patients with septic shock.

Non-invasive Prenatal Gene Diagnosis: Progress through Cell-free Fetal DNA and RNA in Maternal Plasma and Urine

Non-invasive prenatal gene diagnosis has been developed rapidly in the recent years, and numerous medical researchers are focusing on it. Such techniques could not only achieve prenatal diagnosis accurately, but also prevent tangential illness in fetuses and thus, reduce the incidence of diseases. Moreover, it is non-invasive prenatal gene diagnosis that prevents potential threaten and danger to both mothers and fetuses. Therefore, it is welcomed by clinical gynecologist and obstetrian, researchers of medical genetics, and especially, pregnancies. This review article touches briefly on the advanced development of using cell-free DNA, RNA in maternal plasma and urine for non-invasive prenatal gene diagnosis.

Multiple z-Score Based Method for Noninvasive Prenatal Test Using Cell-Free DNA in Maternal Plasma

Objective: To improve the detecting accuracy of chromosomal aneuploidy of fetus by non-invasive prenatal testing (NIPT) using next generation sequencing data of pregnant women’s cell-free DNA. Methods: We proposed the multi-Z method which uses 21 z-scores for each autosomal chromosome to detect aneuploidy of the chromosome, while the conventional NIPT method uses only one z-score. To do this, mapped read numbers of a certain chromosome were normalized by those of the other 21 chromosomes. Average and standard deviation (SD), which are used for calculating z-score of each sample, were obtained with normalized values between all autosomal chromosomes of control samples. In this way, multiple z-scores can be calculated for 21 autosomal chromosomes except oneself. Results: Multi-Z method showed 100% sensitivity and specificity for 187 samples sequenced to 3 M reads while the conventional NIPT method showed 95.1% specificity. Similarly, for 216 samples sequenced to 1 M reads, Multi-Z method showed 100% sensitivity and 95.6% specificity and the conventional NIPT method showed a result of 75.1% specificity. Conclusion: Multi-Z method showed higher accuracy and robust results than the conventional method even at low coverage reads.

Detection of fusion gene in cell-free DNA of a gastric synovial sarcoma

Synovial sarcoma(SS) is genetically characterized by chromosomal translocation, which generates SYT-SSX fusion transcripts. Although SS can occur in any body part, primary gastric SS is substantially rare. Here we describe a detection of the fusion gene sequence of gastric SS in plasma cell-free DNA(cf DNA). A gastric submucosal tumor was detected in the stomach of a 27-year-old woman and diagnosed as SS. Candidate intronic primers were designed to detect the intronic fusion breakpoint and this fusion sequence was confirmed in intron 10 of SYT and intron 5 of SSX2 by genomic polymerase chain reaction(PCR) and direct sequencing. A locked nucleic acid(LNA) probe specificto the fusion sequence was designed for detecting the fusion sequence in plasma and the fusion sequence was detected in preoperative plasma cfD NA, while not detected in postoperative plasma cfD NA. This technique will be useful for monitoring translocation-derived diseases such as SS.

血浆cfDNA甲基化联合检测在肝癌诊断中的价值

目的 探讨血浆循环游离DNA(cfDNA)中GNB4、TCF24、Riplet、ACP1和TSPYL5基因甲基化对肝癌诊断的临床价值。方法 利用滑动窗口技术在公共数据库中筛选肝癌和正常组织间的差异甲基化标志物并分析其甲基化水平。从郑州大学第一附属医院收集肝癌、肝硬化组织和健康人白细胞样本,Sanger测序检测筛选出的标志物的甲基化水平,选出敏感度和特异度较好的标志物。收集24例肝癌、23例肝硬化和99例健康人血浆,通过甲基化特异性PCR检测上述标志物单独和组合时对肝癌的诊断性能。结果 5个标志物(GNB4、TCF24、Riplet、ACP1和TSPYL5)在肝癌样本中显著高甲基化。组织测序结果显示除TCF24外,其他标志物在肝硬化组织中的甲基化阴性率均大于80.0%。在血浆样本验证中,GNB4单独诊断肝癌的曲线下面积(AUC)最大,为0.765,敏感度为66.7%,特异度为91.8%。在多基因组合中,GNB4+TSPYL5的诊断性能最佳,AUC值为0.893,敏感度为83.3%,特异度为90.2%。结论 GNB4、Riplet、ACP1和TSPYL5甲基化可用于肝癌诊断,且联合诊断性能优于单一基因。

Circulating tumor DNA in liquid biopsy: Current diagnostic limitation

With the rapid development of science and technology,cell-free DNA(cfDNA)is rapidly becoming an important biomarker for tumor diagnosis,monitoring and prognosis,and this cfDNA-based liquid biopsy technology has great potential to become an important part of precision medicine.cfDNA is the total amount of free DNA in the systemic circulation,including DNA fragments derived from tumor cells and all other somatic cells.Tumor cells release fragments of DNA into the bloodstream,and this source of cfDNA is called circulating tumor DNA(ctDNA).cfDNA detection has become a major focus in the field of tumor research in recent years,which provides a new opportunity for non-invasive diagnosis and prognosis of cancer.In this paper,we discuss the limitations of the study on the origin and dynamics analysis of ctDNA,and how to solve these problems in the future.Although the future faces major challenges,it also con-tains great potential.

Donor-specific cell-free DNA as a biomarker in liver transplantation:A review

Due to advances in modern medicine,liver transplantation has revolutionised the prognosis of many previously incurable liver diseases.This progress has largely been due to advances in immunosuppressant therapy.However,despite the judicious use of immunosuppression,many liver transplant recipients still experience complications such as rejection,which necessitates diagnosis via invasive liver biopsy.There is a clear need for novel,minimally-invasive tests to optimise immunosuppression and improve patient outcomes.An emerging biomarker in this‘‘precision medicine’‘liver transplantation field is that of donorspecific cell free DNA.In this review,we detail the background and methods of detecting this biomarker,examine its utility in liver transplantation and discuss future research directions that may be most impactful.

Detection of HBV DNA integration in plasma cell-free DNA of different HBV diseases utilizing DNA capture strategy

The landscape of hepatitis B virus(HBV)integration in the plasma cell-free DNA(cfDNA)of HBV-infected patients with different stages of liver diseases[chronic hepatitis B(CHB),liver cirrhosis(LC),and hepatocellular carcinoma(HCC)]remains unclear.In this study,we developed an improved strategy for detecting HBV DNA integration in plasma cfDNA,based on DNA probe capture and next-generation sequencing.Using this optimized strategy,we successfully detected HBV integration events in chimeric artificial DNA samples and HBV-infected HepG2-NTCP cells at day one post infection,with high sensitivity and accuracy.The characteristics of HBV integration events in the HBV-infected HepG2-NTCP cells and plasma cfDNA from HBV-infected individuals(CHB,LC,and HCC)were further investigated.A total of 112 and 333 integration breakpoints were detected in the HepG2-NTCP cells and 22 out of 25(88%)clinical HBV-infected samples,respectively.In vivo analysis showed that the normalized number of support unique sequences(nnsus)in HCC was significantly higher than in CHB or LC patients(P values<0.05).All integration breakpoints are randomly distributed on human chromosomes and are enriched in the HBV genome around nt 1800.The majority of integration breakpoints(61.86%)are located in the gene-coding region.Both non-homologous end-joining(NHEJ)and microhomology-mediated end-joining(MMEJ)interactions occurred during HBV integration across the three different stages of liver diseases.Our study provides evidence that HBV DNA integration can be detected in the plasma cfDNA of HBV-infected patients,including those with CHB,LC,or HCC,using this optimized strategy.

血浆游离胎儿DNA浓度联合血清妊娠相关血浆蛋白-A、β-人绒毛膜促性腺激素检测对孕妇不良妊娠结局的预测价值

目的探讨血浆游离胎儿DNA(cff-DNA)浓度联合血清妊娠相关血浆蛋白-A(PAPP-A)和β-人绒毛膜促性腺激素(β-hCG)检测对孕妇不良妊娠结局的预测价值。方法选取2022年8月至2023年2月佛山复星禅诚医院收治的进行产检、唐氏筛查和无创产前DNA检查(NIPT)的195例孕妇作为研究对象。将发生不良妊娠结局的孕妇设为研究组(n=36),无不良妊娠结局的孕妇设为对照组(n=159)。检测两组血浆cff-DNA浓度、血清PAPP-A和β-hCG水平;采用Spearman分析血浆cff-DNA浓度、血清PAPP-A和β-hCG水平与不良妊娠结局的相关性;采用受试者工作特征(ROC)曲线分析血浆cff-DNA浓度、血清PAPP-A和β-hCG水平对不良妊娠结局的预测价值。结果与对照组比较,研究组血浆cff-DNA浓度和血清PAPP-A水平显著更低,血清β-hCG水平显著更高,差异具有统计学意义(P<0.05);Spearman分析结果显示,血浆cff-DNA浓度、血清PAPP-A和β-hCG水平与妊娠期高血压、子痫前期、早产、死胎、新生儿窒息和胎儿生长受限不良妊娠结局具有显著相关性(P<0.05);ROC曲线结果显示,血浆cff-DNA浓度单独预测孕妇不良妊娠结局的曲线下面积为0.807,截断值为11.82%;血清PAPP-A单独预测孕妇不良妊娠结局的曲线下面积为0.804,截断值为0.41;血清β-hCG单独预测孕妇不良妊娠结局的曲线下面积为0.815,截断值为2.56;预测效能比较结果显示,血浆cff-DNA浓度、血清PAPP-A和β-hCG水平联合预测孕妇不良妊娠结局的特异度(97.48%)和准确度(96.41%)均显著高于三者单独预测(P<0.05),误诊率(2.52%)显著低于三者单独预测(P<0.05)。结论血浆cff-DNA浓度联合血清PAPP-A、β-hCG检测对孕妇不良妊娠结局具有较高的预测价值。

Cell-free mitochondrial DNA quantification in ischemic stroke patients for non-invasive and real-time monitoring of disease status

BACKGROUND Acute ischemic stroke(AIS)is one of the major causes of the continuous increasing rate of global mortality due to the lack of timely diagnosis,prognosis,and management.This study provides a primitive platform for non-invasive and cost-effective diagnosis and prognosis of patients with AIS using circulating cellfree mitochondrial DNA(cf-mtDNA)quantification and validation.AIM To evaluate the role of cf-mtDNA as s non-invasive,and affordable tool for realtime monitoring and prognosticating AIS patients at disease onset and during treatment.METHODS This study enrolled 88 participants including 44 patients with AIS and 44 healthy controls with almost similar mean age group at stroke onset,and at 24 h and 72 h of treatment.Peripheral blood samples were collected from each study participant and plasma was separated using centrifugation.The cf-mtDNA concentration was quantified using nanodrop reading and validated through real-time quantitative polymerase chain reaction(RT-qPCR)of NADH-ubiquinone oxidoreductase chain 1(ND1)relative transcript expression levels.RESULTS Comparative analysis of cf-mtDNA concentration in patients at disease onset showed significantly increased levels compared to control individuals for both nanodrop reading,as well as ND1 relative expression levels(P<0.0001).Intergroup analysis of cf-mtDNA concentration using nanodrop showed significantly reduced levels in patients at 72 h of treatment compared to onset(P<0.01).However,RT-qPCR analysis showed a significant reduction at 24 h and 72 h of treatment compared to the disease onset(P<0.001).The sensitivity and specificity were relatively higher for RT-qPCR than nanodrop-based cfmtDNA quantification.Correlation analysis of both cf-mtDNA concentration as well as ND1 relative expression with National Institute of Health Stroke Scale score at baseline showed a positive trend.CONCLUSION In summary,quantitative estimation of highly pure cf-mtDNA provides a simple,highly sensitive and specific,non-invasive,and affordable approach for real-time monitoring and prognosticating AIS patients at onset and during treatment.

Detection of EGFR-SEPT14 fusion in cell-free DNA of a patient with advanced gastric cancer: A case report

BACKGROUND Gastric cancer is the fifth most diagnosed cancer worldwide and the third most common cause of cancer-related death.In recent decades,increasing application of next-generation sequencing has enabled detection of molecular aberrations,including fusions.In cases where tissue is difficult to obtain,cell-free DNA(cfDNA)is used for detecting mutations to identify the molecular profile of cancer.Here,we report a rare case of EGFR-SEPT14 fusion detected from cfDNA analysis in a patient with gastric cancer.CASE SUMMARY A 49-year-old female diagnosed with advanced gastric cancer in July 2019 received capecitabine and then combination chemotherapy of ramucirumab and paclitaxel,but ascites was detected.The therapy was switched to nivolumab,but disease progression was observed on a positron emission tomography/computed tomography scan in May 2020.Therapy was discontinued,and cfDNA nextgeneration sequencing was immediately evaluated.All genomic variants,including fusions,were analyzed from cfDNA.The following somatic alterations were detected from the patient’s cfDNA:an APC frameshift mutation(NM_000038.5:c.6579del,p.V2194fs)with variant allele frequency of 0.5%,an EGFR amplification with a copy number of 17.3,and an EGFR-SEPT14 fusion with variant allele frequency of 45.3%.The site of the fusion was exon 24 of EGFR fused to exon 10 of SEPT14.The fusion was in-frame and considered to be protooncogenic. Although the patient refused to continue therapy, we suggest thatEGFR-targeted therapies be tried in such future cases.CONCLUSIONThe expanded applications of the cfDNA assay may open a new horizon intreatment of patients with advanced gastric cancer.

The Prognostic Value of Cell-Free DNA in Advanced Non-Small-Cell Lung Cancer

Background: Cell-free DNA (cfDNA) holds promise as a tumor marker of clinical importance. We aimed to investigate the prognostic value of baseline cfDNA in non small-cell lung cancer (NSCLC). Material and Methods: During a three-year period, patients with newly diagnosed, previously untreated advanced NSCLC were included in a consecutive, prospective marker-trial. Plasma was isolated from a pre-treatment peripheral blood sample and the level of total cfDNA was measured by an in-house assay qPCR-method. The treatment comprised carboplatin (AUC 5) intravenously day 1), and vinorelbine (30 mg/m2 intravenously day 1 and 60 mg/m2 perorally day 8) q3w for a maximum of six cycles. The primary end-point was overall survival (OS). Secondary end-points were progression free survival (PFS) and overall response rate (ORR). Results: 245 patients were included and received a minimum of 1 cycle of chemotherapy (median 4). The median OS was 8.9 months, the median PFS by intention to treat 5.4 months and the ORR was 25%. The patients were divided into four groups based on quartiles of cfDNA and subsequently dichotomized by the 75th percentile revealing a significantly worse prognosis for patients in the upper 75th percentile (median OS 4.9 months) compared to patients with lower levels (10.0 months) (HR 2.1, 95%CI 1.4 - 3.1, p 0.0001). A multivariate analysis confirmed the independent prognostic value of cfDNA. A subgroup analysis of patients with high cfDNA and poor performance status (PS = 2) identified a group of patients with even worse prognosis (median OS 2.0 versus 9.1 months, HR 3.6, 95%CI 1.4 - 9.2, p 0.0001). Similar and significant results were found when comparing level of cfDNA and PFS. Conclusions: High pre-treatment level of cfDNA seems to have a strong prognostic impact in patients with newly diagnosed advanced NSCLC. Combined with PS it identifies a patient group with minimal or no benefit of chemotherapy.

Cell-free DNA liquid biopsy for early detection of gastrointestinal cancers:A systematic review

BACKGROUND Gastrointestinal tumors are among the most common cancer types,and early detection is paramount to improve their management.Cell-free DNA(cfDNA)liquid biopsy raises significant hopes for non-invasive early detection.AIM To describe current applications of this technology for gastrointestinal cancer detection and screening.METHODS A systematic review of the literature was performed across the PubMed database.Articles reporting the use of cfDNA liquid biopsy in the screening or diagnosis of gastrointestinal cancers were included in the analysis.RESULTS A total of 263 articles were screened for eligibility,of which 13 articles were included.Studies investigated colorectal cancer(5 studies),pancreatic cancer(2 studies),hepatocellular carcinoma(3 studies),and multi-cancer detection(3 studies),including gastric,oesophageal,or bile duct cancer,representing a total of 4824 patients.Test sensitivities ranged from 71% to 100%,and specificities ranged from 67.4% to 100%.Pre-cancerous lesions detection was less performant with a sensitivity of 16.9% and a 100% specificity in one study.Another study using a large biobank demonstrated a 94.9% sensitivity in detecting cancer up to 4 years before clinical symptoms,with a 61% accuracy in tissue-of-origin identification.CONCLUSION cfDNA liquid biopsy seems capable of detecting gastrointestinal cancers at an early stage of development in a non-invasive and repeatable manner and screening simultaneously for multiple cancer types in a single blood sample.Further trials in clinically relevant settings are required to determine the exact place of this technology in gastrointestinal cancer screening and diagnosis strategies.

Role of cell-free DNA for predicting incidence and outcome of patients with ischemic stroke

Early diagnosis and prognosis of ischemic stroke remains a critical challenge in clinical settings.A blood biomarker can be a promising quantitative tool to represent the clinical manifestations in ischemic stroke.Cell-free DNA(cfDNA)has recently turned out to be a popular circulating biomarker due to its potential relevance for diagnostic applications in a variety of disorders.Despite bright outlook of cfDNA in clinical applications,very less is known about its origin,composition,or function.Several recent studies have identified cell-derived mitochondrial components including mitochondrial DNA(mtDNA)in the extracellular spaces including blood and cerebrospinal fluid.However,the time course of alterations in plasma mtDNA concentrations in patients after an ischemic stroke is poorly understood.DNA is thought to be freed into the plasma shortly after the commencement of an ischemic stroke and then gradually decreased.However,the importance of cell-free mtDNA(cf-mtDNA)in ischemic stroke is still unknown.This review summarizes about the utility of biomarkers which has been standardized in clinical settings and role of cfDNA including cfmtDNA as a non-invasive potential biomarker of ischemic stroke.

Isolation of Cell-Free DNA from Seminal Fluid

Cell-flee DNA （cfDNA） is small short double stranded molecule that is also found in seminal fluid. It is a product ofapoptotic cells in different developmental stages during spermatogenesis. To final concentration of total cfDNA in semen contributesalso cfDNA secreted from living cells and cfDNA that is result of different diseases e.g. prostate cancer or infertility. Amendedconcentration （high or low） can be connected to prostate cancer or male infertility and can represent important non-invasive diagnosticbiomarker for detection and prognosis of these pathological conditions. In this paper, I will discuss different approaches for isolation ofcfDNA from seminal fluid, which includes selection of the samples, separation, isolation, extraction, purification and analysis. Today＇smost popular approach for isolation is the use of commercial kits based on selective binding and elution on silica-membrane technology,magnetic-bead technology or extraction with organic solvents and salting out procedure. Furthermore I will present that I tried to isolatecfDNA from semen with QIAamp DNA Mini Kit to confirm the presence of cell-free DNA in our samples. In the end I will describeproblems we are facing during cfDNA measurement which are mainly associated with low concentration of cfDNA in samples.

Emerging role of cell-free DNA in kidney transplantation

Monitoring kidney transplants for rejection conventionally includes serum creatinine,immunosuppressive drug levels,proteinuria,and donor-specific antibody(DSA).Serum creatinine is a late marker of allograft injury,and the predictive ability of DSA regarding risk of rejection is variable.Histological analysis of an allograft biopsy is the standard method for diagnosing rejection but is invasive,inconvenient,and carries risk of complications.There has been a long quest to find a perfect biomarker that noninvasively predicts tissue injury caused by rejection at an early stage,so that diagnosis and treatment could be pursued without delay in order to minimize irreversible damage to the allograft.In this review,we discuss relatively novel research on identifying biomarkers of tissue injury,specifically elaborating on donor-derived cell-free DNA,and its clinical utility.