Analyzes of cell-free nucleic acids (cfNAs) have shown huge potential in many biomedical applications, gradually entering several fields of research and everyday clinical care. Many biological properties of cfNAs can be informative to gain deeper insights into the function of the organism, such as their different types (DNA, RNAs) and subtypes (gDNA, mtDNA, bacterial DNA, miRNAs, etc.), forms (naked or vesicle bound NAs), fragmentation profiles, sequence composition, epigenetic modifications, and many others. On the other hand, the workflows of their analyzes comprise many important steps, from sample collection, storage and transportation, through extraction and laboratory analysis, up to bioinformatic analyzes and statistical evaluations, where each of these steps has the potential to affect the outcome and informational value of the performed analyzes. There are, however, no universal or standard protocols on how to exactly proceed when analyzing different cfNAs for different applications, at least according to our best knowledge. We decided therefore to prepare an overview of the available literature and products commercialized for cfNAs processing, in an attempt to summarize the benefits and limitations of the currently available approaches, devices, consumables, and protocols, together with various factors influencing the workflow, its processes, and outcomes.

Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes

Gaining a better understanding of the biological properties of cell-free DNA constitutes an important step in the development of clinically meaningful cell-free DNA-based tests. Since the in vivo characterization of cell-free DNA is complicated by the immense heterogeneity of blood samples, an increasing number of in vitro cell culture experiments, which offer a greater level of control, are being conducted. However, cell culture studies are currently faced with three notable caveats. First, the concentration of cell-free DNA in vitro is relatively low. Second, the median amount and size of cell-free DNA in culture medium varies greatly between cell types. Third, the amount and size of cell-free DNA in the culture medium of a single cell line fluctuates over time. Although these are interesting findings, it can also be a great source of experimental confusion and emphasizes the importance of method optimization and standardization. Therefore, in this study, we compared five commonly used cell-free DNA quantification methods, including quantitative polymerase chain reaction, Qubit Double-Stranded DNA High Sensitivity assay, Quant-iT PicoGreen Assay, Bioanalyzer High Sensitivity DNA assay, and NanoDrop Onec. Analysis of the resulting data, along with an interpretation of theoretical values (i.e. the theoretical detection and quantification limits of the respective methods), enables the calculation of optimal conditions for several important preanalytical steps pertaining to each quantification method and different cell types, including the (1) time-point at which culture medium should be collected for cell-free DNA extraction, (2) amount of cell culture supernatant from which to isolate cell-free DNA, (3) volume of elution buffer, and (4) volume of cell-free DNA sample to use for quantification.

https://journals.sagepub.com/doi/pdf/10.1177/1010428319866369

Comparison of methods for the quantification of cell-free DNA isolated from cell culture supernatant:

Comparison of clinical feasibility and oncological outcomes between video endoscopic and open inguinal lymphadenectomy for penile cancer: A systematic review and meta-analysis.

Background Inguinal lymph node dissection (ILND) is an essential step in both treatment and staging of several malignancies including penile and vulvar cancers. Various open, video endoscopic, and robotic-assisted techniques have been utilized so far. In this review, we aim to describe available minimally invasive surgical approaches for ILND, and review their outcomes and complications. Methods The PubMed, Wiley Online Library, and Science Direct databases were reviewed in February 2020 to find relevant studies published in English within 2000–2020. Findings There are different minimally invasive platforms available to accomplish dissection of inguinal nodes without jeopardizing oncological results while minimizing postoperative complications. Video Endoscopic Inguinal Lymphadenectomy and Robotic Video Endoscopic Inguinal Lymphadenectomy are safe and achieve the same nodal yield, a surrogate metric for oncological adequacy. When compared to open technique, Video Endoscopic Inguinal Lymphadenectomy and Robotic Video Endoscopic Inguinal Lymphadenectomy may offer faster postoperative recovery and fewer postoperative complications including wound dehiscence, necrosis, and infection. The relatively high rate and severity of postoperative complications hinders utilization of recommended ILND for oncologic indications. Minimally invasive approaches, using laparoscopic or robotic-assisted platforms, show some promise in reducing the morbidity of this procedure while achieving adequate short and intermediate term oncological outcomes.

Inguinal lymph node dissection in the era of minimally invasive surgical technology

Inguinal lymph node dissection (ILND) is an essential step in both treatment and staging of several malignancies including penile and vulvar cancers. Various open, video endoscopic, and robotic-assisted techniques have been utilized so far. In this review, we aim to describe available minimally invasive surgical approaches for ILND, and review their outcomes and complications.The PubMed, Wiley Online Library, and Science Direct databases were reviewed in February 2020 to find relevant studies published in English within 2000-2020.There are different minimally invasive platforms available to accomplish dissection of inguinal nodes without jeopardizing oncological results while minimizing postoperative complications. Video Endoscopic Inguinal Lymphadenectomy and Robotic Video Endoscopic Inguinal Lymphadenectomy are safe and achieve the same nodal yield, a surrogate metric for oncological adequacy. When compared to open technique, Video Endoscopic Inguinal Lymphadenectomy and Robotic Video Endoscopic Inguinal Lymphadenectomy may offer faster postoperative recovery and fewer postoperative complications including wound dehiscence, necrosis, and infection. The relatively high rate and severity of postoperative complications hinders utilization of recommended ILND for oncologic indications. Minimally invasive approaches, using laparoscopic or robotic-assisted platforms, show some promise in reducing the morbidity of this procedure while achieving adequate short and intermediate term oncological outcomes. 

Objective: To compare the efficacy and safety of video endoscopic inguinal lymphadenectomy through a hypogastric subcutaneous approach (VEIL-H) with a leg subcutaneous approach (VEIL-L) in...

Comparative Study of Video Endoscopic Inguinal Lymphadenectomy Through a Hypogastric vs Leg Subcutaneous Approach for Penile Cancer.

BACKGROUND Postpartum posterior reversible encephalopathy syndrome (PRES) is not uncommon. Its mechanisms and risk factors are not clear. CASE SUMMARY A 28-year-old woman underwent cesarean section but had inadvertent dural puncture during epidural anesthesia. To manage the symptoms of intracranial hypotension, crystalloid fluid was infused. However, the patient developed postpartum preeclampsia and PRES. The patient was treated with diazepam and dehydration therapy. The signs of cerebral lesions on magnetic resonance imaging disappeared on postpartum day 7. CONCLUSION Postpartum preeclampsia and PRES can develop concomitantly. Treating postdural puncture headaches with infusion of crystalloid fluid may precipitate the development of PRES.

https://doi.org/10.12998/wjcc.v10.i28.10332

Postpartum posterior reversible encephalopathy syndrome secondary to preeclampsia and cerebrospinal fluid leakage: A case report and literature review

Background Due to high nuclease activity and complex contents in urine, urinary cell-free DNA (ucfDNA) was prone to degrade. So, we developed standardized urine collection tube (UCT) to prevent ucfDNA degradation and simultaneously maintain urinary cells in their original form during the sample collection process, ensuring stabilization of the original proportion and integrity of ucfDNA. Methods Urine samples were collected from bladder cancer patients and divided into 10-mL normal tubes and 10-mL UCTs, respectively, and kept at ambient temperature. Urine supernatant was separated by centrifuging, and ucfDNA was extracted. Then ucfDNA was quantified by quantitative real-time polymerase chain reaction. UcfDNA fragments distribution was analyzed by Agilent 2200, and the frequency of specific mutations of urinary system disease was detected by next-generation sequencing method. Results Urine collected into UCTs showed no statistically significant changes in their original proportion and integrity of ucfDNA up to 7 days at ambient temperature and also ucfDNA fragments were maintained well. Conversely, urine collected into normal tubes was observed an obviously decline in their original proportion of ucfDNA and ucfDNA fragments changed greatly. The △% of allele fraction (AF) for specific genes of ucfDNA from UCTs was lower than from normal tubes by 3.7-fold. Conclusion Using UCTs, they can maximally keep the original proportion and integrity of ucfDNA and stabilize urinary cells and minimize the background noise caused by urinary cellular DNA releasing, it will be help to open the door of next-generation noninvasive liquid biopsy applications utilizing urine.

/pdf/new-method-to-preserve-the-original-proportion-and-integrity-szyux5hdaf.pdf

New method to preserve the original proportion and integrity of urinary cell-free DNA.

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Comparative Study of Video Endoscopic Inguinal Lymphadenectomy Through a Hypogastric vs Leg Subcutaneous Approach for Penile Cancer.

Postpartum posterior reversible encephalopathy syndrome secondary to preeclampsia and cerebrospinal fluid leakage: A case report and literature review

New method to preserve the original proportion and integrity of urinary cell-free DNA.