Extraction of high quality and high yield RNA from frozen EDTA blood.

Peripheral blood RNA profiling, which can reveal systemic changes in gene expression and immune responses to disease onset and progression, is a powerful tool for diagnosis and biomarker discovery. This technique usually requires high quality RNA, which is only obtainable from fresh blood, or frozen blood that has been collected in special RNA-stabilisation systems. The current study aimed to develop a novel protocol to extract high quality RNA from frozen blood that had been collected in the conventional EDTA tubes. We determined that thawing EDTA blood in the presence of cell lysis/RNA stabilisation buffers (Paxgene or Nucleospin) significantly improved RNA quality (RIN) from below 5 to above 7, which to date has not been shown possible. The EDTA-Nucleospin protocol resulted in 5 times higher yield than the EDTA-Paxgene-PreAnalytix method. The average RIN and mRNA expression levels of five different genes including 18 s, ACTB, MCP1, TNFa and TXNIP using this protocol were also indifferent to those from Paxgene blood, suggesting similar RNA quality and blood transcriptome. Moreover, the protocol allows DNA to be extracted simultaneously. In conclusion, we have developed a practical and efficient protocol to extract high quality, high yield RNA from frozen EDTA blood.

Alkylated EDTA potentiates antibacterial photodynamic activity of protoporphyrin.

Antibiotic resistance has garnered significant attention due to the scarcity of new antibiotics in development. Protoporphyrin IX (PpIX)-mediated photodynamic therapy shows promise as a novel antibacterial strategy, serving as an alternative to antibiotics. However, the poor solubility of PpIX and its tendency to aggregate greatly hinder its photodynamic efficacy. In this study, we demonstrate that alkylated EDTA derivatives (aEDTA), particularly C14-EDTA, can enhance the solubility of PpIX by facilitating its dispersion in aqueous solutions. The combination of C14-EDTA and PpIX exhibits potent antibacterial activity against Staphylococcus aureus (S. aureus) when exposed to LED light irradiation. Furthermore, this combination effectively eradicates S. aureus biofilms, which are known to be strongly resistant to antibiotics, and demonstrates high therapeutic efficacy in an animal model of infected ulcers. Mechanistic studies reveal that C14-EDTA can disrupt PpIX crystallization, increase bacterial membrane permeability and sequester divalent cations, thereby improving the accumulation of PpIX in bacteria. This, in turn, enhances reactive oxygen species (ROS) production and the antibacterial photodynamic activity. Overall, this effective strategy holds great promise in combating antibiotic-resistant strains.

Effect of photodynamic therapy with 5-aminolevulinic acid and EDTA-2Na against mixed infection of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

BACKGROUND: The increasing abundance of drug-resistant bacteria is a global threat. Photodynamic therapy is an entirely new, non-invasive method for treating infections caused by antibiotic-resistant strains. We previously described the bactericidal effect of photodynamic therapy on infections caused by a single type of bacterium. We showed that gram-positive and gram-negative bacteria could be killed with 5-aminolevulic acid and 410 nm light, respectively. However, clinically, mixed infections are common and difficult to treat. OBJECTIVE: We investigated the bactericidal effects of photodynamic therapy on mixed infections of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. METHODS: We compared bacterial growth with and without photodynamic therapy in vitro. Then, in vivo, we studied mixed infections in a mouse skin ulcer model. We evaluated the rates of ulcer area reduction and transitions to healing in treated and untreated mice. In addition, a comparison was made between PDT and existing topical drugs. RESULTS: We found that photodynamic therapy markedly reduced the growth of both methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, in culture, and it reduced the skin ulcer areas in mice. PDT was also more effective than existing topical medicines. CONCLUSION: This study showed that photodynamic therapy had antibacterial effects against a mixed infection of gram-positive and gram-negative bacteria, and it promoted skin ulcer healing. These results suggested that photodynamic therapy could be effective in both single- and mixed-bacterial infections.

Structural and functional basis of bacteriophage K64-ORF41 depolymerase for capsular polysaccharide degradation of Klebsiella pneumoniae K64.

Capsule polysaccharide is an important virulence factor of Klebsiella pneumoniae (K. pneumoniae), which protects bacteria against the host immune response. A promising therapeutic approach is using phage-derived depolymerases to degrade the capsular polysaccharide and expose and sensitize the bacteria to the host immune system. Here we determined the cryo-electron microscopy (cryo-EM) structures of a bacteriophage tail-spike protein against K. pneumoniae K64, ORF41 (K64-ORF41) and ORF41 in EDTA condition (K64-ORF41EDTA), at 2.37 Å and 2.50 Å resolution, respectively, for the first time. K64-ORF41 exists as a trimer and each protomer contains a ß-helix domain including a right-handed parallel ß-sheet helix fold capped at both ends, an insertion domain, and one ß-sheet jellyroll domain. Moreover, our structural comparison with other depolymerases of K. pneumoniae suggests that the catalytic residues (Tyr528, His574 and Arg628) are highly conserved although the substrate of capsule polysaccharide is variable. Besides that, we figured out the important residues involved in the substrate binding pocket including Arg405, Tyr526, Trp550 and Phe669. This study establishes the structural and functional basis for the promising phage-derived broad-spectrum activity depolymerase therapeutics and effective CPS-degrading agents for the treatment of carbapenem-resistant K. pneumoniae K64 infections.

Effect of sodium hypochlorite, ethylenediaminetetraacetic acid, and dual-rinse irrigation on dentin adhesion using an etch-and-rinse or self-etch approach.

The aim was to investigate the influence of endodontic irrigation solutions and protocols on the micro-tensile bond strength (µTBS) to dentin using an etch-and-rinse (ER) or self-etch (SE) adhesive approach. Eighty extracted human molars were ground to dentin. After pretreating for 27 min (21 min-3 min-3 min) with five different endodontic irrigation protocols (Group 1: NaOCl-EDTA-NaOCl; Group 2: NaOCl-NaOCl-EDTA; Group 3: NaOCl-NaCl-NaOCl; Group 4: Dual Rinse-Dual Rinse-Dual Rinse; Group 5: NaCl-NaCl-NaCl), an ER (Optibond FL, Kerr) or a SE (Clearfil SE Bond, Kuraray) adhesive system was applied. After light-curing, composite build-ups were made and cut into dentin-composite sticks. µTBS and failure modes were analyzed. Nonparametric statistical analyses (α = 0.05) were performed for comparison of the five groups within each type of adhesive as well as between the two adhesive systems used. The use of an ER instead of a SE adhesive system resulted in significantly higher µTBS for all irrigation protocols except for group 1 (NaOCl-EDTA-NaOCl) and 2 (NaOCl-NaOCl-EDTA). A statistical difference between the five different endodontic irrigation protocols was only found within the SE adhesive group, where group 1 (NaOCl-EDTA-NaOCl) achieved highest values. The use of an ER adhesive system cancels out the effect of the endodontic irrigation solution. The highest µTBS was achieved when using a NaOCl-EDTA-NaOCl-irrigation protocol in combination with Clearfil SE Bond, which shows that the selection of the endodontic irrigation should match the corresponding SE adhesive system.

Pregnancy and obstetric outcomes of dichorionic triamniotic triplet pregnancies with selective foetal reduction after assisted reproductive technology.

BACKGROUND: It is generally beneficial and recommended that dichorionic triamniotic (DCTA) triplet pregnancies be reduced to monochorionic (MC) twin or singleton pregnancies after assisted reproductive technology (ART). However, some infertile couples still have a firm desire to retain twins. For this reason, the best foetal reduction strategies need to be available for infertile couples and clinicians. Given that data on the elective reduction of DCTA triplet pregnancies to twin pregnancies are scarce, we investigated the outcomes of elective reduction of DCTA triplet pregnancies through the retrospective analysis of previous data. METHOD: Patients with DCTA triplet pregnancies who underwent elective foetal reduction between January 2012 and June 2020 were recruited. A total of 67 eligible patients with DCTA triplet pregnancies were divided into two groups: a DCTA-to-dichorionic diamniotic (DCDA) twin group (n = 38) and a DCTA-to-monochorionic diamniotic (MCDA) twin group (n = 29); the basic clinical data of the two groups were collected for comparison. RESULTS: Compared with the DCDA-to-MCDA twin group, the DCTA-to-DCDA twin group had lower rates of complete miscarriage (7.89% versus 31.03%, p = 0.014), early complete miscarriage (5.26% versus 24.14%, p = 0.034), late preterm birth (25.71% versus 65.00%, p = 0.009) and very low birth weight (0 versus 11.11%, p = 0.025). In addition, the DCTA-to-DCDA twin group had higher rates of full-term delivery (65.71% versus 25.00%, p = 0.005), survival (92.11% versus 68.97%, p = 0.023), and taking the babies home (92.11% versus 68.97%, p = 0.023) than did the DCTA-to-MCDA twin group. In terms of neonatal outcomes, a significantly greater gestational age (38.06 ± 2.39 versus 36.28 ± 2.30, p = 0.009), average birth weight (3020.77 ± 497.33 versus 2401.39 ± 570.48, p < 0.001), weight of twins (2746.47 ± 339.64 versus 2251.56 ± 391.26, p < 0.001), weight of the larger neonate (2832.94 ± 320.58 versus 2376.25 ± 349.95, p < 0.001) and weight of the smaller neonate (2660.00 ± 345.34 versus 2126.88 ± 400.93, p < 0.001) was observed in the DCTA-to-DCDA twin group compared to the DCTA-to-MCDA twin group. CONCLUSION: The DCTA-to-DCDA twin group had better pregnancy and neonatal outcomes than the DCTA-to-MCDA twin group. This reduction approach may be beneficial for patients with dichorionic triamniotic triplet pregnancies who have a strong desire to have DCDA twins.

Multianticoagulant Pseudothrombocytopenia in a Patient with Primary Carcinoma of the Liver with Hypersplenism.

BACKGROUND: Pseudothrombocytopenia (PTCP) is a relatively rare phenomenon in vitro, the mechanism is not completely clear, and there is no unified solution for it. How to identify and solve PTCP accurately is a challenge for laboratory personnel. METHODS: According to the patient's clinical manifestations, thrombocytopenia caused by hypersplenism was excluded. PTCP was confirmed by platelet volume histograms, scattergrams and platelet clumps on the blood smears. Commonly used alternative anticoagulants such as sodium citrate or heparin were used for platelet counting. The corrective effect of the platelet count was not good, so non-anticoagulant blood was collected and tested immediately, and blood smears were used to count platelets manually. RESULTS: The PTCP of the patient could not be solved using sodium citrate and heparin anticoagulation. By collecting non-anticoagulant blood and testing immediately, the platelet count returned to normal (180 x 109/L), which is consistent with the results of manual counting on the patient's blood smears (175 x 109/L). CONCLUSIONS: When PTCP is confirmed, commonly used alternative anticoagulants can be used. If these do not work, non-anticoagulant blood can be collected and tested immediately, and blood smears can be used to count platelets manually.

Five days serum glucose stability at room-temperature in centrifuged fast-clotting serum tubes and the comparability with glucose in heparin-plasma and plasma containing citrate-stabilizer.

Glucose measurement plays a central role in the diagnosis of gestational diabetes mellitus (GDM). Because of earlier reports of overestimation of glucose in the widely used tubes containing granulated glycolysis inhibitor, the study assessed the performance of fast-clotting serum tubes as an alternative sample for the measurement of glucose. Glucose concentration in fast-clotting serum was compared to lithium-heparin plasma placed in an ice-water slurry after sample collection and glucose stability at room-temperature was studied. Blood samples from 30 volunteers were drawn in four different types of tubes (serum separator tubes, fast-clotting serum tubes, lithium-heparin tubes and sodium fluoride, EDTA and a citrate buffer (NaF-EDTA-citrate) tubes, all from Greiner Bio-One). Lithium-heparin tubes were placed in an ice-water slurry until centrifugation in accordance with international recommendations and centrifuged within 10 min. After centrifugation, glucose was measured in all tubes (timepoint T0) and after 24, 48, 72, 96 and 120 h of storage at 20-22 °C. NaF-EDTA-citrate plasma showed significant overestimation of glucose concentration by 4.7% compared to lithium-heparin plasma; fast-clotting serum showed glucose concentrations clinically equivalent to lithium-heparin plasma. In fast-clotting serum tubes, mean bias between glucose concentration after 24, 48, 72, 96 and 120 h and T0 was less than 2.4%. All individual differences compared to T0 were less than 6.5%. The results fulfill the acceptance criteria for sample stability based on biological variation. Fast-clotting serum tubes can be an alternative for the measurement of glucose in diagnosis and management of GDM and diabetes mellitus, especially when prolonged transportation is necessary.

An innovative aluminium foil electrode modified with Al nanoparticles and EDTA for lead detection in biological samples.

This study presents a novel Aluminium foil-based electrode characterized by its affordability, flexibility, and ease of modification with carboxylic moiety-containing organic molecules. Upon foil modification with Aluminium nanoparticles and EDTA (AlNP-EDTA/AlE), the modified electrode exhibits remarkable activity in the oxidation of lead at potentials around -0.4 V. The lead signal is derived from the oxidation of lead deposited on the electrode surface using anodic stripping voltammetry (ASV). The addition of EDTA to AlNP/AlE increased the anodic peak current of lead by more than 500 %. The surface characterization of the electrode was performed by scanning electron microscopy (SEM) and infrared spectroscopy (IR), while its electroactive properties were evaluated by cyclic voltammetry (CV) and electrical impedance spectroscopy (EIS). Optimal operating parameters include pH 2.1, square-wave voltammetry (SWV) with an accumulation time of 60 s and an accumulation potential of -0.8 V. A low detection limit of 0.20 µmol/L and a relative standard deviation (RSD) of 3.0 % were achieved using five different electrodes. The effectiveness of AlNP-EDTA/AlE was further demonstrated with consistent results in biological samples spiked with Pb.

Assessment of the Smear Layer Removal Efficacy of Three Different Agents on Periodontally Compromised Tooth: An In Vitro Study.

AIM: The purpose of the present study was to evaluate the smear layer removal efficacy of three various agents on periodontally compromised tooth. MATERIALS AND METHODS: The current study included 75 molar teeth that were extracted due to periodontal disease. After that, 25 samples were randomly assigned using a simple random technique to the three different agent groups, group A: Scaling and root planing (SRP) and application of SofScale agent, group B: SRP and application of QMix agent, group C: SRP and application of MTAD agent. Using a diamond circular saw, the treated portions were divided into horizontal and vertical halves. All samples were viewed under Scanning Electron Microscope. Every tooth was focused at the coronal third, middle third, and apical third portion with a magnification of 1000×. Data were recorded and statistically analyzed. RESULTS: The smear layer removal efficacy was more in the QMix agent (3.06 ± 0.04) group followed by MTAD agent (3.28 ± 0.09) and SofScale agent (4.14 ± 0.10) group on the root surface. On intra group comparison, there was a statistically significant difference found in all the intra group agents with all the three levels. On inter group evaluation, at coronal third, there was no significant difference found between the different agents. There was a significant difference found between the different agents at middle and coronal third. CONCLUSION: On conclusion, the current investigation found that, the root surfaces treated with QMix shown a greater ability to remove smear layers compared to tooth surfaces treated with MTAD and SofScale agent. CLINICAL SIGNIFICANCE: Conventional therapies such as SRP effectively eliminate calculus, plaque, and necrosed cementum; nevertheless, they leave behind a smear layer that could impede normal healing. In an effort to overcome this, root conditioning agents were applied on the root surface to remove the smear layer. The traditional root conditioning agents such as citric acid have certain disadvantages, though, such as an acidic pH that could harm the root surface. As a result, researchers have been looking for biocompatible root conditioning treatments that are more effective. How to cite this article: Singh DK, BS Raj H, Soans CR, et al. Assessment of the Smear Layer Removal Efficacy of Three Different Agents on Periodontally Compromised Tooth: An In Vitro Study. J Contemp Dent Pract 2024;25(2):156-159.

Understanding boron toxicity in aquatic plants (Salvinia natans and Lemna minor) in the presence and absence of EDTA.

Even though boron is a widely used element in various industries and a contributor to water pollution worldwide, few studies have examined the toxicity of boron in aquatic plants. EDTA is used to maintain aquatic plants cultures, however it is possible to modify the toxicity of metals. The objective of this study is to assess the toxicity of boron in aquatic plants and explore the impact of EDTA presence on the resulting toxic responses. Floating watermoss Salvinia natans and duckweed Lemna minor were exposed to concentrations ranging from 5 to 100 mg/L for 7 days and 1 to 60 mg/L for 3 days, respectively. Growth and photosynthetic activity parameters were investigated in the presence and absence of EDTA. Growth inhibitions in both aquatic plants were observed in a concentration-dependent manner, irrespective of the presence or absence of EDTA. For instance, based on the specific growth rate (leaves coverage), EC10 values for S. natans were calculated as 12.7 (9.9-15.3) mg/L and 8.0 (5.8-10.3) mg/L with and without EDTA, respectively. In the case of L. minor, EC10 values were calculated as 1.3 (0.8-1.89) mg/L and 2.0 (0.4-4.3) mg/L with EDTA without EDTA, respectively. Significant effects were also observed on the photosynthetic capacity, however there was no change in the increase of boron concentration. Generally, negligible effects of EDTA to the toxicity of boron were observed in the present study. By comparing toxicity results based on the presence and absence of EDTA, which is an essential element in the test medium, the results of this study are expected to be utilized for the ecological risk assessment of boron in aquatic ecosystems.

Business continuity plan in the management and operations of hospitals: First experience to certify the PDTA processes with the requirements defined by ISO 22301:2019 in emergency medical services.

BACKGROUND: A business continuity plan (BCP) facilitates the performance of primary functions during emergencies or other situations that can disrupt normal operations. If risk management is done analytically, a business impact analysis (BIA), according to ISO 22301 certification, makes it possible to define the best strategy for supporting the company's assets and image, optimizing the operational efficiency of service recovery and redesigning spaces for health. Since 2015, our healthcare company has embarked on a certification process for all sectors and activities through the implementation and development of diagnostic and therapeutic paths for operational diagnos-tic-therapeutic-assistance pathways (PDTAs). PDTA processes are all certified by the ISO 9001:2015 management system hospital. Our hospital is the first healthcare company to have obtained ISO 22301:2019 certification concerning PDTA processes, offering patients the highest standards of quality and safety of care in emergency medical services. METHODS: The formal BCP process includes several steps prior to the creation of a BCP: create a BCP team, conduct a BIA, determine the continuity plan by using the results of the analyses, and conduct training and exercises to educate staff and improve the BCP. RESULTS: From the BIA analysis, the team identified the time-employee PDTAs in company paths under emergency and urgency: acute ST-elevation myocardial infarction (STEMI), TRAUMA, and STROKE, providing for a planning path that took advantage of the duration of approximately 12 months. This path included the creation of structural procedures, the redefinition and updating of the PDTA in the light of the BCP, the preparation of exercises aimed at guaranteeing the business continuity objectives, and, finally, the awareness of our stakeholders regarding its correct application. CONCLUSIONS: With a business continuity management (BCM) system, companies take preventative measures to ensure they can start operations again quickly in an emergency. An exhaustive BIA in a hospital company reveals the effects when processes fail, how critical each process is for the company, and the amount of time required to get up and running again, thus providing the organization with important information for risk management. The measures for handling risks derived from this analysis are incorporated into a BCM system where the emergency plans are defined, too, so that business operations continue even in the event of an emergency.

Removal of toxic metals from sewage sludge by EDTA and hydrodynamic cavitation and use of the sludge as fertilizer.

Sewage sludge (SS) is rich in plant nutrients, including P, N, and organic C, but often contains toxic metals (TMs), which hinders its potential use in agriculture. The efficiency of removal of TMs by washing with ethylenediamine tetraacetate (EDTA), in combination with hydrodynamic cavitation (HC) and the usability of washed sewage sludge as fertilizer were investigated. The environmental risk was assessed. During 8 wash batches an average 35, 68, 47 and 45 % of Pb, Zn, Cd and Cu, respectively, as well as 22 and 5 % Mn and Fe were removed from the SS. The process solutions and EDTA were recycled at a pH gradient of 12.5-2, which was achieved by adding quicklime (CaO) and then acidification by H2SO4, so that no wastewater was produced, only solid waste (ReSoil® method). The quality of the recycled process solutions (they remained unsaturated with salts) and the efficiency of the washing process were maintained across all batches. On average, 46 % of the EDTA was lost during the process and was replenished. The initial leachability of EDTA-mobilized Pb, Zn, Cu, Cr and Fe remaining in the washed SS increased 6-, 17-, 3-, 11- and 11-fold, respectively, but not to hazardous levels except for Zn. After washing, P and K remained in the SS, plant-available P increased 3.3-fold, while total N and C were reduced by 20.28 and 2.44 %, respectively. Washed SS was used as fertilizer in the pot experiment. The yield of Brassica juncea did not improve, the uptake of TMs by the plants and the leaching of TMs from the soil were minimal. Our study highlighted the drawbacks and potential feasibility of the new SS washing method.

Theranostic Agent Targeting Bone Metastasis: A Novel [68Ga]Ga/[177Lu]Lu-DOTA-HBED-bisphosphonate.

Bone metastasis in cancer patients is a major disease advancement for various types of cancer. Previously, [68Ga]Ga-HBED-CC-bisphosphonate ([68Ga]Ga-P15-041) showed excellent bone uptake and efficient detection of bone metastasis in patients. To accommodate different α- or ß--emitting metals for radionuclide therapy, a novel DOTA-HBED-CC-bisphosphonate (P15-073, 1) was prepared and the corresponding [68Ga]Ga-1 and [177Lu]Lu-1 were successfully synthesized in high yields and purity. Gallium-68 conjugation to HBED-CC at room temperature and lutetium-177 conjugation to DOTA at 95 °C were verified in model compounds through secondary mass confirmation. These bisphosphonates, [68Ga]Ga-1 and [177Lu]Lu-1, displayed high binding affinity to hydroxyapatite in vitro. After an iv injection, it showed excellent uptake in the spine of normal mice, and micro-PET/CT imaging of nude mice model of bone metastasis showed high bone uptake in tumor tissue. The results indicated that [68Ga]Ga/[177Lu]Lu-1 holds promise as a theranostic radioligand agent for managing cancer bone metastases.

COMPARING THE EFFECTS OF DIPOTASSIUM ETHYLENEDIAMINETETRAACETIC ACID AND LITHIUM HEPARIN ON HEMATOLOGIC VALUES IN BLANDING'S TURTLES (EMYDOIDEA BLANDINGII), PAINTED TURTLES (CHRYSEMYS PICTA), AND COMMON SNAPPING TURTLES (CHELYDRA SERPENTINA).

Hematology is a routine component of clinical management in veterinary patients. Anticoagulant choice can profoundly influence morphologic assessment of erythrocytes, leukocytes, thrombocytes, and their subsequent quantification. Previous chelonian studies suggest that lithium heparin (LH) is a superior anticoagulant due to hemolysis resulting from dipotassium ethylenediaminetetraacetic acid (dEDTA) in some species. The aim of this study was to compare the effects of dEDTA and LH on hematologic values in Blanding's turtles (Emydoidea blandingii, n = 35), painted turtles (Chrysemys picta, n = 34), and common snapping turtles (Chelydra serpentina, n = 36). We collected samples from free-ranging turtles and immediately divided whole blood into LH and dEDTA tubes. Packed cell volume, total solids, erythrocyte sedimentation rate, white blood cell counts, and differential leukocyte counts were determined. Hemolysis was observed macro- and microscopically in dEDTA samples from painted turtles and common snapping turtles. Packed cell volume and heterophil:lymphocyte was lower and erythrocyte sedimentation rate was higher in LH samples from painted turtles (p, 0.05). In snapping turtles, the PCV, number of monocytes, and number of eosinophils was lower in LH samples (p, 0.05). In Blanding's turtles, the number of eosinophils and basophils was higher in LH samples, while heterophil counts were lower (p, 0.05). Anticoagulant choice created constant and proportional bias for multiple analytes in a species-dependent fashion. LH is the recommended anticoagulant for hematology in painted turtles and common snapping turtles. Either LH or dEDTA may be used in Blanding's turtles, though anticoagulant-specific reference intervals may be necessary.

Assay Validation of Cell-Free DNA Shallow Whole-Genome Sequencing to Determine Tumor Fraction in Advanced Cancers.

Blood-based liquid biopsy is increasingly used in clinical care of patients with cancer, and fraction of tumor-derived DNA in circulation (tumor fraction; TFx) has demonstrated clinical validity across multiple cancer types. To determine TFx, shallow whole-genome sequencing of cell-free DNA (cfDNA) can be performed from a single blood sample, using an established computational pipeline (ichorCNA), without prior knowledge of tumor mutations, in a highly cost-effective manner. We describe assay validation of this approach to facilitate broad clinical application, including evaluation of assay sensitivity, precision, repeatability, reproducibility, pre-analytic factors, and DNA quality/quantity. Sensitivity to detect TFx of 3% (lower limit of detection) was 97.2% to 100% at 1× and 0.1× mean sequencing depth, respectively. Precision was demonstrated on distinct sequencing instruments (HiSeqX and NovaSeq) with no observable differences. The assay achieved prespecified 95% agreement of TFx across replicates of the same specimen (repeatability) and duplicate samples in different batches (reproducibility). Comparison of samples collected in EDTA and Streck tubes from single venipuncture in 23 patients demonstrated that EDTA or Streck tubes were comparable if processed within 8 hours. On the basis of a range of DNA inputs (1 to 50 ng), 20 ng cfDNA is the preferred input, with 5 ng minimum acceptable. Overall, this shallow whole-genome sequencing of cfDNA and ichorCNA approach offers sensitive, precise, and reproducible quantitation of TFx, facilitating assay application in clinical cancer care.

Assessment of the performance of the plasma separation card for HIV-1 viral load monitoring in South Africa.

Scaling up of newer innovations that address the limitations of the dried blood spot and the logistics of plasma monitoring is needed. We employed a multi-site, cross-sectional assessment of the plasma separation card (PSC) on blood specimens collected from all consenting adults, assenting young and pediatric patients living with HIV from 10 primary healthcare clinics in South Africa. Venous blood for EDTA-plasma samples was collected and analyzed according to the standard of care assay, while collected capillary blood for the PSC samples was analyzed using the Roche COBAS AmpliPrep/Cobas TaqMan (CAP/CTM) HIV-1 Test at the National Reference laboratories. McNemar tests assessed the differences in concordance between the centrifuged plasma and dried plasma spots. The usability of PSC by blood spotting, PSC preparation, and pre-analytical work was assessed by collecting seven-point Likert-scale data from healthcare and laboratory workers. We enrolled 538 patients, mostly adults [n = 515, 95.7% (95% CI: 93.7%-97.1%)] and females [n = 322, 64.2% (95% CI: 60.0%-68.1%)]. Overall, 536 paired samples were collected using both PSC- and EDTA-plasma diagnostics, and 502 paired PSC- and EDTA-plasma samples assessed. Concordance between the paired samples was obtained for 446 samples. Analysis of these 446 paired samples at 1,000 copies per milliliter threshold yielded an overall sensitivity of 87.5% [95% CI: 73.2%-95.8%] and specificity of 99.3% [95% CI: 97.9%-99.8%]. Laboratory staff reported technical difficulties in most tasks. The usability of the PSC by healthcare workers was favorable. For policymakers to consider PSC scale-up for viral load monitoring, technical challenges around using PSC at the clinic and laboratory level need to be addressed. IMPORTANCE: Findings from this manuscript emphasize the reliability of the plasma separation card (PSC), a novel diagnostic method that can be implemented in healthcare facilities in resource-constrained settings. The agreement of the PSC with the standard of care EDTA plasma for viral load monitoring is high. Since the findings showed that these tests were highly specific, we recommend a scale-up of PSC in South Africa for diagnosis of treatment failure.

A facile electrochemical immunosensor based on EDTA-Pb2+ complexation reaction.

The sensitivity of electrochemical (EC) sensors has been improved through the development of multiple approaches. However, the majority of EC sensors were limited in their practical application by high costs or tedious procedures. Herein, based on ethylenediaminetetraacetic acid (EDTA)-Pb2+ complexation reaction, a facile and affordable immunosensor was designed. Pb2+-magnesium silicate hydrate was served as the sensing substrate. The immunorecognition process was carried out in the Eppendorf tube, and antibody-functionalized Pb2+-polydopamine was utilized as immunoprobe. In the tube, the quantitative and appropriate excess of EDTA was introduced to complex with Pb2+ on the immunoprobes. The remaining EDTA was added to the sensing substrate surface to coordinate with some Pb2+ in it. This leaded to the reduction of the EC signal of Pb2+, which was related to the antigen concentration. Using prostate-specific antigen as the model analyte, the sensitive detection was realized with a low limit of detection (30.49 fg mL-1). Remarkably, the assay results were available within 24 min, sensibly faster than the most existing EC sensors.

Evaluation of Automated Magnetic Bead-Based DNA Extraction for Detection of Short Tandem Repeat Expansions With Nanopore Sequencing.

BACKGROUND: Long-read technologies such as nanopore sequencing provide new opportunities to detect short tandem repeat expansions. Therefore, a DNA extraction method is necessary that minimizes DNA fragmentation and hence allows the identification of large repeat expansions. In this study, an automated magnetic bead-based DNA extraction method and the required EDTA blood storage conditions as well as DNA and sequencing quality were evaluated for their suitability for repeat expansion detection with nanopore sequencing. METHODS: DNA was extracted from EDTA blood, and subsequently, its concentration, purity, and integrity were assessed. DNA was then subjected to nanopore sequencing, and quality metrics of the obtained sequencing data were evaluated. RESULTS: DNA extracted from fresh EDTA blood as well as from cooled or frozen EDTA blood revealed high DNA integrity whereas storage at room temperature over 7 days had detrimental effects. After nanopore sequencing, the read length N50 values of approximately 9 kb were obtained, and based on adaptive sampling of samples with a known repeat expansion, repeat expansions up to 10 kb could be detected. CONCLUSION: The automated magnetic bead-based DNA extraction was sufficient to detect short tandem repeat expansions, omitting the need for high-molecular-weight DNA extraction methods. Therefore, DNA should be extracted either from fresh blood or from blood stored in cooled or frozen conditions. Consequently, this study may help other laboratories to evaluate their DNA extraction method regarding the suitability for detecting repeat expansions with nanopore sequencing.

Migration and morphological transformation patterns of heavy metals on sludge cells and extracellular polymeric substances (EPS) under the influence of different treatments.

The impediment of sludge resource utilization stems from the presence of heavy metals within the sludge matrix. To optimize heavy metal removal techniques from undried sludge, it is essential to study the distribution of heavy metals in the sludge flocs structure and the changes in morphology in the sludge cells after different treatments. In this study, the sludge was subjected to chemical treatments using citric acid (CA), EDTA, and saponin, as well as electrokinetic treatment at 2 V/cm. The distribution and migration of Cu, Ni, and Zn in sludge flocs after various treatment methods were analyzed. The heavy metals were found to migrate from intracellular to extracellular polymeric substances (EPS) without causing extensive sludge cell lysis. They gradually diffused outward with the dispersion of the EPS layer. The migration efficiency of the three heavy metals in the sludge flocs was Zn, Ni, and Cu. This was mainly related to the initial distribution and morphology of the heavy metals. Under the influence of chemicals and an electric field, the acid-soluble and reducible heavy metals in the cells partially migrated to the EPS, while the stable heavy metals transformed into an unstable state. Furthermore, the order of chemical reagents in terms of their effect on the migration efficiency of heavy metals was CA > EDTA > Saponin, owing to the varying binding strengths of heavy metals and their impact on the degree of loosening of the EPS. Especially after CA treatment a greater proportion of Cu, Ni, and Zn were transferred from the cells to the EPS. The acidification effect near the anode during electrokinetic treatment intensifies the migration of heavy metals. This study provides basic research for subsequent engineering optimization aimed at removing heavy metals from sludge.