Plasma Centrifuge Research Articles

Plasma centrifugation method for separation of the nuclear waste

The high-throughput, plasma-based, mass separation techniques of nuclear waste are still an active research topic due to the theoretical and practical complexity of processing such waste. The nuclear waste can be separated according to its masses by plasma centrifugation technique. Because of this, the present research has been performed by setting up E → × B → rotation in cold cylindrical plasma surrounded by an ideally conducting homogenous shell inside a plasma centrifuge device. The results obtained are useful in analyzing the magneto-rotational instability (MRI) and trajectories of the plasma particles which are beneficial for improving the throughput and separation factor of the device.

Fabrication of Isotope-Enriched Nanostructures Using Ultrafast Laser Pulses under Ambient Conditions for Biomolecular Sensing.

Recent advances in the use of stable isotopes necessitate novel synthesis techniques for isotope separation and enrichment that are scalable and offer high throughput. Stable-isotope-enriched nanostructures can offer unique advantages as nanomedicines, safe tracers, and labels and are critical for applications in various industrial processes, metabolic research, and medicine. So far, there exists no method to synthesize miniature isotope-enriched materials at the nanoscale. In this study, an ultrafast Laser-induced isotope enrichment at nanoscale (LIIEN) is put forward to synthesize isotope-enriched nanostructures, eliminating the need for large equipment and expenses, thereby demonstrating a lab-scale isotope enrichment process. A significant isotope enrichment for Carbon nanostructures is observed. The isotope enrichment can be attributed to the redistribution of isotope ions in the plasma plume explained by the plasma centrifuge model. The LIIEN synthesized structures exhibit excellent Surface-Enhanced Raman Scattering (SERS)signal enhancement and reproducibility, making them potential candidates for SERS-based biomolecule sensing. This technique is an efficient method to fabricate nanosized isotope-enriched structures of characteristic properties by carefully tuning laser parameters at ambient conditions.

Gravity-driven crossflow membrane filtration and mechanical centrifugation yield plasma with equivalent total and COVID-19-specific immunoglobulin content

While the administration of convalescent plasma is a promising therapy for coronavirus disease 2019 (COVID-19), upfront costs and technological barriers of conventional plasmapheresis have limited the collection and processing of COVID-19 convalescent plasma (CCP) in Low- and Middle-Income Countries (LMICs). We previously reported that bedside plasma separation using gravity-driven microfiltration has made CCP therapy accessible as a treatment option in Suriname. However, the question remains whether the gravity-driven microfiltration method yields similar amounts of immunoglobulins as compared to conventional plasmapheresis. Here, we compared the gravity-driven microfiltration method with conventional plasma centrifugation for the total and COVID-19-specific immunoglobulin content of the obtained CCP. Blood donations from 10 donors recovered from PCR-confirmed COVID-19 were processed using both methods. Samples were collected pre- and post-processing for analysis to allow direct comparison of both methods. There were no differences in COVID-19-specific IgG levels between convalescent plasma obtained by microfiltration and centrifugation for 4 of the 5 assays used. Anti-RBD-Omicron IgG levels were slightly higher in the plasma obtained after filtration (median 274, range 69 to 1258) than after centrifugation (median 249, range 67 to 1175), Wilcoxon P = 0.0488. No significant differences were detected between the two methods for levels of total albumin, total cholesterol, total IgA, IgM and IgG levels. These results indicate that gravity-driven microfiltration and conventional centrifugation yield CCP with equivalent amounts of total and COVID-19-specific antibodies. This makes the gravity-driven microfiltration method a viable option for the collection and treatment with CCP in the LMICs setting.

Simplified Method for Removing Direct Oral Anticoagulant Interference in Mechanical Coagulation Test Systems-A Proof of Concept.

Direct oral anticoagulants (DOACs) cause unwanted interference in various hemostasis assays, including lupus anticoagulant (LA) testing, where false positive and false negative identification may occur. DOAC Stop (DS) is an activated charcoal (AC) product used to specifically and effectively adsorb DOACs from test plasma. This process normally requires plasma treatment, centrifugation and plasma separation prior to tests, but inexperienced operators may also inadvertently transfer residual AC particles, thereby potentially adversely affecting clot detection. We hypothesized that residual DS might not be problematic for mechanical clot detection. We therefore investigated the potential impact of DS and a new DS liquid (DS-L) product on clotting tests using a mechanical clot detection system. Varying concentrations of DS were added to normal and abnormal plasmas with and without DOAC presence. Clotting tests including PT, APTT and dRVVT were performed directly in the analyzer without plasma/DS centrifugation. DS up to double the recommended treatment level had only minor effects on all test results, despite completely obscuring visibility in the plasma/reagent mix. This confirms that the centrifugation step may be able to be omitted when using mechanical detection systems. Should DS carryover into treated plasmas occur, this should not cause issues with testing performed on mechanical clot-sensing devices. Moreover, we hypothesize that DS can be used directly in these systems, without the need for centrifugation, thereby simplifying its many potential applications.

B-378 Cell-free Plasma Preparation Workflow Solution

Abstract Background Liquid biopsy for the study and monitoring of various cancers has become a growing area of study, pre-analytical sample handling can impact the quality and profile of the circulating tumor nucleic acid. Preparing samples like cell-free plasma for downstream assays is imperative to maintaining quality in downstream applications for clinical research in oncology. To obtain cell-free plasma, blood samples must undergo centrifugation at 2000g for 10 min at 4C followed by a second spin for 30 min at 4C. This two-step spin process presents an issue with labs that don’t have a refrigerated centrifuge suitable for higher speeds. Methods Here, we perform a series of studies to investigate the impact of various spin conditions on cell-free nucleic acid recovery and quality. Four plasma centrifugation methods encompassing variables attributing to speed, temperature, and number of centrifugations steps, were evaluated from whole blood tubes obtained from six donors in K2EDTA upfront of nucleic acid isolation with MagMAX™ cell-free TNA nucleic acid isolation chemistry on a KingFisher™ magnetic particle processor. Yield, quality, and functional performance was evaluated from the extracted cell-free nucleic acid and compared across all plasma spin-down methods. Result Study results indicated that centrifugation of plasma at lower speeds for a shorter period at room temperature had no negative impact to nucleic acid recovery and integrity. Conclusion Here, we offer a plasma spin workflow solution to mid- to high- throughput clinical research labs to allow versatility within the laboratory setup. Reducing the need for refrigerated centrifugation allows clinical researchers to obtain high-quality cfNA suitable for their downstream applications in clinical oncology and genomics research.

Virological Follow-up of Adults Living with HIV on Dolutegravir in an Approved Treatment Center in Douala (Cameroon)

Background: Human immunodeficiency virus infection constitutes one of the greatest contemporary human pandemics, lethal in the absence of treatment. WHO 2018 recommendations have placed Dolutegravir-based treatment as first-line treatment, particularly in resource-limited countries. The aim of this work was to study the evolution of virological parameters in people undergoing treatment with this molecule.
 Methodology: This was an analytical cross-sectional study of people aged 25 and over, received since January 2015 at the Approved Treatment center of Laquintinie Hospital in Douala (Cameroon). After plasma collection and centrifugation, viral load was determined on the Abbott m2000TM platform, targeting the Pol gene. The detection threshold was greater than or equal to 40 copies/mL. Data analysis was performed using Excel and SPSS version 20 softwares.
 Results: A total of 504 people were selected, with an average age of 48±11 years and a predominance of women (70.8%). Antiretroviral treatment had been initiated at clinical stage 1 in over 80% of participants. The TDF/3TC/EFV regimen prior to initiation of Dolutegravir was used in 93.5% of cases. Mean weight after initiation of treatment was 75.9 kg. People with a viral load <40 copies/ml before starting Dolutegravir therapy accounted for 332 (65.9%) of cases, compared with 91.1% six months after starting treatment. Weight (>75kg) (95% CI, OR=1.112-2.381, p=0.012) and non-adherence (95% CI, OR=2.790-25.613, p=0.001) were considered factors associated with viral load variation.
 Conclusion: The ARV protocol comprising two nucleoside reverse transcriptase inhibitors and Dolutegravir improves virological response in people living with HIV at all clinical stages.

Plasma from patients with pulmonary embolism show aggregates that reduce after anticoagulation

BackgroundMicroclots, a term also used for amyloid fibrin(ogen) particles and henceforth named aggregates, have recently been reported in the plasma of patients with COVID-19 and long COVID. These aggregates have been implicated in the thrombotic complications of these diseases.MethodsPlasma samples from 35 patients with acute pulmonary embolism were collected and analysed by laser scanning confocal microscopy and scanning electron microscopy before and after clotting.ResultsHere we confirm the presence of aggregates and show that they also occur in the plasma of patients with pulmonary embolism, both before and after clotting. Aggregates vary in size and consist of fibrin and platelets. We show that treatment with low-molecular weight heparin reduces aggregates in the samples of patients with pulmonary embolism. Double centrifugation of plasma does not eliminate the aggregates.ConclusionsThese data corroborate the existence of microclots or aggregates in diseases associated with venous thromboembolism. Important questions are raised regarding their pathophysiological relevance and further studies are warranted to investigate whether they represent cause or consequence of clinical thrombosis.

Ability of donkey sperm to tolerate cooling: Effect of extender base and removal of seminal plasma on sperm parameters and fertility rates in mares.

Artificial insemination using cooled-transported semen has marked importance in equine breeding programs around the world, and the high value of mules has generated avid interest in donkey semen biotechnology. However, donkey semen cools poorly in commercially available equine extenders. Therefore, this study aimed to develop approaches to improve the ability of donkey semen to tolerate cooling. Ejaculates of seven donkeys (n = 21) were cooled at 5°C for 48 h in three different extenders (milk-based, SM; sodium caseinate-based, SC; or egg yolk-based, EY) in the presence or absence of seminal plasma (centrifugation, C). Sperm motility, plasma membrane integrity (PMI), plasma membrane stability (PMS), mitochondrial membrane potential (HMMP), intracellular hydrogen peroxide (H2O2), and intracellular superoxide () were assessed before, 24 h, and 48 h post-cooling. In addition, 15 mares (163 estrous cycles) were randomly inseminated with semen from two jacks (Jack 1, n = 90; Jack 2, n = 73) previously cooled for 24 h under one of the treatments (SM, SC, EY, SM-C, SC-C, or EY-C). Groups EY, SC-C, and EY-C (P < 0.05) demonstrated superior sperm analytical parameters to SM at 24 and 48 h. Centrifugation positively affected sperm analytical parameters in cooled donkey semen extended in SM and SC (P < 0.05). Mares bred with semen extended in SC (67%, 18/27), SC-C (89%, 24/27), EY (89%, 25/28), or EY-C (74%, 20/27) had significantly greater conception rates than mares bred with SM (33%, 9/27; P < 0.05). Mares bred with SM-C had intermediate conception rates (59%, 16/27). In conclusion, SC and EY improved the cooling ability and fertility of donkey semen in horse mares, and centrifugation positively affected donkey semen extended in SM.

Isolation of Intermediate-Mass Components of Spent Nuclear Fuel in a Direct-Flow Plasma Centrifuge

Isolation of Intermediate-Mass Components of Spent Nuclear Fuel in a Direct-Flow Plasma Centrifuge

Using platelet-rich plasma for skin rejuvenation

The skin is the largest organ of the body, and it is affected by both intrinsic and extrinsic ageing factors. Platelet-rich plasma is an effective treatment for skin ageing, whether as a standalone treatment or in combination with other treatment modalities. The results depend on the skin quality of the patient, compliance and aftercare. Other factors to consider include the platelet-rich plasma kits and centrifuge used by the practitioner, as well as whether it is activated or not. In this article, Claudia McGloin will discuss skin ageing and the use of platelet-rich plasma for skin rejuvenation.

Characterization of pre-analytical sample handling effects on a panel of Alzheimer's disease-related blood-based biomarkers: Results from the Standardization of Alzheimer's Blood Biomarkers (SABB) working group.

IntroductionPre‐analytical sample handling might affect the results of Alzheimer's disease blood‐based biomarkers. We empirically tested variations of common blood collection and handling procedures.MethodsWe created sample sets that address the effect of blood collection tube type, and of ethylene diamine tetraacetic acid plasma delayed centrifugation, centrifugation temperature, aliquot volume, delayed storage, and freeze–thawing. We measured amyloid beta (Aβ)42 and 40 peptides with six assays, and Aβ oligomerization‐tendency (OAβ), amyloid precursor protein (APP)699‐711, glial fibrillary acidic protein (GFAP), neurofilament light (NfL), total tau (t‐tau), and phosphorylated tau181.ResultsCollection tube type resulted in different values of all assessed markers. Delayed plasma centrifugation and storage affected Aβ and t‐tau; t‐tau was additionally affected by centrifugation temperature. The other markers were resistant to handling variations.DiscussionWe constructed a standardized operating procedure for plasma handling, to facilitate introduction of blood‐based biomarkers into the research and clinical settings.

Thiol Isomerase Activity of β3 Integrin Psi Domain and L33P Polymorphism: Implications in Blood Coagulation and Anti-Thrombotic Therapy

Background: Integrins are α and β subunit heterodimeric receptors expressed ubiquitously on metazoan cells that play key roles in cell adhesion, movement, and signaling. The αIIbβ3 integrin on platelets is essential for thrombosis and hemostasis through its binding of fibrinogen and other ligands to mediate platelet adhesion and aggregation. The plexin-semaphorin-integrin (PSI) domain is an approximately 54 amino acid sequence at the N-terminus of the β3 subunit located in close proximity to the “knee” region. Our lab recently discovered that two “CXXC” motifs within PSI domains in the integrin family exert endogenous thiol isomerase activity, and blockade of this activity in β3 integrin with our novel anti-PSI domain monoclonal antibodies (mAbs) significantly attenuated platelet adhesion and aggregation without impairing hemostasis (Blood, 2017). Interestingly, intravital microscopy studies demonstrate that our anti-PSI antibodies inhibited thrombosis in vivo 10-30 fold more potent than their effects in vitro under anti-coagulant conditions, suggesting a possible inhibitory effect on blood coagulation. Notably, A L33P polymorphism (HPA-1b) within the β3 integrin PSI domain is linked to an approximately two-fold increased risk for cardiovascular disease (CVD), however, the underlying mechanism for this remains unclear.Methods/Results: To investigate the role of PSI domain in blood coagulation, we first employed thromboelastography (TEG) to compare our anti-PSI domain mAbs with other anti-β3 mAbs that do not directly bind to β3 PSI domain. Results show that anti-PSI domain mAbs inhibited blood coagulation in human and murine whole blood or platelet-rich plasma (PRP) significantly more than anti-αIIbβ3 antibodies JAN-D1, M1, and Abciximab precursor 7E3. To address whether the L33P polymorphism affects PSI domains thiol isomerase activity, we generated L33P PSI domain via site-directed mutagenesis in E. coli. Using a scrambled RNase assay, we found that L33P polymorphism enhanced thiol-isomerase activity relative to WT PSI domain. We further corroborated these findings through an insulin β chain reduction assay, and a MPB (N-Maleimidopropionyl-biocytin) western blot assay, which quantifies thiol isomerase activity through MPB binding to free thiols that have not been oxidized into RNase. Interestingly, TEG results show that recombinant human PSI domain enhanced blood coagulation in platelet-microparticle (PMP) free plasma, which was generated through high-speed centrifugation (17,000 x g) of platelet poor plasma (PPP)for 15 minutes that removed residual platelets and microparticles .Conclusion: We have discovered a novel role of integrin β3 PSI domain in blood coagulation, which is enhanced by the L33P polymorphism (HPA-1b). These data highlight the β3 PSI domain as a suitable therapeutic target for its roles in both platelet adhesion/aggregation, and blood coagulation. Furthermore, these data may explain the increased risk of CVD such as myocardial infarction and deep vein thrombosis for individuals with the L33P polymorphism. DisclosuresNo relevant conflicts of interest to declare.

The non-isothermal Bödewadt problem applied to an advanced plasma centrifuge

Several valuable isotopes used in medicine, industry, and science cannot be separated by a gas centrifuge with a driven mechanical rotor due to the lack of a suitable working gas that has a sufficiently high vapor pressure at room temperature. Such isotopes are typically produced by more costly separation processes. An advanced plasma centrifuge (PC) has been proposed that can separate specific isotopes at a lower cost. The PC achieves increased separation performance by the excitation of an axial counterflow in the plasma medium. In this work, we consider the non-isothermal Bödewadt problem for the PC with thermally driven axial circulation created by cooling one end of the PC's separation chamber. Due to the imbalance between the centrifugal force and the radial pressure gradient, we show that a secondary flow appears, which is imposed on the medium's rotation motion. The effect is analogous to the classical Bödewadt problem, where a rotational flow decelerates over a fixed surface.

Incorporación salival y plasmática de ácidos grasos luego de su ingesta inmediata en ratas

Objective: the aim of this study was to analyze salivary and plasma fatty acid (FA) levels after their immediate intake in rats.Methods: 6 adult male Wistar rats were fed on commercial diet until the 12th week of life, in which it was replaced by a laboratory diet containing corn oil (6%) as a lipid source (linoleic 18: 2 n-6 FA). At 12 and 24 hours after the diet change, rats were anesthetized and salivary secretion was induced by an intraperitoneal injection of isoproterenol / pilocarpine (5 mg/kg each) before their sacrifice. Total saliva (S) was collected for 20 min by using intraoral cotton rolls. Blood was obtained (cardiac puncture) to separate plasma (P) (centrifugation). S and P lipids were extracted for FA methylation and analysis by gas chromatography - mass spectrometry. Spearman correlation and linear regression coefficients (p≤0.05) were applied. Results: 18:2 n-6 FA was detected in S and P at 12 and 24 hours, being higher at 24 hours and in P. A higher number of FA was also found at 12 and 24 hours both in P than in S. 24/12 hour concentration ratios in S were 16:0 (1.20); 16:1 (1.65); 18:0 (0.33); 18:1 n-9 (1.55); 18:2 n-6 (1.70); 20:4 n-6 (1.20) and 22:6 n-3 (1.19) and in P: 16:0 (0.49); 16:1 (4.84); 18:0 (3.43); 18:1 n-9 (3.14); 18:2 n-6 (1.75); 20:4 n-6 (0.52) and 22:6 n-3 (0.73). A significant positive correlation between S and P 16:1 n-9 FA and a high regression between 16:0;16:1 n-9 and 18:0 salivary/plasma FA was found. Conclusion: this preliminary study showed that the 18:2 n-6 FA intake is rapidly reflected in rat S and P, with a time increase, and that there is a correlation between some FA of both fluids.

Isoagglutinin removal by plasma centrifugation or filtration (single or double): A prospective study in a single center.

ABO-incompatible (ABOi) kidney transplantation, a well-established procedure, has good long-term results provided pretransplant desensitization that includes immunosuppression and apheresis. To compare, within the first pretransplant apheresis session given to 29 ABOi kidney-transplant candidates, the effect on isoagglutinin titers (both IgG and IgM isotypes) of three modalities: centrifugation therapeutic plasmapheresis (cTP; n = 10), filtration TP (fTP; n = 9), and double-filtration plasmapheresis (DFPP; n = 10). The three groups were comparable according to baseline demographics. Treated plasma volumes were similar across the three groups, that is, 4111 ± 403 mL (cTP), 3861 ± 282 mL (fTP), and 3699 ± 820 mL (DFPP): that is, 54 ± 7, 53 ± 7, and 53 ± 10 mL/kg respectively. One session of centrifugation or filtration TP reduced IgG anti-A/anti-B isoagglutinin titer by ~4, whereas one DFPP session reduced it by ~2. One session of cTP reduced IgM anti-A isoagglutinin titer by a little less than 4, whereas fTP and DFPP sessions reduced it by ~3. There were no statistical differences across the three groups regarding isoagglutinin rebound (IgG and IgM). However, isoagglutinin IgG rebound was >4 dilutions for anti-B titers compared with ~2 dilutions for anti-A titers. The median decreases in IgG level were -3.9 g/L (DFPP), -5.9 g/L (cTP), and - 6.06 g/L (fTP) (p = ns). Median fibrinogen depletions were ~ 60% (fTP), 64% (DFPP), and 76% (cTP). Isoagglutinin depletions within the first apheresis session were similar across cTP, fTP, and DFPP: this was numerically lower for DFPP.

Plasma Centrifuge With Crossed E × B Fields and Thermally Driven Countercurrent Flow

It is well established from gas centrifuge (GC) development that isotope separation in a rotating, multispecies gaseous medium increases with axial circulation of the rotating gas in a long separation chamber. This article aims at the development of a commercially viable plasma centrifuge (PC) for specific, nonuranium isotopes. The proposed model PC employs crossed constant radial electric and axial magnetic fields to provide radial separation of a partially ionized gas, combined with a thermal method to establish axial circulation in the rotating medium. The proposed thermal method creates an axial temperature gradient by cooling one end of the separation chamber. By calculation, the inclusion of circulation flow is shown to create axial redistribution of the target component concentration, increasing the longitudinal separation. An analysis of the hydrodynamic and thermal problems is performed for an extended end disk PC in the framework of the boundary layer theory. Radial velocity profiles of the conducting gas in the boundary layer are calculated with respect to the temperature ratio in the external flow ( $T_{1}$ ) and the disk surface ( $T_{0}$ ) for various Prandtl (Pr) numbers. The results demonstrate that for small values of the Pr number, cooling of an end surface within the PC separation chamber results in an intensive inflow of the conducting medium to the solid surface. It is shown that in a PC with crossed fields, cooling one of its ends achieves an effective multiplication of the separation effect along the length of the column.

Comediation of Erythrocyte Haemolysis by Erythrocyte-Derived Microparticles and Complement during Malaria Infection.

Background Due to the sustained morbidity and mortality that malaria-associated anaemia imposes on patients, malaria is still a global threat, most especially, to residents in sub-Saharan Africa. Merozoite invasion and destruction of erythrocytes, a target for this study, have been necessary due to its unique nature and also since the erythrocytes suffer the most brunt of malarial infection leading to anaemia. The issue of malaria anaemia has to do with why uninfected RBCs get destroyed and even more so than infected ones. Studies have proposed that cytophilic anti-RSP2 (ring surface protein 2—merozoite rhoptry protein 2) antibodies present in sera enhance phagocytosis of RSP2-tagged RBCs by macrophages either directly or via complement, while others have proposed transfer of RSP2 to both infected and uninfected RBCs which may render them susceptible to phagocytosis. What is missing is the agent involved in the transfer of these parasite-induced surface proteins onto the uninfected RBCs, i.e., the mediator molecules. Considering the intracellular location of the parasite in the parasitophorous vacuolar membrane and the absence of a transport mechanism such as the Golgi apparatus within the mature RBC, since the latter has no nucleus, we propose that erythrocyte-derived microparticles (EMPs) may be the possible mediators. Aim This study aimed at examining the immunological interactions between EMPs released during malarial infections and host erythrocytes that may lead to their lysis possibly through complement mediation. Methods This was an experimental study during which malarial EMPs were isolated by differential centrifugation of malaria-positive plasma. This was followed by cell-based in vitro assays where malaria-positive EMPs were added to uninfected blood group “O” negative erythrocytes in the presence of complement and haemolysis checked for. Results and Conclusion. At a fixed volume of 50 μL complement, there were statistically significant (p < 0.01) increases in mean percentage haemolysis as the volume of EMPs increased. Similarly, at a fixed volume of 50 μL EMPs, there were statistically significant (p < 0.01) increases in mean percentage haemolysis with increasing volumes of complement. This was an indication that both complement and EMPs contribute significantly to uninfected erythrocyte haemolysis during malaria infection.

Thermally driven countercurrent plasma centrifuge

ABSTRACT Renewed interest in plasma isotope separation stems from the need to produce certain isotopes more inexpensively. These include isotopes of groups I and II, rare earth and some other elements of the periodic table. While gas centrifuge cascades, developed for uranium enrichment, are capable of a wider range of separation applications, they are limited to elements that can form volatile gaseous compounds at room temperature. In the absence of a suitable compound, more expensive separation techniques are used, consuming considerable electrical power for relatively low throughputs. We believe that a new type of plasma centrifuge can be a viable alternative.

Abstract A66: Analytical validation of 7 droplet digital PCR assays detecting TERT, BRAF, and NRAS hotspot mutations in plasma-derived circulating tumor DNA (ctDNA)

Abstract Introduction: Serial monitoring of sensitive and specific blood-based biomarkers of disease burden in cancer patients could enable clinicians to adjust treatments at the earliest signs of disease progression when the likelihood of improved outcomes is probably greatest. Such assays require preanalytical, analytical, and clinical validation prior to use. We conducted analytical validation studies of 7 droplet digital PCR (ddPCR) assays to determine their performance characteristics in detecting mutant TERT, BRAF, and NRAS ctDNA. Methods: Probe-based ddPCR assays (Bio-Rad) detecting BRAF V600E, K, NRAS Q61K, R, L, and TERT C228T, C250T were studied. Preanalytical validation experiments using plasma collected in EDTA tubes investigated processing variables including plasma volumes, centrifugation speeds, and DNA extraction efficiencies, and evaluated the necessity of internal controls using spike-in experiments. Analytical validation studies assessed accuracy using plasma from healthy donors spiked with known amounts of mutant nucleosomal DNAs from control cell lines. Potentially interfering substances were evaluated using the AcroMetrix inhibition panel (Thermo Scientific). Precision/reproducibility was assessed using combinations of multiple reagent lots and operators. Assay specificity was determined using templates with different mutations to check cross-reactivity. Limits of Blank (LoB) and Limits of Detection (LoD) were calculated for each assay using 8 replicate wells of healthy donor plasma or mutant cell line DNA, respectively. Three-phase inter-laboratory harmonization was conducted using: 1) extracted DNA, 2) plasmas spiked with control DNAs, and 3) melanoma patient samples shipped in PAXgene Blood ccfDNA tubes (Qiagen). Results: Preanalytical validation yielded a double centrifugation protocol to reduce contaminating large DNA. Assays were highly accurate and precise with 80-90% recovery of spiked-in mutant templates and coefficients of variation (CVs) &amp;lt;10%, respectively. BRAF and NRAS mutation-specific assays also detected alternative specific mutations in their respective targets that were easily discernible from the perfect-match mutation as discrete clusters in the ddPCR output software. The LoBs ranged from 0.24 to 1.68 copies/mL and 0.016% to 0.058% fractional abundance; LoDs ranged from 0.01% to 0.05%. Inter-laboratory harmonization demonstrated CVs ranging from 1.5% to 15%. Conclusion: This ddPCR-based plasma analysis platform demonstrates low background levels in normal plasma, yielding high sensitivities to detect small amounts of ctDNAs with these mutations. The assays also demonstrate highly reproducible results between laboratories. These assays could be integrated into biomarker-driven clinical trials to evaluate their potential to inform more personalized clinical decision-making. Citation Format: Mahrukh M. Syeda, Broderick Corless, Iman Osman, Cindy Spittle, George Karlin-Neumann, David Polsky. Analytical validation of 7 droplet digital PCR assays detecting TERT, BRAF, and NRAS hotspot mutations in plasma-derived circulating tumor DNA (ctDNA) [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A66.

Analysis of Plasma Products for Cellular Contaminants: Comparing Standard Preparation Methods

Recent reports suggest that component plasma products contain significant quantities of cellular contamination. We hypothesized that leukoreduction of whole blood before preparation of derived plasma is an effective method to prevent cellular contamination of stored plasma. Samples of never-frozen liquid plasma prepared by standard methods (n= 25) were obtained from 3 regional blood centers that supply 3 major trauma centers. Samples were analyzed for leukocyte and platelet contamination by flow cytometry. To determine if leukoreduction of whole blood before centrifugation and expression of plasma prevents cellular contamination of liquid plasma, 1 site generated 6 additional units of liquid plasma from leukoreduced whole blood, which were then compared with units of liquid plasma derived by standard processing. Across all centers, each unit of never-frozen liquid plasma contained a mean of 12.8 ± 3.0 million leukocytes and a mean of 4.6 ± 2 billion platelets. Introduction of whole blood leukoreduction (LR) before centrifugation and plasma extraction essentially eliminated all contaminating leukocytes (Non-LR: 12.3 ± 2.9 million vs LR: 0.05 ± 0.05 million leukocytes) and platelets (Non-LR: 4.2 ± 0.3 billion platelets vs LR: 0.00 ± 0.00 billion platelets). Despite widespread belief that stored plasma is functionally acellular, testing of liquid plasma from 3 regional blood banks revealed a significant amount of previously unrecognized cellular contamination. Introduction of a leukoreduction step before whole blood centrifugation essentially eliminated detectable leukocyte and platelet contaminants from plasma. Therefore, our study highlights a straightforward and cost-effective method to eliminate cellular contamination of stored plasma.