HD Target Research Articles

Abstract 5018: Evaluation of whole blood fractionation conditions for optimal liquid biopsy NGS applications

Abstract “For research use only. Not for diagnostic procedures.” Introduction: Liquid biopsy is a non-invasive alternative to tissue biopsies for cancer profiling using cell-free total nucleic acids (cfTNA; including cell free DNA (cfDNA) and cell free RNA (cfRNA)), the biological analytes derived from tumor cells floating in blood. However, it is crucial to optimize the whole blood fractionation workflows, since limited access to a suitable centrifuge at blood collection sites poses a challenge to this critical fractionation step, causing delays in processing the blood, leading to decreased sensitivity of variant detection and increased occurrence of false positives. Current study shows a systematic comparison of the effects of 8 different blood fractionation conditions for spin speeds, temperatures and duration on the levels of cfTNA derived from K2-EDTA-derived blood from multiple healthy and non-small cell lung cancer (NSCLC) donors, using next generation sequencing (NGS). Methods: CfTNA from K2-EDTA derived plasma was isolated using the MagMAXTM Cell-Free Total Nucleic Acid kit and the Genexus™ Cell free total nucleic acid kit, utilizing the KingFisher TM Flex and GenexusTMpurification systems. Yield and integrity of cfTNA isolated was evaluated by Qubit™, Agilent Tapestation and quantitative real time PCR (qRT-PCR). Furthermore, the impact of these test conditions on quality control specifications of DNA and RNA was assessed by performing sequencing on an Ion TorrentTM GenexusTM sequencing instrument using the AmpliSeqTM HD target amplification assay, that reports sequence variations in DNA for 42 cancer related genes and RNA in 7 cancer related genes. Results: Results showed comparable performance for all the 8 blood fractionation conditions tested, for yield and cfDNA profiles compared to control condition, indicating that there is no significant impact of these conditions on either yield or integrity of the cfDNA isolated. In addition, NGS data also showed that on target reads and median molecular coverage for DNA are comparable for all the conditions tested. However, sequencing data demonstrated that one test condition showed significantly better performance for RNA quality control specifications in terms of RNA mapped reads and molecular coverage for process controls used in the assay, compared to all other conditions tested. Conclusion: Data suggests that one of the whole blood fractionation conditions tested impact the cfRNA recovery from plasma without negatively affecting the yield, and integrity of isolated cfDNA and shows increased sensitivity for RNA process controls in NGS. Citation Format: MADHU JASTI, Sergio Hernandez, Nick Siepert, Lokesh Kodandaramaswamy, Thilanka Jayaweera, Jeoffrey Schageman, Angie Cheng, Susanne Lopez, Miao Der Chen. Evaluation of whole blood fractionation conditions for optimal liquid biopsy NGS applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5018.

Abstract 5030: Evaluation of different blood collection tubes for liquid biopsy NGS applications

Abstract Intro: Liquid biopsy is fast becoming a standard practice in oncology research due to recent advancements in Next Generation Sequencing (NGS). To detect genetic variants, whole blood samples are collected in blood collection tubes (BCTs) and fractionated to obtain plasma which is then used to isolate cell free nucleic acid (cfNA) for analysis. While the limited availability of cfNA is a challenge in liquid biopsy, storage condition and duration between blood collection and fractionation have a significant impact on the quantity and quality of the cfNA due to degradation and genomic DNA and RNA contamination. There are several BCTs available on the market offering stabilization of cfNA profiles to overcome the above challenges. Methods: Here we compare the NGS performance of cfNA derived from three commonly used BCTs on the market, BD Vacutainer™ K2EDTA Tubes, Streck™ Cell-Free DNA, and PAXgene® Blood ccfDNA tubes. Matched whole blood samples collected in the above-mentioned tube types were obtained from commercial vendors which were derived from 38 healthy donors as well as 6 cancer research samples. Whole blood samples were fractionated as per manufacturer recommendations and cfNA was isolated and quantified using Genexus™ Cell-Free Total Nucleic Acid Purification Kit on Genexus™ purification system. Isolated cfNA was then sequenced using Ion Ampliseq™ HD target amplification assay on Genexus™ Integrated sequencer. Results: Data shows that K2EDTA collected samples contain slightly higher amounts of cfNA compared to PAXgene® and Streck™ samples. When the cfDNA profiles were analyzed by Agilent™ BioAnalyzer™, K2EDTA derived cfDNA showed varying degrees of genomic DNA contamination which can inflate the cfDNA concentration. However, both Streck™ and PAXgene® derived cfDNA showed little to no detectable genomic DNA contamination. As a result, a higher percentage of PAXgene® and Streck™ samples did not meet the minimum input requirement (5ng) for the sequencing assay. Sequencing data for DNA showed that K2EDTA samples had higher total reads and median molecular coverage compared to the other two BCT types. However, all samples had passing QC metrics, and mean read length, on target reads, and uniformity were comparable between the three tube types. When DNA variant detection was evaluated using cancer research samples, no significant difference was observed in performance between K2EDTA and PAXgene® samples. Interestingly, sequencing data for RNA shows that PAXgene® samples had slightly higher mapped reads on average, and significantly higher internal process control coverage compared to both K2EDTA and Streck™ samples. Whereas Streck™ samples had significantly lower internal process control coverage. Conclusion: In summary, this study demonstrates the advantages and disadvantages of using different BCTs for liquid biopsy applications, in terms of cfNA yields, isolated DNA profiles, and sequencing performance. Citation Format: Nicholas Siepert, Emilia Ostrowska, Luming Qu He, Madhuri Jasti, Kris Lea, Thilanka Jayaweera, Angie Cheng. Evaluation of different blood collection tubes for liquid biopsy NGS applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5030.

Experimental Test of the Polarization Persistence in Inertial Confinement Fusion

The complete deuteron and triton polarization in the DT fusion increases the reactivity by 50%. For Inertial Confinement Fusion (ICF), due to the dynamics of the fusion reaction process, the fusion rate could even be further increased. It has been argued that the polarization would survive as well in magnetic as in inertial confinements. Recently, we have proposed an experiment to test the persistence of the polarization in a fusion process, using a powerful laser hitting a polarized HD target.The polarized deuterons heated in the plasma induced by the laser can fuse. The corresponding reaction is: D + D → 3He + n. The angular distribution of the emitted neutrons and the change in the corresponding total cross section are signatures to estimate the polarization persistency. A proposal to test the persistence of the polarization in ICF has been accepted at ILE: the POLAF project (POlarization in LAser Fusion Process). It uses the polarized HD targets produced at RCNP and the powerful ILE lasers, as well as the neutron detectors existing there. Both institutions are on the same campus at Osaka University. The description of the POLAF experiment and of the corresponding set-up is given.

A cryostat to hold frozen-spin polarized HD targets in CLAS: HDice-II

The design, fabrication, operation, and performance of a 3/4He dilution refrigerator and superconducting magnet system for holding a frozen-spin polarized hydrogen deuteride target in the Jefferson Laboratory CLAS detector during photon beam running is reported. The device operates both vertically (for target loading) and horizontally (for target bombardment). The device proves capable of maintaining a base temperature of 50mK and a holding field of 1T for extended periods. These characteristics enabled multi-month polarization lifetimes for frozen spin HD targets having proton polarization of up to 50% and deuteron up to 27%.

DD fusion from a polarized HD target

AbstractRecently, we have proposed an experiment to test the persistence of the polarization in a fusion process (D+D→3He+n), using a powerful laser hitting a polarized HD target. The purpose of the present contribution is to examine in more detail the experimental constraints, to move from a principle proposal to a doable experiment. Some of the difficulties are as follows: Production of a windowless cryogenic HD target and target cryostat vacuum breakdown, identification of thermal fusion or accelerated deuterons, inducing nuclear reactions, and finally, a clear signature of the polarization persistence of the fused deuterons must be found. Those points will be reviewed and discussed in the scope of the new results presented at this conference.

A06 Chdi-00340246, A Kynurenine Monooxygenase Inhibitor As Potential Therapeutic Agent For The Treatment Of Huntington's Disease

<h3></h3> Deregulation of the Kynurenine Pathway, has been causally implicated in the pathophysiology of HD. Metabolites of this pathway have been shown to be neurotoxic (3-Hydroxy kynurenine (3-HK) and Quinolinic Acid (QA), while kynurenine (KYN) and kynurenic acid (KYNA) have been described as neuroprotective. Pathway deregulation showing increased in the neurotoxic metabolites has been reported in postmortem striatum and cortex samples of early grade HD brains as compared to controls, and in HD mice models R6/2, YAC128 and HdhQ111. The collective evidence for the implication of these metabolites in the pathophysiology of HD highlights the KP pathway as an attractive therapeutic intervention point in this disease. Inhibition of KMO, a key enzyme in the pathway, should shunt the pathway away from the toxic metabolites 3-HK and QA and towards the formation of the protective metabolite KYNA. The potential of KMO as HD target has been shown in a yeast, flies and mice models of HD. This talk will describe CHDI’s efforts towards obtaining a series of potent and selective inhibitors of KMO. Will speak to the PK/PD effects that we have obtained with our lead compound CHDI-00340246 (‘246), a compound that warrants further consideration for pharmaceutical development in HD. ‘246 has been declared a clinical candidate and an upcoming GLP-tox study in rodents and NHPs is planned. In addition we will present the methods development efforts that have been undertaken in support of a clinical biomarker study.

In-Line Calibration of Raman Systems for Analysis of Gas Mixtures of Hydrogen Isotopologues with Sub-Percent Accuracy

Highly accurate, in-line, and real-time composition measurements of gases are mandatory in many processing applications. The quantitative analysis of mixtures of hydrogen isotopologues (H2, D2, T2, HD, HT, and DT) is of high importance in such fields as DT fusion, neutrino mass measurements using tritium β-decay or photonuclear experiments where HD targets are used. Raman spectroscopy is a favorable method for these tasks. In this publication we present a method for the in-line calibration of Raman systems for the nonradioactive hydrogen isotopologues. It is based on precise volumetric gas mixing of the homonuclear species H2/D2 and a controlled catalytic production of the heteronuclear species HD. Systematic effects like spurious exchange reactions with wall materials and others are considered with care during the procedure. A detailed discussion of statistical and systematic uncertainties is presented which finally yields a calibration accuracy of better than 0.4%.

Polarization: A must for fusion

The complete polarization of DT fuel would increase the fusion reactivity by 50% in magnetic as well as in inertial confinements. The persistence of polarization in a fusion process could be tested, using a terawatt laser hitting a polarized HD target. The polarized deuterons heated in the plasma induced by the laser can fuse producing a 3He and a neutron in the final state. The angular distribution of the emitted neutrons and the change in the corresponding total Cross Section (CS) can sign the polarization persistence. The polarization of solid H2, D2 or T2 Hydrogen isotopes is very difficult. However, it has been possible to polarize HD, a hetero-molecular form of Hydrogen, by static polarization, at very low temperature and very high field. The radioactivity of DT molecules forbids there high polarization by the static method, therefore one has to develop the Dynamic Nuclear Polarization (DNP) by RF transitions. The DNP of HD has been investigated in the past. The magnetic properties of HD and DT molecules are very similar, it is therefore expected that any polarization result obtained with HD could be extrapolated to DT.

HDice, Highly-Polarized Low-Background Frozen-Spin HD Targets for CLAS experiments at Jefferson Lab

Large, portable frozen-spin HD (Deuterium-Hydride) targets have been developed for studying nucleon spin properties with low backgrounds. Protons and Deuterons in HD are polarized at low temperatures (∼10mK) inside a vertical dilution refrigerator (Oxford Kelvinox-1000) containing a high magnetic field (up to 17T). The targets reach a frozen-spin state within a few months, after which they can be cold transferred to an In-Beam Cryostat (IBC). The IBC, a thin-walled dilution refrigerator operating either horizontally or vertically, is use with quasi-4π detector systems in open geometries with minimal energy loss for exiting reaction products in nucleon structure experiments. The first application of this advanced target system has been used for Spin Sum Rule experiments at the LEGS facility in Brookhaven National Laboratory. An improved target production and handling system has been developed at Jefferson Lab for experiments with the CEBAF Large Acceptance Spectrometer, CLAS.

Polarized Fusion on a Table Top Test of the Persistence of the Polarization in a Fusion Process

We propose an experiment to test the persistence of the polarization in a fusion process, using a terawatt laser hitting a polarized HD target. The polarized protons and deuterons heated in the plasma induced by the laser have a significant probability to fuse producing a 3He and a γ ray or a neutron in the final state. The angular distribution of the radiated γ rays and the change in the corresponding total cross section are related to the polarization persistence, but the resulting signal turns out to be weak. By comparison, the neutrons are produced hadronically with a larger cross section and it is much easier to detect them. A significant reduction of the cross section by parallel polarization of the deuterons as well as a structured angular distribution of the emitted neutrons is reliably predicted by the theory. Therefore, it is expected that the corresponding signal on the neutron counting rate could be seen experimentally.

Nuclear spin imaging with hyperpolarized nuclei created by brute force method

We have been developing a polarized HD target for particle physics at the SPring-8 under the leadership of the RCNP, Osaka University for the past 5 years. Nuclear polarizaton is created by means of the brute force method which uses a high magnetic field (∼17 T) and a low temperature (∼ 10 mK). As one of the promising applications of the brute force method to life sciences we started a new project, "NSI" (Nuclear Spin Imaging), where hyperpolarized nuclei are used for the MRI (Magnetic Resonance Imaging). The candidate nuclei with spin ½ℏ are 3He, 13C, 15N, 19F, 29Si, and 31P, which are important elements for the composition of the biomolecules. Since the NMR signals from these isotopes are enhanced by orders of magnitudes, the spacial resolution in the imaging would be much more improved compared to the practical MRI used so far. Another advantage of hyperpolarized MRI is that the MRI is basically free from the radiation, while the problems of radiation exposure caused by the X-ray CT or PET (Positron Emission Tomography) cannot be neglected. In fact, the risk of cancer for Japanese due to the radiation exposure through these diagnoses is exceptionally high among the advanced countries. As the first step of the NSI project, we are developing a system to produce hyperpolarized 3He gas for the diagnosis of serious lung diseases, for example, COPD (Chronic Obstructive Pulmonary Disease). The system employs the same 3He/4He dilution refrigerator and superconducting solenoidal coil as those used for the polarized HD target with some modification allowing the 3He Pomeranchuk cooling and the following rapid melting of the polarized solid 3He to avoid the depolarization. In this report, the present and future steps of our project will be outlined with some latest experimental results.

Spin Physics at SPring-8 – Recent Results

We have been carrying out hadron photoproduction experiments at SPring-8 since the completion of the LEPS beam-line in 2000. The photoproduction of the ϕ, K, η and π0 mesons is studied by using linearly polarized photon beams with energies of Eγ = 1.5–2.9 GeV. Introducing a polarized nucleon target is expected to upgrade the LEPS experiments to a new level. The measurement of double spin polarization asymmetries provides important information to investigate the hidden nucleon structure, hadron photoproduction dynamics, and exotic hadron properties. We started developing a polarized HD target in 2005. Five large refrigerators necessary for the polarized target have been already obtained. We plan to carry out an experiment using polarized photon beams and the polarized HD target in 2011–2012. We also started constructing a new beam-line (LEPS2) at SPring-8 in 2010. The large acceptance E949 spectrometer, which has been used at BNL, will be transported to the LEPS2 beam-line in 2011. With these new developments, advanced studies of spin physics are expected in future experiments.

POLARIZED HD TARGET FOR FUTURE LEPS EXPERIMENTS AT SPRING-8 IN JAPAN

We are carrying out hadron photoproduction experiments by using polarized photon beams with energies of 1.5-2.9 GeV at SPring-8 in Japan. In 2005, we started developing a polarized HD target for future LEPS experiments using both the polarized photon beams and the polarized target. To introduce a polarized target plays an important role in upgrading the LEPS experiments to the next higher step. The measurement of double polarization asymmetries is expected to give important informations to investigate the nucleon hidden structure, hadron photoproduction dynamics, and exotic hadron property. We report on the present status of the development of the polarized HD target at RCNP. We will carry out a test experiment to confirm that the polarized HD target can be transported from Osaka university to SPring-8 without any problems in 2010. If we succeed in the test experiment, we will carry out a long experiment to take physics data in 2011-2012.

Persistence of the Polarization in a Fusion Process

We propose an experiment to test the persistence of the polarization in a fusion process, using a petawatt laser hitting a polarized HD target. The polarized protons and deuterons heated in the plasma induced by the laser have a significant probability to fuse producing a 3 He and a γ ray or a neutron in the final state. The angular distribution of the radiated γ rays and the change in the correponding total cross section are related to the polarization persistence, but the resulting signal turns out to be weak. By comparison, the neutrons are produced hadronically with a larger cross section and are much easier to detect experimentally. A significant reduction of the cross section by parallel polarization of the deuterons is reliably predicted by the theory. Therefore, it is expected that the corresponding signal on the neutron counting rate could be seen experimentally.

Static and dynamic polarization of HD

The static polarization of HD samples has been achieved using “brute force”, for HD samples purified by double distillation. Proton polarization in excess of 60% and deuteron vector polarization higher than 14% have been reached. It has been demonstrated that the ageing technique allows to get relaxation times at 1.5 K and 1 T larger than a week. It is advocated that the conventional dynamic polarization of HD should be feasible for the proton and the deuteron contained in the HD molecule. This would simplify considerably the machinery presently necessary to perform nuclear physics experiments with HD targets polarized by the static method.

A polarized HD target factory in Europe

Highly polarized hydrogen molecules in the solid phase are of primary interest as “polarized targets” for Nuclear Physics experiments and polarized fuel for Inertial Confinement Fusion (ICF). Within the French–Italian GRAAL collaboration, we are constructing a new Polarized HD Target Factory, implanted at IPN Orsay. It consists of a powerful Dilution Refrigerator (12 μW at 10 mK) equipped with a 16.5 T superconducting magnet and several ancillary cryostats for condensation, storage, transfer and transport of polarized HD samples. The primary objective is to produce polarized HD targets (HYDILE project) for the photoproduction experiments with the GRAAL polarized high energy real photon beams, obtained from backscattering of laser light against the electron stored in the ESRF ring at Grenoble. Production of targets for other sites is forseen. The system will be described and the results already obtained for the static polarization of a HD sample will be given. The possibility to investigate dynamic polarization methods for HD and apply them to DT in order to produce polarized DT fuel for fusion will be discussed.

Measurement of the resonant d(mu)t molecular formation rate in solid HD.

Measurements of muon-catalyzed dt fusion ( d(mu)t-->4He + n + mu(-)) in solid HD have been performed. The theory describing the energy dependent resonant molecular formation rate for the reaction (mu)t + HD-->[(d(mu)t)pee](*) is compared to experimental results in a pure solid HD target. Constraints on the rates are inferred through the use of a Monte Carlo model developed specifically for the experiment. From the time-of-flight analysis of fusion events in 16 and 37 microg x cm(-2) targets, an average formation rate consistent with 0.897+/-(0.046)(stat)+/-(0.166)(syst) times the theoretical prediction was obtained.

Asymmetric branching ratio for the dissociation ofHD+(1sσ)

Single ionization of the HD molecule by fast ion impact is used to populate the vibrational continuum of the ${\mathrm{HD}}^{+}$ electronic ground state $(1s\ensuremath{\sigma}).$ The resulting dissociation leads to two final states, the lower $1s\ensuremath{\sigma}$ state or the first excited $2p\ensuremath{\sigma}$ state, which is 3.7 meV higher at the separated atom limit due to the finite nuclear mass correction to the Born-Oppenheimer approximation. We find dissociation to the lower ${\mathrm{H}}^{+}+\mathrm{D}(1s)$ state to be about 7% more likely than to the upper $\mathrm{H}(1s)+{\mathrm{D}}^{+}$ final state. The major experimental difficulty in this measurement is the determination of the ${\mathrm{H}}_{2}$ contamination in the HD target. Two different methods of determining this contamination are presented, and the details of the measurement of the relative yields of the final two ground-state dissociation channels are discussed. The experimental results are compared to our coupled channels calculation and to a model based on Meyerhof's approximate formulation.

Polarization of hydrogen molecules HD, D 2 and DT

Highly polarized Hydrogen molecules in the solid phase are of primary interest as “polarized targets” for Nuclear Physics and “polarized fuel” for Inertial Confinement Fusion (ICF). We are developing new techniques to polarize Hydrogen molecules. There are 2 objectives: production of polarized HD targets by static polarization, in order to perform nuclear physics experiments; and investigation of dynamic polarization processes for HD, D 2 and DT, for fusion research. These approaches are described and the results already obtained for the static polarization of HD are given. The possibility to investigate dynamic polarization methods for HD and apply them to DT in order to produce polarized DT fuel for fusion is discussed.

An experimental method for evaluating the H 2 and D 2 contamination levels in a HD bottle

Studies of one- and two-electron processes in collisions with hydrogen molecules using the time-of-flight technique are simplified by using the heteronuclear HD isotope because the H + and D + fragments can be easily distinguished from each other. The difference in the time-of-flight of H + and D + enable the fragments to be measured in coincidence with each other. However, it is hard to determine the purity of the HD target, in particular the H 2 contamination, because the H 2 + molecular ions coincide with the D + fragments. A method is suggested to determine the purity of a HD target to a precision of a few percent. This method is based on measuring the yield of very slow (< 0.5 eV) H + and D + fragments of HD bombarded by fast protons ( E > 1 MeV). The H 2 contamination is then determined by subtracting the theoretically evaluated contribution of the ground-state dissociation of the HD + molecular ion from the measured sum of both H + and D + low-energy fragments.