Functionalized Polysaccharides Improve Sensitivity of Tyramide/Peroxidase Proximity Labeling Assays through Electrostatic Interactions.

High-throughput assays that efficiently link genotype and phenotype with high fidelity are key to successful enzyme engineering campaigns. Among these assays, the tyramide/peroxidase proximity labeling method converts the product of an enzymatic reaction of a surface expressed enzyme to a highly reactive fluorescent radical, which labels the cell surface. In this context, maintaining the proximity of the readout reagents to the cell surface is crucial to prevent crosstalk and ensure that short-lived radical species react before diffusing away. Here, we investigated improvements in tyramide/peroxidase proximity labeling for enzyme screening. We modified chitosan (Cs) chains with horseradish peroxidase (HRP) and evaluated the effects of these conjugates on the efficiency of proximity labeling reactions on yeast cells displaying d-amino acid oxidase. By tethering HRP to chitosan through different chemical approaches, we localized the auxiliary enzyme close to the cell surface and enhanced the sensitivity of tyramide-peroxidase labeling reactions. We found that immobilizing HRP onto chitosan through a 5 kDa PEG linker improved labeling sensitivity by over 3.5-fold for substrates processed with a low turnover rate (e.g., d-lysine), while the sensitivity of the labeling for high activity substrates (e.g., d-alanine) was enhanced by over 0.6-fold. Such improvements in labeling efficiency broaden the range of enzymes and conditions that can be studied and screened by tyramide/peroxidase proximity labeling.

68Ga-labeled amphiphilic polymer nanoparticles for PET imaging of sentinel lymph node metastasis.

Precise diagnosis of lymph node metastasis is important for therapeutic regimen planning, prognosis analysis and probably better outcomes for cancer patients. In this work, 68Ga-labeled amphiphilic alternating copolymers nanoparticles with different rigid ligands were synthesized as positron emission tomography (PET) probes for lymph node metastasis imaging. The labeling efficiency and stability of nanoparticles was improved with increased rigidity of coordination unit. PU(68Ga-L-MDI-PEG) nanoparticles (PU(68Ga-L-MDI-PEG) NPs) with the strongest rigidity of coordination unit exhibited the lowest critical micelle concentration, the best 68Ga labeling efficiency and stability. During in vivo lymph node metastasis imaging, PU(68Ga-L-MDI-PEG) NPs led to different accumulations in normal lymph nodes (N-LN) and tumor metastasized sentinel lymph nodes (T-SLN), which resulted in different PET signal presentation, making it feasible to differentiate N-LN from T-SLN. In comparison, small molecule probe 68GaL had poor lymph node accumulation, not only making it difficult to find lymph nodes on PET/computed tomography scan, but also tough to distinguish N-LN from metastatic ones. Overall, this work provides a reference for design of 68Ga labeled polymeric nanoparticles with high chelation efficiency and stability, as sensitive PET probes for lymph node imaging.

Dyestuff chemistry auxiliary instant immune-network label strategy for immunochromatographic detection of chloramphenicol.

Commercial immunochromatographic assay (ICA) is a convenient tool for controlling antibiotic abuse. Although great efforts have been made to improve the detection performance and quantitative capabilities, simplify the manufacturing process of commercial ICA is rarely mentioned. Here, a proof-of-principle work are developed to solve the above problem. Inspired by dyestuff chemistry, we developed an instant immune-network label strategy by dynamic protonation capacity of neutral red (NR), achieving a desirable labeling efficiency (＜1min), and applying for ICA detection of chloramphenicol (CAP). Benefits from the efficiently protonation of NR, lengthy probe production time and organic reagents can be avoided, displaying excellent strip production efficiency and detection performance. Eventually, this strategy presents a visual limit of detection (vLOD) at 3 ng/mL, cut-off value is 9 ng/mL. The assay recoveries in milk and honey were 74.45-107.15 %, with the total RSD of 1.62-6.90 %. We envision that this strategy raises the possibility of commercializing of laboratory prototype products.

Optimized sample preparation and data analysis for TMT proteomic analysis of cerebrospinal fluid applied to the identification of Alzheimer's disease biomarkers.

BACKGROUND: Cerebrospinal fluid (CSF) is an important biofluid for biomarkers of neurodegenerative diseases such as Alzheimer's disease (AD). By employing tandem mass tag (TMT) proteomics, thousands of proteins can be quantified simultaneously in large cohorts, making it a powerful tool for biomarker discovery. However, TMT proteomics in CSF is associated with analytical challenges regarding sample preparation and data processing. In this study we address those challenges ranging from data normalization over sample preparation to sample analysis. METHOD: Using liquid chromatography coupled to mass-spectrometry (LC-MS), we analyzed TMT multiplex samples consisting of either identical or individual CSF samples, evaluated quantification accuracy and tested the performance of different data normalization approaches. We examined MS2 and MS3 acquisition strategies regarding accuracy of quantification and performed a comparative evaluation of filter-assisted sample preparation (FASP) and an in-solution protocol. Finally, four normalization approaches (median, quantile, Total Peptide Amount, TAMPOR) were applied to the previously published European Medical Information Framework Alzheimer's Disease Multimodal Biomarker Discovery (EMIF-AD MBD) dataset. RESULTS: The correlation of measured TMT reporter ratios with spiked-in standard peptide amounts was significantly lower for TMT multiplexes composed of individual CSF samples compared with those composed of aliquots of a single CSF pool, demonstrating that the heterogeneous CSF sample composition influences TMT quantitation. Comparison of TMT reporter normalization methods showed that the correlation could be improved by applying median- and quantile-based normalization. The slope was improved by acquiring data in MS3 mode, albeit at the expense of a 29% decrease in the number of identified proteins. FASP and in-solution sample preparation of CSF samples showed a 73% overlap in identified proteins. Finally, using optimized data normalization, we present a list of 64 biomarker candidates (clinical AD vs. controls, p < 0.01) identified in the EMIF-AD cohort. CONCLUSION: We have evaluated several analytical aspects of TMT proteomics in CSF. The results of our study provide practical guidelines to improve the accuracy of quantification and aid in the design of sample preparation and analytical protocol. The AD biomarker list extracted from the EMIF-AD cohort can provide a valuable basis for future biomarker studies and help elucidate pathogenic mechanisms in AD.

Optimization of pseudo-continuous arterial spin labeling using off-resonance compensation strategies at 7T.

PURPOSE: The sensitivity of pseudo-continuous arterial spin labeling (PCASL) to off-resonance effects (ΔB0 ) is a major limitation at ultra-high field (≥7T). The aim of this study was to assess the effectiveness of different PCASL ΔB0 compensation methods at 7T and measure the labeling efficiency with off-resonance correction. THEORY AND METHODS: Phase offset errors induced by ΔB0 at the feeding arteries can be compensated by adding an extra radiofrequency (RF) phase increment and transverse gradient blips into the PCASL RF pulse train. The effectiveness of an average field correction (AVGcor), a vessel-specific field-map-based correction (FMcor) and a vessel-specific prescan-based correction (PScor) were compared at 7T. After correction, the PCASL labeling efficiency was directly measured in feeding arteries downstream from the labeling location. RESULTS: The perfusion signal was more uniform throughout the brain after off-resonance correction. Whole-brain average perfusion signal increased by a factor of 2.4, 2.5, and 2.1, respectively, with AVGcor, FMcor and PScor compared to acquisitions without correction. With off-resonance correction, the maximum labeling efficiency was ~0.68 at mean B1 (B1mean ) of 0.70 µT when using a mean gradient (Gmean ) of 0.25 mT/m. CONCLUSION: Either a prescan or a field map can be used to correct for off-resonance effects and retrieve a good brain perfusion signal at 7T. Although the three methods performed well in this study, FMcor may be better suited for patient studies because it accounted for vessel-specific ΔB0 variations. Further improvements in image quality will be possible by optimizing the labeling efficiency with advanced hardware and software while satisfying specific absorption rate constraints.

99m Technetium-HMPAO-labeled platelet scan in practice: Preparation, quality control, and biodistribution studies

Abstract There is no biodistribution or imaging data on 99mtechnetium (Tc)-hexamethyl propylamine oxime (HMPAO)-labeled platelets in the literature. The current study aimed to present updated information about the clinical procedures for preparation and use of labeled platelets. Following two-step centrifugation at 1500 and 2500 rpm, the platelets were extracted from whole blood into platelet-rich plasma (PRP) above the buffy coat and then from PRP into a platelet pellet at the bottom of the tube. The 99mTc-HMPAO-labeled platelets were inspected for purity, viability, release of 99mTc from platelets, and sterility. Also, microscopic examination and thin layer chromatography (TLC) were performed. Biodistribution was assessed following necropsy in BALB/c mice and through imaging of New Zealand rabbits. The separation ratio was estimated at 98%, and radiochemical purity was measured to be 80%. The labeling efficiency was above 30% in more than half of the assays (range: 17-43%). The release of 99mTc from platelets was 9% per hour at 37ºC. After 24 hours, stability was estimated at 54% in the human serum. The target organs of mice included the spleen and liver. In rabbits, the imaging results indicated liver as the target organ. Thyroid uptake was negligible up to 90 minutes. Based on the findings, extraction of platelets and labeling them with 99mTc-HMPAO is a feasible and safe approach in routine practice.

[Factors influencing platelets labelling with indium-111 oxine]. / Facteurs influençant le marquage des plaquettes à l'oxinate d'indium-111.

Autologous indium-111 labelled platelets can be used for kinetic studies in patients with autoimmune thrombocytopenic purpura [AITP]. The objective of this study was to evaluate some biological and clinical factors influencing the labeling efficiency. METHODS: We studied incubation media (Plasma media [MP] or dry media [MS]), platelet concentration [NP], mean platelet volume [VPM], hemoglobin level and pathology associated with AITP. RESULTS: This was a retrospective study of 93 platelets labelling (43 in MS and 50 in MP), 38 primary AITP (41%) and 55 secondary AITP (59%). The labeling efficiency was 72% (78% in MS versus 53% in MP; p < 0.0001). The labeling efficiency was correlated with VPM (p = 0.0004), NP (p = 0.03), hemoglobin level (p = 0.037) and type of AITP (p = 0.0036). The incubation medium, hemoglobin level and type of the AITP have an independent predictive value on the labeling efficiency. CONCLUSION: These data confirm the influence of the incubation medium on the labeling efficiency and identify two other predictive criteria, hemoglobin level and type of AITP.

Determining the Efficiency of Single Molecule Quantum Dot Labeling of HER2 in Breast Cancer Cells.

Quantum dots exhibit unique properties compared to other fluorophores, such as bright fluorescence and lack of photobleaching, resulting in their widespread utilization as fluorescent protein labels in the life sciences. However, their application is restricted to relative quantifications due to lacking knowledge about the labeling efficiency. We here present a strategy for determining the labeling efficiency of quantum dot labeling of HER2 in overexpressing breast cancer cells. Correlative light- and liquid-phase electron microscopy of whole cells was used to convert fluorescence intensities into the underlying molecular densities of the quantum dots. The labeling procedure with small affinity proteins was optimized yielding a maximal labeling efficiency of 83%, which was applicable to the high amount of ∼1.5 × 106 HER2 per cell. With the labeling efficiency known, it is now possible to derive the absolute protein expression levels in the plasma membrane and its variation within a cell and between cells.

Supporting measurements or more averages? How to quantify cerebral blood flow most reliably in 5 minutes by arterial spin labeling.

PURPOSE: To determine whether sacrificing part of the scan time of pseudo-continuous arterial spin labeling (PCASL) for measurement of the labeling efficiency and blood T1 is beneficial in terms of CBF quantification reliability. METHODS: In a simulation framework, 5-minute scan protocols with different scan time divisions between PCASL data acquisition and supporting measurements were evaluated in terms of CBF estimation variability across both noise and ground truth parameter realizations taken from the general population distribution. The entire simulation experiment was repeated for a single-post-labeling delay (PLD), multi-PLD, and free-lunch time-encoded (te-FL) PCASL acquisition strategy. Furthermore, a real data study was designed for preliminary validation. RESULTS: For the considered population statistics, measuring the labeling efficiency and the blood T1 proved beneficial in terms of CBF estimation variability for any distribution of the 5-minute scan time compared to only acquiring ASL data. Compared to single-PLD PCASL without support measurements as recommended in the consensus statement, a 26%, 33%, and 42% reduction in relative CBF estimation variability was found for optimal combinations of supporting measurements with single-PLD, free-lunch, and multi-PLD PCASL data acquisition, respectively. The benefit of taking the individual variation of blood T1 into account was also demonstrated in the real data experiment. CONCLUSIONS: Spending time to measure the labeling efficiency and the blood T1 instead of acquiring more averages of the PCASL data proves to be advisable for robust CBF quantification in the general population.

Practical considerations for territorial perfusion mapping in the cerebral circulation using super-selective pseudo-continuous arterial spin labeling.

PURPOSE: This paper discusses several challenges faced by super-selective pseudo-continuous arterial spin labeling, which is used to quantify territorial perfusion in the cerebral circulation. The effects of off-resonance, pulsatility, vessel movement, and label rotation scheme are investigated, and methods to maximize labeling efficiency and overall image quality are evaluated. A strategy to calculate the territorial perfusion fractions of individual vessels is proposed. METHODS: The effects of off-resonance, label rotation scheme, and vessel movement on labeling efficiency were simulated. Two off-resonance compensation strategies (multiphase prescan, field map), cardiac triggering, and vessel movement were studied in vivo in a group of 10 subjects. Subsequently, a territorial perfusion fraction map was acquired in 2 subjects based on the mean vessel labeling efficiency. RESULTS: Multiphase calibration provided the highest labeling efficiency (P = .002) followed by the field map compensation (P = .037) compared with the uncompensated acquisition. Cardiac triggering resulted in a qualitative improvement of the image and an increase in signal contrast between the perfusion territory and the surrounding tissue (P = .010) but failed to show a significant change in temporal and spatial SNR. The constant clockwise label rotation scheme yielded the highest labeling efficiency. Significant vessel movement (>2 mm according to simulations) was observed in 50% of subjects. The measured territorial perfusion fractions showed good agreement with anatomical data. CONCLUSION: Optimized labeling efficiency resulted in increased image quality and accuracy of territorial perfusion fraction maps. Labeling efficiency depends critically on off-resonance calibration, cardiac triggering, optimal label rotation scheme, and vessel location tracking.

TMT Labeling for the Masses: A Robust and Cost-efficient, In-solution Labeling Approach.

Isobaric stable isotope labeling using, for example, tandem mass tags (TMTs) is increasingly being applied for large-scale proteomic studies. Experiments focusing on proteoform analysis in drug time course or perturbation studies or in large patient cohorts greatly benefit from the reproducible quantification of single peptides across samples. However, such studies often require labeling of hundreds of micrograms of peptides such that the cost for labeling reagents represents a major contribution to the overall cost of an experiment. Here, we describe and evaluate a robust and cost-effective protocol for TMT labeling that reduces the quantity of required labeling reagent by a factor of eight and achieves complete labeling. Under- and overlabeling of peptides derived from complex digests of tissues and cell lines were systematically evaluated using peptide quantities of between 12.5 and 800 µg and TMT-to-peptide ratios (wt/wt) ranging from 8:1 to 1:2 at different TMT and peptide concentrations. When reaction volumes were reduced to maintain TMT and peptide concentrations of at least 10 mm and 2 g/l, respectively, TMT-to-peptide ratios as low as 1:1 (wt/wt) resulted in labeling efficiencies of > 99% and excellent intra- and interlaboratory reproducibility. The utility of the optimized protocol was further demonstrated in a deep-scale proteome and phosphoproteome analysis of patient-derived xenograft tumor tissue benchmarked against the labeling procedure recommended by the TMT vendor. Finally, we discuss the impact of labeling reaction parameters for N-hydroxysuccinimide ester-based chemistry and provide guidance on adopting efficient labeling protocols for different peptide quantities.

The effect of surface charge on the cytotoxicity and uptake of carbon quantum dots in human umbilical cord derived mesenchymal stem cells.

Carbon quantum dots (CQDs) are emerging as an ideal agent for efficient stem cell labeling. In current study, we synthesized a series of CQDs carrying different surface charges by changing the mass ratio of diammonium citrate (DC) and spermidine (Spd), and evaluated the effects of different surface charges on the cytotoxicity, cellular uptake, stability in human umbilical cord derived mesenchymal stem cells (hUCMSCs). We ascertained the optimal labeling time (24 h) and subtoxic concentration (50 µg/mL) of all different charged CQDs. Our results demonstrated that, although positively charged CQDs are more cytotoxic and have lower photoluminescence (PL) compared to negative CQDs, they still have higher labeling efficiency for their higher uptake capacity. We found that relatively weak positive surface charges enabled CQDs to possess good biocompatibility and labeling efficiency in hUCMSCs. This work will helpfully contribute to the design and optimization of CQDs for tracking stem cells and further benefit to clinical research and application.

Simultaneous measurement of brain perfusion and labeling efficiency in a single pseudo-continuous arterial spin labeling scan.

PURPOSE: The aim of this study was to propose, optimize, and validate a pseudo-continuous arterial spin labeling (pCASL) sequence for simultaneous measurement of brain perfusion and labeling efficiency. METHODS: The proposed sequence incorporates the labeling efficiency measurement into the postlabeling delay period of a conventional perfusion pCASL sequence by using the time-encoding approach. In vivo validation experiments were performed on nine young subjects by comparing it to separate perfusion and labeling efficiency sequences. Sensitivity of the proposed combined sequence for measuring labeling efficiency changes was further addressed by varying the flip angles of the pCASL labeling radiofrequency pulses. RESULTS: The proposed combined sequence decreased the perfusion signal by ∼4% and a lower labeling efficiency (by ∼10%) was found as compared to the separate sequences. However, the temporal signal-noise-ratio of the perfusion signal remained unchanged. When the pCASL flip angle was decreased to a suboptimal setting, a strong correlation was found between the combined and the separate sequences for the relative change in pCASL perfusion signal as well as for the relative change in labeling efficiency. High correlation was also observed between relative changes in perfusion signal and the measured labeling efficiencies. CONCLUSION: The proposed sequence allows simultaneous measurement of brain perfusion and labeling efficiency with high time-efficiency at the price of only a small compromise in measurement accuracy. The additional labeling efficiency measurement can be used to facilitate qualitative interpretation of pCASL perfusion images. Magn Reson Med 79:1922-1930, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Pseudo continuous arterial spin labeling quantification in anemic subjects with hyperemic cerebral blood flow.

PURPOSE: To investigate possible sources of quantification errors in global cerebral blood flow (CBF) measurements by comparing pseudo continuous arterial spin labeling (PCASL) and phase contrast (PC) MRI in anemic, hyperemic subjects. METHODS: All studies were performed on a Philips 3T Achieva MRI scanner. PC and PCASL CBF examinations were performed in 10 healthy, young adult subjects and 18 young adults with chronic anemia syndromes including sickle cell disease and thalassemia. CBF estimates from single and two compartment ASL kinetic models were compared. Numerical simulation and flow phantom experiments were used to explore the effects of blood velocity and B1+ on CBF quantification and labeling efficiency. RESULTS: PCASL CBF underestimated PC in both populations using a single compartment model (30.1±9.2% control, 45.2±17.2% anemia). Agreement substantially improved using a two-compartment model (-8.0±6.0% control, 11.7±12.3% anemia). Four of the anemic subjects exhibited venous outflow of ASL signal, suggestive of cerebrovascular shunt, possibly confounding PC-PCASL comparisons. Additionally, sub-study experiments demonstrated that B1+ was diminished at the labeling plane (82.9±5.1%), resulting in suboptimal labeling efficiency. Correcting labeling efficiency for diminished B1+, PCASL slightly overestimated PC CBF in controls (-15.4±6.8%) and resulted in better matching of CBF estimates in anemic subjects (0.7±10.0% without outflow, 10.5±9.4% with outflow). CONCLUSIONS: This work demonstrates that a two-compartment model is critical for PCASL quantification in hyperemic subjects. Venous outflow and B1+ under-excitation may also contribute to flow underestimation, but further study of these effects is required.

Characterization of pseudo-continuous arterial spin labeling: Simulations and experimental validation.

PURPOSE: To characterize pseudo-continuous arterial spin labeling (pCASL) through simulations of spin inversion and to discuss suitable parameter settings for measuring cerebral perfusion. METHODS: Simulations of arterial spin inversion in pCASL were performed based on the Bloch equation. Both the labeling and the control condition of pCASL were analyzed separately, and the labeling efficiency, α, was calculated depending on the averages of both, the radiofrequency (RF) field amplitude and labeling gradient strength. The influence of additional parameters characterizing the pCASL pulse sequence, such as the interpulse interval, the RF duty cycle, and the labeling gradient, also were studied. An echo-planar imaging protocol utilizing a short repetition time was developed for experimental validation by estimating α in the internal carotid artery. RESULTS: The effectiveness of the control condition of balanced pCASL crucially depends on both the labeling gradient amplitude and the RF duty cycle. The use of large values for both quantities improves the insensitivity to off-resonance gradients caused by magnetic field inhomogeneities. In addition, balanced and unbalanced pCASL become comparably effective. CONCLUSION: By use of appropriate parameter settings, labeling efficiencies of around 90% are feasible, independent of expected off-resonance gradients at 3T. Magn Reson Med 79:1638-1649, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Atomic mass tag of bismuth-209 for increasing the immunoassay multiplexing capacity of mass cytometry.

Mass cytometry (or CyTOF) is an atomic mass spectrometry-based single-cell immunoassay technology, which has provided an increasingly systematic and sophisticated view in basic biological and clinical studies. Using elemental reporters composed of stable heavy metal isotopes, more than 50 cellular parameters are measured simultaneously. However, this current multiplexing does not meet the theoretical capability of CyTOF instrumentation with 135 detectable channels, primarily due to the limitation of available chemistries for conjugating elemental mass tags to affinity reagents. To address this issue, we develop herein additional metallic mass tag based on bismuth-209 (209 Bi) for efficient conjugation to monoclonal antibody. This enables the use of an addtional channel m/z = 209 of CyTOF for single-cell immunoassays. Bismuth has nearly the same charge-to-radius ratio as lanthanide elements; thus, bismuth(III) cations (209 Bi3+ ) could coordinate with DTPA chelators in the same geometry of O- and N-donor groups as that of lanthanide. In this report, the coordination chemistry of 209 Bi3+ with DTPA chelators and Maxpar® X8 polymers were investigated in details. Accordingly, the protocols of conjugating antibody with bismuth mass tag were provided. A method based on UV-Vis absorbance at 280 nm of 209 Bi3+ -labeling DTPA complexes was developed to evaluate the stoichiometric ratio of 209 Bi3+ cations to the conjugated antibody. Side-by-side single-cell analysis experiments with bismuth- and lanthanide-tagged antibodies were carried out to compare the analytical sensitivities. The measurement accuracy of bismuth-tagged antibody was validated within in vitro assay using primary human natural killer cells. Furthermore, bismuth-tagged antibodies were successfully employed in cell cycle measurements and high-dimensional phenotyping immunoassays. © 2017 International Society for Advancement of Cytometry.

Influence of binding mechanism on labeling efficiency and luminous properties of fluorescent cellulose nanocrystals.

Cellulose nanocrystals (CNCs) have excellent properties, such as reproducibility, low biodegradability and a large amount of reactive hydroxyl groups on the surface. This study focused on the labeling efficiency and fluorescent properties of the fluorescent labeling of CNCs by means of electrostatic adsorption and covalent bonding. The CNCs in the sample were approximately 94.76% successfully labeled with dyes, and the number of dye molecules adsorbed by per CNC was approximately 208 by electrostatic adsorption. For the sample covalently linked, the efficiency of the fluorescent labeling was 95.51%, and the number of dye molecules attached to per CNC was 1038. The quenching mode of the fluorescent CNCs was dynamic quenching. The fluorescence lifetime and quantum yield of the fluorescent CNCs increased by 1-2 times compared to the free dye. A thorough investigation of the relation between the binding mechanism and the fluorescent properties in fluorescent CNCs was conducted.

Improving labeling efficiency in automatic quality control of MRSI data.

PURPOSE: To improve the efficiency of the labeling task in automatic quality control of MR spectroscopy imaging data. METHODS: 28'432 short and long echo time (TE) spectra (1.5 tesla; point resolved spectroscopy (PRESS); repetition time (TR)= 1,500 ms) from 18 different brain tumor patients were labeled by two experts as either accept or reject, depending on their quality. For each spectrum, 47 signal features were extracted. The data was then used to run several simulations and test an active learning approach using uncertainty sampling. The performance of the classifiers was evaluated as a function of the number of patients in the training set, number of spectra in the training set, and a parameter α used to control the level of classification uncertainty required for a new spectrum to be selected for labeling. RESULTS: The results showed that the proposed strategy allows reductions of up to 72.97% for short TE and 62.09% for long TE in the amount of data that needs to be labeled, without significant impact in classification accuracy. Further reductions are possible with significant but minimal impact in performance. CONCLUSION: Active learning using uncertainty sampling is an effective way to increase the labeling efficiency for training automatic quality control classifiers. Magn Reson Med 78:2399-2405, 2017. © 2017 International Society for Magnetic Resonance in Medicine.

Measuring the labeling efficiency of pseudocontinuous arterial spin labeling.

PURPOSE: Optimization and validation of a sequence for measuring the labeling efficiency of pseudocontinuous arterial spin labeling (pCASL) perfusion MRI. METHODS: The proposed sequence consists of a labeling module and a single slice Look-Locker echo planar imaging readout. A model-based algorithm was used to calculate labeling efficiency from the signal acquired from the main brain-feeding arteries. Stability of the labeling efficiency measurement was evaluated with regard to the use of cardiac triggering, flow compensation and vein signal suppression. Accuracy of the measurement was assessed by comparing the measured labeling efficiency to mean brain pCASL signal intensity over a wide range of flip angles as applied in the pCASL labeling. RESULTS: Simulations show that the proposed algorithm can effectively calculate labeling efficiency when correcting for T1 relaxation of the blood spins. Use of cardiac triggering and vein signal suppression improved stability of the labeling efficiency measurement, while flow compensation resulted in little improvement. The measured labeling efficiency was found to be linearly (R = 0.973; P < 0.001) related to brain pCASL signal intensity over a wide range of pCASL flip angles. CONCLUSION: The optimized labeling efficiency sequence provides robust artery-specific labeling efficiency measurement within a short acquisition time (∼30 s), thereby enabling improved accuracy of pCASL CBF quantification. Magn Reson Med 77:1841-1852, 2017. © 2016 International Society for Magnetic Resonance in Medicine Magn Reson Med 77:1841-1852, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Investigation of control scans in pseudo-continuous arterial spin labeling (pCASL): Strategies for improving sensitivity and reliability of pCASL.

PURPOSE: To investigate the performance of control scans in pseudo-continuous ASL (pCASL) and propose strategies for improving sensitivity and reliability of pCASL. METHODS: The labeling efficiencies of pCASL with conventional control scan and distal control scan were investigated at various radiofrequency (RF) duration/spacing of 0.5/1-2/4 ms, mean slice-selection gradients (GSS ) of 1 and 0 mT/m, and total labeling durations of 1.5-3 s, through Bloch equation simulations and in vivo experiments. In addition, the feasibility of three-dimensional (3D) pCASL with the distal control scan and control scan with no RF preparation was demonstrated in a wide brain area, by suppressing the magnetization transfer (MT) effects with high GSS while maintaining the GSS /mean GSS ratio. RESULTS: The distal control scan provided pCASL signals approximately 40% higher and more robust to variations in the labeling conditions than those from the conventional control scan. The distal and no RF control scans with high GSS provided uniform pCASL signals in approximately 8-cm-thick imaging region with MT contributions <10% of the perfusion signals. CONCLUSIONS: pCASL perfusion signals can be enhanced (∼40%) and become more stable by using the distal or no RF control scan, which can be applied in a wide area by increasing GSS while maintaining GSS /mean GSS . Magn Reson Med 78:917-929, 2017. © 2016 International Society for Magnetic Resonance in Medicine.