Assessing Vascular Inflammation with Bioluminescence Imaging

Cardiovascular disease (CVD) is the leading cause of morbidity, mortality, and health care costs in the developed world and is often undiagnosed until clinical presentation with a major adverse cardiovascular event (MACE). Plasma myeloperoxidase (MPO) content is an emerging biomarker for risk, progression, and prognosis at different stages of CVD. Enzyme-linked immunosorbent assays (ELISAs) are currently used to measure clinical plasma MPO concentration, but ELISAs are costly and time-intensive. Luminol is a chemiluminescent compound with specificity for MPO activity in vivo, but is not sensitive enough for use as a bioluminescence reporter of plasma MPO oxidation. The luminol derivative L-012 is more sensitive to oxidation, but little is known about its sensitivity or specificity to reactive oxygen species (ROS) produced by MPO and its interactions with the myriad components of whole plasma. Therefore L-012 was first characterized as an MPO-dependent bioluminescence reporter in whole blood and plasma. The data indicated that L-012 is not a reporter of all MPO-dependent reactions, but specifically reports halogenation with chloride or bromide. Additionally, plasma components inhibited MPO both specifically and non-specifically and prevented precision measurement of plasma MPO activity with L-012 bioluminescence. To overcome this, a method was developed to isolate MPO on a solid substrate, remove small molecule antioxidants, and eliminate specific inhibition by plasma proteins. With this method, MPO activity from human samples could be assayed using bovine plasma supplemented with purified human MPO as calibration standards. The assay can be performed in under an hour, comprises only two steps, and uses no costly immunologic reagents. The assay was validated with a pilot study using 72 plasma samples from cardiology patients undergoing elective catheterization. Individuals in this cohort assayed by the bioluminescence method were concordant with a parallel ELISA within 2 ± 11 μg/L MPO and overall the measurements from the two assays were not significantly different. To further validate the assay, an outside laboratory also measured MPO from 67 of the same plasma samples with an approved, clinical MPO ELISA. The newly developed MPO assay agreed with the outside ELISA on par with the parallel ELISA. In conclusion, a clinical assay for plasma MPO activity was developed with comparable sensitivity and specificity to current ELISAs which can be performed at a fraction of the time and cost

A rapid bioluminescence assay for measuring ​myeloperoxidase activity in human plasma

Myeloperoxidase (MPO) is a circulating cardiovascular disease (CVD) biomarker used to estimate clinical risk and patient prognosis. Current enzyme-linked immunosorbent assays (ELISA) for MPO concentration are costly and time-intensive. Here we report a novel bioluminescence assay, designated MPO activity on a polymer surface (MAPS), for measuring MPO activity in human plasma samples using the bioluminescent substrate L-012. The method delivers a result in under an hour and is resistant to confounding effects from endogenous MPO inhibitors. In a pilot clinical study, we compared MAPS and two clinical ELISAs using 72 plasma samples from cardiac catheterization patients. Results from parallel MAPS and ELISAs were concordant within 2±11 μg l(−1) MPO with similar uncertainty and reproducibility. Results between parallel MAPS and ELISA were in better agreement than those between independent ELISAs. MAPS may provide an inexpensive and rapid assay for determining MPO activity in plasma samples from patients with CVD or potentially other immune and inflammatory disorders

Cerenkov Radiation Energy Transfer (CRET) Imaging: A Novel Method for Optical Imaging of PET Isotopes in Biological Systems

Positron emission tomography (PET) allows sensitive, non-invasive analysis of the distribution of radiopharmaceutical tracers labeled with positron (β(+))-emitting radionuclides in small animals and humans. Upon β(+) decay, the initial velocity of high-energy β(+) particles can momentarily exceed the speed of light in tissue, producing Cerenkov radiation that is detectable by optical imaging, but is highly absorbed in living organisms.To improve optical imaging of Cerenkov radiation in biological systems, we demonstrate that Cerenkov radiation from decay of the PET isotopes (64)Cu and (18)F can be spectrally coupled by energy transfer to high Stokes-shift quantum nanoparticles (Qtracker705) to produce highly red-shifted photonic emissions. Efficient energy transfer was not detected with (99m)Tc, a predominantly γ-emitting isotope. Similar to bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), herein we define the Cerenkov radiation energy transfer (CRET) ratio as the normalized quotient of light detected within a spectral window centered on the fluorophore emission divided by light detected within a spectral window of the Cerenkov radiation emission to quantify imaging signals. Optical images of solutions containing Qtracker705 nanoparticles and [(18)F]FDG showed CRET ratios in vitro as high as 8.8±1.1, while images of mice with subcutaneous pseudotumors impregnated with Qtracker705 following intravenous injection of [(18)F]FDG showed CRET ratios in vivo as high as 3.5±0.3.Quantitative CRET imaging may afford a variety of novel optical imaging applications and activation strategies for PET radiopharmaceuticals and other isotopes in biomaterials, tissues and live animals

Abdominal Imaging Utilization during the First COVID-19 Surge and Utility of Abdominal MRI

We sought to determine relative utilization of abdominal imaging modalities in coronavirus disease 2019 (COVID-19) patients at a single institution during the first surge and evaluate whether abdominal magnetic resonance imaging (MRI) changed diagnosis and management. 1107 COVID-19 patients who had abdominal imaging were analyzed for modality and imaging setting. Patients who underwent abdominal MRI were reviewed to determine impact on management. Of 2259 examinations, 80% were inpatient, 14% were emergency, and 6% were outpatient consisting of 55% radiograph (XR), 31% computed tomography (CT), 13% ultrasound (US), and 0.6% MRI. Among 1107 patients, abdominal MRI was performed in 12 within 100 days of positive SARS-CoV-2 PCR. Indications were unrelated to COVID-19 in 75% while MRI was performed for workup of acute liver dysfunction in 25%. In 1 of 12 patients, MRI resulted in change to management unrelated to COVID-19 diagnosis. During the first surge of COVID-19 at one institution, the most common abdominal imaging examinations were radiographs and CT followed by ultrasound with the majority being performed as inpatients. Future COVID-19 surges may place disproportionate demands on inpatient abdominal radiography and CT resources. Abdominal MRI was rarely performed and did not lead to change in diagnosis or management related to COVID-19 but needs higher patient numbers for accurate assessment of utility

CRET imaging of pseudotumor phantoms in live animals <i>in vivo</i>.

<p>(<b>A</b>) Subcutaneous pseudotumors of 500 nM Qtracker705-impregnated Matrigel (closed arrow) and PBS-impregnated Matrigel (open arrow) in opposing flanks of <i>nu/nu</i> mice were imaged with an IVIS 100 using open, <510 nm (blue), 500–570 nm (green), and >590 nm (red) filters 30 minutes following tail-vein injection of [¹⁸F]FDG (17.6 MBq; 475 µCi). (<b>B</b>) The calculated CRET image.</p

CRET <i>in vitro</i> was dependent on [¹⁸F]FDG radioactivity.

<p>(<b>A</b>) IVIS 100 images of 96-well assay plates using either a <510 nm filter (left) or a >590 nm filter (right). (<b>B</b>) Plot of photon flux from either the <510 nm filter (□) or the >590 filter (▪) with increasing amounts of [¹⁸F]FDG radioactivity. (<b>C</b>) Plot of CRET ratios versus [¹⁸F]FDG radioactivity.</p

CRET ratios of pseudotumors <i>in vivo</i>.

<p>CRET ratios of pseudotumors <i>in vivo</i>.</p

Spectral analysis.

<p>(<b>A</b>) UV/vis emission spectra of [⁶⁴Cu]CuCl₂ in PBS containing various concentrations of Qtracker705 nanoparticles (Qdots ) demonstrate Cerenkov radiation energy transfer (CRET); (blue) [⁶⁴Cu]CuCl₂ without Qdots, (green) [⁶⁴Cu]CuCl₂ with 49 nM Qdots, (orange) [⁶⁴Cu]CuCl₂ with 222 nM Qdots, (red) [⁶⁴Cu]CuCl₂ with 400 nM Qdots, (black) non-radioactive CuCl₂ without Qdots, (brown) non-radioactive CuCl₂ with 400 nM Qdots, (gray) decayed [⁶⁴Cu]CuCl₂ with 400 nM Qdots. (<b>B</b>) Fluorescence emission spectrum (350 nm excitation) of decayed (>8 half-lives) [⁶⁴Cu]CuCl₂ in PBS containing 400 nM Qtracker705. (<b>C</b>) UV/vis emission spectra of [^99mTc]NaTcO₄ in PBS without (black) and with (red) 400 nM Qtracker705 nanoparticles.</p