Quantitative bioimaging of copper in frozen liver specimens from cats using laser ablation-inductively coupled plasma-mass spectrometry.

OBJECTIVES: The aim of this study was to assess laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) as a tool for measuring concentrations and determining accumulation of copper in frozen liver specimens from cats. METHODS: Six frozen liver specimens were evaluated by qualitative copper staining and quantitative flame atomic absorption spectroscopy. Tissue specimens were cryo-sectioned and quantitative bioimaging of copper was performed using LA-ICP-MS. Results were compared with those obtained using conventional methods. RESULTS: Of the six specimens, only one showed positive staining for copper with rhodanine. Using flame atomic absorption spectroscopy (FAAS), one specimen showed a deficient copper level (<100 µg/g dry weight), two specimens had copper within the reference interval (RI; 150-180 µg/g) and three specimens had copper concentrations above the RI. Bioimaging from LA-ICP-MS showed inhomogeneous distribution of hepatic copper. The areas with dense copper accumulation were represented as hotspots in the liver specimens. Hepatic copper quantification by LA-ICP-MS correlated well with copper quantified by FAAS (r = 0.96, P = 0.002). CONCLUSIONS AND RELEVANCE: Our findings suggest that quantitative bioimaging by LA-ICP-MS could be used to demonstrate the distribution and concentration of copper in frozen liver specimens from cats. The distribution of copper in these specimens was inhomogeneous with dense accumulation represented as hotspots on tissue sections. A positive correlation of hepatic copper concentrations determined by LA-ICP-MS and FAAS was found. Further studies to establish an RI for hepatic copper using this technique and to further determine its clinical utility are warranted.

Elemental Imaging of Long-term Gadolinium Retention in Rodent Femur.

Background The use of gadolinium-based contrast agents (GBCAs) is linked to gadolinium retention in the skeleton of healthy individuals. The mechanism of gadolinium incorporation into bone tissue is not fully understood and requires spatially resolved analysis to locate the gadolinium. Purpose To compare the quantitative distribution of gadolinium retained over time in rodent femur following the administration of gadodiamide and gadobutrol at three different time points. Materials and Methods In this animal study conducted between May 2018 and April 2020, 108 9-week-old healthy rats were repeatedly injected with either gadodiamide, gadobutrol, or saline solution and were killed 1, 3, or 12 months after the last injection. The femurs of six female and six male rats per each group and time point were collected. Quantitative elemental imaging of gadolinium in longitudinal thin sections was performed on one sample per sex with use of laser ablation inductively coupled plasma mass spectrometry (ICP-MS). Gadolinium concentration was determined with use of ICP-MS on the samples of all animals (six per group). Mann-Whitney U tests were applied on pairwise comparisons to determine potential sex effect and GBCA effect on gadolinium concentrations. Results The highest gadolinium retention was observed in the gadodiamide group (concentration, 97-200 nmol · g-1), exceeding the mean concentration in the gadobutrol group (6.5-17 nmol · g-1). However, the gadolinium distribution pattern was similar for both contrast agents, showing prominent gadolinium retention at endosteal surfaces, in the bone marrow, and in small tissue pores. Gadolinium distribution in cortical bone changed over time, initially showing a thin rim of higher concentration close to the periosteum, which appeared to grow wider and move toward the interior of the femur over 1 year. Conclusion For both gadolinium-based contrast agents, gadolinium retention in rat bone was initially located close to the periosteum and bone cavities and changed with bone remodeling processes. The relevance to long-term storage of gadolinium in humans remains to be determined. © RSNA, 2022 Online supplemental material is available for this article.

Long-term Gadolinium Retention in the Healthy Rat Brain: Comparison between Gadopiclenol, Gadobutrol, and Gadodiamide.

Background Safety concerns caused by gadolinium retention call for the development of high-relaxivity gadolinium-based contrast agents (GBCAs) allowing minimal dosing. Purpose To investigate brain gadolinium retention in healthy rats after exposure to gadopiclenol (Elucirem, Guerbet; macrocyclic GBCA) compared with gadobutrol (Gadovist or Gadavist, Bayer; macrocyclic GBCA) and gadodiamide (Omniscan, GE Healthcare; linear GBCA) over 1 year. Materials and Methods In this study conducted between May 2018 and April 2020, 9-week-old healthy Sprague Dawley rats received five injections of either gadopiclenol, gadobutrol, or gadodiamide (2.4 mmol of gadolinium per kilogram of body weight for each), or saline (control animals) over a period of 5 weeks. Rats were randomly assigned to different groups (six female and six male rats per group). MRI examinations were performed before euthanasia at 1, 3, 5, or 12 months after the last injection. Brains were sampled to determine the total gadolinium content via inductively coupled plasma mass spectrometry (ICP-MS), to characterize gadolinium species with size exclusion chromatography (SEC)-ICP-MS, and to perform elemental mapping with laser ablation (LA)-ICP-MS. Mann-Whitney tests were performed on pairwise comparisons of the same time points. Results For both macrocyclic agents, no T1 signal hyperintensities were observed in the cerebellum, and approximately 80% of gadolinium washout was found between 1 month (gadobutrol, 0.30 nmol/g; gadopiclenol, 0.37 nmol/g) and 12 months (gadobutrol, 0.062 nmol/g; gadopiclenol, 0.078 nmol/g). After 12 months, only low-molecular-weight gadolinium species were detected in the soluble fraction. Gadodiamide led to significantly higher gadolinium concentrations after 1 month in the cerebellum (gadodiamide, 2.65 nmol/g; P < .001 vs both macrocyclics) combined with only 15% washout after 12 months (gadodiamide, 2.25 nmol/g) and with gadolinium detected bound to macromolecules. Elemental bioimaging enabled visualization of gadolinium deposition patterns colocalized with iron. Conclusion Gadopiclenol and gadobutrol demonstrated similar in vivo distribution and washout of gadolinium in the healthy rat brain, markedly differing from gadodiamide up to 12 months after the last injection. © RSNA, 2022 Online supplemental material is available for this article.

Combined speciation analysis and elemental bioimaging provide new insight into gadolinium retention in kidney.

This study uses a leaching approach in combination with elemental bioimaging and speciation analysis to obtain insight into the gadolinium species present in the kidney of rats that were treated with either a linear or a macrocyclic gadolinium-based contrast agent. Fresh frozen thin sections of the harvested kidneys were immersed halfway into water to wash out hydrophilic species and subsequently analyzed by laser ablation-inductively coupled plasma-mass spectrometry. The water-extracted gadolinium species were analyzed by means of hydrophilic interaction liquid chromatography-inductively coupled plasma-mass spectrometry. Information on the water-soluble species could not only be obtained from the full kidney, but also be traced back to its localization in the tissue. On longitudinal kidney sections treated with gadobutrol, it was found that water-insoluble, permanent Gd depositions were mainly located in the renal cortex, while water-soluble species were found in the medulla, which contains the intact contrast agent up to 1 year after injection. Moreover, kidney samples from gadodiamide-treated rats showed more water-insoluble Gd deposition in both the cortex and medulla, while the concentration of intact contrast agent in the water-soluble fraction was below the limit of detection after 12 months. In conclusion, this rapid approach allowed the spatially resolved differentiation between water-soluble and insoluble gadolinium deposition and is therefore capable of generating new insight into the retention and transportation behavior of gadolinium.

Weighted Linear Regression Improves Accuracy of Quantitative Elemental Bioimaging by Means of LA-ICP-MS.

The application of ordinary least squares (OLS) linear regression is widely used in order to approximate linear external calibration data. However, the assumption of homoscedasticity is often not considered as a requirement for correct data approximation, which can result in a poor regression fit that is often more prominent in the lower concentration range. Heteroscedasticity in inductively coupled plasma-mass spectrometry (ICP-MS) data has been discussed in literature as an intrinsic problem and was found to be addressed better by the use of weighted least squares (WLS) regression in multiple studies. In this study, the effects of OLS and WLS linear regression models have been investigated for quantitative imaging experiments by means of laser ablation (LA)-ICP-MS using matrix-matched standards. The calibration data produced by this technique was found to be heteroscedastic in all 60 analyzed datasets, which yielded poor regression fits for OLS linear regression. In comparison to conventional ICP-MS analysis, the resulting negative effects were found to become even more visible in imaging LA-ICP-MS due to an inaccurate estimation of the regression line's intercept. Also, the calculation of average concentrations in selected regions of interest (ROIs) yields incorrect quantification results at the lower end of the calibration range. The application of WLS linear regression resulted in an improved goodness of fit (GOF), although the weighting factor should be selected carefully. Besides the reciprocal of the variance of each calibration standard (1/si2), more empirical weighting factors that have been discussed in the literature were also evaluated regarding the GOF.

Plug-and-play laser ablation-mass spectrometry for molecular imaging by means of dielectric barrier discharge ionization.

The plug-and-play hyphenation of UV-laser ablation (LA) and mass spectrometry is presented, using dielectric barrier discharge ionization (DBDI). The DBDI source employed here is characterized by its unique geometry, being directly mounted onto the inlet capillary of a mass spectrometer. In the literature, this particular kind of DBDI source is also referred to as active capillary plasma ionization. It has been commercialized as soft ionization by chemical reaction in transfer (SICRIT) and will be addressed as DBDI in this study. LA-DBDI-MS was used for the direct, molecule-specific and spatially resolved analysis of various solid samples, such as coffee beans and pain killer tablets without extensive sample preparation. The combination of fast washout UV-laser ablation and the principle of the DBDI source used here allowed for highly efficient soft ionization as well as high spatial resolution down to 10 µm for molecular imaging.

Targeted cell entry of lentiviral vectors.

Retargeting of lentiviral vector entry to cell types of interest is a key factor in improving the safety and efficacy of gene transfer. In this study we show that the retargetable envelope glycoproteins of measles virus (MV), namely, the hemagglutinin (H) responsible for receptor recognition and the fusion protein (F), can pseudotype human immunodeficiency virus 1 (HIV-1) vectors when their cytoplasmic tails are truncated. We then pseudotyped HIV-1 vectors with MV glycoproteins displaying on H either the epidermal growth factor or a single-chain antibody directed against CD20, but without the ability to recognize their native receptors. Gene transfer into cells that expressed the targeted receptor was several orders of magnitude more efficient than into cells that did not. High-target versus nontarget cell discrimination was demonstrated in mixed cell populations, where the targeting vector selectively eliminated CD20-positive cells after suicide gene transfer. Remarkably, primary human CD20-positive B lymphocytes were transduced more efficiently by the CD20-targeted vector than by a vector pseudotyped with the vesicular stomatitis virus G (VSV-G) protein. In addition, the CD20-targeted vector was able to transduce even unstimulated primary B cells, whereas VSV-G pseudotyped vectors were unable to do so. Because MV enters cells through direct fusion at the cell membrane, this novel targeting system should be widely applicable.

Retroviral display and high throughput screening.

Retroviruses distinguish themselves from all other mammalian viruses by their abilities to infect and propagate in mammalian cells without causing a cytopathic effect and to stably integrate their genetic information into the genome of the host cell. These unique properties make them an ideal platform for the display and directed evolution of proteins in a mammalian cell environment. This review will describe the essentials about retrovirus biology and then discuss in detail display and screening strategies that have been developed during the past 15 years of retroviral display technology.