Given its ideal decay characteristics (Eβ+average: 632 KeV) and a half-life (T1/2 = 3.97 h) that is well matched to the pharmacokinetic profile of small molecules targeting angiogenesis, scandium-44 (44Sc) has gained meaningful attention as a promising radiometal for positron emission tomography (PET) imaging. More recently, intensive research has been centered around the investigation of 44Sc-labelled angiogenesis-directed radiopharmaceuticals. Previous studies dealt with the evaluation of 44Sc-appended avb3 integrin–affine Arg-Gly-Asp (RGD) tripeptides, GRPR-selective aminobenzoyl–bombesin analogue (AMBA), and hypoxia-associated nitroimidazole derivatives in the identification of various cancers using experimental tumour models. Given the tumour-related hypoxia- and angiogenesis-targeting capability of these PET probes, 44Sc seems to be a strong competitor of the currently used positron emitters in radiotracer development. In this review, we summarize the preliminary preclinical achievements with 44Sc-labelled angiogenesis-specific molecular probes.Angiogenesis-related cell-surface molecules, including integrins, aminopeptidase N, vascular endothelial growth factor, and gastrin-releasing peptide receptor (GRPR), play a crucial role in tumour formation. Radiolabelled imaging probes targeting angiogenic biomarkers serve as valuable vectors in tumour identification. Nowadays, there is a growing interest in novel radionuclides other than gallium-68 ( The formation of new blood vessels from pre-existing ones—denoted as angiogenesis—is required for tumour maintenance and development as well as for metastasis formation ,2. Angiovβ3 integrins, aminopeptidase N (APN/CD13), fibronectin, nitroimidazole, matrix metalloproteinases, gastrin-releasing peptide receptor (GRPR), and E-selectin represent the most important angiogenic molecules Sc-PSMA-617-based (PSMA (prostate-specific membrane antigen)) PET/CT imaging serves as a useful tool for the calculation of normal organ-absorbed doses and the maximum allowable activity in prostate-cancer patients prior to lutetium-177 (177Lu)-labelled PSMA-617 ([177Lu]Lu-PSMA-617) radiotherapy [44Sc-labelled somatostatin analogue [44Sc]Sc-DOTATOC and [44Sc]Sc-PSMA-617 as valuable radiotracers in dosimetry settings [44Sc enables better pre-therapeutic dosimetry for [177Lu]Lu-PSMA-617 radiotherapy amino-6-methylperhydro-1,4-diazepine) AMBA: aminobenzoyl–bombesin analogue; AR42J: rat exocrine pancreatic tumour; BBN: bombesin; BN: bombesin; c(RGD)vβ3 integrin has a central role in angiogenesis, neovascularisation, tumour growth, and related metastatic spread F-galacto-RGD, [18F]F-fluciclatide, [18F]F-RGD-K5, [18F]F-FPPRGD2, [18F]F-alfatide, [68Ga]Ga-NOTA-RGD, and [68Ga]Ga-NOTA-PRGD2 Sc-DOTA-c(RGD)2 at both the in vivo and in vitro levels [vβ3-targeting capacity of [44Sc]Sc-DOTA-c(RGD)2, U87MG human glioblastoma-bearing female athymic nude mice were intravenously (iv.) administrated with 5.5–7.4 MBq of the radiotracer and then underwent microPET/microCT acquisition at different time points . The αvβ3 integrin overexpression of the tumour cells and the vasculature of the U87MG experimental models is well established. In a bid to assess the αvβ3 selectivity of cRGD, (cRGD)2, and DOTA-(cRGD)2 in vitro, a competitive cell-binding assay was performed using integrin-targeting radioligand 125I-echistatin. Moreover, in vivo and ex vivo receptor-blocking studies were further conducted with the simultaneous application of 3.7 MBq of [44Sc]Sc-DOTA-c(RGD)2 and 50 mg/kg (approximately 1 mg) of c(RGD)2 to confirm receptor selectivity. During the competitive assays, continuously elevating amounts of cRGD, (cRGD)2, and DOTA-(cRGD)2 were administered to 1 × 105 U87MG cells that were pre-incubated with 125I-echistatin. As part of the ex vivo experiments, the weight and radioactivity of the harvested major tissues and organs, the blood, and the tumour were measured. The tracer-uptake values were obtained as mean %ID/g ± SD. Due to the significantly elevated tracer accumulation of the tumours, along with the negligible background activity, high-contrast PET images could be obtained at each investigated time point.Hernandez et al. used o levels . The tum44Sc]Sc-DOTA-c(RGD)2 uptake in the tumourous lesions, with %ID/g values of 3.93 ± 1.19, 3.07 ± 1.17, and 3.00 ± 1.25 measured 0.5, 2, and 4 h after the injection, respectively. Similar to the PET data, insignificant ex vivo non-target activity was detected. Receptor-blocking studies showed the reduction of the radiotracer uptake of the tumours both in vivo and ex vivo. However, a (cRGD)2-derived decrease in the tracer concentration was recorded ex vivo in the background organs/tissues, which could be attributed to their physiological αvβ3 expression.Quantitative in vivo analyses corresponding to the ex vivo data strengthened prominent Sc/[68Ga]Ga-DOTA/NODAGA-RGD)  complexed with the same chelators ([44Sc]Sc/[68Ga]Ga-DOTA/NODAGA-NOC). Besides in vitro stability examinations, biodistribution and in vivo PET-/CT-imaging studies of female athymic nude (CD-1 nude) mice bearing U87MG (human glioblastoma) and AR42J (rat exocrine pancreatic tumour) tumours were conducted. DOTA chelator-derivatized peptides (both RGD and NOC) labelled with either 44Sc or 68Ga showed high in vitro stability. Although 68Ga-labelled NODAGA-RGD and NODAGA-NOC were stable during the examination time, NODAGA-conjugated peptides radiolabelled with 44Sc did not present such results. The in vitro stability was evaluated in the presence of Fe3+ and Cu2+, as well. The instability of [68Ga]Ga-DOTA-RGD and [68Ga]Ga-DOTA-NOC was registered upon Cu2+ administration, whereas 44Sc-labelled molecules coordinated with NODAGA were less stable in the case of the addition of both metal cations. The 44Sc-labelled RGD peptides conjugated with either DOTA or NODAGA showed an identical tumour-distribution profile . Intriguingly, the hepatic uptake of [44Sc]Sc-DOTA-RGD was more elevated in comparison to the accumulation of [44Sc]Sc-NODAGAD-RGD 0.5 h after the tracer injections; however, at later time points similar liver activities were measured. As for other selected organs and tissues, Domnanich et al. experienced the same radiopharmaceutical accretion. The biodistribution profile of the 44Sc-labelled and 68Ga-labelled compounds was compared, as well. The liver showed increased uptake of the 68Ga-labelled DOTA-chelated peptides relative to the 44Sc-appended DOTA matches. This finding was in accordance with the in vivo PET data. Regarding other organs, the uptake of the 68Ga and 44Sc peptides was mostly identical. Since the 44Sc-appended molecules complexed with NODAGA demonstrated a largely identical tracer-accumulation pattern compared to the DOTA-chelated ones, NODAGA seems to be an attractive alternative chelator to more established DOTA for the development of 44Sc-labelled PET radiotracers Sc-B28110) was confirmed for the PET imaging of prostate cancer Sc-B28110 and [68Ga]Ga-B28110) by performing dynamic (0–90 min) and 20 min-long static (150 min post tracer injection) in vivo PET/MRI examinations in male CB17 SCID mice bearing PSMA-positive LNCaP tumours. Furthermore, the diagnostic feasibility of these novel AAZTA-based imaging probes was compared with that of the DOTA analogue PSMA-617, which contains the same binding sequence. Analysing the PET/MRI scans, increased radiotracer accumulation was registered in the tumorous regions 20 min after the tracer administration. Based on the time-activity curves, [44Sc]Sc-B28110 accumulation was statistically more enhanced in the LNCaP tumours in comparison to the other assessed tracers regarding all time points; however, the tumorous lesions could be clearly detected with the use of all three probes . Based on the ex vivo biodistribution data three hours post tracer application, [44Sc]Sc-B28110 and [68Ga]Ga-B28110 showed the highest concentration in the LNCaP tumours, although no remarkable difference was noted between the accumulation of these two compounds. The ex vivo results also revealed an uptake two times higher of the labelled B28110 derivatives relative to that of the [44Sc]Sc-PSMA-617. Therefore, the findings of Ghiani et al. also strengthened the suitability of AAZTA for the conjugation of 44Sc PET probes in diagnostic settings amino-6-methylperhydro-1,4-diazepine) for conjugation with ratified . The out cancers . Positroe cancer . For thesettings . G-protein-coupled gastrin-releasing peptide receptors (GRPR) can be encountered in a diverse array of malignancies, including prostate, breast, colon, pancreatic, and pulmonary tumours ,101,102.2, AMBA) is regarded as a highly valuable imaging agent in the nuclear medical diagnostics of GRPR-upregulated tumours Sc-NODAGA-AMBA and its in vivo and in vitro stability were determined. Based on the in vitro serum-stability measurements, the RCP of the 44Sc-appended compound was over 98% at both the 15 and 90 min time points, whereas the RCP of the 68Ga-tagged AMBA derivative showed a gradual decrease from 92% to 85% between 15 and 90 min. To authenticate the receptor-binding affinity of [44Sc]Sc-NODAGA-AMBA and [68Ga]Ga-NODAGA-AMBA, in vitro cellular-uptake studies were executed by applying GRPR-overexpressing human PCa PC-3 and receptor-negative human immortal keratinocyte HaCaT cell lines. Followed by a 60 or 120 min-long incubation with 0.37 MBq of [44Sc]Sc-NODAGA-AMBA or [68Ga]Ga-NODAGA-AMBA, the samples were measured for radioactivity in a gamma counter to provide uptake values in %ID/million cell units. Receptor-upregulated PC-3 cells presented notably higher (25 times) accumulation of both [44Sc]Sc-NODAGA-AMBA and [68Ga]Ga-NODAGA-AMBA than receptor-negative HaCaT cells (p < 0.01). Although from statistical point of view no considerable difference was encountered between the accumulation of the 44Sc-appended and the 68Ga-labelled radiotracers in the tumour cells, they expressed relatively more elevated [44Sc]Sc-NODAGA-AMBA uptake compared to [68Ga]Ga-NODAGA-AMBA (p < 0.05). Furthermore, in vitro blocking investigations performed with 200 nM BBN as blocking agent revealed that the tracer uptake of the PC-3 cells was remarkably diminished, whereas the radioactivity of the control cells remained unchanged. Analytical radio-HPLC-based in vivo serum-stability calculations conducted in healthy mice proved outstanding metabolic stability in the case of both probes. According to the pharmacokinetic investigations—similar to [68Ga]Ga-NODAGA-AMBA—[44Sc]Sc-NODAGA-AMBA possessed a circulatory half-life shorter than 30 min.Investigating the GRPR selectivity of [44Sc]Sc-NODAGA-AMBA or [68Ga]Ga-NODAGA-AMBA static PET acquisition were conducted with the application of a miniPET device . Standardized uptake values (SUV) and T/M ratios were registered to quantify the radiopharmaceutical uptake. Both qualitative analyses and quantitative SUV data confirmed the GRPR-targeting adequacy of the investigated radiolabelled AMBA derivatives. Although 60 min post administration of [44Sc]Sc-NODAGA-AMBA higher SUVmean (0.90 ± 0.17), SUVmax (1.54 ± 0.18), T/M SUVmean (6.16 ± 1.24), and T/M SUVmax (6.71 ± 1.08) values of the sc. growing PC-3 tumours were registered compared to the 68Ga-labelled counterpart, this was not statistically significant (p ≤ 0.05). Moreover, 120 min after the injection of the radiotracers, decreased non-target activity was observed, which led to excellent T/M ratios.In a bid to conduct in vivo and ex vivo measurements, 12-week-old, severe combined immunodeficient (SCID) CB17 mice bearing PC-3 prostate cancer in their left-shoulder area and healthy control mice were examined. For the assessment of the biodistribution pattern of the probes, 60 and 120 min post injections of 11.3 ± 1.4 MBq of [44Sc]Sc-NODAGA-AMBA or [68Ga]Ga-NODAGA-AMBA. The radioactivity of the explanted tissues and organs was registered with a calibrated gamma counter, and the counts-per-minute (CPM) values of all samples were converted to the percentage of administered dose per gram of tissue. The radiotracer concentration was presented as %ID/g. The biodistribution pattern of the control small animals displayed lower accumulation of [44Sc]Sc-NODAGA-AMBA relative to [68Ga]Ga-NODAGA-AMBA; however, this difference was not statistically significant (p < 0.05). After analysing the tracer uptake of the organs separately, the blood, liver, spleen, small and large intestines, stomach, heart, and lungs were depicted with moderate radioactivity, whereas the kidneys, urine, adrenal glands, and pancreas demonstrated significant tracer accretion. The outstanding urinary activity could have been due to the renal method of elimination or the physiological presence of GRPR in the urogenital smooth muscle [44Sc]Sc-NODAGA-AMBA uptake of the PC-3 tumours was more prominent in comparison to the 68Ga-labelled derivative.Ex vivo biodistribution studies of both the healthy control and the tumour-bearing mice were accomplished 30, 60, 120, and 180 min after the administration of 11.3 ± 1.4 MBq of Sc-NODAGA-AMBA established by Kálmán-Szabó et al., along with the better imaging characteristics of 44Sc over 68Ga, 44Sc-labelled BBN-based radiotracers, will hopefully be embraced in the diagnostic armamentarium of GRPRpos. tumours.Given the GRPR selectivity of NH2 and 68Ga-DOTA-BN[2-14]NH2 were assessed. Another investigation, executed by Koumarianou et al., focused on the comparison of l medium . In a bi2 complexed with natGa and natSc were used as non-radioactive derivatives to study in vitro GRPR-binding competence in competition with radiolabelled BBN analogue [125I-Tyr4]-BN in the human androgen-independent prostate-cancer PC-3 cell line. Thereafter, organ distribution was investigated in healthy male Sprague-Dawley rats one and two hours after the intravenous (iv.) administration of 11 and 3 MBq 68Ga-DOTA-BN[2-14]NH2 and 44Sc-DOTA-BN[2-14]NH2, respectively, via the jugular vein. As part of the biodistribution studies, blocking experiments were also performed with the iv. application of native BBN (100 µg/100 µL) 15 min before the radiotracer injection. The organ-distribution pattern of the control cohort—solely receiving the radiolabelled analogue—and that of the blocked group was compared. Androgen-independent Dunning R-3327-AT-1 prostate-cancer tumour-bearing small animals were generated by the subcutaneous (sc.) transplantation of GRPR-overexpressing R-3327-AT-1 cells  into the dorsal aspect of the hind foot of male Copenhagen rats [68Ga- or the 44Sc-appended imaging probes. natGa-DOTA-BN[2-14]NH2 showed higher specificity towards GRPR receptors than the natSc-complexed counterpart. The pancreas was presented with considerable uptake using both labelled compounds with ID/g values of 0.64 ± 0.00% and 0.58 ± 0.05% for 68Ga-DOTA-BN[2-14]NH2 1 and 2 h post injection, respectively, whereas the subsequent data were obtained in the case of the 44Sc-labelled probe: 2.67 ± 0.53% ID/g and 1.51 ± 1.19% ID/g one and two hours after tracer administration, respectively. The physiological pancreatic GRPR expression could explain these results. Upon visual assessment of the in vivo PET images, the peripheral tumour areas demonstrated more enhanced tracer uptake compared to the central neoplastic regions. The heterogeneity of the receptor expression within the tumour and the unevenness regarding the radiopharmaceutical binding ability of GRPR could underpin the difference between the tracer accretion of the external and inner tumour regions. Followed by prompt radiotracer uptake post injection, Koumarinaou et al. noted a gradual decline in the accumulation kinetics of the 68Ga-labelled compound throughout the entire examination period, whereas they observed a stable albeit lower tracer uptake in the case of the 44Sc-appended match. Of note, the relative tumour radioactivity-based kinetics  did not reveal any meaningful differences between the two tracers. Overall, the GRPR selectivity of 68Ga and 44Sc differed. However, given the similar distribution pattern, uptake, and excretion times of both the 68Ga- and the 44Sc-labelled radiopharmaceuticals, as well as their comparable accumulation in neoplastic tissues, these radiometals seemed equally effective in the detection of GRPRpos. tumours [DOTA-BN[2-14]NHgen rats . DynamicBeyond the above-mentioned radiopharmaceuticals, hypoxia-associated PET tracers are also embraced in tumour diagnostics ,125,126.18F-labelled NI-derivatives such as [18F]F-fluoromisonidazole ([18F]F-FMISO) and [18F]F-fluoroazomycin arabinoside ([18F]F-FAZA) in hypoxia imaging [18F]F-fluoroetanidazole ([18F]F-FETA), [18F]F-fluoroerythronitroimidazole ([18F]F-FETNIM), and [18F]F-[2-(2-nitro-1[H]-imidazol-1-yl)-N--acetamide] ([18F]F-EF5), respectively, for the non-invasive detection of cancer-related hypoxia Sc-DO3AM-NI) and its comparison to the 68Ga-labelled diagnostic match Ga-DO3AM-NI concentration was visually depicted 90 min post injection. The 44Sc-labelled NI-based counterpart accumulated rapidly in the sc. developing KB tumours and produced satisfactory images at 90 min and 240 min post tracer injection . Although the quantitative tumour uptakes of [44Sc]Sc-DO3AM-NI and [68Ga]Ga-DO3AM-NI were comparable, the liver, spleen, kidneys, intestines, lung, heart, and brain were depicted with higher [68Ga]Ga-DO3AM-NI uptake compared to the 44Sc counterpart 90 and 240 min post injection. More elevated accretion of the 68Ga-linked tracer in the non-target organs led to T/M ratios 10–15 times lower relative to the 44Sc-labelled derivative. In the case of [68Ga]Ga-DO3AM-NI, T/M SUVmean and T/M SUVmax values of 85.66 ± 6.06 and 41.72 ± 13.61 were experienced, respectively, whereas for [44Sc]Sc-DO3AM-NI the following parameters were defined: T/M SUVmean: 182.67 ± 23.50 and T/M SUVmax: 129.68 ± 26.11 4 h after the injection. Hence, outstanding lesion identification, which could be derived from the suitable T/M ratios of the 44Sc-labelled derivative, warrants a place for 44Sc in the targeted PET imaging of hypoxia-related tumour diagnostics. Unlike these findings, previous studies dealing with 18F, iodine-131 (131I), technetium-99m (99mTc), and 64Cu-appended NI imaging probes reported substantial tracer accumulation in the abdominal organs, mainly in the intestines, along with hepatic metabolization, which could lead to difficulties regarding the detection of abdominal malignancies Sc-DO3AM-NI than [68Ga]Ga-DO3AM-NI (p ≤ 0.01). In addition, the ex vivo organ-distribution pattern also confirmed notably lower [44Sc]Sc-DO3AM-NI uptake in the evaluated organs compared to the 68Ga-labelled sister compound (p ≤ 0.01). The biodistribution results of Hoigebazar et al. and Seelam et al. regarding NI compounds labelled with 68Ga were in accordance with those of Szücs et al. [44Sc]Sc-DO3AM-NI seemed to be superior to the 99mTc-, 64Cu-, 18F-, 131I-, and 68Ga-linked derivatives. Based on the exceptional tumour-homing capability, appropriate contrast and favourable quantitative parameters of the 44Sc-appended molecular agent [44Sc]Sc-DO3AM-NI could be a highly valuable hypoxia-targeting PET probe in the diagnostic algorithm of tumour identification.In line with the in vivo findings, the ex vivo figures revealed that the KB tumour uptake of the two investigated probes did not differ significantly between the two assessed time points; however, the accumulation of the s et al. ,139. In s et al. ,139. As VEGFs, including VEGF-A, -B, -C, -D, and -E, and placenta growth factor (PlGF) also have a pivotal role in blood-vessel formation ,141. The44Sc-labelled VEGF-VEGFR-NRP-1-directed radiopharmaceuticals have been published so far, Masłowska et al. examined the potential diagnostic and therapeutic feasibility of such molecules under in vitro circumstances Sc-DOTA-Ahx-K4R) could be effectively used to prevent the development of VEGF-A165/NRP-1 complex. Hence, they could widen the existing scale of the diagnostic and therapeutic probes applied in cancer-related angiogenesis. In contrast, retro–inverso peptide DR7A-based compounds exerted low NRP-1 specificity. However, due to the inadequate human-serum stability of the evaluated agents, future studies are required for the optimization of their usage for therapeutic purposes.With the aim of inhibiting the formation of the vascular endothelial growth factor/neuropilin-1 co-receptor complex VEGF-Aotracers . In thei2+-dependent membrane-bound ANP/CD13 and cell differentiation, motility, proliferation, cell death, and angiogenic processes is well-established [68Ga in the detection of tumours with APN/CD13 upregulation [64Cu, rhenium-188 (188Re), and 177Lu, respectively, for labelling purposes of NGR radiopharmaceuticals [44Sc-labelled NGR-based radiotracers are available so far, 44Sc could be a potential radionuclide for the labelling of APN/CD13-targeting imaging vectors.Out of the neo/angiogenesis-related molecules, another intensively investigated one is the aminopeptidase N receptor (APN/CD13) . The assablished . Accordiablished ,151,152.ablished . Consideablished ,155,156.gulation ,158,159.euticals ,161,162.44Sc-labelled, angiogenesis-targeting imaging probes, along with the favourable physicochemical properties of the radiometal, capitalize on the relevance of this isotope for the development of highly specific PET radiotracers. Their transition into clinical routine will inevitably herald a new era in personalized cancer diagnostics. Furthermore, the non-invasive assessment of angiogenesis-/neo-angiogenesis-related molecules is of outstanding importance to identifying patients who may benefit from targeted antiangiogenic therapy. Together with its therapeutic surrogate, the 44Sc/47Sc isotopic pair could be effectively employed in dosimetry estimations, cancer treatment, and therapeutic follow-up.The in vivo tumour-targeting capability of 44Sc-labelled molecular vectors from preclinical level to human application could be hampered by some constraints regarding the usage of 44Sc. First, the limited accessibility of the radiometal is a meaningful hurdle to its centralized distribution. In addition, the high-energy concomitant gamma rays (1157 keV) of 44Sc could lead to reduced image quality; therefore, future studies to optimize PET-imaging parameters are required [43Sc lacking in accompanying gamma co-emission may serve as a valuable substitute for 44Sc in imaging settings [44Sc.However, the future progression of required . Furthersettings . Global


Giant cells (GCs) are found in many different tissues and among different backgrounds, including reactive, and neoplastic settings. It is not yet fully understood whether GCs in similar settings or with related functions share the same surface antigen expression. We performed antigen profiling of GCs via immunohistochemistry in multiple reactive and neoplastic lesions. We were able to characterise distinct groups of GCs with similar expression patterns, as well as GCs in some lesions that had a unique antigen expression pattern. These findings may help in the diagnosis of histologically similar GC-rich lesions and provide further insight into the function and origin of these cells.n = 47). We studied the expression of 15 molecules including HLA class II molecules those relevant to the cell cycle, bone metabolism and lineage affiliation. HLA-DR was detectable in the GCs of sarcoidosis, sarcoid-like lesions, tuberculosis, and foreign body granuloma. Cyclin D1 was expressed by the GCs of neoplastic lesions as well as the GCs of bony callus, fibroid epulis, and brown tumours. While cyclin E was detected in the GCs of all lesions, p16 and p21 showed a heterogeneous expression pattern. RANK was expressed by the GCs of all lesions except sarcoid-like lesions and xanthogranuloma. All GCs were RANK-L-negative, and the GCs of all lesions were osteoprotegerin-positive. Osteonectin was limited to the GCs of chondroblastoma. Osteopontin and TRAP were detected in the GCs of all lesions except xanthogranuloma. RUNX2 was heterogeneously expressed in the reactive and neoplastic cohort. The GCs of all lesions except foreign body granuloma expressed CD68, and all GCs were CD163- and langerin-negative. This profiling points to a functional diversity of GCs despite their similar morphology.Giant cells (GCs) are thought to originate from the fusion of monocytic lineage cells and arise amid multiple backgrounds. To compare GCs of different origins, we immunohistochemically characterised the GCs of reactive and neoplastic lesions ( Since the early description of multinucleated giant cells (GCs), these cells have been observed to emerge against different backgrounds ,2. OsteoThroughout the decades, different theories of GC formation have been put forward. Two main paths of multinucleation have been suggested: acytokinetic cell division, and cell fusion . In myxoToday, a fusion of monocytes that have developed into macrophages is widely accepted as the main mechanism of formation for GCs . This seSo far, two distinct categories of GCs have been established: osteoclasts of the bone as well as osteoclast-like GCs of GC-rich bone lesions; and so-called macrophage polykaryons of extraosseous GC-containing lesions. These include Langhans GCs of tuberculosis and sarcoidosis, as well as foreign body GCs . The comFor this study, tissue samples were selected by systematic screening of the tissue bank of the Institute of Pathology Ulm University, Germany. Samples were provided as formalin-fixed and paraffin-embedded (FFPE) tissue sections. All analysed samples are listed in The neoplastic cohort included samples of eleven GC tumours of the bone, four of which represent the core biopsies and resected tissues of the same case respectively (samples 24a and b as well as 25a and b), and two of which represent resected tissue of a primary tumour and a local recurrence in the same patient (samples 26a and b). Seven samples of aneurysmal bone cysts were included, among them one incision biopsy and resected tissue of the same tumour (samples 32a and b). We further included three chondroblastomas, three non-ossifying fibromas, three tenosynovial GC tumours and one xanthogranuloma.All diagnostic procedures were performed in accordance with histological subtyping as defined by the WHO classification 2020 .This study was approved by the Ulm University Ethics Committee (06/2017), which allowed the usage of archived human material. Appropriate clinical information was obtained from case notes, and patient material and samples were treated in a coded manner. All analyses were performed in accordance with the Declaration of Helsinki.Immunohistochemistry was performed on giant cell-containing tissue sections (1.5 µm). Following deparaffination in xylol and rehydration in a graded alcohol series, antigen retrieval was performed to further unmask the antigen. To achieve this, the sections were either placed in target retrieval solution (TRS pH 6.1) or EDTA (pH 8 or 9) and heated in a steamer for 25 min. Alternatively, the sections were heated in citrate buffer (pH 6) for 25 min in a microwave or pressure cooker.For the primary antibody reaction, sections were first marked with a PAP pen and pre-incubated with phosphate-buffered saline (PBS), then covered with 50–100 µL primary antibody in the specified dilution and incubated in a humidified chamber for 30 min. The utilised primary antibodies are listed in For the secondary antibody reaction, Dako REAL Detection System Alkaline Phosphatase/RED rabbit/mouse (cat# K5005) was used. Counterstaining followed, using Meyer’s hematoxylin with an incubation time of 5 min. Evaluation was performed in a blinded, neutral manner and by consensus diagnosis using a multi—head microscope. Immunoreactivity of giant cells was categorised into four categories, depending on the proportion of positively stained cells: “no immunoreactivity detected”, “immunoreactivity in up to 30%”, “immunoreactivity in more than 30% and up to 70%” and “immunoreactivity in more than 70%” of the total number of analysed cells. A typical example of categorisation is given in The pattern of expression of all studied antigens is shown in HLA-DR was positive in the GCs of sarcoidosis, sarcoid-like lesion, and foreign body granuloma. The GCs of tuberculosis stained positive for HLA-DR in up to 70%. The GCs of bony callus, fibroid epulis, and brown tumour were HLA-DR-negative.All GCs within neoplastic lesions were HLA-DR-negative.Cyclin D1 was positive in the GCs of bony callus, fibroid epulis and brown tumour, while the GCs of foreign body granuloma were cyclin D1 positive in about 70% of the GCs. The GCs of sarcoidosis, sarcoid-like lesion, and tuberculosis were consistently cyclin D1-negative.Cyclin D1 was positive in the nuclei of the GCs of the studied neoplastic lesions throughout.Cyclin E was found to be expressed in the GCs of all lesions, although in varying percentages. In sarcoidosis, tuberculosis, and in one brown tumour, the percentage of stained GCs was up to 70%, while it was above 70% in all other studied reactive lesions. In the GCs of two samples of foreign body granuloma, the staining pattern of cyclin E was strictly cytoplasmatic, while it was nuclear restricted in one foreign body granuloma and in all other studied lesions.The GCs of all studied neoplastic lesions showed nuclear expression of cyclin E. More than 70% of GCs were positive in GC tumour of the bone, aneurysmal bone cyst, chondroblastoma, and non-ossifying fibroma. In tenosynovial GC tumour, up to 70% of the GCs were positive, and the GCs of xanthogranuloma showed up to 30% immunoreactivity.p16 was positive in over 70% of the GCs of fibroid epulis, over 30% of the GCs of foreign body granuloma, and in up to 30% of the GCs in bony callus. The GCs of sarcoidosis, sarcoid-like lesion, tuberculosis, and brown tumour were p16-negative.The GCs of aneurysmal bone cyst and tenosynovial GC tumour were positive for p16 in up to 70%, while the GCs of chondroblastoma were up to 30% positive.In one sample of GC tumour of the bone, p16 was detected in the incision biopsy but not in the resected tissue (samples 25a and b). In another case, p16 was positive in up to 70% of GCs in the primary occurrence of the tumour, while the recurrence only showed positivity in up to 30% of GCs (samples 26a and b). In the GC tumours of the bone and aneurysmal bone cysts, the reaction was cytoplasmatic in some analysed samples, and strictly nuclear in others, pointing to a mutually exclusive expression of p16. The GCs of non-ossifying fibroma and xanthogranuloma were p16-negative.p21 was positive in bony callus and sarcoidosis in up to 30% of GCs, in up to 70% of GCs in tuberculosis, and the mandibular brown tumour, and in more than 70% of the GCs of foreign body granuloma, fibroid epulis and the brown tumour of the femur. The GCs of sarcoid-like lesion were p21-negative.Many of the neoplastic lesions were partially positive in GCs between 30% and 70%, namely GC tumour of the bone, aneurysmal bone cyst, chondroblastoma, non-ossifying fibroma, and tenosynovial GC tumour. In one sample of GC tumour of the bone, the GCs of the core biopsy and resected tissue showed a reaction in above 70% (24a and b), while results were incongruent in another sample of GC tumour of the bone (25a and b) and a sample of aneurysmal bone cyst (32a and b). The GCs of one sample stained partially positive, while the other remained negative, respectively. The GCs of xanthogranuloma were p21-negative.More than 70% of GCs showed immunoreactivity for RANK in bony callus, foreign body granuloma, fibroid epulis, and brown tumour. The GCs of sarcoidosis and tuberculosis were positive in 30–70%, while the GCs of sarcoid-like lesion were negative.The GCs of almost all neoplastic lesions were positive for RANK in more than 70%, namely the GCs of GC tumour of the bone, aneurysmal bone cyst, chondroblastoma, non-ossifying fibroma, and tenosynovial GC tumour. The GCs in xanthogranuloma showed no immunoreactivity for RANK.Up to 30% RANK-L-positive GCs were found in bony callus, fibroid epulis, and in one of two brown tumours studied. The GCs of sarcoidosis, sarcoid-like-lesion, tuberculosis, foreign body granuloma, fibroid epulis, and one localisation of brown tumour were RANK-L-negative.The GCs of aneurysmal bone cyst, chondroblastoma, and tenosynovial GC tumour showed immunoreactivity for RANK-L in up to 30%. The GCs of GC tumour of the bone, non-ossifying fibroma, and xanthogranuloma were RANK-L-negative throughout.OPG-expression was detected in the GCs of all reactive lesions at a varying percentage. The GCs of bony callus, sarcoid-like lesion, tuberculosis, fibroid epulis, and one brown tumour expressed OPG in more than 70% of GCs, while sarcoidosis, foreign body granuloma and one brown tumour showed OPG-positivity in 30–70% of GCs.The GCs of all studied neoplastic lesions were more than 70% OPG-positive throughout.The GCs of almost all studied reactive lesions were osteonectin-negative throughout, namely bony callus, sarcoidosis, sarcoid-like lesion, tuberculosis, foreign body granuloma, fibroid epulis, and brown tumour.The GCs of neoplastic lesions were mostly negative throughout, including the GCs of GC tumour of the bone, non-ossifying fibroma, tenosynovial GC tumour, and xanthogranuloma. The GCs of aneurysmal bone cyst were immunoreactive in up to 30% in most studied samples. In one case, the GCs of the incisional biopsy of aneurysmatic bone cyst (sample 32a) were positive in up to 70% of cells, while the GCs of the tissue resected shortly thereafter (sample 32b) were osteonectin-negative.In the GCs of all studied reactive lesions, osteopontin was present at different levels. In bony callus, sarcoid-like lesion, tuberculosis, foreign body granuloma and one brown tumour, osteopontin was expressed in over 70% of GCs. In sarcoidosis and one brown tumour, osteopontin was present in 30–70% of the studied GCs.The GCs of GC tumour of the bone and tenosynovial GC tumour expressed osteopontin in over 70%, while chrondroblastoma and non-ossifying fibroma showed up to 70% osteopontin-positive GCs. In one chondroblastoma, the reaction observed was strictly cytoplasmatic, while it was strictly nuclear in the other two chondroblastomas. Most aneurysmal bone cysts were up to 70% osteopontin-positive in the GC compartment. However, sample 32a (incision biopsy) was osteopontin-negative, while sample 32b (resected tissue of the same lesion) was over 70% osteopontin-positive. The GCs of xanthogranuloma were osteopontin-negative.TRAP was expressed in the GCs of all reactive lesions to different degrees. In bony callus, sarcoid like lesion, foreign body granuloma and fibroid epulis, more than 70% of the analysed GCs were TRAP-positive. In sarcoidosis, tuberculosis and brown tumour, between 30% and 70% of GCs were TRAP-positive.All analysed neoplastic lesions expressed TRAP in the GC compartment. In GC tumour of the bone, aneurysmal bone cyst, chondroblastoma, non-ossifying fibroma, and tenosynovial GC tumour, the GCs were TRAP-positive throughout. The GCs of xanthogranuloma were TRAP-positive in up to 30%.The GCs of sarcoid-like lesion, foreign body granuloma and fibroid epulis were above 70% RUNX2-positive, while the GCs of bony callus and sarcoidosis were between 30% and 70% RUNX2-positive. In tuberculosis, up to 30% of GCs were RUNX2-immunoreactive, and the GCs of brown tumour were RUNX2-negative throughout.The studied neoplastic lesions show a heterogenous profile, with lesions like GC tumour of the bone revealing different patterns within one group. Six samples of GC tumour of the bone  reveal between 30% and 70% RUNX2-positive GCs. Sample 24a (core biopsy) shows 30–70% immunoreactive GCs, and the resected tissue 24b is above 70% RUNX2-positive in the GC compartment. Sample 26 is similar, with the biopsy showing up to 30% immunoreactivity, and the GCs of the resected tissue being RUNX2-positive throughout. The GCs of aneurysmal bone cyst were up to 30% RUNX2-positive, with the exception of sample 32a (incision biopsy), which was RUNX2-negative.The GCs of tenosynovial GC tumour were RUNX2-positive throughout, while the GCs of non-ossifying fibroma were up to 30% RUNX2-positive, and the GCs of chondroblastoma and xanthogranuloma did not show immunoreactivity for RUNX2.Almost all reactive lesions showed a high percentage (>70%) of CD68-positive GCs, one exception was GC of foreign body granuloma, with up to 30% positive GCs.All neoplastic lesions showed a varying percentage of reactive CD68-positive GCs. 30–70% of the GCs of chondroblastoma stained CD68-positive, while the GCs of all other lesions stained positive above 70%.None of the GCs in the studied reactive and neoplastic lesions showed immunoreactivity for CD163.None of the GCs in the studied reactive and neoplastic lesions showed immunoreactivity for Langerin.We performed a comprehensive immunohistochemical analysis of the GCs of various reactive and neoplastic lesions and detected characteristic profiles of GCs in the lesions analysed. An overview of the tested molecules and their function is given in HLA-DR- one of the major histocompatibility complex class II molecules- is a surface molecule that presents exogenously derived antigens to CD4⁺ T-lymphocytes, thereby alerting the immune system to pathogens like viruses or bacteria . SubsequCyclin D1 plays a key role in cell cycle progression from G1- to S-phase. It acts as a regulatory subunit of cyclin-dependent kinases (CDK) 4 and 6, that in turn phosphorylate the retinoblastoma protein, allowing the cell cycle to progress to the phase of DNA replication . The GCsCyclin E, a common term used for cyclins E1 and E2, is another key player in cell cycle regulation: it binds protein kinase CDK2 and allows the cell to progress from G1- to S-phase by contributing to the phosphorylation of the retinoblastoma protein . ConsequCDKN2A, the gene coding for p16, has been called the second most common tumour suppressor gene after TP53, the gene coding for p53, as it is altered or inactivated in a large variety of neoplasms [p16 binds to and inhibits cyclin-dependent kinases (CDK) 4 and 6 that will otherwise bind to cyclin D1, leading to the phosphorylation of the retinoblastoma protein and enabling cell cycle progression. Thereby, the cell cycle is arrested at the G1/S-transition, which contributes to cellular senescence . CDKN2A,eoplasms . Causingeoplasms . This haeoplasms . Howevereoplasms . Cytoplaeoplasms . This iseoplasms . Expresseoplasms . In someeoplasms . For theeoplasms , which wTP53 can occur through cellular stress like DNA-damage, and leads to increased transcription of CDKN1A, the gene coding for p21 [KRAS-mutation [KRAS is a defining molecule, constitutes a signalling cascade from the cell surface to the nucleus and is frequently mutated in various cancers [KRAS-mutation is a logical consequence. The GCs of tenosynovial GC tumour have been found to express p21 before [p21 is a CDK-inhibitor, binding to and inhibiting every cyclin/CDK-complex involved in the hyperphosphorylation of the retinoblastoma protein . These e for p21 . Further for p21 . Since t for p21  To our k for p21 . This mamutation . The MAP cancers . Activat cancers . This ma cancers . As cycl cancers . In the  cancers . In chon cancers . This wa1 before . This waRANK is one of the three critical molecules for bone homeostasis studied here, along with RANK-L and OPG. RANK is a transmembrane protein that leads to osteoclast development in the presence of M-CSF (macrophage colony stimulating factor) ,73. The RANK-L is the ligand for RANK and constitutes the only ligand that binds to this receptor. It also binds to the decoy-receptor OPG with high affinity . RANK-L OPG acts as a soluble decoy receptor for RANK-L and prevents interaction with osteoclast precursors, thereby inhibiting the formation and activation of osteoclasts . Like RASPARC has been identified as one of the possible fusion partners in the characteristic UPS6-rearrangement of the lesion [Osteonectin is a glycoprotein present in the extracellular matrix of bone and a variety of non-mineralised tissues . During e lesion . Howevere lesion . The ostLike osteonectin, osteopontin is considered to be a matricellular glycoprotein that interacts with different cell types, proteins, molecules, and soluble factors of the extracellular matrix . It compTRAP is an iron-containing enzyme present in macrophages, dendritic cells and epithelial cells in many different tissues ,131. HigRUNX2 gene has been discovered by next generation sequencing to be one of the fusion partners of the gene encoding ubiquitin-specific peptidase 6 (USP6), possibly contributing to the osteoblastic as well as osteolytic features of this neoplasia. RUNX2-expression has been shown in the neoplastic, mononuclear compartment of chondroblastoma, but not in the GCs, confirming our data [RUNX2 is a member of the RUNX family of DNA-binding transcription factors and is expressed by osteoblasts and chondrocytes . Studiesour data . There aCD68 is a transmembrane protein that is heavily glycosylated and belongs to the family of lysosomal-associated glycoproteins (lamp-1). Unlike the other broadly expressed members of the lamp-family, CD68 is mainly present on the lysosomal and plasma membranes of macrophages and other cells of the monocytic lineage, like osteoclasts . HoweverCD163 is a scavenger receptor for haemoglobin (Hb) molecules and the haemoglobin-haptoglobin-complex (Hb-Hp), that leads to their phagocytising and degradation upon haemolysis or haemorrhage, reversing the pro-inflammatory effect of these processes . CD163 iLangerin (CD207) is a transmembrane, C-type lectin receptor that binds the carbohydrate fraction of many pathogens . These lWhile analysing the expression pattern of GCs of different antigen categories in the multitude of lesions studied here, a broad profile emerged see . Most noNotably, the GCs of xanthogranuloma were negative for molecules like cyclin E, RANK, and osteopontin, that are common among the GCs of the other analysed lesions. TRAP was confirmed as a general marker of all GCs, except xanthogranuloma. This unique profile may be explained by the specific formation mechanism of the Touton-GCs of xanthogranuloma: they are derived from activated blood monocytes that have phagocytised large amounts of lipids, to the point of inducing hypocholesterinaemia in patients . On top As osteoclast-like GCs show largely similar expression profiles to osteoclasts, the specific anti RANK-L-antibody Denosumab may be beneficial in pathologies beyond GC tumour of the bone, such as aneurysmal bone cyst and chondroblastoma, for which off-label uses have been reported . RecurriA weakness of our study is its purely descriptive nature, albeit at a detailed level. In addition, the sample size for many of the rarer analysed lesions is small and therefore only suitable for portraying a tendency. For these reasons, the data present the platform for further studies, and an even broader panel of analysed molecules:In physiological bone, osteoclast formation depends on the presence of macrophage-colony stimulating factor (M-CSF) . HoweverConsequently, it is of interest to study the prevalence of and susceptibility to VEGF and other growth factors in the GCs of different lesions, as giant cell tumours of the bone have been shown to be sensitive to antiangiogenetic tyrosine kinase inhibitor treatment combined with Denosumab .To sum up, GCs derive from various lines of differentiation and seem to reflect the respective microenvironment , origin, and function. The antigen expression pattern of GCs mirrors these differences, gives further insight into the common and distinctive features of this cell type, and provides a platform for further studies.


There remains a need for pre-exposure prophylaxis against SARS-CoV-2 infection in vulnerable patients in whom response to vaccination is often sub-optimal. The PROTECT-V platform trial is testing pre-exposure prophylactic interventions for COVID-19 in vulnerable patient populations: transplant recipients, individuals with oncological/haematological diagnoses, immune deficiency, autoimmune diseases requiring immunosuppression, and individuals receiving dialysis.Sotrovimab is a dual-action monoclonal antibody and the second agent to be added to the PROTECT-V platform . Although a single sotrovimab 500mg intravenous (IV) dose has been widely used for early treatment, data on a 2000mg IV dose are limited. Tolerability data in the first 143 participants randomized to this arm of the trial are presently available. Patients are randomized 1:1 sotrovimab to placebo. Data remain blinded.Median age was 66 years (range 21 – 86) and 82 (57%) patients were female. 132 (96%) had received ≥3 doses of SARS-CoV-2 vaccine. Patient populations were: 82 (57%) autoimmune disease; 26 (18%) haematological/oncological diseases; 21 (15%) transplant recipients; 12 (8%) immunodeficiency; 2 (1%) on dialysis.Two (1%) participants experienced a mild infusion related reaction (IRR). The infusion was briefly interrupted, but completed. Thirty (21%) participants experienced at least one symptom in the 24 hours post-infusion, but none were severe or required hospital admission. The most common symptoms were dizziness (7 [5%]), headache (7 [5%]), rigors (5 [4%]) and fever (4 [3%]). No severe adverse events were reported within 72 hours of IMP infusion at the time of writing.The first 55 patients underwent routine hematological and biochemical blood test evaluation 72 hours post-infusion. Ten events from 9 participants exhibited worsening of laboratory parameters, meeting at least grade two DAIDS criteria or worse. None of these were clinically significant.A 2000mg IV dose of sotrovimab was tolerated well in this blinded analysis of immunocompromised participants, with no severe IRRs or significant change in haematological or biochemical markers up to 72 hours post infusion.Davinder Dosanjh, n/a, Astrazeneca: Honoraria|Astrazeneca: Employee|Boehringer Ingelheim: Advisor/Consultant|Boehringer Ingelheim: Honoraria|Gilead: Advisor/Consultant|GSK: Grant/Research Support|Synairgen: Advisor/Consultant Louise Crowley, n/a, GSK: Grant/Research Support Michael Chen-Xu, n/a, GSK: Grant/Research Support Rona M. Smith, MD MRCP, GSK: Grant/Research Support|Union Therapeutics: Grant/Research Support


The prognosis of upper urinary tract urothelial carcinoma (UTUC) is associated with tumour grade (G) and stage. Despite preoperative risk stratification and radical treatment, recurrence and progression are common. Thus, prognostic and monitoring biomarkers are needed. This feasibility study aimed to investigate if targeted analyses on circulating tumour DNA (ctDNA) in plasma could identify tumour-specific gene variants, and thus have potential for further evaluation as a biomarker in UTUC.Nine UTUC patients with genetically characterised tumours were included in this prospective pilot study. Two tumour-specific variants were chosen for targeted analyses with multiplex droplet digital PCR on cell-free DNA (cfDNA) from plasma at diagnosis or from recurrence.2 in size. Three of these patients progressed in their disease and the fourth had the largest G3 tumour at sampling. In contrast, the two patients with undetectable ctDNA in diagnostic plasma had a G1 tumour and G3 carcinoma in situ (CIS), respectively. The patient with G3 CIS had detectable ctDNA later during follow-up and progressed thereafter with aggressive intravesical recurrence and CT-scan-verified CIS progression in the upper urinary tract. In three patients with small recurrent G1 or G2 tumours, none had detectable ctDNA in plasma and all were progression free.Of six patients with diagnostic plasma samples, ctDNA was detected in four with G2 or G3 tumours and tumours > 300mOur early findings demonstrate that ctDNA in plasma can be detected by targeted analysis in patients with UTUC. However, further studies are needed to determine its role as a potential biomarker.The online version contains supplementary material available at 10.1007/s00345-023-04583-w. Although upper urinary tract urothelial carcinoma (UTUC) is rare, the incidence has been increasing during the last decades . The proWhen cell death occurs by apoptosis or necrosis, small fragments of cell-free DNA (cfDNA) are released into body fluids such as plasma . In cancThis prospective study included nine patients diagnosed with UTUC at ureterorenoscopy (URS). Six patients underwent organ-sparing surgery; one patient (PP-6) received radiotherapy as palliative treatment, one patient received BCG-instillation in a solitary kidney with CIS (PP-7) and one patient (PP-8) underwent nephroureterectomy. Patients were diagnosed between 2018 and 2020 and were followed-up routinely  to censor date (February 2023) or death. A next-generation sequencing (NGS) gene panel consisting of 387 genes was performed on barbotage specimens obtained at URS at diagnosis or local recurrence for genetic characterisation of all tumours prior to inclusion . The NGSExtraction of cfDNA from plasma and droplet digital PCR (ddPCR) was performed as previously published and described in Supplementary methods . TargetsAll patients had verified UTUC at the time of first plasma sample collection, 6/9 at initial diagnosis of UTUC and 3/6 at recurrence. The follow-up time after UTUC diagnosis was 4–55 months and no patient was lost to follow-up. Clinical characteristics including age at diagnosis, sex, tumour grade (G) and follow-up data of each patient are summarised in Table Two SNVs or indels were selected as targets for each patient based on NGS gene panel data on barbotage tumour cells. Multiplex ddPCR assays were performed on same tumour cell DNA and also cfDNA from plasma samples . VAF, false positive rate, concentration of mutant and wild type allele and total cfDNA are listed in Table n = 2) or G2 (n = 1) tumours and were progression free at follow-up. None of these three patients had detectable ctDNA in plasma.Tumour-specific variants in cfDNA from plasma were analysed by multiplex ddPCR in 6/9 patients at initial UTUC diagnosis and at recurrent disease for the remaining 3/9 patients. In addition, more than one plasma sample was analysed for three patients. The results from ddPCR in relation to clinical status and outcome are presented in Fig. 2. This specific plasma sample was taken at diagnosis in a patient with G1 tumour. In contrast, in patients where ctDNA was detected, the smallest tumour size at sampling was 300 mm2. The only exception was the third sampling time point that was positive for ctDNA in the patient with G3 CIS where the tumour was macroscopically undetectable. Amongst the patients with detectable ctDNA at diagnosis, the largest tumour size at sampling was 2491 mm2. This specific patient did not suffer disease progression, but had a large inoperable G3 tumour at the time of sampling and later received palliative treatment with radiotherapy.Tumour size was estimated by URS at the time point of each plasma sampling Table . We obseAlthough a number of studies have been performed assessing the utility of ctDNA as a prognostic and monitoring biomarker in urothelial carcinoma with promising results –17, onlyIn our pilot cohort, where all had detectable tumour-specific gene variants in tumour cells received from focal barbotages, 5/9 patients had detectable ctDNA in plasma during their clinical course. Four of them, including one patient with CIS, later progressed in their disease with either distant metastasis, progression to higher tumour grade or death from UTUC 4–12 months after the sampling time point, which might suggest that ctDNA could have potential as a biomarker for disease progression. These results are consistent with previous findings where ctDNA was found in patients with metastatic UTUC and was shown to predict UTUC patients with poor prognosis , 8. The TERT promoter mutation C228T, which highlights the need to perform genetic testing on tumour material to select patient-specific genetic variants for monitoring tests. NGS analysis of tumour cells will likely become part of future clinical routine as the cost of sequencing comes down which will simplify the selection of tumour-specific gene targets. Tumour genetic heterogeneity and subclonal genetic evolution during progression affect the genetic landscape and level of ctDNA [In this study, targeted multiplex ddPCR was used to detect known patient-specific gene variants in plasma. This method offers a fast, high-sensitivity, minimally invasive and easy-to-interpret approach to detect a few patient-specific variants at once and could, thus, serve as a promising method for surveillance of UTUC if deemed clinically relevant. The only recurrent mutation analysed in the cohort was the of ctDNA  and are Detection of tumour-specific gene variants in ctDNA from plasma by targeted multiplex ddPCR is a feasible method in patients with UTUC. Our early observations suggest that detection of ctDNA by this minimally invasive approach could have potential as a biomarker in UTUC. However, no clinical conclusions can be drawn and further studies are warranted to determine its role as a biomarker.Supplementary file1 (PDF 5674 KB)Supplementary file2 (PPTX 81 KB)Supplementary file3 (DOCX 15 KB)Supplementary file4 (DOCX 14 KB)Below is the link to the electronic supplementary material.


Re). The results show that the mixing index increases with baffle length and obstacle length-to-width ratio and decreases with gap width at Re = 0.1, 1, 10, 20, 40, and 60. The mixing index can reach more than 0.98 in the range of Re ≥ 20 when the baffle length is 150 μm, the obstacle length-to-width ratio is 600/100, and the gap width is 200 μm. The pressure drop of the microchannel is proportional to baffle length and obstacle length-to-width ratio. Combining baffles and obstacles can further improve the mixing performance of square cavity micromixers. A longer baffle length, larger obstacle length-to-width ratio, narrower gap width, and a more symmetrical structure are conducive to improving the mixing index. However, the impact of pressure drop must also be considered comprehensively. The research results provide references and new ideas for passive micromixer structural design.As an essential component of chip laboratories and microfluidic systems, micromixers are widely used in fields such as chemical and biological analysis. In this work, a square cavity micromixer with multiple structural parameters  has been proposed to further improve the mixing performance of micromixers. This study examines the comprehensive effects of various structural parameters on mixing performance. The impact of baffle length, obstacle length-to-width ratio, gap width, and obstacle shape on the mixing index and pressure drop were numerically studied at different Reynolds numbers ( Microfluidic chip  is a scientific technology that operates fluids on a micrometer scale ,2,3,4. TPassive micromixers can induce convection by optimizing and designing the microchannel structures, thereby breaking the laminar state of the fluid and increasing the contact area between fluids to achieve rapid mixing. The passive micromixer has the advantages of easy processing, easy integration, and low cost , ensurinRe = 0.1, 1, 10, 20, 40, and 60. The optimal design parameters are obtained by considering both the mixing index and pressure drop.In this paper, a square cavity micromixer with rib structures is presented, in which multiple baffles and obstacles are arranged in the square cavity to form rib structures and connecting different square cavities through gaps. The squeezing effect of the narrow space produced by the baffles and obstacles on the fluid is combined with the throttling effect of the gaps, and thus generates chaotic convection between the two fluids in the mixing area, which increases the contact area between the fluids and achieves vortex enhancement. Visualization experiments and numerical simulations are used to study fluid flow and mixing characteristics. The effects of the parameters of baffles, the parameters and shapes of obstacles, and parameters of gaps on mixing performance are discussed through numerical calculations within the range of 1 is defined as the outlet of the second rectangular square cavity; the mixing index of section S1 is taken as the research object in this research. The mixing area length of the micromixer is 1.13 mm, and the total length is 3.35 mm. Reducing the size of microchannels can effectively improve the mixing efficiency of molecular diffusion. It has been reported that in microchannels with a size of only a few microns, the mixing time of different components of the fluid is less than 100 ms, and the diffusion rate is fast. However, the presence of microchannels with excessively small dimensions can result in significant pressure drops and the possibility of channel blockages [1, and the width of Inlet 1 is W1 = 2W2 = 2W3. In this paper, a three-dimensional geometric model of a square cavity micromixer with baffles, obstacles, and gaps is established. The height of the micromixer is uniform throughout and fixed at 50 μm. The two-dimensional sketch is shown in lockages ,31. To e3 and −4 Pa·s in this model.This study employs COMSOL Multiphysics 6.1 software to conduct three-dimensional numerical simulations of fluid flow and mixing properties. The physical field adopts laminar flow and transport of diluted species. To avoid numerical instability in convection-dominated transport, the default Consistent Stabilization is used for stability in the transport of diluted species. This method employs streamline diffusion and crosswind diffusion techniques. Additionally, to save computation, the equation residual is set to approximate residual. The fluid flow is presumed to be steady and laminar, and the fluid being treated as an incompressible Newtonian fluid. The influence of gravity is disregarded. Therefore, the governing equations for fluid flow within the micromixer adhere to the continuity equation and the Navier–Stokes equation, namely:Regarding the solution of the velocity field, a no-slip wall is set on the wall of micromixer. The fully developed flow is set as the velocity boundary condition at the three inlets of the microchannel. The outlet is set the pressure boundary condition; the pressure is 0.C represents the concentration of the solute to be mixed, and D represents the diffusion coefficient of the solute, D = 1 × 10−11 m2/s. The concentration boundary conditions are specified at the channel entrance, with Assuming that no chemical reaction occurs between the two mixed fluids and ignoring the heat of dissolution between them, the mixing of solutes satisfies the convection-diffusion equation, which is: 1 is used to evaluate the performance of the micromixer. The formula for calculating the mixing index is as follows [The mixing index of S follows :(5)η=1−∬Re is also an essential parameter, defined as:The To ensure the accuracy of the numerical calculation results, a grid independence analysis is performed. The grid convergence index (GCI)  is used Re = 10, the mixing index at the exit of the second square cavity (section S1) is used as the criterion to determine the Grid Convergence Index (GCI). The GCI is calculated for grid numbers of 26.9w, 77.6w, and 116.5w, with the results shown in The grid convergence index (GCI) has been calculated to ensure the accuracy of the calculation results , and theended in . At Re =To verify the reliability of the numerical model, a microfluidic experimental platform and the Mirco-PIV system , shown in Three micro-injection pumps  inject deionized water containing 1 μm tracer particles  into the micromixer from three inlets. The fluorescent signal of tracer particles moving with the fluid in the channel is collected and processed by computer to obtain the fluid velocity vector field. Re = 0.1, the very slow fluid flow rate makes the fluids of different components have a longer residence time in the channel, thus promoting sufficient mixing; the mixing indexes of the three curves are very good. In the range of Re = 1, 10, 20, 40, and 60, the mixing index gradually increases with Re, the mixing index results from our computational model correspond well with those in [Experimental results and numerical mixing index results from the square cavity micromixer in reference  are usedthose in .Re = 0.1, 1, and 60. Five cross-sections A-A, B-B, C-C, D-D, and E-E were selected to analyze the concentration field, and thus analyze the flow and mixing characteristics of two fluids in the micromixer.Re = 0.1 and 1 in cross-section A-A. The fluid initially contacts, and the interface between the fluids is obvious. At Re = 60, the contact surface between the two fluids is convex because of the different speed at the border and the center of the channel (the flow rate in the center of the channel is faster). In cross-sections B-B, C-C, D-D, and E-E, at Re = 60, the form of mixing changes from molecular diffusion to convection-dominated transport as the fluid velocity increases. The boundary between different component fluids becomes a curve due to the influence of the micromixer structure; fluid generates lateral movement by the action of baffles and obstacles. The fluid flows upward to the walls on both sides of the microchannel in the B-B section. After passing through the obstacles, the fluid flows to the center and confluence in the middle of the micro-channel in the D-D section. The boundary between different component fluids is blurred and curved. At Re = 60, the fluid contact surface is fuzzier and more tortuous than at Re = 0.1 and 1. The fluid is basically completely mixed when the fluid reaches the section E-E.The mixing effect is not obvious within the range of Re = 0.1 and 1, the concentration distribution and obvious fluid boundaries in each section is basically consistent. The mixing of fluids mainly relies on molecular diffusion and is less affected by structure, resulting in poor mixing effect with obvious boundaries between different fluids. However, compared with Re = 1, the fluid velocity is slower and the residence time of the fluid in the channel is longer, resulting in longer mixing time and better mixing efficiency at Re = 0.1. At Re = 60, fluid boundaries in each section are curved and blurred; fluid mixing is mainly dominated by convective diffusion, with the fluid mixing thoroughly at the extrusion effect of the channel structure.At Re = 20 as an example, Re = 0.1, 1, 10, 20, 40, and 60.To further study the mixing and pressure drop characteristics of the micromixer, a comparative analysis was conducted on a straight channel micromixer, a square cavity micromixer, a square cavity micromixer with baffles, a square cavity micromixer with obstacles, and the micromixer designed in this paper in order to investigate the combined effects of baffle structure, obstacles, and gaps. Taking Re = 20 after two units of square cavity.As shown in Re = 20, the mixing index can only reach 60%. In the micromixer only with straight channel shown in As presented in Re = 0.1 and 1, the mixing index of the five micromixers shows a downward trend with Re. Fluid mixing is mainly dominated by molecular diffusion, which is less affected by structure. As Re changes from 0.1 to 1, the fluid velocity increases, resulting in shorter residence time of the fluid in the channel and shorter mixing time. Compared with ordinary square cavity micromixers , the mixing index of the new micromixer has increased by 0.30~0.42. At Re = 10, 20, 40, and 60, the mixing index shows an upward trend with Re, the influence of natural diffusion on mixing efficiency gradually decreases, and chaotic convection dominated by structural influence plays a dominant role. Compared with micromixers with only baffles or only obstacles, the mixing index of the new micromixer has increased by 0.14~0.56. The pressure drop curves all show an approximately linear growth trend with the increase of Re and are positively correlated with mixing index.As presented in b at Re = 0.1, 1, 10, 20, 40, and 60. The mixing index shows a trend of decreasing and then increasing with Re, which is due to the transition from molecular diffusion to chaotic convection. The mixing index and pressure drop increase significantly with the gradual increase of lb. When lb ≥ 150 μm, within the range of Re = 0.1 and Re ≥ 10, the mixing index reaches over 90%. At Re ≥ 20, the mixing index reaches over 95%, with good mixing efficiency in a wide range of Re. Compared with lb = 150 μm, when lb = 170 μm, the maximum increase in mixing index is 0.007, while the pressure drop increases by 3.99 kpa. After comprehensive consideration, lb = 150 μm is selected as the design parameter.To explore the influence rule of baffle length on mixing index and pressure drop, eight values were selected within the range of 30~170 μm for baffle length. Re increases, fluid velocity gradually increases and obstruction effect on fluid by baffles and obstacles enhances, and thus the pressure drop also increases exponentially with Re. At the same time, the space formed between baffles and obstacles decreases with the increase of baffle length, which is also resulting in greater pressure drop.As Re = 1 and 60 as an example, b = 30, 110, and 170 μm. At Re = 1, the fluid flow velocity is slow, and thus there is no vortex generation in the channel. At Re = 60, fluid flow is significantly affected by structure; fluid generates backflow on both sides of the narrow space and expansion vortices between baffles due to the jetting effect of the narrow space between baffles and obstacles. However, the expansion vortex is close to the corner of the channel, and thus has a poor promotion effect on mixing.Taking Re = 60, at lb = 30 μm, only a small amount of backflow occurs behind obstacles with general mixing effect; the jetting effect of narrow spaces is enhanced with increases of lb. At lb = 110 μm, the vortex generated by backflow is more obvious and a vortex is gradually formed in the space area between obstacles and walls with obvious mixing effect. It can be seen that this eddy current has obvious promoting effect on fluid mixing by comparing with Re = 1 (no eddy in the space area). At lb = 170 μm, new vortices are generated on right side baffles of square cavity, fluid is basically completely mixed when flowing out of the first rectangular cavity. The obstruction effect of baffles and obstacles and throttling effect of formed narrow spaces can significantly form vortices in micromixer to efficiently promote fluid mixing by breaking fluid laminar state and boundaries.When −8 m2. Length-to-width ratios of Lo/Wo = 750/80, 600/100, 500/120, 400/150, 375/160, and 300/200 were selected to discuss the influence of different obstacle width-to-length ratios on mixing performance.To study the influence of obstacle structural parameters on mixing performance, the total area of obstacles is defined as 0.6 × 10o/Wo and Re. At the same Lo/Wo, the mixing index shows a trend of first decreasing and then increasing with Re, and pressure drop shows an increasing trend. In micromixers with different Lo/Wo, as Re increases, the maximum mixing index gradually moves toward a larger pressure drop (higher Lo/Wo). At Re ≥ 20, when Lo/Wo = 600/100 and 500/120, the mixing index can reach over 90%. At Lo/Wo = 750/80, mixing efficiency can reach over 90%, with a mixing index of 99.7% at Re = 60. However, compared with Lo/Wo = 600/100 and 500/120, pressure drop increases by 79.1~169.9%, reaching to 231.6 kpa at Re = 60. Compared with Lo/Wo = 500/120, Lo/Wo = 600/100 has better mixing index and smaller pressure drop. Considering comprehensively the adverse effects of excessive pressure drop on pipelines, this paper selects obstacle length-to-width ratio Lo/Wo = 600/100 for design.Re = 60. When Lo/Wo = 750/80, the space between obstacles and walls is only 50 μm, resulting in a small cross-sectional area of fluid flow and strong squeezing effect on fluid but also resulting a large pressure drop. When Lo/Wo = 600/100 and 500/120, the overall concentration distribution and velocity streamline distribution in square cavities are consistent with similar mixing performance. The expansion vortices gradually form and grow between baffles on the side walls close to the wall as the length-to-width ratio gradually increases. However, these vortices have no significant effect on promoting mixing because they are too close to the wall. The squeezing effect on fluid weakens and mixing performance gradually decreases with the spaces between baffles and obstacles gradually increases. Therefore, compared with Lo/Wo = 500/120, micromixers with Lo/Wo = 600/100 have better mixing performance.Re values under specific baffle lengths and obstacle length-width ratio. There is no significant improvement on the mixing index by continuing to change structural parameters. To discuss the impact of different gap widths on mixing performance, the effect of gap width is examined in the range of 50–200 μm under the condition of baffle length of 70 μm.The micromixers have been able to achieve good mixing performance within a wide range of Re at different gap widths. It can be found that the mixing index increases as the gap width decreases. The overall mixing efficiency is best at Wg = 50 μm, reaching more than 95% in the range of Re = 0.1, 10, 20, 40, and 60. However, the pressure drop has increased by 1.29–2.19 times compared to when the gap width is wider (Wg = 200 μm). The transition point from molecular diffusion to chaotic convection is advanced under the decrease of Wg.g) is 50, 110, and 200 μm, Re = 0.1 and 60. At Re = 0.1, there are no vortices in micromixers under different gaps, but the difference in mixing effects under different gap widths is obvious. At Re = 60, due to the increase in flow rate, the fluid impacts the obstacles and generates a backflow vortex at the gap outlet, and the size of the vortex gradually increases with the decreases of the gap width. The throttling effect of the gap is strongest at Wg = 50 μm, and the fluid is basically completely mixed at the gap area. However, a smaller gap width also produces a larger pressure drop. The pressure drop generated by the fluid is 2.19 times that of Wg = 200 μm, which causes a greater burden on the micromixer.When the gap width , with d = 150 μm having the best mixing efficiency. As Re increases , the mixing index increases as the symmetry of the obstacle increases (the distance d decreases), and d = 0 μm has the best mixing performance and the smallest pressure drop.Re = 0.1 and 60. At d = 150 μm, the shape of the concave obstacles is the same as the convex obstacles, and both the concentration distribution and velocity streamline are consistent. In the micromixers with concave obstacles the mixing effect in the upper part of the micromixer (the concave part of the obstacle) is worse at d = 150 μm compared to d = 0. The reason is that the space narrows between the baffles and the obstacle at the concave part becomes larger, thus weakening the squeezing effect on the fluid and reducing vortex generation on the upper side wall of the obstacle. When d = 0, obvious vortices are generated in both upper and lower wall areas of the obstacle, resulting in a blurred fluid interface and obvious mixing effects.Re = 0.1, d = 150 μm and 0, there are generally consistent mixing trends in the upper half part of micromixers due to the larger space narrow between the concave groove of the obstacles and baffle. When d = 150 μm, there is a significant improvement in mixing effect due to well squeezing effect of obstacles squeezing baffles in lower half area, although the difference is not much compared with that when d = 0, where the overall mixing performance is better. At Re = 60, fluid flow is large and basically completely mixed after passing through the first rectangular cavity. Under asymmetric obstacles (d = 150 μm), the mixing in lower half part of the microchannel is further enhanced, and the mixing in upper half part of the microchannel is further weakened because of the asymmetrical eddy currents on the back side of the obstacle. Therefore, the overall mixing index is worse compared to symmetric obstacles (d = 0). As such, symmetric obstacles are more conducive to overall mixing performance in the micromixers designed in this paper.In the micromixers with convex obstacles at In this paper, a new square cavity micromixer that incorporates baffles, obstacles, and gaps is proposed to examine the impact of various structural parameters on mixing performance through numerical analysis of the flow field, concentration field, pressure drop, and mixing index within the micromixer. Based on experimentally verifying the accuracy of the numerical model, the combined effects of baffle parameters, gap parameters, obstacle parameters, and shape on the micromixer were discussed.Re = 0.1 and 1, molecular diffusion is the primary mixing mechanism, and the mixing index is related to the residence time of fluid in the channel. At Re = 10, 20, 40, and 60, fluid convection becomes dominant, and the channel structure is the main factor affecting mixing performance.Numerical results indicate that within the range of Re (Re ≥ 20). Although increasing the length of the baffle and length-to-width ratio of obstacles or reducing gap width can improve the mixing index further, it also produces excessive pressure drop that affects fluid flow characteristics. Excessive pressure drop is not conducive to design and sealing of microchannel. Designing an asymmetric obstacle structure to generate asymmetric vortices in the channel can enhance mixing in half of the area but reduce it in the other half area, thereby reducing overall mixing performance.A comprehensive study of multiple structural parameters reveals that the narrow space generated by the combination of baffle and obstacle will squeeze and jet the fluid, which forms a series of vortices and backflow areas on the wall and inside the micromixer. This effectively reduces the cross-sectional area of fluid flow and increasing the lateral contact area, and thus significantly strengthens mixing. The mixing index can exceed 97.9% in a wide range of In this paper, the effects of multiple structural parameters  on mixing performance are studied, and a micromixer is designed based on this analysis and comprehensively analyzed for mixing index and pressure drop. This provides a clear reference for passive micromixer structural design.


This paper describes a locomotion strategy for robots based on the interaction between two fluids, through the development of an untethered mobile robot. The fundamental principle of robot locomotion is to exploit the active deformations of ferrofluid caused by internal magnetic fields, which generate reaction forces to the surrounding fluid . The developed robot is equipped with two permanent magnets (PMs), two electromagnets (EMs), two clusters of ferrofluid, and a control unit with batteries. It has a length, width, and mass of 107 mm, 94 mm, and 127 g, respectively. In the robot, PMs are used to hold clusters of ferrofluid. The activation of EMs by the controller achieves forward and rotational movements of the robot. Experimental results show the forward speed and rotational speed in water to be 2.7 mm/s (at a driving frequency of 9 Hz) and 1.2°/s (at a driving frequency of 7 Hz), respectively. The measured thrust force of the robot is 2 mN, further supporting the concept of robot locomotion by fluid–fluid interaction. Robots locomote based on the physical interaction between their body and the surrounding environment. Normally, the bodies of robots are solid; therefore, the majority of robotic locomotion on Earth is explicated as solid–solid  and solid–fluid  interaction. The former is employed in, for instance, legged robots that move via interaction between the legs and the ground . The latter includes flying robots and swimming robots that locomote through the interaction between their body and the surrounding fluid.Here, we present a robot that can locomote by fluid–fluid interaction. The idea is to use ferrofluid as the body of the robot, which can be actively deformed by magnetic fields. When the robot is surrounded by an immiscible fluid, the deformations of ferrofluid generate movement, as shown in Fig. 1. Researchers have demonstrated movement and manipulation of a liquid cluster based on ferrofluids4 and liquid metals9. Furthermore, ferrofluids have also been reported to enable in-memory computation10. These robotic systems usually move by applying external electromagnetic fields. Moreover, their movements are the result of a perfect balance between the surface tension of the fluid, the static electromagnetic field, magnetization, including that caused by the fluid itself, and the strength of the externally applied field. An exceptional work presented in Ref.11 demonstrates ground locomotion  using liquid metal. Our robot essentially differs from them in the sense that it locomotes using an internal magnetic field and reaction forces resulted from fluid–fluid interaction.Our robots may be classified as liquid robotsIn this study, we focus on investigating the locomotion characteristics derived by fluid–fluid interaction through characterization of the robot. Specifically, we measure the forward speed, rotational speed, and thrust force generated by fluid–fluid interaction as functions of the switching frequency of the internally generated magnetic field and discuss on the results.A ferrofluid changes its shape in response to an applied magnetic field. The deformation follows the magnetic flux lines that appear as spikes as shown in Fig. The speed of shape change of ferrofluid in the water is ~ 0.1 s, as can be seen from the sequence of large deformation represented in Fig. Various locomotion patterns can be formed by the combination of large deformations and small deformations. Figure Rotational movement can also be achieved by setting up the PMs to have the same polarity on one side and the opposite polarity on the other, as shown in Fig. From a systematic point of view, the polarity of EMs can be changed by switching the direction of the electrical current. The polarity of PM, as can be seen in Fig. The robot developed in this study is displayed in Fig. Under the fuselage is the moving unit composed of EMs, PMs, and acrylic plates. This unit is underwater when the robot is placed on the water surface. The EMs are fabricated by coiling a copper wire (diameter 1 mm) onto a hollow steel core (diameter 8 mm and length 25 mm) followed by inserting an M6 steel bolt (length 35 mm). These steel parts are made of chromium–molybdenum steel (SCM435). The number of turns and the electrical resistance of the coil are 63 and 0.45 Ω, respectively. With the control circuit, the EM can generate a magnetic flux density up to 81.8 mT on the surface of the pole and 69.7 mT on the acrylic plate where the ferrofluid is held. These values were acquired using a Tesla meter . The distance between the two EMs is 26 mm. The PMs used in this study are neodymium magnets having a diameter and thickness of 8 and 4 mm, respectively. Each of them generates a magnetic flux density up to 380.0 mT on the surface and 219.0 mT on the acrylic plate (measured by the Tesla meter). They are attached to an acrylic plate having a thickness of 1 mm. The planar dimensions of the robot are 107 mm long and 94 mm wide. It has a mass of 127 g without ferrofluid. In the robot, each component, e.g., the control circuit, moving unit, and batteries, is placed to ensure that the center of mass is positioned at the center of the robot, thereby avoiding mass imbalance. This minimizes any potential bias that could occur in the trajectory of the robot.During preliminary tests, we found the robot acts like a compass because of the environmental magnetic fields such as geomagnetism or magnetic field caused by the iron components of the building. Therefore, the direction of the water tank and the robot was set carefully during the characterization to minimize the influence of environmental magnetic fields and to clearly visualize the locomotion by fluid–fluid interaction. To determine the suitable initial condition for the characterizations of the robot, we set the permanent magnets of the robot as Fig. After setting up the initial condition, we observed a forward and rotational movement as expected from the working principle previously discussed. The sequence of these movements is displayed in Fig. We then quantified the speed of the robot (see “Method” section for more detail). The measured forward speed as a function of the driving frequency is plotted in Fig. There are two possible reasons associated with the locomotion without ferrofluid (up to ~ 0.5 mm/s). The first reason is the presence of Lorentz force generated in the robot. As seen in Fig. The measured rotational speed as a function of the driving frequency is plotted in Fig. Peak frequencies observed in the forward and rotational movements are 9 Hz and 7 Hz, respectively. As mentioned previously, these peak frequencies suggest a resonance mode that increases the deformation of the ferrofluid. The difference in peak frequency values for forward and rotational motions may be attributed to the relative speed of the surrounding water and the ferrofluid. At the peak, the forward speed is 2.7 mm/s and the rotational speed is 1.1 mm/s on the side of the robot with the ferrofluid . We assume that water inhibits the ferrofluid's deformation by acting as a load on it. Due to the direction of deformation in which the ferrofluid generates thrust and the direction of water flow alignment, in the resonant state, the ferrofluid faces a smaller load at the forward movement and, conversely, a larger load at the rotational movement, which is thought to have caused the resonant frequency to be higher in the former case and lower in the latter.We next measured electric power consumption as a function of the driving frequency as displayed in Fig. We further quantified the movements of the robot to support that the movements are consequence of fluid–fluid interaction. To do so we looked at force acting on the robot, with hypothesis that if robot moves the net force is not zero and vice versa. Figure We have demonstrated robot locomotion by fluid–fluid interaction. The robot developed in this study employs ferrofluid, whose active deformations generate movements in water. During the experiments, we observed the influence of environmental magnetic fields on the movements of the robot. The presence of these external fields may cause an error in the measured data. To mitigate this issue, magnetic shielding will be implemented in the experimental setup, and further characterization of the robot will be carried out. A variety of locomotion patterns, a wider range of driving frequency, and force are tested. Through these experiments, the characteristics of the robot will be clearly determined.Investigation of liquid combinations for locomotion of fluid–fluid interaction remains an aspect of future work. The current study employs water as the surrounding fluid, but other liquids such as seawater and blood can be considered in the future. In addition, the robot body could also be changed from ferrofluid to liquid metals. To use different liquids as the surrounding fluid, the effects of their electrical conductivity and magnetic permeability on locomotion should be investigated carefully. As the experimental results suggest, the conductivity may contribute to the generation of induced currents; therefore, the induced magnetic fields in the surrounding liquid due to the magnetic field generated by the robot ultimately affect the way it moves. In a liquid with high magnetic permeability, the magnetized liquid may cling around the ferrofluid, which may inhibit deformation when the EMs are active. However, the collision of the moved ferrofluid with the magnetized liquid attracted by the EM could generate a large reaction force, which may conversely increase the locomotion speed of the robot. Modeling of fluid–fluid interaction should also be conducted. It is important to reveal the mechanism and condition of proceeding direction reverse to occur which can be utilized for more rational hardware setup and controller. For this purpose, computational fluid dynamics coupled with electromagnetism is a powerful tool. The outcome of these works will contribute to define the application of fluid–fluid robots.As suggested in the experimental result discussed above, locomotion efficiency may be an important issue. One potential approach to address this issue is to investigate optimal design parameters  to maximize the locomotion performance through experimental and computational environments. Especially, electromagnets in the experiments consume roughly 10 A of current. Replacing the electromagnets to one with more thin and dense wire would be effective.Based on the results of the aforementioned research efforts, fluid–fluid robots of diverse configurations and size scales could be designed for various applications. We are interested in the configuration of robots that are completely covered with ferrofluid or other liquids able to function as the body. This type of robot would exhibit a wide range of locomotion patterns and act as a cell of modular robots. Potential applications of fluid–fluid robots include inspection, delivery, and sampling in fluid environments, such as rivers, seas, and inside the human body. In these environments, the liquid nature of the robots assures the safety of the surrounding objects. This is an expected advantage of robotic systems that locomote based on fluid–fluid interaction.Measurement and subsequent calculation of the translation and angular displacement was carried out with the fiducial markers on the robot and the corners of the water tank Fig. b. These The locomotion of the robot in terms of forward speed and rotational speed was characterized as follows. 1.0 g of ferrofluid  was put on each acrylic plate on both sides. The ferrofluid consists of isoparaffin (40–60%) and triiron tetraoxide (40–60%). Its physical properties include a specific gravity ranging from 1.3 to 1.5, a typical viscosity of 50 to 150 mPa s, and a saturation magnetization typically between 40 and 50 mT (measured at 20 °C). The robot was then placed on the water surface in a tank (475 mm × 250 mm × 140 mm) equipped with fiducial markers on each the corners. The moving unit of the robot was activated, and subsequent locomotion was captured using the camera. The recorded video was then processed to extract the moving speed of the robot. The forward speed was calculated by dividing the travel distance by the measurement time of 20 s. To prove that the translational speed was caused by the ferrofluid, the translational speed using the robot without the ferrofluid was also measured. The rotation speed was calculated by dividing the maximum angular displacement by the time to reach that angle. During the experiment, the applied voltage to the EMs was set to 4.35 V, which induces an electric current of ~ 9.5 A. The driving frequency were 1, 3, 5, 7, 8, 9, and 11 Hz. At each frequency, three measurements were taken, and their average and standard deviation were reported. After each measurement, the electric power consumed was calculated using a battery charger .The thrust force generated by the ferrofluid was measured with a low capacity, high precision force sensor , signal amplifier , and multimeter . A fixture made of 3D printer was used to connect the sensor to the robot directly. An additional float was added to the robot to offset the mass of the fixture (~ 50 g). The thrust force was measured with the translational mode (Fig. Supplementary Video S1.Supplementary Video S2.


Candida auris is deemed an urgent threat because it spreads easily in healthcare facilities, can cause severe infections with high mortality rates, and is resistant to antifungal agents. Since the onset of the COVID-19 pandemic, we detected a rise in inpatient C. auris cases. We describe a series of C. auris cases at a tertiary hospital in Washington, DC.Cases of Candida auris by date of initial sample collection, GW Hospital, Washington, DC, March 2020 - April 2023.C. auris between 3/2020-4/2023. Patients transferred from a single long-term care acute hospital (LTACH) with high C. auris prevalence were placed in empiric isolation on admission and screened for C. auris colonization via axilla and groin surveillance swabs. C. auris was identified by MALDI-TOF MS and/or Biofire BCID Panel v2 and strain typing was performed by Fourier-Transform Infrared Spectroscopy. Antifungal susceptibility testing (AST) was performed via broth microdilution and interpreted using tentative breakpoints from CDC.We reviewed charts of inpatients with C. auris . 29 patients (71%) were transferred from the LTACH. 29 (71%) patients had either a tracheostomy or gastrostomy tube. 22 (54%) were identified through surveillance culture (21 from the LTACH) and 19 (46%) via clinical culture . 2/32 (6%) patients with C. auris detected initially via non-blood specimens later developed C. auris candidemia. 8/11 (73%) cases of candidemia were CLABSIs. Overall, 8/41 (20%) patients either died or were placed on comfort care during hospitalization. Among 23 isolates available for AST, 22 (96%) were resistant to fluconazole, 15 (65%) to amphotericin B, and 1 (4%) to anidulafungin. Preliminary strain typing suggested grouping of isolates into 2 main clusters denoting distribution into 2 distinct clades; further characterization is undergoing.We identified 41 cases of C. auris infection or colonization died/were placed on comfort care underscores the poor prognosis associated with C. auris. To combat the spread of C. auris, further development of antifungals, rapid diagnostics, and effective infection prevention strategies are needed.The COVID-19 pandemic placed healthcare systems under unprecedented strain which has accelerated the spread of antimicrobial resistant pathogens. The finding that 1 of 5 patients with Tara Palmore, MD, Abbvie: Grant/Research Support|Gilead: Grant/Research Support|Rigel: Grant/Research Support Jennifer Dien Bard, PhD, Abbott Molecular: Grant/Research Support|BioMerieux: Advisor/Consultant|BioMerieux: Grant/Research Support|BioMerieux: Honoraria|Genetic Signature: Advisor/Consultant|Genetic Signature: Grant/Research Support|Luminex: Grant/Research Support|Salve: Stocks/Bonds|Thermo Fisher: Honoraria