Here we report on the first application of 3D printing for the fabrication of monolithic devices suitable for capturing and imaging tumor spheroids under dynamic perfusion flow. Resolution of the printing process has been refined to a level sufficient to obtain high precision features that enable capture and retention of tumor spheroids in a perfusion flow stream that provides oxygen and nutrient requirements sufficient to sustain viability over several days. Use of 3D printing enables rapid design cycles, based on optimization of computational fluid dynamic analyses, much more rapidly than conventional techniques involving replica molding from photolithographic masters. Ultimately, these prototype design and fabrication approaches may be useful in generating highly multiplexed monolithic arrays capable of supporting rapid and efficient evaluation of therapeutic candidates in the cancer drug discovery process.Microfluidic systems for the analysis of tissue models of cancer and other diseases are rapidly emerging, with an increasing recognition that perfusion is required to recapitulate critical aspects of the  Additive manufacturing techniques such as 3D printing enable the construction of microfluidic devices with complex vertical features, including high aspect ratio pillars and channels that are challenging to fabricate using conventional techniques , 2. HistExisting fabrication of microfluidic devices for cell and tissue culture often require laborious and time-consuming optimization of channel geometry, particularly when bonding together separate layers. Indeed, achieving strong adhesion between micromolded features and the substrate is challenging, particularly when the contact footprint is relatively small . Furtherin vitro model for pre-clinical testing of targeted therapies and immunotherapies on human cells , and to be able to be retrieved for off-chip evaluation such as single cell analyses. Previously, a number of groups have used soft lithography to prepare PDMS microfluidic devices for spheroid culture –25. We ain vivo-relevant manner.In this brief report, we demonstrate 3D printing of monolithic microfluidic devices that integrate high aspect ratio pillars for spheroid trapping, as well as multilevel vertical features such as bubble traps and tubing connectors. We characterize these devices using finite element modeling to optimize flow conditions and capture efficiency. As a proof-of-concept, tumor spheroids were prepared and introduced into these devices and imaged with time-lapse fluorescence microscopy over periods of up to 3 days. The device reported here was capable of being printed and prepared for use within an hour and a half, constructed as a single unit with novel integrated connectors to minimize burdensome post-fabrication assembly. Within each channel, an array of 3D printed microposts was incorporated to capture and hold tumor spheroids in a perfusion flow stream for periods of several days, extending viability well-beyond what is typically observed for spheroids in static culture. Ultimately, these microfluidic devices will enable researchers and clinicians alike to test various cancer therapeutics such as immunotherapies in a scalable, rapid, and Photopolymerizable resin GR-1 was purchased from Pro3dure Medical GmbH . Perfluoroalkoxy tubing (1514L), polyetheretherketone tubing connector screws (XP-235X), and polyoxymethylene stopper screw (P-309) were purchased from IDEX Health Science LLC . Twenty milliliters sterile syringes (309661) were purchased from Becton Dickinson. A syringe pump (Standard Infuse/Withdraw PHD ULTRA) was purchased from Harvard Apparatus .CT26 colon cancer cells (CDL-2638), RPMI-1640 media, fetal bovine serum, and Trypsin EDTA solution 1X (30-2101) were purchased from ATCC. CellTracker Green CMFDA Dye (C7025), APC Annexin V (A35110), and UltraPure Agarose (16500500) were purchased from Invitrogen. PBS was purchased through ThermoFisher Scientific. 3D Petri Dishes  were purchased from Microtissues, Inc.The Asiga Max X27 was used for all the light-directed 3D printed devices presented here. This printer uses a high power ultraviolet light emitting diode (385 nm wavelength) with digital light processing and has a pixel resolution of 27 by 27 μm. Computer aided designs were prepared in SolidWorks 2016  as an STL file at highest resolution (“fine”), then imported into Asiga Composer to optimize layer thickness and build orientation. Three types of test structures were printed to optimize feature resolution.Fluid flow and shear conditions in the microfluidic capture region were computationally modeled in COMSOL 5.3 Multiphysics . The geometry was initialized from a top-down snapshot of the device design exported from SolidWorks 2016. This simulation solves for laminar flows based on the Navier-Stokes equation with boundary conditions of a 1 mm entrance length and thickness, with no-slip wall conditions, and assuming a fluid viscosity of 1 cP and flow rates of 5 uL/min. The mesh was set to a physics-controlled mesh setting, with normal element size with a minimum mesh quality measure of 0.1537 μm, chosen to be sufficiently small for modeling a characteristic feature size of 76 μm.6/mL.CT26 cells were subcultured from 1:4 to 1:10 ratios with media replacement every 2–3 days, based on the recommended ATCC protocol. Prior to seeding, CT26 cells were trypsinized and stained with CellTracker Green CMFDA Dye (Invitrogen C7025) at a concentration of 20 μM in PBS for 15 min, then resuspended in media to a cell density of at least 1 × 10Molten agarose was cast against the 3D Petri Dish elastomeric masters, then allowed to cool and solidify. The cooled agarose molds were placed in a 12 well plate and prepared for the introduction of cells by soaking in 2.5 mL complete growth media twice for 15 min. To seed 200 μm diameter spheroids, the 24–96 3D Petri Dish geometry was used with a 75 μL droplet of containing 96,000 cells. To seed 300 or 500 μm diameter spheroids, the 24–35 3D Petri Dish geometry was used with a 75 μL droplet of containing 118,000 and 547,000 cells, respectively.A 5 mL syringe with a gauge 12 blunt tip was used to obtain the spheroids for transfer into the device's bubble trap port. Transfer into the channel was done by gently pushing the syringe's plunger with the syringe inside the bubble trap and angled toward the capture geometry. Validation of sample capture was completed using a fluorescence microscope for visual verification.2). Using Zen 2.3 Blue software, images were acquired every 600 min using a 10X objective (imaging 14 slices at 25 μm steps). Images were recorded under consistent acquisition parameters .A total of five CT26 spheroids were set up for imaging over a course of 3 days. Controls for this test consisted of a spheroid in a device without flow to evaluate the rate of cell death caused by the device. The loaded devices were placed in a Zeiss LSM 710 confocal microscope with 488 and 594 nm Argon lasers and environmentally controlled conditions . Mean gray values and integrated density functionalities values along with area of the outlined spheroid were used to calculate the corrected total cell fluorescence (CTCF) relative to a background region at the bottom of the image.Analysis of each set of images was done Corrected total cell fluorescence was used to calculate the percent decrease in brightness when compared between the values at 10 and 70 h. The 10 h time point was chosen as the baseline due to unintended inconsistencies in brightness associated with the 0 h images.Light-directed 3D printing occurs through spatially selective photopatterning of successive layers. The vertical spatial resolution is primarily determined by the layer thickness, while the lateral spatial resolution of light-directed 3D printing is dependent on light exposure. Thus, underexposure results in uncured resin, while overexposure results in larger features than expected. Indeed, light exposure must be carefully optimized for patterning hollow microfluidic channels with an overhanging ceiling. Overexposure of the channel walls could result in narrower or occluded channel cross-sections, while underexposure of the ceiling could result in poor mechanical strength and collapse.In order to optimize the printed features to achieve sub-100 μm resolution, we used a scanning white light interferometer to characterize the layer thickness using a series of steps designed with vertical offsets of 10 μm . A vertiThe design and features of the 3D printed device improved on previous reports of tumor microenvironment structures , 21, in Next, a bubble trap was designed to prevent any bubbles from reaching the capture geometry, by creating a tall curved chamber that allowed bubbles to rise up and out of the path of flow along the bottom of the chamber . Any bubThe capture region is the most crucial feature of this design, since it must capture a biological sample without damaging it throughout the testing process. In prior reports, capture was achieved through the design of intersecting channels with opposing flow currents, along with a depressed feature to entrain a tissue in the flow stream . HoweverThe micropost array design was derived based on several considerations, including prior reports , 28 the An additional unique feature of this device is the modular nature of the connector geometry located around its perimeter. The goal of these modular connectors is to permit precise assembly of multiple components in an arrayed format, allow for any number of configurations to be achieved with this device, and to remove any restrictions on testing sample size or setup. As long as these devices have a flat surface to rest on, they are able to connect to each other on each side without restriction . The conFluid flows and shear stress within the capture region of the microfluidic device were simulated using finite element modeling (COMSOL), based on laminar flow with no-slip boundary conditions at the channel walls. For an unoccupied chamber with a flow rate of 5 μL/min, the fluid flow was fastest between the three top pillars, near the center of the channel (180 μm/s) . Increas−6 mol/cm2/s  . The corol/cm2/s , as repool/cm2/s , 33. Forol/cm2/s , 33. At ol/cm2/s . This isol/cm2/s , 5.t = 0. Normalization of the data at the 10 h time point likely understates the difference between the flow and no-flow control conditions, due to the steepness of the no-flow curve at this point. Further analysis involving perfusion and permeation of a live-dead stain such as Annexin V will be required to further validate extended viability of captured spheroids in this system.As an initial proof of concept, multicellular spheroids were prepared (average diameter of 375 μm and a standard deviation of 8 μm) using CT26 colon carcinoma cells, labeled with CellTracker Green, captured in the device, then imaged in the absence of fluid flow or with perfusion (constant flow rate of 5 μL/min) over 70 h with media containing APC Annexin V. In the absence of fluid flow, the normalized fluorescence intensity of the spheroid decreased by 70% over 40 h, reaching a plateau that remained consistent from the 40 to 70 h time points . With pevia 3D printing with multiscale vertical features, including arrays of posts for spheroid capture, bubble traps, and tubing connectors. The manufacturing time and scalability of the device presented here surpasses that of existing platforms (We report a microfluidic device to capture and culture multicellular spheroids under dynamic perfusion over periods of several days. This monolithic structure was constructed solely latforms , 21. Spein vivo-like immune microenvironment. Future work will expand the device's capability to further expand the viability of biological samples and explore retrieval of samples from the device for further testing. Ultimately, this device will enable researchers and clinicians to test various immunotherapies in a scalable, rapid, and in vivo-relevant manner.Initial perfusion studies suggest that this device, under dynamic perfusion, has the potential of extending tumor spheroid viability compared with static culture conditions. These tests, along with the simplified design and short production time, position this platform as promising development in oncology drug testing platforms. This advance is particularly relevant to immunotherapy-based testing, as static culture devices cannot fully simulate the The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.AM, IW, and JB designed research. AM performed experiments. AM analyzed data with feedback from IW and JB. All authors wrote the paper and approved the final submission.JB was a co-inventor on patent applications that address aspects of the microfluidic technology reported here; those patent applications are assigned to Draper. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


R2 above 0.95 p < 0.001. Yield was measured at the end of the end of the growing season. The results revealed that yield had a high correlation with water stress at R2 = 0.9758 and 0.9816 p < 0.01 for sandy loam and silty loam soils, respectively. Leaf temperature (LT °C), relative leaf water content (RLWC), leaf chlorophyll content (LCC), Leaf area index (LAI), were measured at each growth stage at the same time spectral reflectance data were measured throughout the growth period. Spectral reflectance indices used were grouped into three: (1) greenness vegetative indices; (2) water overtone vegetation indices; (3) Photochemical Reflectance Index centered at 570 nm (PRI570), and normalized PRI (PRInorm). These reflectance indices were strongly correlated with all four water stress indicators and yield. The results revealed that NDVI, RDVI, WI, NDWI, NDWI1640, PRI570, and PRInorm were the most sensitive indices for estimating crop water stress at each growth stage in both sandy loam and silty loam soils at R2 above 0.35. This study recounts the depth of 858 to 1640 nm band absorption to water stress estimation, comparing it to other band depths to give an insight into the usefulness of ground-based hyperspectral reflectance indices for assessing crop water stress at different growth stages in different soil types.Drought and water scarcity due to global warming, climate change, and social development have been the most death-defying threat to global agriculture production for the optimization of water and food security. Reflectance indices obtained by an Analytical Spectral Device (ASD) Spec 4 hyperspectral spectrometer from tomato growth in two soil texture types exposed to four water stress levels  was deployed to schedule irrigation and management of crops’ water stress. The treatments were replicated four times in a randomized complete block design (RCBD) in a 2 × 4 factorial experiment. Water stress treatments were monitored with Time Domain Reflectometer (TDR) every 12 h before and after irrigation to maintain soil water content at the desired (FC%). Soil electrical conductivity (Ec) was measured daily throughout the growth cycle of tomatoes in both soil types. Ec was revealing a strong correlation with water stress at  Plant response to water stress is articulated by a variety of physiological and biophysical changes as well as soil characteristics . Due to climate change and increasing global water scarcity, water stress has been the most perilous abiotic stressor to plant growth. However, to increase food production using a minimal amount of water, timely detection and quantification of crop water status in crop production are very vital in precision agriculture technology ,2,3.Remote sensing, as a tool in precision agriculture to effectively schedule irrigation in vegetable production, has not been extensively developed, especially in greenhouse vegetable production. For sustainability of agricultural water management, timely identification of plant abiotic stress is required , to imprA remote sensing approach in assessing crop water status is fast, accurate, less laborious, and non-destructive at different scales  of measurements ,9,10,11.750, and many other VIs  is based on the following spectra band regions: (i) the ultraviolet region (UV), which ranges from 10 to 380 nm; the visible spectra region, which ranges from 380 to 740 nm; the near-infrared band 740–1400 nm), and 1400 to 2500 nm ranges for the short wavelength region ,25,26. Aramootoo , Nemeské00 nm, anramootoo , Roujeanramootoo , Gitelsoramootoo , Sakamotramootoo , Peñuelaramootoo , Chen etramootoo , Chen etramootoo , Liu et ramootoo , Mzid etramootoo , Rouse eramootoo , Zeng etramootoo ), to evagreen help researchers to better understand the biophysical and biochemical processes of crop leaves and also help in predicting crop yield , yield, and end of season biomass within 980 and 1240 nm. The photochemical index is a very good indicator of photosynthetic rate and spatiotemporal scales, this has been used by . Accords et al. , water in et al. , Magney n et al. , Wong ann et al. , Zhang en et al. ), for th2 assimilation has also been efficiently estimated by PRI under severe drought conditions and this has established that changes in PRI were correlated with water stress in maize . Photosg et al. , Magney g et al. , Garbulsg et al. , Wong ang et al. , Zhang eg et al. , Gamon eg et al. , Massonng et al.  and Pedrg et al. ). For opg et al. , preciseg et al. , or leacg et al. ,48,49. TSolanum Lycopersicum) cv. Xiang Sheng seed was nursed in a 6 × 12 nursery tray in cocopeat on 15 October 2020 and transplanted on 10 November 2020 into truncated black plastic pots (single plant per pot) with 1 m × 0.4 m plant spacing within a block and 1.5 m between block spacing; black plastic mulch was used to cover the ground to control weeds.Pot experiments were conducted in a greenhouse at Tea Research Academy, South China Agricultural University, Guangzhou—China from October 2020 to March 2021. The research site is located between 23°157′826″ N and 113°350′668″ E, with an elevation of 30 m. A simple arch double-span greenhouse was used with a gutter length of 26 m, a width of 20 m, a gutter height of 3 m, gable height of 4.5 m. The top was covered with diffused polyethylene film with 95% light transmission, the sides covered with 50 mesh insert net with the floor covered with black woven polyethylene mulch. The experimental plot was a 5 m × 8 m field and the experimental design was a 2 × 4 factorial experimental design arranged in a randomized complete block design (RCBD) with four replications in each sub-block. Water stress treatments applied ranged from 70–100, 60–70, 50–60, and 40–50% field capacity with each replicated four times to give a total of 16 tomato plants in each sub-block (32 plants for the main block). Fertilizer was applied based on the available N:P:K observed from the soil analysis in each soil type to meet crop nutrient requirement. Soil physical and chemical properties are shown in I is the irrigation water (mm), Q is the volume of ponding water (mm), SWC is the saturated water content of the soil (%), AWC is the actual water of the soil when irrigating (%), DSM is the dry soil mass (kg), and A which is the area of a truncated cone is given by the equationR is the radius of the bottom base; r is the radius of the top base and h is the slant height of the truncated cone. High-quality irrigation water was applied through a drip system, with emitters placed in each pot. Water stress treatment was introduced 2 weeks after transplanting based on soil % FC.Irrigation water was applied through a gravity-driven drip system, with a discharge of 1.2 L/h, and the flow rates were calibrated in the field. Water stress treatment was applied based on the soil field capacity. Soil moisture content (SMC) in each pot was continuously measured with soil moisture sensors Time Domain Reflectometers (TDR). The water stress treatment levels for each treatment used were 70–100% FC, 60–70% FC, 50–60% FC, and 40–50% FC. The upper irrigation and lower irrigation thresholds were set at 10% depletion of soil FC% for each treatment. The volumetric soil water content and the observed soil moisture content measured by a Time Domain Reflectometry (IMKO. TRIME. PICO TDR HD2 64) every 12 h before and after irrigating the plants was used to determine the soil moisture content before water application. The probes of TDR were inserted throughout the soil medium in the bucket at a depth of 30 cm. The volume of water applied to soil to bring the soil back to field capacity was calculated based on the following equation by Hamouda et al. (2019),Soil water content was measured using the TDR and the Gravimetric technique. These two techniques measured different values of soil water content for the same soil type at the same field capacity. A correlation was used to establish a relationship between the TDR readings and the gravimetric reading to give a good reflection of Available Soil Water Content (AWC) for the two soils used for the experiment to maintain continuous and constant FC% for each treatment . The comFertilizer applications were performed twice during the planting season (on the 20 November onset of the vegetative stage) with 20-20-20 N-P-K water-soluble fertilizer, at a rate of 3.5 kg of N per hectare for both soils due to the high levels of NPK already available in the soil. This was changed to calcium nitrate after the first fruits were noticed and later changed to potassium nitrate during the fruits’ expansion stage. Organic pesticide (neem oil plus emulsifying soap) was applied in a ratio of 2:1 in 15 L of water biweekly. Weeds were handpicked from pots and woven polyethylene plastic mulch was used to control weeds on the greenhouse floor.® handheld non-contact infrared thermometer with an emissivity of 0.95 Wm−2 and a display resolution of 0.2 °C (0.5 °F). The thermometer was held 30 cm above the tomato leaf with the laser point set at an angle of 90° to the horizontal 1640 weren et al. , Chen etn et al.  and Huetn et al. . PRInormg et al. , Wong ang et al. , and Gamg et al. . However1640, PRI570, and PRInorm were the most sensitive indices for detecting water stress within all the growth stages. This implies that spectra reflectance from 531 to 1640 nm band depth is ideal for estimating water stress in tomato growth in sandy loam and silty loam soils in a greenhouse. This technique is capable of timely and accurately estimating actual crop water status from leaves to scheduling irrigation . This technique is also unique in determining the onset of water stress in crops grown in sandy loam and silty loam soils. In this study, these VIs have accurately estimated water stress in tomato growth in sandy loam and silty loam soils in a greenhouse even though the relationships between water stress indicators and the selected indexes were high. Further studies on tomato growth in different agroecological zones need to be conducted. This was beyond the scope of this study.This study assessed very high-resolution narrow-band hyperspectral thermal imagery acquired from tomato growth using ASD SPEC 4 in an experiment comprising four water stress treatments  in sandy loam and silty loam soils for crop water stress assessment. However, the use of reflectance indices for sensing the effects of water stress in tomato plants at different growth stages in sandy loam and silty loam soils revealed that plants grown in each of these soils have different spectral signatures at the different growth stages but displayed similar trends. Quantifying crop leaf water status with remote sensing from the spatial and temporal resolutions it provides imparts highly valued information for making good irrigation management decisions towards optimizing crop productivity. Testing different spectral reflectance indices between 531 to 2130 nm to estimate water stress in tomato growth by comparing various reflectance indices and their relationship with water stress indicators showed that the NDVI, RDVI, WI, NDWI, NDWI


Measurements of allergen-specific IgE antibodies with different manufacturers’ assays show modest or poor agreement. This study compares analytical performance of specific IgE tests for whole allergen extracts and individual allergen components of two assay systems, IMMULITE and ImmunoCAP, using human sera as well as monoclonal antibodies.Comparisons were performed for specific IgE to house dust mite , egg white  and the allergen components rDer p 1, rDer p 2, nGal d 1, nGal d 2 and nGal d 4 in human sera and with monoclonal mouse/human chimeric IgE antibodies specific for the same allergen components. Competitive interference with IgE measurement was investigated using allergen-specific monoclonal IgG and IgG4 antibodies.Measurements of IgE to HDM and EW in serial dilutions of human sera revealed weaker dilution linearity with IMMULITE than with ImmunoCAP. Analysis of five different monoclonal IgE antibodies with total and specific IgE assays, expected to return similar levels, gave an average specific/total IgE ratio of 0.96 (range 0.71–1.14) with ImmunoCAP and 1.89 (range 0.76–2.85) with IMMULITE, indicating overestimation of specific IgE by IMMULITE. With the EW IgE tests of both assay systems, measurements of a chimeric anti-Gal d 2 IgE antibody were unaffected by a competing mouse IgG antibody. While the same was true for measurement of a chimeric anti-Der p 1 IgE antibody using the HDM test in ImmunoCAP, a suppression of measured concentrations by up to 42% was observed in IMMULITE. Similarly, measurement of HDM-specific IgE in human sera by ImmunoCAP was unaffected by a competing monoclonal anti-Der p 2 IgG4 antibody while IMMULITE displayed a reduction of HDM-specific IgE values by up to 30%.In this evaluation of analytical performance of two widely used assay systems, ImmunoCAP showed higher accuracy in quantitation of specific IgE and greater resistance against competing allergen-specific non-IgE antibodies which may arise through natural or dietary exposure, or as a result of allergen immunotherapy treatment. House dust mite (HDM) allergy has a significant clinical effect on lung function in children . Among pAs far as food allergy is concerned, egg white is one of the most common causes in children. Prevalence figures of 0.5–2.5% have been reported  and its A/L (ImmunoCAP) failed an egg OFC while 95% of those with a level less than 0.9 kUA/L passed an egg OFC. Similar results were reported by Ando et al. [A/L and 1.6 kUA/L, respectively. Thus, when measuring Gal d 1-specific IgE antibodies, high test precision is clinically important, not only in the high concentration region but also at low concentrations. In addition to Gal d 1, there are other important EW components such as ovalbumin  and lysozyme .Allergic reactions can occur either to any form of egg or, in some patients, mainly to raw or lightly cooked / baked egg , 14. Clio et al. , defininIt is of utmost importance in sIgE testing that measurements are truly specific and accurate. Currently, there are three commercially available specific IgE immunoassay platforms in Japan, of which IMMULITE® 2000  and ImmunoCAP™  are dominant. Both platforms provide analysis of IgE to a wide range of allergens, including crude HDM and EW extracts and the HDM and EW allergen components Der p 1 and Der p 2 and Gal d 1, Gal d 2 and Gal d 4, respectively. Earlier comparisons of the two systems have revealed both significant differences in test results as well as similar performances –19. The Gradually declining levels of egg specific IgE and milder symptoms on accidental exposure or in food challenge are signs of tolerance development. At the age of 16 years, around 70% of egg allergic children will have developed tolerance .Forty-four HDM-positive and 36 EW-positive serum samples were included in the study. For the EW, samples were selected from a patient pool where all patients had been diagnosed as having egg allergy while for the HDM, samples were selected from sensitized patients but with no access to their clinical diagnosis. Of each sample a series of three dilutions  was prepared using the ImmunoCAP IgE/ECP/Tryptase Sample Diluent. Hence, including undiluted specimens, all samples were available at four different concentrations for the assay comparisons. In a dilution linearity study, the observed (O) IgE concentration measured in each sample dilution point was evaluated against the expected (E) value derived from the measured IgE concentration in the undiluted sample, divided by the dilution factor, as an O/E ratio. For each of HDM and EW, an average O/E ratio was calculated from the O/E ratios of all dilution measurements. Weighted linear regression lines were fitted to the data with either ImmunoCAP or IMMULITE ratios as the dependent variables and ImmunoCAP / IMMULITE initial sIgE values as the independent variables.Five allergen-specific mouse-human chimeric monoclonal IgE antibodies were also employed in the evaluation, each consisting of an allergen-specific variable domain from a mouse monoclonal antibody and the constant part of the heavy chain of human IgE. The antibodies reacted specifically with Der p 1, Der p 2, Gal d 1, Gal d 2 and Gal d 4, respectively, and could be measured with the specific and total IgE assays of both assay platforms. Furthermore, for each Ab a 6-step dilution series was prepared, up to a dilution factor of 16. Hence, samples with seven different concentrations of each Ab were available for the assay comparisons. The chimeric IgE antibodies to Der p 1, Gal d 1, Gal d 2 and Gal d 4 were obtained from Thermo Fisher Scientific  while the chimeric IgE antibody against Der p 2 was obtained from Indoor Biotechnologies .Competition experiments were carried out using mouse monoclonal IgG antibodies against Der p 1 and Gal d 2 (Thermo Fisher Scientific) and a mouse/human IgG4 antibody against Der p 2 (Indoor Biotechnologies). Each mouse monoclonal antibody was added at three different concentrations  to the anti-Der p 1 or anti-Gal d 2 chimeric IgE antibody samples. Analysis was performed with specific IgE assays for HDM and EW, respectively. The results were evaluated as ratios to a control to which buffer alone had been added instead of the competing IgG antibody.A/L, respectively), followed by measurement of HDM-specific IgE. The results were evaluated as ratios to a control to which buffer alone had been added. The concentration of Der p 2 specific IgG4 antibody was determined using the ImmunoCAP specific IgG4 assay (Thermo Fisher Scientific). A total of 4 comparative studies were thus performed in this work, summarized in Table The anti-Der p 2 chimeric IgG4 antibody was added at three different concentrations  to two HDM-positive serum samples  kU/L for HDM and 58.9–481  kU/L for EW  and the EW sIgE values between 15.8 and 98.6  kUThe ImmunoCAP assays displayed dilution linearity with average O/E ratios of 1.21  and 1.26  for HDM and EW, respectively. The IMMULITE assays displayed dilution linearity with average O/E ratios of 1.29  and 1.44  for HDM and EW, respectively. Weighted linear regression lines were fitted to the data as shown in Fig. Compared with ImmunoCAP, IMMULITE HDM showed greater range of variation in the O/E results (p < 0.001). As demonstrated, the IMMULITE HDM assay ratios increased with increasing levels of sIgE. For both ImmunoCAP and IMMULITE EW, the variation ranges in the O/E result were quite similar while the mean value of the O/E ratio was higher for IMMULITE EW (p < 0.001).In a sample of a monoclonal IgE antibody, where all IgE is represented by a single allergen specificity, measurements of total and specific IgE should yield comparable concentration values. With ImmunoCAP, the levels of sIgE to Der p 1, Der p 2, Gal d 2 and Gal d 4 measured in all 6 dilutions of the chimeric IgE antibodies were comparable to the total IgE levels measured in the same antibody samples, with an average sIgE/tIgE ratio close to 1 (range 0.92–1.14)  in IMMULITE.A robust assay is characterized by its ability to deliver consistent results from specimens regardless of their content of potential interferents. One important cause of potential interference is allergen specific non-IgE antibodies which may be induced by natural allergen exposure or allergen immunotherapy (AIT) treatment. In this study, we assessed the resilience of the ImmunoCAP and IMMULITE HDM and EW IgE assays against such interference by competing with the increasing concentrations of monoclonal IgG antibodies of the same allergen specificity prior to IgE measurement. The results of this experiment are shown in Fig. While the above competition experiment using chimeric IgE antibodies addressed the IgE binding capacity of the assays in an analytical set-up, we also studied their ability to withstand competing antibodies in a situation more closely reflecting their clinical use. This was done by assessing the effect on HDM IgE measurements in patient samples by increasing concentrations of a chimeric Der p 2 specific IgG4 antibody. The results of this experiment are shown in Fig. In this study, the ability of the ImmunoCAP and IMMULITE IgE assay systems to accurately quantify allergen specific IgE antibodies was studied. Main findings were generally poor agreement between the two systems in the measurement of specific IgE levels to HDM and EW. This is especially true for the results in the linearity study where the effect in IMMULITE HDM of the serial dilutions on sIgE recovery was more variable compared to ImmunoCAP. Furthermore, for IMMULITE EW, mean sIgE results from the diluted samples was higher than for the results with ImmunoCAP (1.44 vs 1.29). In a perfect linearity dilution study, the mean O/E ratio should be 1.Differences in assay performance between the two systems were further investigated using chimeric IgE antibodies. In a sample where all IgE antibodies are represented by a single allergen specificity, measurements of total and specific IgE should yield comparable concentration values. Since a close agreement of total IgE concentrations measured with the IMMULITE and ImmunoCAP systems has earlier been demonstrated  and bothThe two interference studies performed showed that IgE measurements with the ImmunoCAP HDM and EW IgE assays were unaffected by the addition of the competing IgG or IgG4 antibodies, as was the IMMULITE EW IgE assay. In contrast, significant interference was observed with the IMMULITE HDM IgE assay, both when the chimeric anti-Der p 1 IgE antibody and human patient samples were measured in competition with anti-Der p 1 IgG and anti-Der p 2 IgG4, respectively, with a reduction of measured specific IgE values by up to 42%. The important implication of the observed interference is that levels of IgE to HDM will not be accurately measured in patients who have mounted an IgG response against HDM, such as during HDM immunotherapy.Taken together, this study demonstrates significant differences in performance of specific IgE assays between the two assay systems with respect to quantitative accuracy. Additionally, different resistance to interference by competing IgG antibodies was demonstrated which may affect IgE measurement in patients treated with allergen immunotherapy. The findings highlight that despite the use of the same concentration units of specific IgE and calibration against the WHO reference preparation of IgE, different test results for a patient sample may be obtained depending on the assay system used by the laboratory. As a consequence, different diagnostic conclusions may be drawn, affecting the management of the patient.A limitation in this study is the relatively small number of sera in the dilution linearity part, with 44 and 36 sera for the HDM and EW group, respectively. A study with a greater number of sera, spread evenly over the entire measuring range, may have provided for a more robust and representative comparison of the assay systems.This study demonstrates significant differences in analytical performance of two specific IgE assay systems for whole extracts and individual allergen components. The results indicate higher accuracy of quantitation of specific IgE and a higher resistance to competing allergen-specific non-IgE antibodies of ImmunoCAP as compared to IMMULITE.


Ventilation monitoring during sleep is performed by sleep test instrumentation that is uncomfortable for the patients due to the presence of the flowmeter. The objective of this study was to evaluate if an innovative type 3 wearable system, the X10X and X10Y, is able to correctly detect events of apnea and hypopnea and to classify the severity of sleep apnea without the use of a flowmeter.40 patients with sleep disordered breathing were analyzed by continuous and simultaneous recording of X10X and X10Y and another certified type 3 system, SOMNOtouch, used for comparison. Evaluation was performed in terms of quality of respiratory signals , duration and classification of apneas, as well as identification and duration of hypopneas.2 = 0.921; p<0.001) was found between the AHI indexes obtained from the two systems. X10X and X10Y devices were able to correctly classify 72.3% of the obstructive apneas, 81% of the central apneas, 61.3% of the hypopneas, and 64.6% of the mixed apneas when compared to SOMNOtouch device.580 periods were evaluated. Mean quality assigned score was 3.37±1.42 and 3.25±1.35 for X10X and X10Y and SOMNOtouch, respectively. The agreement between the two systems was evaluated with grades 4 and 5 in 383 out of 580 cases. A high correlation (rThe X10X and X10Y devices are able to provide a correct grading of sleep respiratory disorders without the need of a nasal cannula for respiratory flow measurement and can be considered as a type 3 sleep test device for screening tests. There is growing evidence regarding the need for ventilation monitoring during sleep –5. IndeeAbnormalities of daytime ventilation, either at rest or during exercise, have also been reported albeit studied to a much lesser extent. Indeed, the ability to monitor ventilation during the daytime is limited and specifically, it is difficult to conceive prolonged ventilation measurements through any sort of flow meter during daytime activity –20. AccoIn a recent study from our group, we evaluated an innovative wearable device for ECG and respiratory Holter monitoring, which allows noninvasive, continuous, simultaneous, prolonged and accurate monitoring of cardiorespiratory signals . HoweverThis is a cross-sectional study conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the ethics committee of the Centro Cardiologico Monzino, IRCCS, Milan, Italy (CCM589). Prior to participating in the study, all patients gave written, signed, and informed consent.Study inclusion criteria were: age 18–90 years, presence of cardiovascular disease requiring hospitalization and previous diagnosis of moderate to severe sleep apnea/hypopnea assessed by standard polysomnography. Patients using continuous oxygen therapy or with neuromuscular diseases were excluded. All subjects who had access to the Centro Cardiologico IRCCS severe heart failure outpatient unit (between May 2018 and January 2019) and meet the study inclusion/exclusion criteria were asked to participate to the present study.X10X and X10Y medical devices  is composed of 10 ink-based dry electrodes to monitor ECG, three respiratory strain sensors, one inertial measurement unit (IMU) and a pulse oximeter  . For thi™ RESP device  is composed of a nasal cannula, a pulse oximeter, two respiratory sensors that are positioned at the level of the manubrium and abdomen, and three thoracic electrodes for ECG recording.The SOMNOtouch™ RESP) in the early afternoon, but data acquisition, of both devices was therefore simultaneous and began according to the patients’ sleep habits . The SOMNOtouch™ RESP nasal cannula and the finger probes of both instruments were checked, and patients were instructed to wear these devices by themselves just before going to sleep.The protocol consisted of a continuous and simultaneous recording of traces from the two devices. All patients wore the SOMNOtouchTM RESP bands and the X10X-Y device in the laboratory at Monzino Hospital where they received practical instructions and where the functioning of the devices was tested by expert medical personnel. After the patients wore the X10X-Y device, signal quality was checked. Thereafter, the SOMNOtouchTM RESP bands were positioned onto the X10X-Y device and all signals were checked again. Only when both devices were judged to provide good quality signals, patients were sent to their home. All recruited patients wore the two devices  were analyzed qualitatively by two experts, and in the case of different scores, they were averaged. More specifically, every 30 minutes, an interval of 8 minutes was analyzed by giving grades that ranged from 1 to 5, i.e. 1 = poor signal and difficult interpretation; 2 = insufficient signal and questionable interpretation; 3 = sufficient signal and reliable interpretation; 4 = good signal and reliable interpretation; 5 = excellent signal and reliable interpretation. An analogous 1 to 5 grading was carried out in order to describe the level of agreement between the quality of SOMNOtouch™ RESP and X10X-Y respiratory sum signals. A set of figures reporting examples of grade 1 to 5 is reported in the supplemental file material as Analysis was divided into two parts. The first analysis was a qualitative one. Respiratory signals from SOMNOtouch™ RESP were automatically processed by SOMNOMedics software for detection of sleep respiratory disorders. Respiratory event scoring was then manually confirmed according to the most recent guidelines [The second analysis, also performed by two expert operators, was a quantitative one. The data derived from SOMNOtouchidelines , consideevent: minimum duration of 10 seconds and ≥3% oxygen desaturation ;hypopnea: signal excursion drop by ≥30% with respect to the previous minute;central apnea: signal excursion drop by ≥80% with respect the previous minute;obstructive apnea: signal excursion drop by ≥50% with respect to the previous minute, in presence of thoraco-abdominal asynchrony (phase angle > 36 degrees);mixed apnea: signal excursion drop by ≥50% with respect to the previous minute, with an initial phase without efforts followed by a second phase with efforts .After automatic analysis performed by the software, respiratory event scoring was successively manually confirmed according to the most recent guidelines . In detaExamples of signals recorded by the two devices during events of hypopnea, central, obstructive and mixed apneas are shown in Figs ™ RESP and X10X-Y devices were compared and classified as true positive , false positive , and false negative .Apnea-hypopnea index (AHI) was graded as mild (≥5 and <15 events/hour), moderate (≥15 and <30) and severe (≥30) . After tData normality was verified using the Shapiro-Wilk test. Metrics of sensitivity (SE) and positive predictivity (+P) were used to summarize the results of comparison as follows:™ RESP and X10X-Y were evaluated using Bland-Altman plot, and the results were presented as bias with limits of agreement (±1.96 STD). In order to observe if the limits were acceptable, the 95% confidence interval was calculated for both bias and limits of agreement according to Giavarina [2) were calculated.A 5x5 confusion matrix was used to cross-validate the detected events during sleep  between devices. Agreement regarding AHI between SOMNOtouchiavarina . CorrelaData analysis was performed using Python scipy library (submodule stats) version 1.2.1. A p-value of <0.05 (2-sided) was considered statistically significant.2 recording (3 patients), the refusal to perform the exams (2 patients) and the failure of the acquisition (1 patient) . Median age was 69 years . Of these, 6 patients were excluded for different reasons: the lack of SpOpatient) . Demograpatient) .™ RESP, respectively. Higher values of quality assessment   and a good agreement (mean bias -0.77±4.39) were found between devices.All included patients AHI were graded as mild, moderate and severe with X10X-Y in 10, 9, and 12 patients, respectively, while SOMNOtouchpatients . Apnea/h™ RESP device . We showed that X10X-Y are able to provide during sleep reliable respiratory signals, reliable classification of patients apnea severity and accurate classification of apnea/hypopnea events.Our study compared a new wearable device for the simultaneous recording of multiple cardiorespiratory signals (X10X-Y devices) to an approved type 3 recording sleep device, a well-established, and widely used nocturnal cardiorespiratory monitoring system in the clinical field  severity disease estimation differed between the two devices and in all cases, the difference was of only one class of severity. Notably in all but one patient with severe apnea/hypopnea as well as in all patients with no sleep abnormalities X10X-Y properly detected the sleep severity of the sleep disorder.As for quantitative analysis, despite the reported technical differences in the system operating features, a good correlation has been found in AHI determination between the two devices. Moreover, the severity grading for respiratory disorders showed a very high agreement between the SOMNOtouch™ RESP event detection, was obtained in the absence of a direct respiratory airflow recorded by a nasal cannula [It is noteworthy that the correct detection of sleep apneas/hypopneas severity with X10X-Y devices, as validated by agreement with SOMNOtouch cannula ,18,22. A cannula ,27.We must recognize, however, that the agreement between the two devices drops if respiratory event classification is taken into account. A good accuracy is still present for central and obstructive apneas, but both sensitivity and positive predictive values fall below 50% for hypopneas. This is not surprising if the operating differences between the two recording systems is considered, as the absence of direct respiratory flow measurement becomes, in this case, a major limitation. Indeed, on the basis of the only respiratory movement analysis, discrimination between an obstructive apnea and a hypopnea substantially depends on the presence of thoraco-abdominal asynchrony, because events with a counter-phase angle close to the cut off value could be easily misdiagnosed.™ RESP were not assessed in the present study. This is due to the simultaneous application of the two systems so that comfort analysis for each device was not possible. Secondly, daily respiratory recordings were not assessed because SOMNOtouch™ RESP is not designed for daily ambulatory monitoring. However in a recent study we analyzed X10X-Y devices continuously for 24 hours in 10 healthy subjects and 30 cardio respiratory patients and observed promising findings [A few study limitations should be acknowledged. Firstly, patients comfort scale while wearing X10X-Y and SOMNOtouchfindings . Thirdly™ RESP, even in the absence of direct measurement of the respiratory flow. At present, the X10X and X10Y devices can be considered as a type 3 sleep test device but it should be principally regarded as a screening test for the detection and quantification of sleep respiratory disorders, as well as a daily monitoring system for early detection, to reveal information that would have otherwise been overlooked. On the other hand, the unrequired need of a nasal cannula for respiratory flow measurement presents an excellent step forward for respiratory analysis during the daytime, and for everyday activities.In conclusion, the X10X and X10Y devices are a reliable, non-invasive device for recording sleep respiratory behavior, able to provide a correct grading of sleep respiratory disorders when compared to a widely used system for nocturnal cardiorespiratory monitoring like SOMNOtouchS1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.


To screen out the predictors of central cervical lymph node metastasis (CLNM) for papillary thyroid carcinoma (PTC) and establish a prediction model to guide the operation of PTC patients (cN0).V600E status, and other characteristics of the two groups, was analyzed and compared using univariate and multivariate logistic regression analyses, and the independent predictors were selected to construct a nomogram. The calibration plot, C-index, and decision curve analysis were used to assess the prediction model’s calibration, discrimination, and clinical usefulness.Data from 296 PTC patients (cN0) who underwent thyroid operation at the Second Affiliated Hospital of Chongqing Medical University were collected and retrospectively analyzed. They were divided into two groups in accordance with central CLNM or not. Their information, including ultrasound (US) features, BRAFA total of 37.8% (112/296) of PTC patients had central CLNM, and 62.2% (184/296) did not. The two groups were compared using a univariate logistic regression analysis, and there were no significant differences between the two groups in sex, aspect ratio, boundary, morphology, hypoechoic nodule, thyroid peroxidase antibody, or tumor location (P>0.05), and there were significant differences between age, tumor size, capsule contact, microcalcifications, blood flow signal, thyroglobulin antibodies (TgAb), and BRAF gene status (P<0.05). A multivariate logistic regression analysis was performed to further clarify the correlation of these indices. However, only tumor size , microcalcifications  and TgAb  were independent predictors of central CLNM and were incorporated and used to construct the prediction nomogram. The model had good discrimination with a C-index of 0.715. An ROC curve analysis was performed to evaluate the accuracy of this model. The decision curve analysis showed that the model was clinically useful when intervention was decided in the threshold range of 16% to 80%.In conclusion, three independent predictors of central CLNM, including tumor size (> 1.0 cm), US features , and TgAb (positive), were screened out. A visualized nomogram model was established based on the three predictors in this study, which could be used as a basis of central cervical lymph node dissection (CLND) for PTC patients (cN0). Papillary thyroid carcinoma (PTC) is the most common endocrine carcinoma. Over the past 30 years, the morbidity of thyroid carcinoma has doubled or more in many countries, including China . Most of131 therapy, and thyroid-stimulating hormone (TSH) suppressive therapy  diagnosis for PTC patients with central cervical lymph node metastasis were 35.3%, 88.6%, 83.2%, 47.4%, and 56.6%, respectively . However, the results were inconsistent. Age is important for PTC staging. Traditionally, patients over 45 years of age have a slightly worse prognosis and an increased recurrence rate , 14. SomV600E mutation tests in addition to history taking. The exclusion criteria were as follows: (1) patients with other pathological types of thyroid cancer, such as medullary carcinoma and follicular carcinoma; (2) patients with other concomitant malignancies, such as breast cancer and nasopharyngeal carcinoma; (3) patients with multiple nodules where at least two of them were diagnosed as PTC; and (4) patients with incomplete medical data. Based on the inclusion and exclusion criteria, all 296 patients included in this study were diagnosed with PTC by fine-needle aspiration biopsy (FNAB) and BRAFV600E mutation analysis prior to surgery between January 2019 and July 2020.All patients were examined and treated at the Second Affiliated Hospital of Chongqing Medical University . The inclusion criteria were as follows: (1) patients who underwent primary thyroid surgery and central CLND; (2) patients with histopathologically confirmed PTC; and (3) patients who underwent preoperative thyroid function tests without history of thyroxine treatment , US, and BRAFPrior to FNAB, nodules were examined with color Doppler US , showing US features such as nodule size , boundary (clear/unclear), shape (regular/irregular), internal echo, aspect ratio (≤1/>1), capsular contact (yes/no), microcalcifications (yes/no) and blood flow (poor/rich).V600E mutation analysis.While the patients were in a supine position with their backs raised and their heads tilted back, US was used to determine the insertion site, angle, and depth. The probe was fixed, with the puncture point on one side of the probe, and the puncture route was set at 30–40° to the long axis of the probe surface  was mixed with 0.4 µl Taq enzyme and later dispensed into a PCR tube (ice bath). Next, 5 µl DNA sample (2-5 ng), 5 µl positive control, and 5 µl negative control were added separately to each PCR tube. The PCR tubes were centrifuged at 2,000 × g for 1 min at room temperature, and a thermocycler  was used. The following thermocycling conditions were used for quantitative PCR: initial denaturation at 95°C for 5 min; 15 cycles at 95°C for 25 sec, 64°C for 20 sec and 72°C for 20 sec; and an additional 31 cycles at 93°C for 25 sec, 60°C for 35 sec and 72°C for 20 sec. Carboxyfluorescein (FAM) and hexachlorofluorescein (HEX) signals were detected at 60°C during the final set of cycling conditions. The 2t of DNA . If the protocol .During the operations, the nodules were sent for frozen sections  and confirmed as PTC. After rediagnosis, these patients underwent thyroid resection  and ipsilateral CLND , and the central lymph nodes were also sent for frozen sections to confirm central CLNM. If there was central CLNM, total thyroidectomy was performed. After the operations, the remaining specimens  were sent for paraffin sections  and finally confirmed as PTC and central CLNM.https://www.r-project.org/). A calibration curve was plotted to assess the calibration of the nomogram. Harrell’s C-index  was measured to quantify the discrimination of the nomogram through the training cohort and further confirmed through two internal validations. The training cohort was divided into two internal validation cohorts  by random sampling. A decision curve analysis was performed to determine the clinical usefulness of the nomogram by quantifying the net benefit at the different thresholds.The statistical analysis was performed using SPSS 26.0. A univariate logistic regression analysis was performed to determine the predictors of central CLNM, and the variables with statistical significance on the univariate analysis were analyzed by a multivariate logistic regression analysis  to indicate a significant difference. The prediction nomogram based on the multivariate logistic regression analysis results was developed using R software  were men, and 221 (74.7%) were women. Of the PTCs, 119 (40.2%) were in the left lobe, 162 (54.7%) were in the right lobe, and 9 (5.1%) were in the isthmus  between the CLNM group and the non-CLNM group. Meanwhile, sex, aspect ratio, boundary, morphology, a low echo, TPOAb, and tumor location were not significantly different between the two groups (P>0.05).The relationships between medical information and central CLNM are presented in The medical information (predictors) with statistical significance in the univariate analysis was analyzed using a multivariate logistic regression analysis. The results confirmed that the following predictors were independently correlated with central CLNM , US features , and TgAb (positive) are given in ’s good discrimination.The calibration curve of the risk nomogram used to predict the risk of central CLNM in PTC patients (cN0) showed good consistency  for the risk nomogram is presented in ’s prognosis. Despite this, prophylactic RCLND is still controversial. Guo et al. suggest that prophylactic RCLND at the time of initial surgery may have benefits, including avoiding the high risk of permanent hypoparathyroidism and RLN injury associated with reoperation  were associated with central CLNM in PTC according to the univariate analysis. In contrast, they were not identified as independent factors through the multivariate logistic analysis. Finally, tumor size (> 1.0 cm), US features , and TgAb (positive) were independent predictors for central CLNM according to the univariate and multivariate logistic regression analyses.Our study included 11 clinical and US characteristics, four hematological indices , and BRAFfor CLMN , 34, 35.Previous studies have shown that thyroid microcalcifications are associated with central CLNM in PTC , 36, 37,Tumor size has been considered an important predictor of central CLNM in PTC, but the thresholds are different. Chen et al. showed that tumor size ≤1 cm was a risk factor for central CLNM . HoweverV600E mutations in TgAb-positive patients than in TgAb-negative patients  was 85.29%, with a higher mutation incidence of 85.29% in the central CLNM group than in the non-CLNM group. However, it was not related to central CLNM according to the univariate and multivariate logistic analyses. Regarding its association with central CLNM, the findings to date have been inconsistent. Studies have shown that PTC combined with BRAFV600E mutation is prone to lymph node metastasis, capsule invasion, and recurrence . Internal validation in the cohort showed good discrimination and calibration. DCA showed that using this nomogram to predict central CLNM would be beneficial if the threshold probabilities ranged from 16% to 80%. The development of this prediction model will enable individualized prediction of CLNM in most PTC patients (cN0), helping surgeons achieve accurate CLND for maximum patient benefit. The clinical application of the nomogram is convenient with simple addition calculation. For example, the PTC patient shown in Our study also has some limitations. First, this was a retrospective study; selection bias is inevitable. Second, the number of PTC patients included in this study was small. In the future, we will accumulate more cases and perform more validation. Third, the US results are largely dependent on the diagnostic experience of the operator. Although experienced sonographers perform US, subjective factors may influence the data.In conclusion, this study screened the following three significant independent predictors for central CLNM in PTC patients (cN0): tumor size (> 1.0 cm), US features , and TgAb (positive). We also developed a prediction nomogram based on the three independent predictors. The quantitative risk assessment and prediction provided by the nomogram can help surgeons assess the status of central cervical lymph nodes preoperatively accurately, avoid insufficient or excessive treatment (such as unnecessary prophylactic CLND), and provide a new strategy for the management of PTC patients (cN0).The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.LY and XG designed the study and wrote the main manuscript text. XG and WL provided the study materials or patients and analyzed all data. JC and LY revised the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (grant no. 81701709), Chongqing Natural Science Foundation (grant no. cstc2020jcyj-msxmX2011), Science and Technology Bureau of Yuzhong District, Chongqing (grant no. 20200145), and Kuanren Talents Program of the Second Affiliated Hospital of Chongqing Medical University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Distraction osteogenesis using the Ilizarov external circular fixator has been applied in lower limb reconstructive surgery widely. The increasing ankle osteoarthritis (OA) progression and severity are often associated with the period of external fixator and the greater relative instability of the ankle joint, but few studies have quantified risk factors directly during this technique.The study was conducted on 236 patients who underwent bone transport surgery for tibias using the Ilizarov external circular fixator from 2008 to 2018. The cumulative incidence of ankle OA diagnoses in patients after the Ilizarov technique treatment was calculated and stratified by risk factors from preoperative and postoperative management. After the data were significant through the Mann-Whitney U test analyzed, odds ratios were calculated using logistic regression to describe factors associated with the OA diagnosis including gender, age, BMI, location of bone defect, diabetes, hypertension, osteoporosis, the history of metal allergy and glucocorticoid intake, the American Orthopaedic Foot & Ankle Society (AOFAS) ankle-HF scale scores, defect size (DS), the type of bone transport, the bone union time, external fixator time (EFT), and external fixator index (EFI).P = 0.005), EFI > 50days/cm , age > 45years , osteoporosis , BMI > 25 . Male, BMI > 25, diabetes, osteoporosis, and AOFAS ankle-HF scale scores are the independent risk factors.There were 199 males and 37 females with a mean age of 47 years (range 28–59 years). Out of 236 patients, 49 had an additional treatment for ankle OA after the Ilizarov technique treatment of bone defects . The incidence of postoperative ankle OA was 20.8 %, with 19 patients classified as K&L grade 3 and seven patients as grade 4. The top five risk factors included double-level bone transport (OR3.79, Ilizarov external circular fixator is a safe and effective method of treatment for critical bone defects. The double level bone transport, EFI > 50days/cm, age > 45years, osteoporosis, BMI > 25 are the top five relevant risk factors of ankle OA. The probability of developing ankle OA among patients having three or more risk factors is 50–70 %. Ilizarov external circular fixator has been an established technique for treating critical bone defects (> 4 cm) caused by trauma, tumor resection, and osteomyelitis debridement widely –7. HowevDespite joint injury is a critical reason for the onset of ankle OA, a way to prevent its progression following these injuries remains elusive. Based on historical consensus, the diagnosis of ankle OA can be based on X-ray characteristics, and the Kellgren and Lawrence (K&L) scale is the accepted reference standard selected by the World Health Organization . Via recSome authors who reported ankle OA rates considered them mostly as an internal fixator complication, with no vital impact on external fixator treatment outcomes , 14–18. This study was a retrospective assessment of 236 patients treated by the same surgery team for bone defects of the tibia between 2008 and 2018. The inclusion criteria were bone defects of the tibia at least 4 cm managed definitively using an external circular fixator of the Ilizarov technique, at least 2 years after treatment ends, observer access to all medical records, and radiological images relating to the patients’ treatment. Other patients older than 60 years, with a poor compliance or any other diseases that can postpone bone healing  were excluded. 199 men and 37 women were assessed in this study according to the inclusion criteria. Bone defects were caused by trauma in 87 cases, tumor resection in 10 cases, and osteomyelitis debridement in 139 cases. Bone defects between 4.5 and 6 cm were managed with single-level bone transport,and more than 6.0 cm with double-level bone transport. The study was conducted by the Declaration of Helsinki, approved by our hospital institutional review board. The analyzed medical and radiological data came from hospital records.At the prior stage of bone transport, all necrotic and infected bone and soft tissues are removed from the hardware completely, debrided thoroughly, and/or implanted with antibiotic-impregnated cement spacers to improve stability. The bacterial culture and antibiotic sensitivity test were conducted in exudation, to instruct the surgeon to apply appropriate postoperative antibiotics. Local tissue flaps or direct sutures were performed to reconstruct small soft tissue defects without tension, yet flap transfers or free skin grafts were applied to cover larger defects.At the bone transport surgery stage, the Ilizarov device with a hinge movement system and 3 or 4 rings was constructed to simplify the operation before the operation, depending on the calculated angle deformity and limb length difference. The patients were performed under general anesthesia and placed in a supine position on the radiolucent operating table. The Kirschner wire  and a half-needle  were used to place the ring perpendicular to the long axis of the proximal and distal segments of the bone defect, and the X-ray was conducted to confirm their position. The distance was maintained up to 1.5-2.0 cm between the inner edge of each ring and the skin, and keep a distance of about 5 cm between the osteotomy site and the adjacent ring. Besides, two or three crossed olive wires  parallel to the joint line to fix each ring near the knee joint or ankle joint. When the distal bone fragment less than 2.0 cm, the ring adjacent to the ankle should be fixed at the level of the calcaneus or talus to provide sufficient stability for the whole frame. Simultaneously, excessive compression of the ankle joint surface should be avoided as much as possible to prevent further degeneration of the cartilage on the joint surface even if the joint was fused. One or more metal spicules were then inserted to ensure that the device has sufficient stability. Two metal pins were inserted so perpendicular to the mechanical axis of the tibia to fix the two rings near the osteotomy. Further, the required retraction of the proximal or distal skin was obtained to avoid skin tension and ensure the correction of the deformity and bone transport. Additionally, two hinges and a motor unit were adjusted to place on the bisector of the deformed convexity, and the bisector divides the true angle of the deformity into two equal halves, which was to ensure that the deformity correction period was normal. Last, a percutaneous osteotomy was performed on the fibula at the apex of the deformity using minimally invasive techniques.2)), location of bone defect , comorbidities  and the history of metal allergy and glucocorticoid intake. Patients’ preoperative conditions such as the duration of disease, the American Orthopaedic Foot & Ankle Society (AOFAS) ankle-HF scale scores, defect size (DS), the type of bone transport (single level and double level) were documented as well.The basic demographics were obtained by standardized self-administered questionnaires at the beginning of in-patient treatment and complemented by extracting relevant data from hospital records, included: age, gender, body mass index (BMI = weight (kg) /height , and external fixator index (EFI) was recorded. EFT referred to the time spent before removing the external fixator. EFI is defined as the ratio of EFT days to the distraction regenerate length (cm). Imaging evaluations were performed every 2 weeks during the distraction phase and monthly during the consolidation phase. All patients were followed up closely for at least 2 years after the external fixator was removed.2, overweight = BMI > 25 kg/m2), location of bone defect , comorbidities such as diabetes, hypertension, and osteoporosis (yes or no), the history of metal allergy (yes or no), glucocorticoid intake (yes or no), and the type of bone transport (single level and double level) were attributed to the categorical variables.Continuous variables included age, defect size (DS), the duration of hospitalization, the AOFAS ankle-HF scale scores, the duration of the bone union, external fixator time (EFT/ET), and external fixator index (EFI). And gender, body mass index  exercises within pain tolerance on the first day after surgery. Besides, standing and walk for at least 2 h per day was recommended, and switch from partial weighting to full weight-bearing as soon as possible. According to the results of exudation culture and antibiotic sensitivity test, select sensitive antibiotics for intravenous application for at least 3 weeks or until the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels return to normal.Slow and stable distraction was commenced on postoperative day 8 with the rate of 0.25 mm per day at the apex of the bone defect and continued in 3 or 4 equal increments until the required angle correction was reached. Subsequently, the distraction rate was maintained at 0.25 mm per day until the bone connection was reached. X-rays of the local bones and the entire length of the lower limb were conducted regularly to evaluate the degree of correction, distraction, and consolidation. Consolidation was considered sufficient when 3 of the 4 visible cortexes in the anteroposterior and lateral radiographs were visible bridging callus formation and the tenderness at the osteotomy site and pain during weight-bearing without connecting rods. After confirming the consolidation, the external circular frame was removed under local anesthesia in the outpatient clinic.All radiographs were evaluated by at least two senior orthopedic doctors and two residents. The radiographs and clinical symptoms were then diagnosed as ankle OA correctly, based on the original version of the K&L scale Table . AdditioStatistical analysis was performed with the SPSS 23.0. Continuous variables, such as age, weight, and height (BMI = weight (kg) /height (m2)), defect size (DS), the duration of hospitalization, the AOFAS ankle-HF scale scores, the duration of the bone union, external fixator time (EFT/ET), and external fixator index (EFI), were analyzed by independent-samples T-tests and expressed as the mean and standard deviation. And the categorical variables included sex, location of bone defect , comorbidities  and the history of metal allergy and steroid hormone intake and the type of bone transport (single level and double level), were analyzed by the Mann-Whitney U test, expressing as the number. Besides, comparing clinical scores in patients with K&L grades < 2 group vs. K&L grades > 2 group to evaluate clinical significance. A statistically significant difference was set at P < 0.05.The variable with a P value of 0.05 or less in the Mann-Whitney U test or T-test was entered to in the multivariate logistic regression model the relationship between the explanatory variable and the ankle OA and control the potential confusion of any included variables. The odd ratio provides a 95 % confidence interval and P value. A P value of less than 0.05 was considered statistically significant. The cumulative number of risk factors was determined for each patient (possible range from 0 to 5) and evaluated the incidence of ankle OA.There were 236 bone defects in 236 patients corresponding to the inclusion criteria with a mean defect size of 5.9 cm (4.1–7.2 cm) and operated upon during the research period. Of these patients, no one died, refused, or did not participate in the follow-up. Out of 236 patients, 49 had an additional surgery for ankle OA after the Ilizarov technique treatment for bone defects  between May 2008 and January 2018. The incidence of postoperative ankle OA was 20.8 %, with nineteen patients classified as K&L grade 3 and seven patients as grade 4. In detail, subtalar joint arthritis in 17 cases (34.6 %), talonavicular joint arthritis in 12 cases (24.4 %), and calcaneocuboid joint arthritis in 18 cases (36.7 %). Fortunately, ankle fusion was achieved in all patients with ankle OA after additional correction surgery, in both clinical and radiographic criteria. Pin tract infection occurred in 18 cases, delayed union on docking site was presented in 7 cases, axial deviation appeared in 12 cases, poor regenerate consolidation was observed in 1 case, and none refracture on docking site after fixator removal occurred. Eighteen pin tract infections were resolved by local saline washes, occlusive dressings, and oral antibiotic therapy. Twelve axial deviations were corrected by adjusting the external fixator manually. Seven delayed unions on docking sites and one poor regenerate consolidation were recovered through surgical treatment of autologous bone grafts.Comparison of the demographic data between those patients seen at follow-up Table  revealedRegarding the accessorial outcome of ankle OA of the ankle, 9 patients 18.3 %) presented with an AOFAS ankle-HF scale score < 50 and a K&L grade 3–4. Additionally, the incidence of ankle OA per individual per risk factor was presented ,delayed union on docking site7 cases), axial deviation12 cases) and poor regenerate consolidation(1 case), which are consistent with the complications related to the Ilizarov technique described by Liu et al. . These c cases, a cases anA multivariate analysis was conducted among the baseline data of ankle OA patients and the variables with statistical significance were analyzed by logistic regression analysis. Subsequently, age > 45years, male, BMI > 25, double level bone transport, diabetes, hypertension, osteoporosis, glucocorticoid intake, duration of disease > 24 months, EFI > 50days/cm, AOFAS ankle-HF scale scores < 50 were associated with a greatly increased risk of ankle OA as defined by the K&L scale. The incidence of ankle OA was 50–70 % in the existence of three or more risk factors. With the help of meticulous multiple logistic regression statistical analysis, independent risk factors for the development of ankle OA were associated with the BMI > 25, male, diabetes, osteoporosis, AOFAS ankle-HF scale scores < 50.Despite the changes and clinical symptoms of osteoarthritis assessed by radiography were controversial , a substObesity (BMI > 25) had been certified as an independent risk factor for OA in previous studies . In our The increasing prevalence of OA with age may be the result of cumulative exposure to various risk factors and biological changes , 27, 33.The published researches had revealed that the double level bone transport shortened the EFT and EFI greatly in the comparison of the single level bone transport , 4, 34. However, although mechanical overload was assuredly a risk factor in the development of ankle OA, it was insufficient on its own to explain why degradation of cartilage also occurred in non-overload affected areas of cartilage. For instance, osteoporosis  and chronic disease subjects, such as diabetes  and hypertension , seemed to sustain more severe types of ankle OA in our study. The majority of patients with osteoporosis in our cohort could be explained by the mechanism of bone defects caused by long-term post-traumatic osteomyelitis (> 24 months), which had been treated for sufficient time before bone transport was initiated. This may make a mess of the microenvironment for bone regeneration and soft tissue coverage, which then delayed docking site union and bone mineralization. In Hannah et al. researchAOFAS ankle-HF Scale scores had been reported to have a greater capacity for diagnosing the degeneration of ankle function , 38. LikFurther, the relationship was also observed between the number of risk factors and risk categories according to logistic regression analysis based on the scale scores. In the presence of 3 or more risk factors, the risk of developing ankle OA appears to increase notably and continued to rise with the number of factors Fig. . More thYet, certain limitations existed in our study. Firstly, given its retrospective nature, small sample size, and exclude the patients older than 60 years, caution should be taken regarding the interpretation of our analytical results. The actual rate of ankle OA could be higher (some ankle insidious pain may not be brought to the attention of patients). Secondly, insufficient postoperative management instruction could give rise to higher numbers of ankle OA due to the complex nature of the postoperative rehabilitation of the Ilizarov technique, and more confounding factors coming into play. Thirdly, to get control of the limitations of our study, further studies with larger sample size, longer follow-up period, and multicenter design should be designed.The study we performed for demonstrating the quantification of risk factors for ankle OA and their role allows one to determine the prognosis of a disease/outcome, which is important for both clinicians and patients. Almost 20.8 % of patients presenting within an average of 2.1 years following Ilizarov technique treatment using an external circular fixator are diagnosed with ankle OA. The double level bone transport, EFI > 50days/cm, age > 45years, osteoporosis, BMI > 25 are the top five relevant risk factors of ankle OA. Male, BMI > 25, diabetes, osteoporosis are the independent risk factors. The risk of ankle OA seems to continue to increase as the number of factors increases. Clinicians should be aware of these risk factors to figure out the patient’s risk condition for collaboration with a view to receiving favorable long-term results after the treatment.


A reliable and accurate estimate of the percentage and distribution of adipose tissue in the human body is essential for evaluating the risk of developing chronic and noncommunicable diseases. A precise and differentiated method, which at the same time is fast, noninvasive, and straightforward to perform, would, therefore, be desirable. We sought a new approach to this research area by linking a person’s relative body fat with their body surface’s areal roughness characteristics.For this feasibility study, we compared areal surface roughness characteristics, assessed from 3D photonic full-body scans of 76 Swiss young men, and compared the results with body impedance-based estimates of relative body fat. We developed an innovative method for characterizing the areal surface roughness distribution of a person’s entire body, in a similar approach as it is currently used in geoscience or material science applications. We then performed a statistical analysis using different linear and stepwise regression models.R2 = 0.55, p < 0.0001). The best results were achieved by calculating the arithmetic mean height, capable of explaining up to three-quarters of the variance in relative body fat .In a stepwise regression analysis of areal surface roughness frequency tables, a combination of standard deviation, interquartile range, and mode showed the best association with relative body fat (This study shows that areal surface roughness characteristics assessed from 3D photonic whole-body scans associate well with relative body fat, therefore representing a viable new approach to improve current 3D scanner-based methods for determining body composition and obesity-associated health risks. Further investigations may validate our method with other data or provide a more detailed understanding of the relation between the body’s areal surface characteristics and adipose tissue distribution by including larger and more diverse populations or focusing on particular body segments. The use of 3D photonic body scans (BS) to assess body shape in epidemiologic studies and for daily fitness tracking is on the rise, thanks to developments in equipment and measurement methods that provide fast, reproducible, and increasingly accurate results –7. HowevIn order to reach a broad target population, measurement methods are needed, which provide a precise and differentiated assessment of %BF and, at the same time, remain straightforward for an application in daily clinical practice. Since this goal is still challenging to achieve, we sought a new approach to this research area by linking a person’s %BF with the roughness of the body surface. We recognize that the influence of the prognostically relevant visceral body fat on body surface characteristics may be somewhat limited. In contrast, the amount and distribution of subcutaneous adipose tissue, located between the musculoskeletal system and the skin, are likely to significantly influence body surface morphology and, thus, have a measurable impact on the areal surface roughness characteristics.To test our approach as a possible starting point for a more accurate assessment of %BF, particularly subcutaneous adipose tissue, we evaluated the roughness of 76 test subjects’ body surfaces and compared the results with BIA estimates of their %BF.3. Comparative %BF (relative body fat) estimates were assessed by BIA . In the present study (Armed Forces basic training), it was not feasible to perform more extensive or time-consuming examinations than BIA. However, the technique and, more specifically, the Seca mBCA 515 device have been validated in various studies and have been widely used for benchmarking body composition measurements derived from photonic 3D BS [3D models used for our study were taken from a prior study in which the correlation of height and WC measured by BS and manual anthropometric measurements in young Swiss men was investigated , 17. Thiic 3D BS , 19. In ic 3D BS . WC was ic 3D BS , 17, 21.A sphere with a user-defined radius is fitted around the point.A best-fitting plane through all the points included in this sphere is determined.The distance between the point (center of the sphere) and the best-fitting plane is calculated .For our study, we used data from the cross-sectional part, for which 104 young male Swiss Armed Forces recruits were examined on the same day, using the same measuring protocol and equipment. We imported available 3D models of 104 study subjects into a 3D modeling software  to calculate total point count, volume, and surface area of each model. To analyze the surfaces of the 3D BS, in a similar approach as currently used for geoscience, material science, and further surface metrology-related applications , we usedFrom these values CloudCompare then creates an areal roughness histogram containing 256 classes, Class 1 being smooth  and Class 256 being rough (largest distances). We excluded Class 1 from statistical evaluations since it typically represented artificial flat surfaces of the 3D model, such as the foot sole. Also, since the absolute number of points contained in the point cloud models of our participants varied, absolute histogram values (number of points), calculated by CloudCompare, had to be divided by the total number of points contained within a particular point cloud, to obtain relative frequency tables and to be able to compare different 3D models statistically.After a visual assessment Fig. , we defiDescriptive statistics , minimum, maximum) of the basic parameters  were calculated for all included study subjects.https://www.r-project.org). In a first analysis step, we verified our initial assumption that the distribution of areal surface roughness levels across the 256 classes in the frequency tables is associated with %BF. For this, we computed different linear regression models (M1–M6) by the measures of statistical dispersion—SD and interquartile range (IQR)—for all three radius values . After a visual assessment, we also assumed an association between the mode, defined as the class with the highest peak, and %BF. Therefore, further linear regression models, including the mode (M7–M9), were computed. Linear regressions have proved suitable for initial evaluations of associations between 3D photonic body scan measurements and body composition  [For further statistical analysis, the software R was used  , 21.In a second analysis step, to find the best association with %BF, we then combined SD, IQR, and mode in stepwise regression models for each radius (M10–M12). To investigate the importance of each explanatory variable  for %BF, we separately excluded each explanatory variable from the model and calculated the Akaike’s information criterion (AIC). Next, we calculated the difference between the AIC when excluding an explanatory variable and the AIC of the complete model. Therefore, the significance of the explanatory variable in the model corresponds to the differences of the respective AICs.In a third and more explorative analysis step, to find an association between the overall roughness of each participant and %BF, we also calculated the arithmetic mean height (Sa), a quantitative roughness parameter (in mm), as defined by ISO 25178, frequently used in surface metrology. Sa is the extension of the concept of the arithmetic mean height of a line (Ra) to a surface. It expresses the average distance of any point of a surface to an arithmetic mean of that surface. For our application, we determined Sa by dividing the sum of all roughness values, as calculated for each point, by the total number of points in the 3D model. Therefore, Sa allows the comparison between participants, despite the point clouds containing different numbers of points, with normalization being part of the measure. Finally, linear regression models for the association between Sa and %BF (M13–M15) were also computed.N = 76) included in our study are displayed in Table 2 (±3.5 kg/m2), with 28% having excess weight (BMI ≥ 25 kg/m2), and 14% of those being obese (BMI ≥ 30 kg/m2) according to the WHO obesity classification . According to BIA estimates, average relative fat mass (%BF) was 14.6% (±7.7%), and absolute fat mass was 11.7 kg (±8.0 kg). Linear regressions showed that BMI was able to explain a high share of the variability in %BF , the same applied for WC .Descriptive statistics about the young men . All associations were negative (as expected). In our second analysis step, M12  showed the best association with %BF among the stepwise regression models M10–M12 presented in Table R2 = 0.69, p < 0.0001) than the models with single parameters (M1–M9). The strongest contributions were coming from mode (AIC = 29.11), followed by IQR (AIC = 45.90). In our more explorative third analysis step . All reported R2 are adjusted R2.In our first analysis step, we assessed the areal surface roughness frequency tables by linear regressions, using the basic measures of statistical dispersion SD, IQR, and mode for the estimation of %BF. Among all basic models, M1–M9  with %BF was observed for Sa (R2 = 0.74), the arithmetic mean of roughness, which compared to other association studies of individual scanner-based measurements  with aspects of body composition, and using comparable samples [Our feasibility study aimed at providing a proof of concept that areal body surface roughness characteristics may be used as a proxy for %BF. To the best of our knowledge, it is the first study analyzing the association between the areal roughness of the body’s surface and body composition. While the individual parameters of statistical dispersion  in the calculated frequency tables only showed moderate associations with %BF, a stepwise regression analysis, considering multiple parameters, showed stronger associations. The best association  may be required to fully understand the relationship between specific body surface parameters and underlying tissue composition.In future investigations, we plan to focus on longitudinal intraindividual observations. In follow-up measurements, we shall, therefore, check whether changes in overall areal surface roughness coincide with changes in %BF. Depending on individual parameters, such as age, sex, or physical activity level, shifts in body compositions may follow distinct distribution patterns identifiable by areal surface roughness measurements.This study shows that 3D body surface roughness characteristics associate well with body composition and represent a viable new approach to improve today’s 3D scanner-based methods to assess body composition and obesity-associated health risks. Although the explanatory variance of some parameters evaluated in our approach was slightly lower than in comparable trials, this first evidence is an auspicious starting point for future studies. The arithmetic mean height reached medium to strong levels of explanatory variance for relative body fat compared to the BMI or WC. Further investigations may provide a more detailed understanding of the association between areal surface roughness characteristics and well-established approaches, such as BIA body fat estimation, by including larger and more diverse study cohorts or by focusing on particular body segments.