In situ detection of organic matter hotspots in ice prevents loss of spatial information and signal dilution when compared with traditional bulk analysis of ice core meltwaters. Our methodology could be useful for detecting microbial and organic hotspots in terrestrial icy environments and on future missions to the Ocean Worlds of our Solar System. We used a deep-ultraviolet fluorescence mapping spectrometer, coupled to a drill system, to scan from the surface to 105 m depth into the Greenland ice sheet. The scan included firn and glacial ice and demonstrated that the instrument is able to determine small (mm) and large (cm) scale regions of organic matter concentration and discriminate spectral types of organic matter at high resolution. Both a linear point cloud scanning mode and a raster mapping mode were used to detect and localize microbial and organic matter “hotspots” embedded in the ice. Our instrument revealed diverse spectral signatures. Most hotspots were <20 mm in diameter, clearly isolated from other hotspots, and distributed stochastically; there was no evidence of layering in the ice at the fine scales examined (100 μm per pixel). The spectral signatures were consistent with organic matter fluorescence from microbes, lignins, fused-ring aromatic molecules, including polycyclic aromatic hydrocarbons, and biologically derived materials such as fulvic acids.  The deep ice of the Outer Solar system satellites would be the first potentially habitable environment encountered by future missions targeting the deep subsurface oceans of those worlds  and low temperatures (−48°C average winter minimum to −11°C average summer maximum) at the top of the ice sheet represent a textbook example of dry  deposition, compression, and modification of grains to form ice  at fine spatial scales in microhabitats . This combination is a departure from traditional ice exploration where a sample is acquired and delivered to an instrument: WATSON brings an instrument to an in situ sample.The overarching objective of our work is to determine the fine-scale distribution of cellular and noncellular organic matter without ice core extraction, transport, melting, and the associated risks of contamination. To address this objective, we developed a deep-ultraviolet (DUV) fluorescence Raman mapping spectrometer for in situ measurements of the borehole wall to determine the distribution of fluorescent organic materials in firn and glacial ice to a depth of 107 m near Summit Station, Greenland. Our investigations at Summit Station provide new information on the processes that affect localization (and concentration) of organic and putative biosignatures during meteoric deposition and compaction. In addition, our efforts advanced detection and characterization techniques that could be used in a laboratory setting to identify hotspots in extracted ice cores for further analysis.In July of 2019, we used WATSON to drill a borehole and make fine-scale et al., et al., Summit Station was established in 1989 as part of the GISP2 drill site. It is located near the apex of the Greenland ice sheet, just inside the southwestern boundary of the North East Greenland National Park  . The Station is currently maintained by the U.S.-based CH2M HILL Polar Services, with support from the National Science Foundation, for the purpose of providing year-round support for scientific research into glaciology, meteorology, atmospheric chemistry, and a wide range of other fields requiring long-term environmental observations. The climate at Summit is currently classified as “ice cap,” with no month during the year having a mean monthly temperature exceeding 0°C, with exceptions in 2019 (after the completion of our expedition), 2012, 1889, and 7 other times between 1250 and 750 AD . During a project led by Ian Baker (Dartmouth College), the borehole was drilled in 2017 with a UWM-IDDO Eclipse-Badger drill. It was “dry drilled”—meaning that no drilling fluid was used to prevent lithostatic collapse of the borehole. Baker  acquired. During et al.  DUV (excitation wavelength: 248.6 nm) hollow-cathode NeCu laser was fired at 40 Hz through a series of optics that cleaned and shaped the laser beam and focused it through a fused silica optical window onto the borehole wall. This DUV illumination causes aromatic-containing organics to fluoresce at electronic transitions diagnostic of the electronic states of the particular molecule or mixture of molecules  The background generally had increased signals at both 324.6 and 341.2 nm and was never larger than 2400 counts in any of the recorded times of any of the point clouds. Maps were acquired by locking WATSON at a constant depth inside the borehole by using metal skids and a serpentine raster line scan of the optical path across a target area. The scanning motor and laser repetition rate were synched such that the maps were prepared with a spatial scale of 100 μm per pixel. Typical map dimensions were 1 cm horizontal × 4 cm vertical and 3 cm horizontal × 4 cm vertical. As for the point cloud, raster maps signal acquisition interleaved dark and active spectra. After data collection, the laser-illuminated instrument counts were subtracted from the dark bias instrument counts and a multiband raster map was created.max) and the main background peaks (either 324.6 or 341.2 nm) were selected for display. During map analysis, the background spectra was removed for each identified spatial region of interest (ROI) in the mapped region. To do this, we selected two multi-pixel blank areas for each feature. These were approximately equidistant and were above and below the feature, usually within 5 pixels (0.5 mm) distance. The blank pixel values for each area were averaged, and the two sets were interpolated (averaged since equidistant to the target feature) to create a synthetic predicted background at the location of the identified feature. The synthetic predicted background was subtracted from the instrument response to determine the actual spectral response. The maximum standard deviation between the two blank areas was set as the noise level. During ROI determination, only those pixels above 3 × noise level for at least one of the bands were used to define the feature outline. For detailed spectral characterization, only those pixels that were 10 × noise level for the lambda max  were obtained by using either a Hitachi F-4500 spectrometer or a Horiba Jobin-Yvon Fluoromax-4 Spectrofluorometer, equipped with an Xe lamp light source and a pathlength of 1 cm. The Hitachi F-4500 had an excitation wavelength scanning from 200 to 400 nm in 5 nm increments and intensity measurements from 200 to 600 nm every 2 nm with a scan speed of 1200 nm/min. Samples scanned by the Hitachi instrument included pure chemical standards as well as whole bacterial cultures. For pure chemical standards, homogeneous aqueous solutions were made and scanned; whereas for whole bacterial samples, culture suspensions in deionized water were used. Samples run on the Hitachi instrument included naphthalene, tryptophan, phenanthrene, anthracene, pyrene, methyl-1-pyrene, fluoranthene, perylene, p-benzoquinone, Suwannee River and Pony Lake fulvic acid samples, and an “Antarctic bacterium” isolate. Samples of the chemical solutions were prepared to a 20 mg/L stock solution, shaken for 24 h, and placed into a quartz cuvette; spectra were collected at 25°C. The “Antarctic bacterium” isolate is an extract from a sample collected from a supraglacial stream on the Cotton Glacier, Antarctica, and is described in the work of Dieser et al. . The recorded spectral emission responses were binned to match the WATSON wavelength binning and average values within the bin used. The spectra were then normalized to the highest response in the WATSON spectral range (275–425 nm), and this was used for comparison of WATSON spectra.We identified points of interest in the point cloud data by searching for a clear discernable pattern when subtracted from the background spectrum. In general, this meant that at least three consecutive band responses were at least 10 times the maximal standard deviation from the two regions used for background determination. An example plot for the 385.3 nm wavelength band of the Point Cloud 17 dataset is shown in Spectral data from the 1039 points showed a distinctive spectral pattern only near 84.9 m. This was very close to the depth at which new drilling into the borehole took place. We deemed this as a possible impurity and, if the 239 points from this region are removed from the bulk analysis, we obtain a corrected bulk hit rate of 0.6%. As can be seen from examination of in situ.It should be noted that the example in et al., The reason for the significantly elevated hit rates at 30–40 and 80–90 m is unclear. We also note an enhancement of hit rate frequency toward shallower depths. From our down-borehole imaging, we noted a melt lens at 3.28 m depth that we interpret as resulting from the surface melting in summer of 2012  . For firn ice, the higher amount of scattering from bubbles and other unconsolidated boundaries can create an effective interrogation volume that is brightly illuminated nearer the surface; this also increases the volume of fluorescence emission that can be received . In contrast, glacial ice may have longer pathlengths (light penetration can travel deeper and straighter into the “clearer” ice) before a fluorophore is detected; only fluorophores that lie directly in the beam path will be illuminated ; Yung et al.  found th, et al.  suggesteOf the 1039 identified points of interest, 308 (30%) were solitary features composed of a single point where consecutive laser shots did not show a significant signal. The majority of points were associated with one or more consecutive shots and had spectrally related signals. We identified 80 clustered features, listed in We calculated the maximal estimated apparent size by assuming that the laser was scanning in a straight line with uniform spacing and that the size extended almost one post-spacing to the neighboring post just before and just after the feature along the line. A potential overestimate of >10.6 mm could occur in cases where the shot post-spacing is 5.321 mm—this overestimate would occur in the case where the detected laser shot was right at the margin of an extended feature but our estimate extended to the next posting.Given this caveat, the binned distribution of maximally estimated size of all the features in the point cloud data is shown in max spanned over four orders of magnitude  the possibility of this material being an introduced impurity. The localization at the depth where previous drilling efforts halted is also consistent with an introduced impurity.During our analysis of Spectral Type occurrence frequency, we present frequency numbers in y axis and depth along the x-axis. The axes in max. Deviations from straight tracks parallel to the y axis show where the instrumented-drill combination rotated during its path down or up the borehole.From initial inspection, the absolute intensity of the peak is not affected by the depth. However, as previously noted, there are a few intense points of interest of Spectral Type L418_d found above 80 m, whereas below 90 m they are some of the most intense points of interest encountered, represented by large nested circles in More than 26 maps were collected spanning depths of 10–106.7 m. For detailed analysis, we selected Map 1, a 1 × 4 cm map acquired at a lower depth of 93.8 m and a rotation of 53 degrees shown in The collected multiband data were visualized and analyzed by ENVI . A list of the identified ROIs from Map 1 is presented in order of increasing depth below the surface in et al. .The areal size of the ROIs ranged from 0.06 to 1.08 mmThe maximum linear dimensions of the ROIs are also indicated; in general, the largest features in After spatial correction, we measured the maximum dimensions as well as the maximum dimension orthogonal to this direction to determine the aspect ratio of the ROIs; most of the features had an aspect ratio between 3.4:1 (oblong) and 1.2:1  with an average aspect ratio of 2:1. From the dimension measurements, we also measured the apparent angle or tilt relative to horizontal. In general, for most of the ROIs where an angle could be recorded, regions were tilted roughly 40–70 degrees counter-clockwise to horizontal, as if they were originally spherical objects that were compressed from the upper left. The sole exception is ROI 17, which has a tilt to the upper right.max wavelengths for each region's assigned Spectral Type. The assumption is that regions may have similar fluorescence intensities and similar quantum yield. We determined the average intensity by summing all the background-subtracted intensities at the λmax, then dividing by the number of pixels for the overall feature. In Map 1, we found that the pixels with the highest intensity were classified as Spectral Type L325_t, specifically, Features 2 and 19.The intensities of the signals across different ROI were compared by using the background-subtracted signal intensity at relative λThe total number of pixels in view for Map 1 is 43,365 pixels, which allows us to compare the hit rate frequency with the point cloud data presented in A summary of data in Map 1 as broken down by Spectral Type is in max. Thus, in Map 1, the Spectral Type 385_m features are numerous, large, and have low signal intensity. The averaged hit rate  for this spectral type is 12.4 per 1000, which is roughly 10 × higher than the overall hit rate determined in Examination of In Map 1, only four ROIs of Spectral Type L325_t are observed. They make up <15% of the total hotspot area yet have more than 30% of the total signal. In general, these signals are bright. In Map 1, they have a hit rate frequency of more than 2 points per 1000 laser shots, but in the point cloud data in The hit rate for Spectral Type L325_s is enhanced relative to that observed in the point cloud dataset for depths below 90 m, whereas for L341_s it is roughly similar . For L372 for the brighter hotspots and they appear as compact <1 mm diameter circular  to ring-like  features with near-uniform spectral signatures of varying intensity. The aspect ratio (1.2–3.4:1) and angle of apparent compression in Map 1 is consistent with a uniform compression amount and direction of compression across the scene that causes any initially spherical objects to appear slightly out of round, but not completely flat.Numerous (178) features were identified in the point cloud data. Most of these were single-point features, although the post-spacing in some point clouds was large enough that a feature as large as 10.6 mm could be detected as a single point. Some larger features were noted, although features larger than 15 mm were rare, comprising <4% of the total number of features. This is in general agreement with our Map 1 data, where all of the ROIs, even after combination of neighboring spectrally similar ROIs, would have dimensions <4 mm. In preliminary analysis of other map data, we saw no evidence of ROIs larger than 15 mm. From the map data presented in in situ WATSON scanning of a section of the borehole 92.8 to 95.1 m with multiple point cloud tracks. Detected hotspots in one track are not repeated on another track. This effect had previously been noted by Rohde et al. (x-axis in figure) of the borehole. Given the 11 cm diameter of our borehole, a dip of 45 degrees would generate a 0.1 m deviation from one side of the borehole to the opposite side and should still be evident in the graphic.There was no clear evidence for layers in the Map 1 data. The point cloud data only provides one dimension; thus, series of consecutive points would look similar to a thick horizontal layer if only the vertical axis were scanned. Some sections of the borehole were scanned with more than one vertical transect, but at a different angle. In many cases, a track with a hotspot at one rotational angle did not show a corresponding hotspot at the same depth but a different rotational angle. This observation is consistent with discrete spots, rather than layers. As an example, e et al.  and RohdIn general, features do not line up with other features on parallel tracks, arguing against consistent layers or bands at a given depth. Although we saw no evidence of layers from the Map 1 or point cloud data, it is possible that some of the detected broad features in the point cloud data occur in layers, zones, or even spotty confined zones, but to fit our observations, most of these would have to be limited to areas that were scanned with only a single track. The data presented in With this method and at this fine resolution, the hotspots we observed appear to be stochastically distributed. The sizes varied from 74 mm to a single pixel in the point cloud data, and from 0.4 to 3 mm in the map data. In both datasets, smaller sizes were more prevalent.et al. , Rohde et al.  showed tOur data allow us to evaluate possible emplacement mechanisms for fluorescent materials that explain our observations. From analysis of our combined point cloud data, the estimated feature size does not appear to vary with depth. Features at the top of the borehole column do not appear significantly larger than those at the base, after the firn-glacial transition, suggesting preservation mechanisms in the ice. This observation from the point cloud dataset is also consistent with preliminary examination of our detailed raster map data—average hotspot feature size does not appear to vary according to depth. These observations suggest that the initial emplacement places a spectrally uniform material at a specific location and size in the near-surface ice, and that the transition from firn to glacial ice does not cause significant migration or transformation.The observed size of the detected particles constrains the origin and emplacement processes. The maximally estimated size for our largest detected ROI is 74.5 mm . HoweverA more likely scenario is that organic material is originally deposited, perhaps as diffuse regions, but is then concentrated into discrete and stochastically located hotspots in the ice at a fine scale. This would happen at the upper surfaces of the ice, as the size of the regions and spectral nature is preserved intact in the ice column. The uniform size may be related to diffusion through the ice, with diffusion occurring quickly at the surface to form an initial ca. 1 mm spot, which is then “locked in” and carried through the firn to glacial ice transition and densification. This may occur at a fine scale that has not been previously observed by bulk analysis. Exact mechanisms are constrained by spectral uniformity, scale that favors smaller hotspots (1 mm being common), favorable energetic requirements, and stochastic emplacement.et al., et al., 2013, 2017, 2020) and includes comparisons with natural organic matter mixtures that have a more characteristically complex fluorescent nature. It is important to note that the standard spectra were acquired at room temperature in aqueous or methanolic solution in the laboratory, whereas WATSON spectra were acquired in an ice matrix at roughly −20°C in field conditions; consequently, our measured signals may not overlap exact regions of laboratory standards. Although a rigorous identification is beyond the scope of this article, our identified spectra compare favorably with likely material targets in the polar environment and with previous studies of fluorescent organic matter of various sources   . Shorternthrene) , are simet al. .The Spectral Types at longer wavelengths >>341 nm are consistent with PAHs or larger aromatic species  could cause changes to the reference spectra  borehole at Summit Station provided a depth transect from the surface down to the base of the Greenland Ice Sheet ice sheet a little more than 3 km below the surface. For microbial concentrations, analysis of the GISP2 ice cores demonstrated that the cell counts varied from 10, et al.  with theet al., et al., et al., 4 cells cm3 to be present. If we take the Map 1 spatial dimensions to be roughly 1 × 4 cm and assume an optical penetration depth of 1 cm , which is a local concentration of 5.5 × 106 cells cm3—more than a 500 × increase compared with the estimated bulk concentration.We can use the microbial concentrations from the literature to speculatively estimate our instrument response based on our measured signal  . We will assume that pyrene is present in the Map 1 data at a concentration of 4 ng/kg snow = ca. 8 ng/kg ice [this is the highest concentration reported in the work of Slater et al. , as a coin situ instrument has the potential to be highly sensitive and is able to detect both microbial and chemical signals at low native concentrations in a field setting at finer scales than can be measured by conventional ice core extraction and laboratory melting.These speculative estimates suggest that our A future deep drilling mission through the icy crust of an Ocean World may sample melted ice during its journey on the way to the ocean, or it could examine hotspots embedded in he borehole walls: Which is better? This is the same question as comparing traditional bulk melt analysis with our fine resolution scanning. During traditional melt analysis, any contained fluorescent microbial or chemical hotspots are diluted into the volume melted and subsequently analyzed. We can examine the effect of dilution by using our collected data from the point clouds and Map 1 to compare detection levels between typical hotspots in the point cloud and map and a scenario if the entire datasets  had been subjected to bulk melt analysis. To do this, we integrated the signals for all the point cloud and map points of interest and ROIs, respectively, and averaged them across all the hotspot pixels in each dataset to create an “average” hotspot spectrum. The averaged spectra per pixel for all the points of interest from the point cloud dataset are shown as a dot-dash brown line in In the same figure, we also plot the average LOD across the map area based on the average extracted backgrounds. We took all the local background spectra used for feature background subtraction and used the average standard deviation of the background signals. These were multiplied by a factor of 3 to generate the LOD (shown as a hashed black line in the figure). Any spectra falling below this limit would be not considered significant. Since all the diluted signals from either the point cloud or map pixels fall under this limit, they would not be detected.In situ ice scanning also maintains spatial integrity and allows more detailed analysis on associations, layering (or lack thereof), and spot distribution. This analysis, thus, provides impetus for future in situ scanning and spatial mapping of ice boreholes compared with traditional bulk analysis.This has important implications for future instrumentation and demonstrates the utility of detecting hotspots at fine scales. By examining hotspots concentrated in borehole ice rather than bulk melt dilution, we can gain 50–100 × in signal intensity. Depending on the instrument threshold, this could be the difference between detecting and not detecting a signal. Our DUV fluorescence mapping spectrometer revealed diverse spectral features from 0 to 107 m in both firn and glacial ice. We used both a linear point cloud scanning mode and a raster mapping mode with a coupled drill-instrument combination. Both instrument modes detected spectrally diverse features consistent with individual, stochastically distributed <20 mm globules of material. Overall, the frequency of the hotspot intensities followed a power law, with intense signals only a few percent of the detected signals. From point cloud analysis, we found that most of the features were small with a few larger features measuring up to 60 mm in dimension. We did not see any of these larger features in our raster maps, instead noting that most features were <2 mm in size. We found Spectral Type-dependent differences in hit rate frequencies as well as differences in frequencies above and below the firn-glacial ice transition. The Spectral Types we identified had spectra that are similar to microbes, lignin-phenols, fused-ring aromatic molecules, including PAHs, and environmentally complex fulvic acids of microbial and multicellular origin.We found no evidence of fluorescent material emplaced in layers, at least at the fine scales examined. There was no apparent correlation between the size of the feature and its depth. Both map and point cloud data had features of roughly the same distribution in shallow firn and in deeper glacial ice. This suggests that the mechanisms of emplacement favor smaller features and may occur at shallow depth, and then “lock in” early in the process. The distribution of most spectral types appears the same in glacial as for firn ice, although some exceptions were noted.in situ detection and spectral analysis of diverse punctate chemical signals similar to microbial and other organic molecules in a drilled ice borehole at Summit Station, Greenland. Data obtained from Summit Station show that our DUV fluorescence mapping spectrometer with 100 μm spacing can spatially resolve spectrally unique features, whereas traditional bulk melt analysis removes spatial context and potentially dilutes the signal below instrumentation limits of detection. Our DUV-based technique can be useful for localizing the emplacement of organic material in icy environments on Earth. This technique could also be key to the detection of astrobiologically relevant organic molecules for a future mission to the icy crusts of the Ocean Worlds such as Europa, Enceladus, and Titan, or perhaps the polar caps of Mars.We successfully demonstrated


For functional analysis of MHC-specific IgE in vivo, a cutaneous hypersensitivity reaction assay was adapted for this purpose using MHC monomers. Humanized RBL-2H3 cells transfected with cDNA coding for the human-high affinity IgE receptor were used for functionality measurement of donor-specific IgE in sensitized transplant patients. For detection of HLA-specific IgE, a bead assay was adapted, using beads expressing single HLA antigens. The aim of this publication is to demonstrate currently established methods for the detection and characterization of MHC-specific IgE in the murine and human setting.Humoral immunity is a major barrier limiting long-term outcome after organ transplantation. Especially, the production of antibodies directed against donor HLA/MHC antigens (i.e. donor-specific antibodies (DSA)) leading to antibody-mediated rejection (ABMR) is considered to be a major factor negatively affecting allograft survival. DSAs of the IgG isotype are routinely measured in transplant patients. However, not all patients diagnosed with IgG-DSA develop ABMR events. Therefore, research in better understanding the mechanisms of ABMR is of great importance. We recently demonstrated the production of MHC-specific IgE upon allograft rejection in mice and in transplant patients. IgE is classically connected with allergy and is known to be important for the humoral defense against helminths and worms. However, its role in autoimmune diseases and cancer has been reported recently as well. The concentration of IgE in blood is extremely low compared to other antibody isotypes. Therefore, detection of MHC-specific IgE from serum requires methods of high sensitivity. Since MHC-specific IgG—typically present at much higher serum levels—develops as well, high specificity is also required of IgE detection methods. In the murine model we developed an enzyme linked immunosorbent assay (ELISA) using MHC monomers for measurement of MHC-specific IgE, allowing us to distinguish between specificities of antibodies against different class I and class II antigens. For measurement of functional activity of MHC-specific IgE  Donor specific antibodies of the IgG isotype are routinely measured in transplant patients . Research in improving long term outcomes, however, still faces huge challenges . Amongstpatients . Althougpatients , 9. Diagpatients , 11. Thepatients , 10, 12.We recently demonstrated the development of donor specific antibodies of the IgE isotype in mice and highly sensitized kidney transplant patients . Antibodvia a MHC-specific ELISA. It is known, that DSA directed against MHC class II are clinically relevant predictors of ABMR . In fact, with plasma concentrations levels of less than 1 µg/ml in healthy humans, a new standard unit (kU/L or IU/ml) was presented in 1981 by ‘The National Institute for Biological Standard and control’ in order to properly express the concentration of serum IgE . Additio of ABMR . As by nvia binding to its effector cells like basophils or mast cells. Once IgE is bound to its effector cells it can lead to inflammation and tissue destruction. By binding to its high affinity receptor FcϵRI on basophils or mast cells and subsequent crosslinking through the specific antigen, cell degranulation is induced releasing inflammatory mediators such as histamine, proteases, heparin and various cytokines , it is possible to identify antibodies binding to an array of defined HLA- antigens in a human serum sample by a single multiplex test  and BALB/c (haplotype H-2d) mice were purchased from Charles River Laboratories, Sulzfeld, Germany. Female C3H/J (haplotype H-2k) mice were purchased from Jackson Laboratory  and bred at the Department of Biomedical Research, Medical University of Vienna (Austria). Mice were kept in a protected environment and a 12-h light-cycle in individually ventilated filter cages on sterile standard beddings. Sterile water and standard pellet diet were given ad libitum. Mice between the ages of 6 to 8 weeks with an average weight of 18 to 22 g were used for the experiments.Mice were anesthetized with ketamine  and xylazin  in 0.9% NaCl. Full thickness tail skin from BALB/c (donor) or C3H (donor) mice was grafted on the left side of the chest and secured with stitches and with band aids for 7 days. Postoperative analgesia consisted of piritramid  and 0.4% glucose in 250 ml drinking water ad libitum for one week. Grafts were visually inspected every day. They were considered rejected if less than 10% of the graft remained viable. Skin grafts from fully mismatched donors without special immunosuppressive treatment of the recipients were rejected about 10 days after transplantation.2CO3, 5.3 g NaHCO3 in 1000 ml ddH2O, pH 9.5) over night at 4°C. Plates were washed twice with washing buffer (PBS/0.05% Tween-20). For saturation of unspecific binding sites, the plate was incubated with PBS/1%BSA/0.05% Tween-20 at room temperature (RT) for 2.5 h, followed by five washing steps with washing buffer. Mouse serum was diluted with dilution buffer (PBS/0.5% BSA/0.05% Tween-20) 1:2.5 for IgE or 1:125 for detection of IgG1. The plate was incubated with 100 µl/well of the respective serum dilution over night at 4°C. After washing of the plate five times using the washing buffer, bound antibodies were detected using monoclonal rat α-mouse IgE (clone R35–72) or α-mouse IgG1 (clone A85-1)  diluted to 0.5 µg/ml in dilution buffer (100 µl/well). After incubation for 2 h at RT or at 4°C overnight, and a further washing step with washing buffer, the plate was incubated for 30 min at 37°C and 30 min at 4°C with HRP-coupled goat anti-rat serum  diluted 1:2000 with dilution buffer (100 µl/well). ABTS  is used as the detection substrate for HRP. This solution has to be prepared freshly and H2O2 is added just before detection. An Infinite microplate reader  was used for absorbance measurements at 405 nm. Background measurement was recorded at 490 nm (A maximum absorbance 96-well plate was incubated with 5 µg/ml (100 µl of per well) of the respective monomeric MHC antigen diluted in coating buffer .MHC I H-2K4 cells were plated on a 96-well tissue culture plate  and incubated for about 18 h at 37°C, 5% CO2. Cells were incubated with mouse serum diluted 1:2.5 with medium (100 µl/well) for 2 h at 37°C, 5% CO2 followed by a washing step with 2× Tyrode’s buffer . Next, the cell layer is incubated with 3 ng/well of a monomeric MHC antigen , for 30 min at 37°C. At least three wells of cultured cells were spared of serum and antigen treatment and were used as positive control for calculation of the percentage of antigen-specific degranulation of the RBL-2H3 cell line. They were incubated with 1% Triton X-100, which leads to cell death and therefore to a maximum (100%) release of β-hexasominidase (Equation 1). The analytic compound 4-methylumbelliferyl-N-acetyl-β-d-glucosamide  is aliquoted beforehand to 10 mM stock solutions in DMSO for storage at −80°C. For measurements of the β-hexasominidase release of degranulated basophils, 80 µl of 4-MUG stock solution is transferred to 5 ml of citrate buffer  and the plate was incubated with this assay solution for 1 h at 37°C. Glycine buffer  was used to stop this reaction and the fluorescence was measured using an Infinite plate reader  at λEx: 360/λEm: 465 nm  with 10% FCS. Upon start of the assay, 4 × 10: 465 nm Figure 2Equation 1 Calculation of the percentage of antigen-specific degranulation of the RBL-23H cells. RFU, relative fluorescence unitsk or rH-2Dk, 2 μg/ml diluted with PBS) were injected into the shaved abdominal skin. 30 µl of the mast cell-degranulating compound 48/80 solution  was injected intra-dermally as positive control. As negative control, PBS was used. After 15 min, mice were sacrificed and their inverted skin was mounted to a foamed polystyrene plate and documented by photography. The degranulation of mast cells, due to crosslinking of bound IgE with the specific antigen, leads to a release of vascular mediators. These mediators increase the permeability of ambient vascular structures. Previously injected Evans Blue is then released into the surrounding tissue leading to a blue discoloration of the skin. Blue discoloration around the respective injection site indicates the presence of MHC-specific IgE against the respective injected MHC monomer.BALB/c mice were injected intravenously (i.v.) with 100 μl 0.5% Evans Blue . 30 μl of MHC monomer  were used to investigate HLA- specific IgE in patient serum and to perform downscaling experiments of the beads. Patient serum was adjusted to 10 mM EDTA (Invitrogen) to prevent complement interference phenomenon (2O) by centrifuging at 1800 x g for 7 min at 4°C and incubated with biotin-conjugated anti- human IgE mab  at a dilution of 1:20 (0.5 µg/well) for 30 min in the dark on a shaker. In the next step, beads were washed twice and PE-conjugated streptavidin (eBioscience) at a dilution of 1:200 (0.02 µg/well) was added and the incubation step was repeated. After incubation, samples were washed twice again, beads were resuspended in 55 µl buffer and then measured on the Luminex flow analyzer 200. The protocol is schematically illustrated in enomenon , 41. NexEquation 2: Threshold definition for positive HLA-specific IgE signals. For calculation of MFI threshold measurements of healthy male donors were used. SD, standard deviation; MFI, median fluorescent intensity.in vitro, a mediator release assay with humanized rat basophil leukemia (RBL-2H3) cells (clone RBL-703/21) was performed (2 and plated in 96 well plates (1 × 105 cells/well) in technical triplicates. The cells were incubated overnight together with sera from highly sensitized kidney transplant patients and as a control with sera from healthy subjects. Beads coated with a single HLA- antigen were used . An HLA antigen was selected to which the patient showed a positive IgE signal in the Luminex assay and one to which he/she was negative on Luminex as a specificity control. Beads were incubated with RBL-2H3 cells and human serum for 1 h at 37°C. The reaction was stopped with 100 µl glycin buffer and basophil degranulation was measured by β-hexosaminidase release by an Infinite plate reader  at λEx: 360/λEm: 465 nm.To test functionality of HLA-specific IgE antibodies erformed . In brieMurine data were statistically analyzed using GraphPad Prism 8.0 . P values were calculated using the 2-sided Mann-Whitney U test. A p value lower than 0.05 was considered statistically significant The detection of allergen-specific IgE using an ELISA is common in allergy research . The estAllergen-specific IgE in isolated murine and human serum has shown to be stable for a period of time also on RT . However1. 96-well plates were coated with MHC class I  monomer concentrations ranging from 0.03 to 5 µg/ml (1 in sensitized serum compared to naïve serum were achieved at the highest monomer concentration (5 µg/ml), without a significant increase in background signal. To ensure binding and therefore detection of IgE despite high levels of donor-specific IgG1 potentially binding to the same MHC epitopes, the highest concentration of MHC monomers was thus selected for use in all further experiments.As a first step, MHC-monomers were titrated using an antibody against IgG 5 µg/ml Figure 4Although levels of MHC-specific IgE were very low, serum had to be diluted for detection, as using undiluted serum leads to high unspecific background signals. Different serum dilutions for detection of MHC-specific antibodies are shown in d, H-2Dd) with serum of different recipient and donor strain combinations. A positive signal was only expected for C57BL/6 mice after rejection of a BALB/c skin allograft (haplotype d). Serum was isolated three and five weeks after rejection and diluted, as mentioned before, 1:2.5 using PBS/Tween/BSA. Positive signals were only detected against monomers of the haplotype d. No positive signals were seen after incubation with serum of C57BL/6 mice or BALB/c mice sensitized with C3H skin allografts (haplotype k), demonstrating the specificity of this method to detect MHC-specific IgE [for these specificity data see Figure E2 in (The specificity of this method of detection was verified by incubating MHC monomers (H-2Kre E2 in ].via heat (via this procedure (1 were not lost in treated serum (data not shown). This further confirms the detection of IgE specific for donor MHC, ruling out positive signals due to unspecific binding or MHC-specific IgG.In contrast to IgG, free IgE in plasma can be inactivated via heat . To provrocedure . As showin vitro, we adapted the RBL-2H3 assay, primarily used in allergy research. The cells used for this assay are a basophilic leukemia cell line derived from rats (RBL-2H3), binding murine IgE . As positive control of specific basophil degranulation, serum from mice sensitized with a major grass pollen allergen (Phl p 5) was used. No significant difference in degranulation compared to naïve serum was achieved with serum dilutions from 1:10 to 1:5  needed for the detection and functional analysis of MHC-specific IgE via possible epitope sharing of IgG and IgE. We do not recommend changing the monomer concentration, but it is possible to first test different serum concentrations when using these assays on other mouse models.Adaptions in terms of serum concentrations should be taken into consideration when using these protocols in different mouse strains. Due to the high concentration of donor specific antibodies of the IgG isotypes in the used serum, it might come to saturation of the presented epitopes in vivo, we adapted the cutaneous type I hypersensitivity assay used in allergy research to measure anaphylaxis in mice . Each kit contains about 100 different single antigen-coated beads to maintain wide coverage of different HLA specificities present in the population. Routine laboratories are screening mainly for the HLA- specific IgG subtype and are using standardized 5 µl of beads according to manufacturer’s instructions. In order to optimize the protocol, we performed a downscaling experiment of the beads with the goal of maintaining the same sensitivity but using lower amounts of beads. We used human serum of two highly sensitized kidney transplant recipients and the serum of one healthy male control who never received an organ transplant or a blood transfusion.In order to test the optimal bead volume, two experiments were performed for each HLA class I and class II. Maintaining a ratio of 1:4 beads to serum, we scaled the beads to 1.5, 2, and 5 µl. Downscaling of the beads for HLA class I and II is shown in For our downscaling experiments, we used the MFI of each single bead, subtracted the background from each bead value and compared it to the background-subtracted MFI of one healthy control serum. For a positive classification this bead value had to be higher than the respective value of the healthy control. Additionally, a minimum requirement for a positivity was a MFI of >25 .The change of bead reactivities and specificities is shown in Each HLA kit contains a negative control bead (bead #1), which is not coated with any antigen and demonstrates the background of the assay, and also a positive control bead (bead #2) which is coated with human IgG.We analyzed the MFIs of the two positive IgE sera signals and compared positive signals for each downscaling attempt.A strong and robust bead signal was observed by using 1.5 µl of beads with a bead count of 40 to 100/single antigen bead which led us to the conclusion that downscaling to 1.5 µl beads is sufficient and still effective for HLA- specific IgE Luminex measurements.In the first experiment we had a bead count of >40 beads compared to the second experiment with less than 40 counts/bead resulting in 13 changed bead specificities . The changes of positive antigen specificities from those two experiments are shown in As human IgE is relatively unstable and sensitive to heat, samples should not be thawed more than two times before measuring on the Luminex instrument. Therefore, serum samples need to be stored in small aliquots at −80°C.It is also necessary to vortex the bead suspension very well before starting to pipette them into the V-bottom plate. While using 1.5 µl of the bead suspension, it is essential to work very precisely and to resuspend the stock again after pipetting about 10 samples to maintain homogenous bead distribution as they tend to sediment. Furthermore, in order to preserve an acceptable bead count, after centrifugation the supernatant of each washing step needs to be removed with a pipette instead of inverting and flicking the plate.de novo DSA post-transplantation. This assay is a very sensitive method based on the principle of flow cytometry which we modified for measuring IgE DSA. In The single-antigen bead Luminex assay is routinely used in clinical diagnostic laboratories for testing sensitization of the recipient towards the donor prior to transplantation and to check for the development of Functionality of HLA-specific IgE antibodies was assessed in a basophil release assay and we could successfully demonstrate that IgE triggered basophils release and confirmed IgEs unique effector mechanisms (not shown) .We established different methods for measuring donor specific IgE antibodies in the murine transplant setting and in human transplant patients. Although methods for measuring DSA in the murine setting are already available, they cannot directly discriminate MHC specificities . Here weThe establishment of these assays for measurements of MHC-specific antibodies in the murine transplant models was done with monomers of the MHC class I isotype. However, ensuing experiments on antibody-mediated rejection done by our group were regularly performed using monomers of MHC class I and class II antigens.in vitro in an adapted rat basophil leukemia cell degranulation assay by using monomers to trigger mediator release.Sensitivity for detecting IgE was assessed through several experiments. An important issue was the occurrence of masking IgG. Therefore, it was especially important to experiment with different antigen concentrations on the solid phase in order to increase the sensitivity of this assay. This prevents the issue of MHC-specific IgG competing with IgE for binding sites. Signals for MHC-specific IgE were lost, when serum was treated with heat and inactivating temperature-sensitive IgE. Moreover, cross-reactivity of the detecting antibody with other isotypes was ruled out by an immunoblot assay [see Figure E2 from ]. This dOne major limitation of measuring MHC-specific IgE in mice is the large amount of serum which has to be used for these assays. We have already demonstrated previously that MHC-specific IgE is present in murine serum until at least 12 months after transplant rejection . TherefoUsing these assays on other mouse models or with different modes of sensitization, we recommend to test different serum concentrations in order to achieve as little background noise as possible in combination with a proper signal for measurement of MHC-specific IgE.in vivo in sensitized BALB/c mice. Due to the light skin color of this mouse strain, the degranulation of mast cells in the skin shown by the blue discoloration could be presented clearly. Therefore, we used this strain for demonstration purposes. However, this method is also expected to work in other donor-recipient combinations.The cutaneous hypersensitivity type I reaction assay was used to show the functional activity of IgE specific antibodies Luminex-based bead assays are routinely used in clinics for HLA-specific antibody screening and we could successfully establish a method for detection of HLA-specific IgE . In thisThe heat sensitivity of IgE was used to demonstrate the specificity of this bead based assay , 43. Incin vitro by an adapted humanized RBL assay using beads presenting a selected single antigen against which the patients were sensitized  are ongoing which will give us more insight into the production and potential role of HLA-specific IgE in transplant patients. Furthermore, the mechanistical background and role of MHC-specific IgE in cardiac transplant rejection is studied at the moment using a mouse model of antibody mediated rejection (CCR5 KO) established by the group of Fairchild et al. , 50.In conclusion, we established various assays to measure MHC-specific IgE and to demonstrate its functional activity in murine and human transplant recipients which might be a valuable tool for further studies in antibody mediated rejection.The study participants were recruited at the Vienna General Hospital/Medical University of Vienna. All human samples were collected after written informed consent. The study was approved by the ethics review board of the Medical University of Vienna .All murine experiments were approved by the Ethics and Animal Welfare Committee of the Medical University of Vienna and were performed in strict accordance with national and international guidelines of laboratory animal care. All animals received humane care in compliance with FELASA and ARRIVE and were approved by the Austrian Federal Ministry of Science, Research and Economy BMWF GZ: 66.009/0295-1I/3b/2011 and GZ 66.009/0230- II/3b/2011.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics review board of the Medical University of Vienna . The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Ethics and Animal Welfare Committee of the Medical University of Vienna .AW, JM, and TW designed the research. AW, JM, AF, UB, NP, AC, MM, and SH performed the research. MW, BL, and RV contributed new reagents/analytic tools. AW, JM, AF, and MW analyzed the data. AW, JM, and TW wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by the Austrian Science Fund , and with institutional support from TEVA Pharmaceuticals Europe (for the investigator-initiated study “HLA-specific IgE antibodies in organ transplantation”). AW is a recipient of a DOC Fellowship of the Austrian Academy of Sciences at the Department of Surgery, Medical University of Vienna (DOC/25556). In addition, the study was funded by the Danube Allergy Research Cluster by the country of Lower Austria. Rudolf Valenta is a recipient of a Megagrant of the Government of the Russian Federation, grant No 14.W03.31.0024. The funder bodies were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.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.


The magnesium screw had the highest pull-out force of 399 N (cracked without the screw out being pulled out), followed by the titanium screw, with a force of 340 N, and the PLGA screw, with a force of 138 N . ANOVA was performed for the maximal torques before damage to the screw , revealing that the maximal torque of the magnesium screw was 16 N·cm, while that of the titanium screw was 19 N·cm. The magnesium screw was significantly weaker than the titanium screw (p < 0.05). The measured torque and pull-out force were not related to each other (p > 0.05). Conclusion: Among the screws compared, the metal biodegradable magnesium screw seems to be the most suitable material for multiscrew mandibular head osteosynthesis, considering the condition of the fragile screwdriver socket.Recently, magnesium alloys have gained a significant amount of recognition as potential biomaterials for degradable implants for craniofacial bone screws. Purpose: The aim of this work was to compare screws made specifically for mandibular head osteosynthesis from different materials. Materials and Methods: Screws measuring 14 mm made by one manufacturer specifically for mandibular head osteosynthesis out of the following materials were selected: magnesium (MgYREZr), titanium (Ti6Al7Nb), and polymer (PLGA). The axial pull-out strength and torsional properties were investigated. Results: Each type of screw presented different pull-out forces (Kruskal–Wallis test,  Recently, magnesium alloys have gained a significant amount of recognition as potential biomaterials for degradable implants for craniofacial bone screws. Since the first study of magnesium alloys was published, which included five magnesium osteosynthesis cases in the mandibular condyle head , a few mThe current knowledge on biomaterials is being transformed by the development of corrosion-resistant resorbable metals. The role of biodegradable implants is to heal a specific trauma; support tissue regeneration; and, finally, disappear through degradation in the biological environment. In the last decade, magnesium alloys, as a new class of materials, have shown great potential to be used for maxillofacial surgery and have received much attention owing to their biodegradability ,3,4, antUnfortunately, the mechanical properties of medical magnesium alloys are poorer than those of titanium. It is possible to damage screws during fixation . Becausewww.chm.eu; Juchnowiec Koscielny, Poland) from the following materials: magnesium alloy (14 pieces), titanium alloy (14 pieces), and polymer (7 pieces). The magnesium alloy utilized for manufacturing screws was MgYREZr (WE43MEO). The chemical composition of the magnesium screw by percentage of weight was 3.5–4.5% yttrium, 2.5–3.5% rare earth elements, 0.6% zirconium (max.), <200 ppm manganese, <200 ppm aluminum, <100 ppm silicon, <100 ppm copper, <80 ppm iron, <30 ppm nickel, and <20 ppm beryllium, and the remainder was magnesium. Titanium reference screws were made of Ti6Al7Nb alloy according to standard ISO 5832-11. The polymer screws used for the test were made of poly(L-lactide-glycolide), i.e., a copolymer of L-lactide and glycolide in a molar ratio of 85:15.The tests included 35 screws  made spewww.astm.org/Standards/F1839.htm). Foam blocks have consistent material properties that are similar to those of human bone. Solid polyurethane foam is widely used to mimic and is an ideal medium for mimicking human bone, and the American Society for Testing and Materials  and maximal torque [N·cm] results are presented in ly 400 N . In addiThree types of screws can be chosen for mandible condyle head osteosynthesis: Low-profile titanium screws ,21,22, pEarly monomeric forms of biodegradable implants, namely, poly-L-lactide, have been shown to be associated with delayed degradation (>5 years), leading to occurrences of foreign-body reactions, local fistulas, osteolytic lesions, and peri-implant fluctuant swelling ,31,32. HWE43 magnesium alloy has been used in the clinic in screws ,26,39, aTitanium small fragment screws 1.7–1.8 mm in diameter  have beeThe pull-out force being higher for the magnesium screw than for the titanium screw results from the screw design (shaft diameter and thread depth) and not the material differences. It is known  that theIt is worth pointing out a few clinical issues. For MgYREZr alloy , the operator must handle the screw gently. The mechanical properties of this magnesium alloy are quite similar to those of bones. It is worth imagining how much effort is required to screw a screw made of cortical bone into such a bone. It is possible to damage the screwdriver socket or bend a 1.7 mm solid screw (without cannulation) designed with a slim shaft . Next, iMaxillary artery laceration during maneuvers can occur with impacted bone segments or during careless proximal head fragment reduction with substandard lateral pterygoid muscle preparation. Drilling with too long a drill is another problem. It may be observed in mandible head fixation as operator perforates higher part of the neck trying to put a screw too low, below the mandible head. Known mechanical properties of the screws allow one to avoid iatrogenic damage and extension of the procedure. Moreover, in patients with increased risk of venous thromboembolism or haemorrhagic diathesis, shortening the time of the procedure is very important in order to avoid stroke/bleeding complications. This is the second reason for using proven screws and using an effective insertion technique ,46.Because the solubility of alloying elements in magnesium is limited and biocompatibility and biodegradation must be considered for the design of new screws, the ability to improve the mechanical properties is very restricted . MagnesiAmong the screws compared, the metal biodegradable magnesium screw seems to be the most suitable material for multiscrew mandibular head osteosynthesis, considering the condition of the fragile screwdriver socket.


When the application of a free vascularised flap is not possible, a segmental mandibular defect is often reconstructed using a conventional reconstruction plate. Mechanical failure of such reconstructions is mostly caused by plate fracture and screw pull‐out. This study aims to develop a reliable, mechanically superior, yet slender patient‐specific reconstruction plate that reduces failure due to these causes.Eight patients were included in the study. Indications were as follows: fractured reconstruction plate (2), loosened screws (1) and primary reconstruction of a mandibular continuity defect (5). Failed conventional reconstructions were studied using finite element analysis (FEA). A 3D virtual surgical plan (3D‐VSP) with a novel patient‐specific (PS) titanium plate was developed for each patient. Postoperative CBCT scanning was performed to validate reconstruction accuracy.SD: 0.85, R: 0.10–3.19), and mean screw angular deviation was 5.76° . FEA indicated decreased stress and screw pull‐out inducing forces. No mechanical failures appeared .All PS plates were placed accurately according to the 3D‐VSP. Mean 3D screw entry point deviation was 1.54 mm (Reconstructing mandibular continuity defects with bookshelf‐reconstruction plates with FEA underpinning the design seems to reduce the risk of screw pull‐out and plate fractures. Maurer et al. . However, when a patient's general medical condition does not allow for this type of reconstructive surgery, the mandibular continuity defect can be bridged using solely a conventional reconstruction plate (RP). This type of RP usually needs manual bending to match the contour of the mandible. This method, however, has been reported to fail due to screw loosening or plate fracture  to prevent plate fracture by changing the material or design of conventional RP  and FEM supported individual design.22.1All the patients included in this study required a reconstruction of either a primary or a secondary mandibular continuity defect due to a fractured or loosened conventional RP. None of these patients' mandible could be reconstructed with a free vascularised bone flap due to the poor quality of the donor site vascularisation, an impaired medical condition or refusal to undergo major free vascularised bone flap surgery. The study was approved by the Medical Ethical board of our centre (no. METc‐2019/301), and informed consent was obtained for all patients.In the secondary cases, we started by assessing the mechanically failed conventional RPs retrospectively, following plate fracture or screw pull‐out. Postoperative CT scans were used for the segmentation, and 3D models of the mandibular segments were obtained using Mimics 19.0 software . In order to perform finite element analysis (FEA) on the failed primary reconstructions, we digitalised the conventional reconstruction plates, which had been bent to follow the mandible's contour. Using the postoperative CT scans of primary reconstructions, we obtained the contours the plates were bent to, and using the manufacturer's dimensions, we designed matching plates for analysis. This was necessary since the quantity of metal artefacts or scatter in the CT data would not allow for proper segmentation of the osteosynthesis material.3D FEA  of the conventional reconstructions enabled assessment of the local stress values in the conventional reconstruction plates. The typical defect size used in our general design process was a continuity defect spanning from the mandibular angle up to the approximate midline of the mandible while not crossing it. This represents a L‐defect according to Jewer's HCL classification  that combines both CT data  and fused with MRI‐based tumour delineation for osteotomy placement  from medical grade polyamide powder Figure . Cylinde2.3All the patients underwent a postoperative CBCT scan (120 kV/5 mA with a field of view of 130–230 mm and 0.2–0.4 mm voxel size) to assess the accuracy of implant placement by means of screw entry point deviation and angular screw deviation. Manual alignment of the planned 3D objects of the mandibular segments and screw cylinders with the postoperative CBCT was performed with the Mimics Medical 19.0 software. Two observers executed the alignment independently (BJM and JK). The in situ plate was segmented in order to assess angular screw deviation and subsequently matched to the plate's design file, while the manually aligned screw cylinders were moved along. The Geomagic Studio 2012 (3D Systems) software was used for the matching through a best‐fit surface alignment procedure.Screw entry point deviation was measured between the entry points in the virtual planning, and the cylinders were matched to the postoperative CBCT by means of Euclidean distance (3D) measurements in the 3‐Matic Medical 11.0 software. Prior to these measurements, all the manually aligned mandibular segments and corresponding cylinders were matched to the virtual planning using the global alignment function in 3‐Matic Medical 11.0.Data analysis was performed using MedCalc for Windows, version 19.0.5 . The inter‐observer variability was supported by the calculation of the interclass correlation coefficient (ICC) for every screw entry point, placed by both observers. A value of <.40 is reported as poor, .40–.59 fair, .60–.74 good and .75–1.00 as excellent . This group required either primary treatment of tumours (n = 5) or replacement of a mechanically failed conventional reconstruction plate (n = 3). The latter consisted of two patients with a broken conventional 2.7 RP and one patient with pulled out screws, causing loosening of the conventional 2.7 RP and the locking screws. Table A total of eight patients who had either already undergone reconstruction of a continuity resection of the mandible or were scheduled to undergo one presented to our centre , by 42% up to 153%, indicating plastic deformation would occur on applying the load case. The plates would therefore not return to their original shape after loading. Furthermore, the stress in these plates exceeded the material's ultimate strength (US) value by 13% up to 100%, indicating a high risk of plate fracture. The application of our PS reconstruction to the latter patient resulted in a decrease in the YS and US percentages, going from 253% to 59% and 200% to 55%, respectively  would lower the resultant screw forces to 184 N, 144 N and 90 N, respectively, which represents a minimisation of 58%, 66% and 20%. The axial pull‐out force components, the forces along the longitudinal screw directions, did not exceed 180 N for the conventional reconstructions or 90 N for the PS reconstructions.3.4All the PS bookshelf‐reconstruction plates were inserted in accordance with the 3D‐VSP. The surgical procedures were uneventful. During surgery, prior to the drilling of screw pilot holes, the mandibular bone was denuded and the guides were positioned and fixed using 1.5‐mm mini screws (KLS Martin). Subsequently, the osteotomies were performed. Thereafter, the plate was inserted and fitted to the mandibular segments using several centring pins. Once properly aligned, these pins were replaced one by one by 2.3‐mm locking screws with a length in agreement with the surgical planning. Primary closure was performed according to plan in two patients. In the remaining six patients, a pectoralis major flap was used to reconstruct the soft tissue defect and to cover the plate.3.5SD: 9.3, R: 3–29). One patient, however, patient 5 in Table R: 7–29) and was uneventful with regard to plate failure or screw pull‐out.Recovery was uneventful from a mechanical point of view for seven patients with a mean of 11.4 days of hospitalisation  for a total of 64 screws. The mean angular screw deviation was 5.76 degrees . The 95% confidence interval of the inter‐observer variability for our measurements was 0.15–0.26 mm with a P‐value of .05. The interclass correlation coefficient (two‐way mixed) was 0.97, indicating an excellent match of measurements by both observers.Screw entry point deviation (3D) resulted in a mean value of 1.54 mm , and the surrounding soft tissue could be closed Figure . By comp4In this pilot study, we present a unique patient‐specific bookshelf‐reconstruction plate for accurate bridging of mandibular continuity defects. The novel design was based on the FEA of the conventional reconstruction plate failure, with regard to plate fractures and screw loosening. Application of bookshelf‐like flanges, with a screw fixation in the osteotomy sites, resulted in substantial reduction of resultant screw pull‐out inducing forces and, in combination with a change in material, lowered plate stress to within satisfactory levels. Therefore, the chances of the widely reported mechanical problems seen with conventional RPs, which are responsible for 5%–10% of reconstruction failures, were reduced  and angular screw deviation of 5.77 degrees (SD 3.33) indicate this method could be applied as a reliable one‐phase procedure for resection and direct reconstruction of a tumour in the mandible. These results represent the accumulation of errors in all visualisation and segmentation steps as well as geometrical errors of the guides and plate and the actual surgical procedure.The addition of our bookshelf‐flange concept is only of value, in terms of mechanical stability and reduction of the risk of failure, when applied accurately to the planned position. First, a per‐operative visual inspection after guided placement confirmed that no gap remained between the flange and the mandibular bone at the osteotomy sites. Second, postoperative analysis of CBCT scans showed a high accuracy of placement, with 3D deviations comparable to our prior studies and of others, using surgical guides (Kraeima, Dorgelo, et al., Over the last two decades, a rapidly increasing number of PS‐RPs have been presented in the literature with an equal increase in varying finite element analysis models. Applying FEA is of great importance in the design process, since PS‐RPs can still fail mechanically when designed using incorrect assumptions (Li et al., We expect the dehiscence of the cranial section of the ventral flanges, that occurred in two patients, to be caused by the height of the flanges in combination with contraction of the covering soft tissue. Minimising the bulkiness and height of these flanges might exclude this occurrence. A gradual remodelling of the mandibular angle was observed in one patient past the implant border. The reconstruction plate could have shielded this particular region of the mandible mechanically, causing bone remodelling to occur. Stress‐shielding, also seen in conventional reconstruction plate reconstructions, might explain this resorption. Future application of the topology optimisation technique (Iqbal et al., 5Using the finite element method, we retrospectively analysed mechanically failed conventional reconstruction plates and developed an alternative reconstruction plate for the mandible that reduces the chance of screw pull‐out and plate fracture. During this phase‐one study, we successfully reconstructed mandibular continuity defects using our bookshelf‐reconstruction plate concept in eight patients and no mechanical failures have occurred in the study cohort. This novel design of reconstructive plates seems to reduce the risk of screw pull‐out and plate fractures.There is no conflict of interest to report.All authors were involved in the study design and critical reading of the manuscript. Merema and Kraeima analysed the data and Merema, Kraeima and Witjes dr.Video S1Click here for additional data file.


Human-induced  salinization affects aquatic biodiversity and ecosystem functioning worldwide. While agriculture or resource extraction are the main drivers of secondary salinization in arid and semi-arid regions of the world, the application of deicing road salt in winter can be an important source of salts entering freshwaters in cold regions. Alpine rivers are probably affected by salinization, especially in highly populated mountain regions, although this remains to be explored. In this study, we analyzed multi-year conductance time series from four rivers in the European Alps and demonstrated that the application of deicing road salt is linked to peaking rivers’ salinity levels during late winter/early spring. Especially in small catchments with more urban surfaces close to the rivers, conductance increased during constant low-flow periods in late winter and was less correlated with discharge than in summer. Thus, our results suggest that small rivers highly connected to urban infrastructures are prone to considerable salinity peaks during late winter/early spring. Given the low natural level of salinities in Alpine rivers, the aquatic biodiversity might be significantly affected by the recorded changes in conductance, with potential consequences on ecosystem functioning. Thereby, we urge the research community to assess the impact of secondary salinization in Alpine rivers and call for an implementation of management practices to prevent the degradation of these pristine and valuable ecosystems.The online version of this article (10.1007/s11356-020-11077-4) contains supplementary material, which is available to authorized users. Freshwater habitats  are threatened by secondary  salinization worldwide . The ecological effect of relative increases in salinity needs to be considered within the context of biodiversity conservation in Alpine freshwaters, since aquatic organisms in these ecosystems have evolved under relatively low salinities  and agriculture for economic development. During winter, snowfall and cold conditions can cause icy roads. In most regions of the European Alps, the use of deicing salts to maintain clear road and other urban pavements free of ice during winter is a common practice. For example, in the Alpine region of Tyrol, Austria, an average of 30,000 tons of salt (sodium chloride) are applied annually to approximately 2240 km of roads . During periods of snow-removal from the roads (which is partly poured into rivers and stored next to them), and especially during melting and precipitation events, these salts might enter the rivers, as it has been shown for a northern Italian catchment , 5243 km2 (site B), 327 km2 (site C), and 198 km2 (site D), respectively. The catchment geology of sites A, B, and C is dominated by non-limestone bedrock, while stream D drains a catchment with calcareous bedrock (Brandner 3/s (1983–2015) at site A, 11.3 m3/s at site C (1951–2015), and 7.6 m3/s at site D, while it was much higher (165 m3/s) at site B (1971–2015) , that drain catchments with and without glacial influence Fig. . The monBrandner . BesidesMean daily discharge data were obtained from the Hydrographisches Jahrbuch 2015  can often be used as a proxy for the concentration of dissolved ions , wetlands, water bodies, and glaciers.ts function). Consequently, a “Seasonal and Trend decomposition using Loess” (STL) was performed . We performed regression diagnostics to check model performance . Such time series decomposition was only possible for site A (2012–2019), while records from the other sites were too short. Within years, we discriminated between winter and summer periods. “Winter” was defined as the period when winter tires were mandatory for cars in the region (November 1 until April 15), while “summer” was set as the period with high meltwater runoff (May 15 until September 15). The discrepancy in specific conductance between these seasons was expressed in absolute and relative differences. Cross-correlation on previously pre-whitening time series was used to assess the relationship between discharge and conductivity . Given its small size, and contrary to relative differences, site D had the smallest catchment area covered by urban surfaces  was identified as the dominant land cover type in all catchments (73–92%). The catchments of sites C and D had higher proportions of non-natural areas, with 18% and 20.4% of agricultural surfaces, and 7.9% and 5.7% of urban surfaces for sites C and D, respectively  for 11 μs cm−1, while long-term discharge showed a non-significant decrease  and 196.8 μs cm−1 (August 2018). After removing the multi-year trend and averaging monthly differences of all the studied years, we found that electrical conductance was up to 38.0 μs cm−1, 85.7 μs cm−1, or 40.9 μs cm−1 higher than the running average in March or February for sites A, B, and C, respectively. In contrast, the highest positive deviation of mean monthly conductance occurred in November at site D (44.9 μs cm−1 above running average) and the highest negative in April (with − 74.3 μs cm−1 below running average). Mean monthly deviations during summer were low or negative in all streams, with the highest negative deviations observed between April and June, depending on the study site . Conductance and discharge exhibited antagonist seasonal fluctuations, with high discharge and low conductance during summer in most streams, and vice versa during winter  in streams at much higher levels than reported here can reduce the biomass of autotrophic standing crops and the diversity of benthic algae and invertebrates  in soils , provides evidence that ion concentrations in Alpine streams peak during the end of winter and underline the importance of continuous and high-frequent salinity measurements in Alpine rivers. The changes in electrical conductance in winter during long and constant low-flow periods, especially in smaller catchments with low pollutant dilution capacity


Axial length was positively correlated and keratometry and lens power were negatively correlated with age in non-syndromic craniosynostosis and controls, while these correlations between age and ocular biometric parameters were not present in the FGFR-related syndromic craniosynostosis. In conclusion, ocular biometric parameters in FGFR-related syndromic craniosynostosis differed from those of non-syndromic craniosynostosis and age-matched controls, and did not show the relations with age, suggesting this cohort may have abnormal refractive growth.Ametropia is reported as a common ophthalmic manifestation in craniosynostosis. We retrospectively compared childhood refractive error and ocular biometric features of fibroblast growth factor receptor (FGFR)-related syndromic craniosynostosis patients with those of non-syndromic craniosynostosis and control subjects. Thirty-six eyes (18 patients) with FGFR-related syndromic craniosynostosis, 76 eyes (38 patients) with non-syndromic craniosynostosis, and 114 eyes (57 patients) of intermittent exotropes were included in the analysis. Mean age at examination was 7.82 ± 2.51  years and mean spherical equivalent was -0.09 ± 1.46 Diopter. Mean age and refractive error were not different between groups, but syndromic craniosynostosis patients had significantly longer axial length, lower corneal power, and lower lens power than other groups ( Other forms of craniosynostosis are genetic syndromes caused by genetic mutations in fibroblast growth factor receptors (FGFRs), TWIST1, EFNB1, TCF12, MSX2, or ERF.3 Syndromic craniosynostosis is characterized by complex craniofacial deformities with or without malformation of the extremities and neurocognitive defects3. Because the incidence of syndromic craniosynostoses such as Crouzon and Apert syndromes are as low as 16.5 and 12.5 per million births, respectively5, only a few studies have evaluated the ophthalmological features of these patients.Craniosynostosis is a rare and heterogeneous disorder characterized by premature fusion of the skull plates and subsequent cranial deformities. The majority of craniosynostosis patients are isolated form (up to 85%), which is thought to be multifactorial in nature9. According to prior studies, hypermetropia is the most common type of ametropia in both non-syndromic and syndromic craniosynostosis10. However, the age at examination and definition of significant refractive error  differ between studies. Moreover, prior studies were not controlled for environmental factors and ethnic differences, which can affect refractive error13. Thus, the impact of syndromic and non-syndromic craniosynostosis on refractive error remains to be elucidated.Ophthalmic pathologies including optic atrophy, refractive error, amblyopia, and strabismus had been described in both isolated and syndromic craniosynostosis patientsIn the present study, we aimed to compare the refractive error and ocular biometric characteristics of pediatric FGFR-related syndromic craniosynostosis patients with those of non-syndromic craniosynostosis and control subjects. Furthermore, we analyzed the correlations of ocular biometric measurements with age to determine differences in the age related distribution of refractive components between the three groups.FGFR2 gene according to the 2015 American College of Medical Genetics and Genomics and Association for Molecular Pathology guidelines14. The patient with Muenke syndrome had a pathogenic or likely pathogenic mutation in FGFR3. Genetic mutations in syndromic craniosynostosis patients are summarized in Table A total of 113 patients (226 eyes) with mean age of 7.82 ± 2.51  years were included: 36 eyes from 18 FGFR-related syndromic craniosynostosis patients, 76 eyes from 38 non-syndromic craniosynostosis patients, and 114 eyes from 57 intermittent exotrope control subjects. Of the FGFR-related syndromic craniosynostosis, 14 were Crouzon Syndrome, two were Pfeiffer syndrome, one was Apert syndrome, and one was Muenke syndrome. All patients with Crouzon, Pfeiffer, and Apert syndrome carried pathogenic or likely pathogenic mutations in the p = 0.17). The mean age at cranial vault remodeling procedure in syndromic craniosynostosis and non-syndromic craniosynostosis group were 20.9 ± 24.6 and 18.1 ± 20.0 months respectively, and there was no difference in the mean age at cranial vault remodeling procedure between the two groups (p = 0.98). Overall, 10 out of 18 patients with syndromic craniosynostosis and 19 out of 38 patients with non-syndromic craniosynostosis patients underwent expansion cranioplasty before the first year of age. The proportion of patients who underwent expansion cranioplasty before the first year of age was not different between syndromic and non-syndromic craniosynsostosis groups (p = 0.78).In patients with non-syndromic craniosynostosis, lamdoid suture (3/38), metopic suture (3/38), sagittal suture (12/38), coronal suture (14/38), and combined suture involvement (6/38) were observed. Cranial vault remodeling procedure was performed in all of the syndromic craniosynostosis patients and in 35 out of 38 non-syndromic craniosynostosis patients. The proportion of cranial vault remodeling surgery was not significantly different in two groups (p = 0.94). The frequency of optic atrophy (p = 0.11) and amblyopia (p = 0.39) was also not different in each group. However, corneal opacity (p < 0.01) and strabismus (p < 0.01) were more frequently observed in syndromic craniosynostosis than in non-syndromic craniosynostosis group. In this series, all cases of corneal scarring were visually insignificant because corneal scarring in syndromic craniosynostosis only involved the inferior cornea.We’ve compared the visual acuity and presence of ophthalmic complications such as amblyopia, optic atrophy, corneal opacity and strabismus in FGFR-related syndromic craniosynostosis and non-syndromic craniosynostosis patients . Mean keratometric value was significantly smaller in syndromic craniosynostosis patients (42.55 ± 1.26 Diopter) than that in non-syndromic ones  and lens power was also lower in syndromic group (23.11 ± 2.18 Diopter) than in non-syndromic group . Then we performed a subgroup analysis according to the age at cranial vault remodeling procedures: early surgery group  versus late surgery group . In early surgery group, mean keratometric value (p = 0.02) and lens power (p < 0.01) was still significantly lower in syndromic craniosynostosis patients than in non-syndromic group. However, mean value of axial length was not significantly different between the syndromic and non-syndromic craniosynostosis patients (p = 0.14). In late surgery group, axial length was significantly longer (p = 0.02) and mean keratometric value was significantly lower (p < 0.01) in syndromic craniosynostosis patients than in non-syndromic group. But, mean keratometric value was not significantly different between the syndromic and non-syndromic craniosynostosis patients (p = 0.13).When we performed subgroup analysis for biometric values of the patients who underwent cranial vault remodeling procedures, axial length was significantly longer in syndromic craniosynostosis (23.73 ± 0.84 mm) than that in non-syndromic group  and control , while there was no significant difference between the mean value of axial length from young subgroups of non-syndromic craniosynostosis and control subject (p = 0.66). In contrast, axial length did not differ between the groups in older patients. The mean axial length in older subgroup of syndromic craniosynostosis, non-syndromic craniosynostosis, and control subjects were 23.57 ± 0.75 mm, 23.86 ± 0.88 mm, and 23.44 ± 0.90 mm, respectively . In younger subgroup, mean keratometric value and mean lens power were significantly lower in syndromic craniosynostosis patients than that in non-syndromic craniosynostosis  and control . Contrastingly, in older subgroup, there was no significant difference between the mean keratometric value and mean lens power of syndromic, non-syndromic craniosynostosis, and control subjects .When patients were subgrouped into younger (4–8 years) and older (≥ 9 years) age groups, mean axial length was significantly longer in the young syndromic craniosynostosis subgroup (23.79 ± 0.88 mm) relative to the young subgroups of non-syndromic craniosynostosis ] and control subjects [spherical equivalent = -0.13*Age + 0.95 ]. However, no such correlation was detected in syndromic craniosynostosis patients [spherical equivalent = 0.06*Age – 0.24 ]. The distribution of axial length was positively correlated with age in the non-syndromic craniosynostosis [axial length =  + 0.19*Age + 21.60 ] and control group [axial length =  + 0.14*Age + 21.99 ]. However, no significant age-axial length correlation was detected in syndromic craniosynostosis patients [axial length = 0.05*Age + 23.38 ]. Age and mean keratometric value were negatively correlated in the non-syndromic craniosynostosis [mean keratometry = -0.16*Age + 44.78 ] and control groups [mean keratometry = -0.13*Age + 44.64 ], while no significant age-related decrease of keratometric value was noted in the syndromic craniosynostosis group [mean keratometry =  + 0.10*Age + 41.77 ]. Age and lens power were negatively correlated in non-syndromic craniosynostosis [lens power = -0.27*Age + 26.89 ] and control subjects [lens power = -0.19*Age + 25.63 ]. In contrast, age and lens power were not correlated in syndromic craniosynostosis subjects [lens power = -0.11*Age + 23.92 ].To delineate the differences in age-specific distribution of refractive error and ocular biometric values from the syndromic craniosynostosis, non-syndromic craniosynostosis, and control groups, linear regression analysis was performed Fig. . Spheric10. To the best of our knowledge, this is the first comparative study of refractive errors in FGFR-related syndromic and non-syndromic craniosynostosis. In the present study, we demonstrated that pediatric FGFR-related syndromic craniosynostosis is characterized by longer axial length, lower mean keratometric value, and lower lens power than the non-syndromic craniosynostosis and control groups, while mean refractive error was not significantly different between the groups.Ametropia is consistently suggested as one of the major ophthalmic manifestations of both syndromic and non-syndromic craniosynostosis, but age and ethnic group-controlled comparative studies evaluating the refractive errors of syndromic and non-syndromic craniosynostosis patients have not reported yetMean anterior chamber depth and anterior chamber depth/axial length ratio were not significantly different between the groups. Taken together, the lower refractive index of the cornea and lens dampen the potential myopic defocus due to longer axial length in syndromic craniosynostosis.16. Although syndromic craniosynostosis patients had longer mean axial length, this age-related trend was not observed in the syndromic craniosynostosis group. Moreover, inter-group axial length difference was only significant in the younger (4–8 years) subgroup. During longitudinal axial growth, rapid axial growth occurs before 5 years of age, and a slower growth phase then ensues until 13 years of age17. Therefore, FGFR-related syndromic craniosynostosis patients could have had a more prominent early axial growth spurt than non-syndromic craniosynostosis and control groups.Age-dependent myopic shift was not prominent in syndromic craniosynostosis patients compared to non-syndromic craniosynostosis and control subjects. In terms of age-related distribution of ocular biometric components, the non-syndromic craniosynostosis and control groups exhibited age-dependent axial elongation as previously reported19. Age-related elongation of axial length and accompanying deterioration of corneal and lens power could be explained by emmetropization. According to our study, unlike the non-syndromic craniosynostosis and control groups, the age-related deterioration of keratometric value and time-dependent loss of lens power were not noted in the syndromic craniosynostosis group. Therefore, the authors have assumed that the emmetropization mechanism is involved in the stable axial length and corneal/lens refractive indices observed in syndromic craniosynostosis subjects.Loss of cornea and lens power in school-age children has been well documented by several longitudinal studiesFGFRs (FGFR1-3), TWIST, and MSX2 gene mutations are the major causes of syndromic craniosynostosis. Common genetic syndromes associated with craniosynostosis, including Crouzon-Pfeiffer syndrome, Apert syndrome, and Muenke syndrome, are caused by pathogenic mutations in FGFRs. Most of the pathogenic FGFR mutations in syndromic craniosynostosis are gain-of-function mutations21. Fibroblast growth factors (FGFs) are a family of growth factors that exert diverse physiological roles via FGFRs. FGFs are essential factors for ocular development during both prenatal and postnatal periods. FGFRs are well known to be expressed in the mammalian and avian sclera23. Thus, FGFs are thought to be involved in the axial growth of ocular dimensions. However, there are contradictory results on whether excessive FGFs could induce axial refractive growth25. FGFs are also known as crucial factors for lens epithelial growth and differentiation28, but the role of FGFs in refractive growth of the crystalline lens is largely unknown. In the present study, all of the syndromic craniosynostosis subjects were genetically proven to have pathological or likely pathological mutations of FGFR2/3. Considering that non-syndromic craniosynostosis patients shared the same refractive growth pattern as control patients, the unique ocular biometric characteristics of FGFR-related syndromic craniosynostosis could be at least partly attributed to overactivation of FGFRs.There are some limitations of the study that should be acknowledged to avoid overinterpretation of the findings. First, due to the cross-sectional nature of the study, a head-to-head comparison of time dependent refractive growth patterns between study groups could not be performed. A longitudinal study analyzing the time-dependent change of refractive error and ocular biometric values should be conducted to confirm the characteristic refractive growth pattern of syndromic craniosynostosis patients suggested by the present study. Second, FGFR-related syndromic craniosynostosis is a rare disease entity and is often accompanied by cognitive dysfunction, which is an obstacle for measurement of ocular biometric values. Therefore, fewer syndromic craniosynostosis subjects were available for analysis than non-syndromic craniosynostosis and control subjects.In conclusion, although mean refractive error of pediatric FGFR-related syndromic craniosynostosis patients did not differ from that of non-syndromic craniosynostosis and control subjects, pediatric FGFR-related syndromic craniosynostosis patients had longer axial length, flatter keratometry, and lower crystalline lens power than other two groups. Age-associated distribution of refractive error and ocular biometric values in pediatric FGFR-related syndromic craniosynostosis patients was also strikingly different from that of non-syndromic craniosynostosis and control subjects. Early axial elongation and stable biometric values throughout adolescence can be characteristic of pediatric FGFR-related syndromic craniosynostosis patients.We retrospectively reviewed the medical records of craniosynostosis patients who were referred to a pediatric ophthalmology clinic of Ajou university hospital for ophthalmological evaluation between March 2019 and February 2020. The institutional review board (IRB) of Ajou university hospital reviewed and approved the study . This study was performed in accordance with institutional guidelines of Ajou University Hospital and Informed consent was waived by the IRB of Ajou university hospital because of the retrospective nature of the study.FGFR1, FGFR2, and FGFR3 hot spot mutations was performed for genetic diagnosis of FGFR-related syndromic craniosynostosis. In patients with craniosynostosis, a comprehensive ocular examination, including ocular alignment, refractive errors, biometric measurements, slit-lamp examination, and fundus examination, were performed. Refractive errors were measured by cycloplegic refraction and ocular biometry  using a partial coherence interferometry, IOL Master 500 device . During the study period, cycloplegic refraction and ocular biometric measurements were routinely performed in all new patients that were diagnosed with intermittent exotropia at the pediatric ophthalmology clinic. These patients with intermittent exotropia were served as non-craniosynostosis controls.The diagnosis of craniosynostosis was made at the Department of Medical Genetics and Department of Pediatric Neurosurgery in Ajou university hospital after detailed clinical, radiological examination, and genetic evaluation. Systemic abnormalities such as maxillofacial dysmorphism, ear anomaly, cleft lip and palate, hearing problem and anomalies of the phalanges were carefully checked by medical geneticists to rule out syndromic craniosynostosis. Sanger sequencing analysis of Craniosynostosis patients and intermittent exotropia patients that underwent measurement of refractive error under cycloplegia and ocular biometry during the study period were included. Clinical information, including diagnosis, history of systemic and ocular disease, gender, age, refractive error, axial length, keratometric value, and anterior chamber depth, were collected. We’ve reviewed the visual acuity and presence of ophthalmic complications such as amblyopia, optic atrophy, corneal opacity and strabismus in FGFR-related syndromic craniosynostosis and non-syndromic craniosynostosis patients. For patients with FGFR-related syndromic craniosynostosis, genetic information was also collected. Patients with a history of systemic disease other than craniosynostosis syndrome or with a history of ocular disease other than intermittent exotropia were excluded. Patients in which biometric values were not measured due to poor cooperation or with low signal-to-noise ratio values (less than 2.1) in IOL master measurement were also excluded.29. We comprehensively reviewed the literature and ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/) to determine the functional consequences of the FGFR mutations detected in each patient.Crystalline lens power was estimated based on refractive error and ocular biometric values using Bennett–Rabbetts methodsCategorical clinical characteristics of syndromic craniosynostosis, non-syndromic craniosynostosis, and control eyes were compared by chi-square test. Mean value of refractive errors and biometric measurements from each groups compared by Mann–Whitney U test. The mean value of each variable was represented as mean ± standard deviation. A one-way ANOVA test assuming unequal variances was performed to compare the rates of refractive growth between the three groups. Linear regression was used to determine if the rate of refractive growth was correlated with changes in biometric values in the three groups.Supplementary Information