Aim of this study is to investigate bacterial growth on non-infected devices and compare antibiotic-coated and non-coated implants.The charts of 71 patients who underwent revision surgeries for penile prosthesis between 1995 and 2013 were reviewed. Of those, 31 devices were antibiotic-coated prostheses, while 40 of the implants were non-coated. Swab cultures were routinely obtained from corporal, pump or reservoir site during the operation. If a bacterial biofilm was determined on the prosthesis, it was also cultured.A total of 5 different organisms were cultured from 18 patients. Of them, 4 devices were antibiotic-coated and the other 14 were non-coated devices. Staphylococcus epidermidis was the most common organism, while Staphylococcus hominis, beta hemolitic streptococcus, Escherichia coli and Proteus mirabilis were also cultured. All patients who had positive cultures were treated with appropriate antibiotics for four weeks postoperatively. Median follow-up time was 41 months, ranging between 8 and 82 months. One prosthesis (non-coated) became clinically infected in the follow-up period with a totally different organism. Culture positivity rates of antibiotic-coated and non-coated devices were 13% and 35% respectively and the result was significant (p=0.00254).Positive bacterial cultures are present on non-infected penile prostheses at revision surgeries in some of the patients. Antibiotic coated prostheses have much less positive cultures than non-coated devices. Penile implants have been used for more than 40 years and are the standard of care for erectile dysfunction in patients who do not respond to medical treatment. The morbidity rate associated with penile implants is relatively low, and patient satisfaction is high owing to improvements in techniques, materials and design. Mechanical failure, which is the primary cause of reoperations following penile prosthetic surgery, has an incidence ranging from 0–56% in large series –4. The mTreatment of PPIs is difficult and costly, and the prevention of infections is accordingly of utmost importance. In the last 10 years, there have been some technical and device-related modifications that have been adopted to reduce the risk of infection associated with penile prosthesis surgery . After 2In this study, we aimed to investigate bacterial growth on non-infected penile prostheses. We also compared antibiotic-coated and non-coated implants during the revision surgery.This study was a retrospective evaluation of prospectively followed patients who underwent penile prosthesis revision surgery due to non-infected causes. We prospectively obtained cultures in a standard manner from patients who underwent either revision or explantation of clinically non-infected penile prostheses. We focused on a total of 71 consecutive patients who underwent revisions for clinically non-infected reasons between 1995 and 2013. The patients underwent revision surgery for mechanical failure in 57 cases (80.2%), cylinder aneurysms in 3 cases (4.2%), impending erosion of the device in 4 cases (5.6%), patient dissatisfaction in 4 cases (5.6%), and undersizing in 3 cases (4.2%). There was no evidence of clinical infection in any patients prior to the revisions, and all of the patients had negative urine cultures. Thirty-one of the devices were coated with antibiotic, while 40 were non-coated. Swab cultures were routinely obtained from the corporal, pump or reservoir site during the operation. The cultures were obtained from all of the accessible components after removal using swabs of the surface of the device and peri-prosthetic space. If a biofilm was detected on any part of the prosthesis, an additional swab culture was obtained from that area. The collected samples were enclosed in air-tight plastic tubing and then transported to the microbiology test laboratory. The swabs were inoculated on blood sheep agar and EMB agar plates and incubated for 48 hours. The agar plates were examined after 24 and 48 hours. Bacteria exhibiting growth were identified using standard techniques based on morphological, cultural and biochemical characteristics. Antimicrobial sensitivities were determined using the standard disc diffusion technique. Bacterial colony counts of the identified organisms were quantitated by our microbiology laboratory and reported.All of the surgical procedures were performed by a single surgeon (MMC) according to standard surgical protocols . There were no complications during any of the surgeries. On the day of the revision surgery, all of the patients underwent a 10-minute skin preparation procedure with a povidone-iodine scrub. Furthermore, all of the patients received perioperative intravenous antibiotics (a combination of amikacin 500mg and teicoplanin 400mg). During the procedure, antibiotic irrigation was routinely performed on the surgical sites. A spray solution containing antibiotics was only used on the non-coated devices. All of the patients continued to receive intravenous antibiotics postoperatively until discharge. Fluoroquinolones were also prescribed for 7 days after discharge. All of the patients who had positive cultures were treated with appropriate antibiotics for four weeks postoperatively. After the revision surgery, urine and blood cultures were obtained from the symptomatic patients to evaluate the incidence of postoperative infections.Different types of three-piece hydraulic prostheses were evaluated in this study. Either AMS  or Coloplast  penile prostheses were used in all of the patients. The prostheses not coated with antibiotics included AMS-CX, AMS-Ultrex and Mentor Alpha devices; the antibiotic-coated prostheses included AMS-CX and AMS-LGX-devices. The non-coated implants were old devices; they were used prior to 2000 and did not absorb antibiotics.All of the data were collected from the hospital record system, and the study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.All of the collected data were entered and analyzed using the SPSS 17.0 statistical program . We used an independent sample t-test and Fisher's exact tests to compare the groups. Statistical significance was set at a p value of <0.05.A comparison between the antibiotic-coated and non-coated groups is listed in Within the antibiotic non-coated group, 20 patients had AMS-CX devices, 8 patients had AMS-Ultrex devices, and 12 patients had Mentor Alpha devices; in the antibiotic-coated group, 26 patients had AMS-CX devices and 5 patients had AMS-LGX devices.While design modifications have decreased the incidence of mechanical failure, prosthesis infection remains a significant complication of penile implants. The incidence of PPIs associated with primary implantation is estimated to be between 1% and 3% for first implantation; the incidence of infection is higher after reoperation for revision or re-implantation , 7. BecaDespite reducing the risk of infection with preoperative skin preparations, perioperative prophylactic antibiotics, copious irrigation with antibiotics and minimizing device-skin contact, the bacteria remains dormant on implants and may result in late prosthesis infection at later times. If revision of the device is necessary, the sequestered bacteria may be released and result in increased risks of infection in the new device . This siClinically apparent infections are related to the virulent species of bacteria and the host defense mechanisms. Patients with diabetes mellitus are more susceptible to developing infections as a result of high glucose levels that encourage bacterial growth. However, the number of patients with diabetes mellitus was not different significantly between our two study groups.It has been shown that antibiotic-coated penile prostheses are associated with a lower risk of infection than non-coated devices. A recent study reported that antibiotic impregnated devices have a 1.62% infection-related revision rate; non-impregnated penile prostheses have a 4.24% infection-related revision rate at a 7 year follow-up. This difference was statistically significant . The autBacteria are also cultured from the penile prostheses that were explanted during a revision surgery owing to non-infected causes. Licht et al. obtained cultures from 65 clinically non-infected penile prostheses and 22 artificial urinary sphincters during revision. These authors found that 43% of clinically non-infected penile prostheses (28 out of 65) exhibited a positive culture during revision surgery . StaphylSkin flora are the most common source of PPIs; those organisms are introduced at the time of device implantation. Staphylococcus epidermidis is the most common organism cultured in infected prostheses, and it accounts for 35–80% of all positive cultures . ParsonsThis study had a few limitations. First, although the patients were followed prospectively, the data were collected retrospectively from hospital records. Second, there have been no similar and comparable studies in the literature with a large volume of data pertaining to non-infected penile prosthesis revision surgery and comparing antibiotic-coated and non-coated devices. Despite these limitations, this study for the first time clearly showed that antibiotic-coated devices result in less bacteria colonization on the implant and peri-prosthetic space compared with non-coated devices, a situation that may decrease the incidence of PPIs.Based on our results, positive bacterial cultures are present on non-infected penile prostheses during the revision surgeries of some patients. Our results also suggest that, antibiotic-coated penile prostheses have less bacteria seeding on prostheses in revision surgeries. Therefore, antibiotic coating may prevent late PPIs, particularly after revision surgeries. We also confirmed that, Staphylococcus epidermidis was the most common cultured organism from uninfected penile prostheses during revisions.


Characterization of land cover change in the past is fundamental to understand the evolution and present state of the Earth system, the amount of carbon and nutrient stocks in terrestrial ecosystems, and the role played by land-atmosphere interactions in influencing climate. The estimation of land cover changes using palynology is a mature field, as thousands of sites in Europe have been investigated over the last century. Nonetheless, a quantitative land cover reconstruction at a continental scale has been largely missing. Here, we present a series of maps detailing the evolution of European forest cover during last 12,000 years. Our reconstructions are based on the Modern Analog Technique (MAT): a calibration dataset is built by coupling modern pollen samples with the corresponding satellite-based forest-cover data. Fossil reconstructions are then performed by assigning to every fossil sample the average forest cover of its closest modern analogs. The occurrence of fossil pollen assemblages with no counterparts in modern vegetation represents a known limit of analog-based methods. To lessen the influence of no-analog situations, pollen taxa were converted into plant functional types prior to running the MAT algorithm. We then interpolate site-specific reconstructions for each timeslice using a four-dimensional gridding procedure to create continuous gridded maps at a continental scale. The performance of the MAT is compared against methodologically independent forest-cover reconstructions produced using the REVEALS method. MAT and REVEALS estimates are most of the time in good agreement at a trend level, yet MAT regularly underestimates the occurrence of densely forested situations, requiring the application of a bias correction procedure. The calibrated MAT-based maps draw a coherent picture of the establishment of forests in Europe in the Early Holocene with the greatest forest-cover fractions reconstructed between ∼8,500 and 6,000 calibrated years BP. This forest maximum is followed by a general decline in all parts of the continent, likely as a result of anthropogenic deforestation. The continuous spatial and temporal nature of our reconstruction, its continental coverage, and gridded format make it suitable for climate, hydrological, and biogeochemical modeling, among other uses. Determining the spatial structure of land cover and its variation through time is essential in order to understand the interplay between biosphere, atmosphere, and human societies. Knowledge of past vegetation dynamics is of great interest to a range of disciplines dealing with landscape, climate, human development, and their reciprocal interactions. For example, information concerning past forest cover influences the archeological narrative e.g.,  and playRecent landscape history makes use of different mapping technologies, ranging from historical and cartographic sources e.g.,  to satelSemi-quantitative models have been developed by grouping individual taxa into plant functional types (PFTs) and biomes , thus prA first attempt to achieve a real quantification was pioneered by Pollen-based vegetation reconstructions produced with the LRA are currently available for most of Europe outside of the Mediterranean e.g., . A widerIn the present study, we test the capabilities of the MAT for continuous forest-cover reconstructions at a continental scale and from the Pleistocene/Holocene transition to the present day. We follow the methods used by r2 and Root Mean Square Error of Prediction (RMSEP) from cross-validation exercises]. In addition, down core evaluation was also undertaken through a comparison between the MAT and the REVEALS-based forest-cover reconstructions for specific sites, thus testing the performance of our method against methodologically independent forest-cover reconstructions.Our results are presented here as both maps for the whole of Europe at 1,000-year intervals, and as regional area-average time-series. The full set of 49 maps, each one covering an ∼250-year interval, is available as Supplementary Material. Each map displays interpolated forest-cover data with continental coverage at a resolution of five arc-minutes. The predictive ability of our model was assessed though standard statistical indicators based on analysis of the modern training set [1 and 14C chronologies, with those for EPD sites based on the latest available chronologies from The fossil pollen dataset used in our analysis is the same as presented in The European Modern Pollen Database EMPD;  was selePollen spectra in both the modern and fossil datasets were converted into PFTs following the approach developed by Modern forest-cover values were extracted from the Global Forest Change dataset , which iThe average forest-cover values for the modern pollen data set were extracted by placing a search window around the location of each sample. We applied a general search radius of 50 km to approach a more consistent comparison between MAT and REVEALS estimates, because the REVEALS method suggests that its reconstructions are representative of a 50-km radius around the sites in question (see section “Comparison between MAT and REVEALS Estimates of Past forest cover”). A circular search window was preferred over a square one , between ∼30 and >80% of successful pollinations were ascribed to pollen coming from within the stand . Computational constraints, the wide diversity of the EMPD samples and, above all, the current availability of metadata prevent the calculation of sample-specific search windows and weight factors within this study. The application of narrow distance-weighting parameters (100 m and 500 m) was prompted by empirical and simulation studies on pollen deposition dynamics e.g., . Local ff ∼200 m . When smhe stand . Surfacend sensu. In the Study-specific definitions of forest cover should be kept in mind when interpreting and comparing the results from different quantitative approaches. REVEALS-based estimates of forest cover correspond to the overall regional tree abundance expressed in relative percentage cover. Age and size of trees are not assumed to be critical factors influencing the results, although they can have an influence on pollen productivity estimates and pollen dispersal and deposition . Analog-2, accessed through the R package “elevatr”  in the filtered EMPD were then used to find potential inaccuracies in surface samples with insufficient metadata. Any sample with arboreal pollen > 80% associated to forest-cover values < 25% was considered as potentially carrying low-quality information and excluded from the model. Beside geolocation and chronological inaccuracies, this filter addresses also samples collected under isolated trees, or in small thickets not detectable by the forest-cover data set. These threshold values are drawn from Figure 1 and find support in the available literature. As an example, in open or semi-open contexts such as alpine meadows or arid shrublands, AP values are in the range of 40–50%  through multiple lines of evidence. After duplicate removal, samples with estimated geolocation errors higher than 100 m were excluded from further elaborations. An additional reliability check was performed by comparing the elevation of each sample as recorded in the EMPD with the elevation for the same latitude and longitude extracted from a high-resolution Digital Elevation Model. Control elevation values were obtained from Mapzen’s Elevation Serviceelevatr” . Any difelevatr”  was used0% e.g., . Even ab0% e.g., . Nonethe0% e.g., , samplesThe filtering process led to the removal of ∼52% of the EMPD content, resulting in 2,526 usable samples  cross-validation. Reconstructed forest-cover percentages were then recalculated as the weighted average of the forest-cover values of the best analogs whose chord distance did not exceed a threshold T. The value of T is not straightforward to define since it is data dependent . Each timeslice covers a 250-year window with the exception of the most recent one, which is asymmetric as it cannot project into the future, and therefore covers an interval of 185 years centered on 0 BP.The reconstructed forest covers from each pollen record  within each timeslice have been interpolated onto a uniform spatial and temporal grid using a four-dimensional  Thin Plate Spline algorithm (TPS) fitted to a Digital Elevation Model (five arc-minutes resolution) using the R package ‘fields’ . A threeThe maps of forest cover also include changes in European coastlines and ice sheets throughout the Holocene following To provide a comparison with traditional approaches based on the interpretation of arboreal pollen (AP) percentages, we also applied the same interpolation procedure to AP values  for every fossil timeslice. The complete series of 49 forest-cover maps and 49 AP maps is included in the Supplementary Materials  divided by the square root of n. This first interpolated error estimate was then added to the standard error generated by the interpolation process itself  and area-average estimates of forest cover were calculated for each region based on the gridded maps. The region borders are loosely based on the European biogeographic areas proposed by In order to provide regional summaries of forest cover and AP changes, the study area was divided into eight regions  into 200-vertical-meter elevation bands, except for those below 400 m which were grouped into a single band. This process was repeated for each of the 49 paleo-timeslices, as well as for the present day using both the LOO-based reconstruction and the map from The Alpine region provides an excellent context to test the performance of the 4-D interpolation due to its high topographic variability, the abundance of fossil pollen archives, and the numerous studies concerning local tree line and timberline dynamics. Therefore, as an additional test, we compared the MAT-based forest-cover values reconstructed at different elevations in the Alpine region with the data from As with the maps, areas with low site/sample density were excluded from the analysis. A lack of pollen sites at the very highest altitudes meant that for this test the analysis was limited to below 2,800 m throughout the Holocene, with a slightly lower limit of 2,400 m before 11,000 BP and 2,600 m from 10,000 to 11,000 BP when there were fewer sites above those altitudes.A basic definition of “forest” in alpine contexts includes a minimum canopy cover above 40% e.g.,  or 50–60Forest-cover reconstructions based on MAT have been evaluated against methodologically independent REVEALS reconstructions for selected fossil pollen records  in central and northern Europe. The REVEALS model  providesWe used five REVEALS target sites published in Several tests have been done to decrease the 500-year time intervals of the reconstructions in Abies, Alnus, Betula, Carpinus, Corylus, Fagus, Fraxinus, Juniperus, Picea, Pinus, Quercus, Salix, Tilia, and Ulmus, and represent the regional cover within 50 km around each target site.The REVEALS estimates for the 25 taxa were grouped into open-land and forest categories, and the associated standard errors were calculated again. The REVEALS forest covers include covers of r2 = 0.49) and that the RMSEP (20.7%) is lower than the standard deviation of the data set (29%). These values reflect the wide array of uncertainties affecting the model , yet they suggest that it can identify and reproduce a relationship between remote sensing data and pollen percentages. An analysis of the residuals of the two-fold cross-validation  highlights negligible bias in the 0–70% forest-cover range, with the median error lying well in the range of ±10% points. A larger discrepancy between observations and the reconstruction is especially visible at sites with very high forest cover (>80%), where the median reconstructed values tend to be more than 25% points lower than the observed forest cover. These differences might depend on a combination of factors. For instance, high-forest-cover samples (>80%) are the least represented in the calibration dataset, likely making them more prone to be affected by poor analogs. The lowest residuals are concentrated in southern Europe  shows generally similar trends . This level of agreement is notable, considering the quite different approaches of the two methodologies toward landscape reconstruction. Disagreements in absolute values constitute the main difference between the two models. The MAT assigns lower forest covers to early pioneering pine and birch woodlands ; during this phase, most of the closest modern analogs are chosen within the tundra biome or in proximity of the forest-tundra ecotone , pointing to a limited performance of the MAT in densely forested contexts. As noted in section “Performance of the Cross-validation Exercises,” the reasons behind this behavior might depend on a lack of suitable analogs in the training data set, in turn resulting from the low residual forest cover across present-day Europe. Different approaches toward forest-cover quantification might play a notable role too, i.e., sum of all forest-forming taxa, regardless of their habitus (REVEALS) vs. tree-height detections limits of the underlying forest cover map (MAT). Undetectable trees lower than 5 m might then account for at least part of the difference between the MAT and REVEALS curves.The comparison between MAT and REVEALS estimates  are recorded at around 8,000 BP, testifying further to limits in the training data set concerning the reconstruction of early Mid-Holocene forests. The occurrence of no-analog situations in our reconstructions is addressed in section “Quantification of No-analog Occurrences.”The agreement between MAT and REVEALS curves improves in the Late Holocene, when forest-cover decline brings the REVEALS values below 80%. The average difference in absolute values between the two reconstructions is reduced to ∼10–17 percentage points in the last 4,000 years of the sequences , highlighting the occurrence of missing analogs and prompting us to explore the extent of this issue. For an appropriate comparison, we ran the MAT algorithm for the whole fossil and modern data sets using selected pollen taxa  and PFT scores (as described in section “Fossil and Modern Pollen Data”) separately. The full list of taxa used in this simulation is presented in Supplementary Table Figure 6. Consistently with the parameters of our model, a no-analog situation is detected when the closest analog to a fossil sample has a squared chord distance higher than 0.3 . The period between ∼9,000 and 6,000 BP shows the highest values, with over 30% of the samples in each timeslice having no close analogs in the training set. The effect of the PFT approach is visible in Figure 6B: the highest no-analog percentages are still concentrated between ∼9,000 and 6,000 BP, but with values rarely exceeding 2%. A more detailed region-by-region subdivision is presented in Supplementary Figure The taxa-based approach results in large portions of the fossil data sets presenting no-analog situations  and used as a calibration curve for the modeled forest-cover values. The resampling strategy and regression parameters were tested via trial-and-error, opting for a solution that minimizes the bias for high forest-cover classes. The application of the bias correction curve to the two-fold cross-validation iterations is visible in Figure 8, where it shows a largely corrected median bias across all forest-cover classes. The correction of MAT values in the MAT–REVEALS comparison  shows how the calibration curve succeeds in closing the gaps between the two models during the Mid-Holocene .The results of the two-fold cross-validation exercise highlight a mild overestimation of forest cover at values ≲ 30% and, above all, a growing bias for values ≳ 50%. This systematic underrepresentation of high forest-cover values is clearly visible in the MAT–REVEALS downcore comparison, possibly suggesting a comparable bias spread across all Holocene reconstructions. Considering the seemingly systematic occurrence of this modeling error, we employ an empirical distribution correction approach  to rectiThe forest-cover reconstructions presented in the following sections are based on the application of this bias correction procedure.Figure 9A) and the MAT-based, interpolated modern forest-cover map  is used to evaluate the ability of the interpolation procedure to reproduce the main patterns in forest cover at the European scale. Higher forest densities are correctly reconstructed in mountain areas, notably at mid-low elevations in the Alps and along the Apennines, in the Balkan Peninsula and in the Carpathians, and partly in northern Iberia. In northern Europe, extensive forested areas are reconstructed around the Gulf of Bothnia and in western Russia. By comparison, the interpolated AP map displays similar distributions in central and southeastern Europe, but differs substantially in the remaining areas. Particularly visible is the pattern in northern Europe, where high AP values are present well beyond the northernmost limit of densely forested areas and show a generally limited spatial variability. A region-by-region comparison is presented in Figure 10, together with the full extent of the fossil-database reconstructions. The synthesis in Figure 10 highlights how a combination of MAT and interpolation succeeds in producing forest-cover values that are within the range derived from A visual comparison between actual modern cover from Figure 11. The overall lowest forest-cover values are reconstructed at the end of the Younger Dryas period, before 11,700 BP, when values > 50% are not recorded in the regional synthesis. A sharp increase in forest-cover percentages is immediately visible after 11,500 BP, rapidly leading to the maximum altitudinal development of dense Alpine forests in our reconstruction . A gradual negative trend is then recorded between ∼7,000 and 2,250 BP, lowering the 50% threshold from ∼2,600 m to ∼1,800 m. Eventually, after ∼500 BP, values below 800 m decrease noticeably too, bringing the overall forest-cover situation close to the modern one across all elevation bands. In Figure 11, the 50% boundary in both the 0 BP timeslice and LOO-based modern timeslice is located at 1,800 m. By comparison, the satellite-based modern forest cover is placed 400 m lower, at 1,400 m. It should be noted that the satellite-based 1,400–1,600-m elevation class has a forest cover value only slightly lower than the threshold value (48.9%), causing a seemingly abrupt difference in elevation between the satellite-based and MAT-based forest covers. Comparing the 0 BP timeslice with the satellite-based and the LOO-based forest covers returns a correlation coefficient r = 0.98 in both cases. Similarly, comparing the satellite-based and LOO data results in r = 0.97.A summary of the average MAT-derived forest cover in the Alps at different altitudes during the Holocene is presented in Figure 12A). The beginning of the Holocene is marked by a general increase in forest cover, with particularly steep trajectories in the British Isles, in the Atlantic region and, resulting in an isolated peak, in Eastern Europe.During the last stage of the Pleistocene, the average forest-cover ranges from ∼6% (British Isles) to ∼48% , with an overall study area average of ∼25%. The highest percentages are reconstructed along a diagonal belt spanning from the circum-alpine area to central-eastern Europe , an arctic/cool temperate low shrub, is not distinguishable from arboreal birches through grain size alone, requiring instead to calculate the ratio of equatorial diameter to pore height . Surface samples with unknown location reliability amount to >50% of the total. Despite this lack of metadata, they were included in the elaboration in order not to depopulate the analog pool.An additional issue revolves around the availability of remote-sensing data. While satellite-based forest-cover data are available only for selected time periods, i.e., the year 2000 for All of these examples are meant to highlight how surface-pollen datasets would benefit from both enhanced sample density and metadata quality, ideally not only covering a wider range of vegetation assemblages, but also containing redundancy that would allow for stricter and task-oriented selection of samples. Furthermore, improvements in remotely sensed land-cover data would be desirable, e.g., using new sensors that directly integrate the spectral and physical characteristics of the vegetation into a single dataset. It is important to remember that good transfer function performance may equally be dependent on having a reliable calibration dataset as on having good pollen data.2 have a direct effect on the production of flowering shoots, leading to an increase in pollen production with growing CO2 values  proves the effectiveness of the PFT approach, and suggests that no-analogs do not affect the performance of the model. Yet a concentration of no-analogs between ∼9,000 and 6,000 BP remains notable. It occurs during the phase of maximum European forest development and highlights from a different perspective the noted model weakness with dense forest covers.Changes in pollen productivity within the same species represent an issue that is difficult to address and common to all pollen-based models. On the other hand, the sensitivity of the MAT to migration lags, extinction events, and gaps in the training data set can be lessened by avoiding a taxa-based approach. The use of PFT scores, as used in the present study, groups pollen taxa in broader classes defined by physical, phenological and climatic factors. The absence of specific taxa from an area does not hinder the performance of a PFT-based reconstruction as long as their potential bioclimatic space is occupied by species with comparable traits. The low occurrence of no-analog situations in our reconstructions (Fagus), lime (Tilia), and elm (Ulmus) share the same PFT , likely hindering a fine distinction between moderately and highly forested contexts.The results of the two-fold cross-validation exercise and the MAT–REVEALS comparison  highlight a distinct issue with the reconstruction of high forest covers. A visible contribution to this issue is given by the limited availability of EMPD samples from highly forested contexts, but additional factors could play important roles too. Notably, the modern European landscape is largely deforested. As a consequence, any sample misplacement or incorrect distance-weighting could more likely lead to pair pollen samples with low forest-cover values. The use of PFTs could impact the model performance too, as noted in section “No-Analog Situations and the PFT Approach.” Most importantly, the MAT does not have any intrinsic ability to differentiate between local pollen sources and long-distance transport, an issue exacerbated by the generally poor pollen dispersal capabilities of open-landscape species . The under-representation of non-arboreal species results in samples with comparably high AP values but widely different forest-cover percentages .Unraveling the fine reasons behind the regionally diverse forest cover trends remains beyond the scope of this paper. We will mostly constrain our analysis to a comparison between our data and the land cover dynamics emerging from qualitative interpretations and quantitative models. The interpolated results of our reconstruction draw a coherent picture of forest advancement and retreat spanning the last 12 millennia. While the limits of the method constitute an undeniable source of approximation, it is important to note that the reconstructions are often consistent with existing narratives of vegetation development from a regional to a continental scale, although these have virtually all been based on a qualitative interpretation of pollen data.Figure 12A appear to be fitting for high-elevation sites and areas affected by a decrease in precipitation , occurring together with visibly higher error estimates . Additional similarities between the PBM and MAT can be found at a smaller scale too. During the second half of the Holocene, forest-cover percentages are characterized by negative trends that bring the curves to present-day values. Increasing human pressure is regarded as the primary driver behind such behavior , althougThe differences between Eastern and Western Europe are not limited to absolute land-cover values: the two regions show different forest-loss dynamics too. It is worth noting that quantitative landscape reconstructions by In the British Isles, The Mediterranean region shows an overall weak and gradual increase in open areas when compared with other more dramatic regional dynamics. The largely open landscape reconstructed during the Early/Middle Holocene is likely to play a primary role in lessening any evidence of radical human impact, yet other factors might contribute to this modest change in land cover too. As an example, even severe disturbance events e.g.,  might siIt has been widely argued that pollen percentages alone are not sufficient to infer the local presence of tree populations in extreme environments at the limits of tree growth e.g., . Under tFigure 11). Differences in the scale of the reconstructions, chronological uncertainties and temporal interpolation might explain the slight offset between the two events. Speculatively, the forest-cover stagnation seen in our model could be ascribed to the combined effects of the cool Preboreal oscillation . Notably, estimates for maximum forest limits range from 2,500 m . The 50% threshold never exceeds 2,400 m after 5,750 BP, possibly reflecting the origin of anthropogenic pastures above 2,300 m during the Copper Age , alpine forests became rather open even in the lowlands . This si 2,500 m  to 2,800 2,500 m  in the F 2,500 m , while iUltimately, a comparison between our model and independent palaeo-ecological studies suggests that a combination of MAT and interpolation can reproduce broad vertical vegetation patterns  at a resolution sufficient for large-scale vegetation models. The reconstruction of sharper forest-vs.-no-forest cutoffs remains beyond the capability of the interpolation procedure. Finer results could be achieved through the incorporation of complementary proxy data , although with the caveat that such data remain much less widely available than pollen records.In this study we applied the MAT to generate a continuous, continental-scale reconstruction of European forest-cover spanning the last 12,000 years. Our reconstruction follows the general methodology published by The reliability of our reconstructions is supported by a comparison with methodologically independent data sets. forest-cover reconstructions based on the MAT (this study) and on the LRA were compared for selected sites  in the cThe resulting continental-scale maps draw a coherent picture of vegetation development across Europe since the end of the Younger Dryas period. forest cover trends fit well in accepted narratives based on qualitative and quantitative interpretations of palaeovegetation data.As a further test, we evaluated the vertical performance of the interpolation procedure against macrofossil-based tree line/timberline studies in the Alps. Detecting ecotones in alpine environments via pollen percentages is a task affected by wide margins of error. Consequently, we focused our comparison on broader vegetation patterns . A combination of MAT and spatio-temporal interpolation proved able to track major tree cover fluctuations in proximity of the timberline, thus reinforcing the broad reliability of the interpolation algorithm across a highly dynamic landscape.MZ and BD designed the research. MZ performed the data analysis and presentation. LM contributed the independent (REVEALS) forest-cover reconstructions to aid method validation. MZ, BD, LM, SB, and JK were involved with substantial contributions in refining the methodology, writing, and reviewing the manuscript.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.


This study investigates the impact of ice-templating conditions on the morphological features of composite polysaccharide aerogels in relation to their mechanical behavior and aims to get a better insight into the parameters governing these properties. We have prepared polysaccharide aerogels of guar galactomannan (GM) and tamarind seed xyloglucan (XG) by enzymatic oxidation with galactose oxidase (GaO) to form hydrogels, followed by conventional and unidirectional ice-templating (freezing) methods and lyophilization to form aerogels. Composite polysaccharide aerogels were prepared by incorporating nanofibrillated cellulose (NFC) into polysaccharide solutions prior to enzymatic oxidation and gel formation; such a cross linking technique enabled the homogeneous distribution of the NFC reinforcement into the gel matrix. We conducted phase-enhanced synchrotron X-ray microtomography (XMT) scans and visualized the internal microstructure of the aerogels in three-dimensional (3D) space. Volume-weighted pore-size and pore-wall thickness distributions were quantitatively measured and correlated to the aerogels’ mechanical properties regarding ice-templating conditions. Pore-size distribution and orientation depended on the ice-templating methods and the NFC reinforcement that significantly determined the mechanical and shape-recovery behavior of the aerogels. The results obtained will guide the design of the microporous structure of polysaccharide aerogels with optimal morphology and mechanical behavior for life-sciences applications. Later, ry state , cryogelry state , or foamry state . Currentry state ,10. The ry state . We succry state , resultiaerogels ,14. The aerogels . 2 drying [Lyophilization (freeze-drying) is a simple and, compared to supercritical CO2 drying , more ve2 drying ,9. Imita2 drying , researc2 drying ,19,20,212 drying ,19,22,232 drying ,25,26. H2 drying . The por2 drying  because 2 drying . The ver2 drying .Currently deployed probing techniques for the structural characterization of aerogels such as scanning electron microscopy (SEM) and focused ion beam SEM (FIB-SEM) are limited to surface- level characterization. It is still difficult to characterize the actual representation of the 3D structure of the aerogels, the connectivity of the pores, the pore-size distribution, and the interface of the reinforcement with polymers within the aerogel matrix. Recent advancements in experimental techniques enable us to visualize and quantify the 3D morphology of the materials at the micron and sub-micron levels. Synchrotron X-ray microtomography (XMT) is an advanced and non-invasive analytical tool to illustrate the detailed internal structure of a material such as an aerogel. The technique has been used across several disciplines, including physics, materials science, medicine, and powder technology . The porIn this work, enzymatic crosslinking was used to obtain GM and XG hydrogels and NFC-reinforced, completely bio-based composite hydrogels. Both conventional and unidirectional freezing techniques were used as ice-templating methods for hydrogels that were subsequently lyophilized to obtain aerogels. The aim of this study was to see, using synchrotron XMT, the influence of processing conditions (ice-templating methods) and reinforcement regarding polysaccharide types on the qualitative and quantitative morphological features of the aerogels. The qualitative and quantitative morphological features of the aerogels were correlated with the mechanical performance of the aerogels. This information will contribute toward more understanding of the structure-function properties of the biocomposite aerogels and how the XMT technique applies to the characterization of lightweight and highly porous materials.The resolution of XMT images depends on the dimensions of the aerogel samples. Cutting polysaccharide aerogel samples into small sizes  for an XMT scan is difficult due to the elastic interconnected structure of the polysaccharide aerogels, which was experienced in our earlier study . Therefo3. The sample codes and compositions are explained in The XMT reconstruction can be viewed as a stack of two-dimensional grayscale images D. These GMox-CF aerogels exhibited the anisotropic structures of the pores that can easily be seen when the sample is viewed along different axes  A. GMox-CWhen unidirectional freezing was used as an ice-templating method, the GMox-UF and GMox-NFC-UF samples showed a structure oriented in the direction of freezing A,B. The Conventionally frozen XGox samples exhibited notably different morphology compared to that of the GMox aerogels, in which the pore walls consisted of a well-defined thin film and the pores were heterogeneous in shape and size A. A thin3, Length × width × height) were scanned with XMT. All the above mentioned aerogel samples were prepared inside capillaries . Some samples were scanned from cubical aerogels to observe the structural difference compared to the aerogels made inside a capillary. These cubical aerogels were mechanical compressed (MC), as explained in XMT imaging showed noticeable morphological differences between the plain GMox and XGox aerogels and the NFC-reinforced aerogels . These 3The purpose of adding a reinforcing agent to the composite material is to enhance the latter’s mechanical properties. The compatibility and interaction of the reinforcing agent with the polymer are imperative to achieve a high-performance composite material. To locate NFC in the GMox and XGox matrices, we used phase-enhanced XMT for scanning aerogel samples. However, visualizing NFC in the 3D structure was difficult due to the similar attenuation of X-rays by NFC and the matrix polysaccharides. Therefore, we did not distinguish NFC from the biocomposite aerogel structure. In earlier studies, Sedighi-Gilani et al. reported that XMT successfully revealed NFC in the NFC-silica hybrid aerogel because of difference in the attenuation of the X-ray energy of NFC and silica .The viscosity/gel stiffness of the polymer is, in particular, a key parameter in the ice-templating approach because it governs the critical freezing front velocity at which the polymer structure is trapped during solidification . The mecUnidirectional freezing aligned the aerogel’s structure in the direction of freezing and it also favored the formation of small pores due to the very low temperature of the freezing medium (−196 °C). The rapid-freezing kinetics resulted in a higher nucleation rate of ice crystals compared to their growth speed . The fasThe 3D-rendered images give a detailed overview of the internal morphology of the aerogels. The digital nature of the XMT data enables further quantitative analyses such as computing the pore size and wall thickness distributions.GMox aerogels showed a random pore size distribution; although they had some large pores of over 300 μm, most of the pores were distributed in the range of 100 μm to 250 μm A. The adThe XGox aerogel also exhibited pore size distribution similar to that of the GMox aerogel when prepared by the conventional freezing method. Most pores were distributed between 50 μm to 200 μm. However, some pores over 400 μm were also observed A. The XGWhen comparing the pore-size distributions of the mechanically compressed cubical aerogel samples, most pores in the GMox-CF-MC were below the 200 μm mark, but some were larger than 400 μm A. The GMThe pore wall thickness of GMox and GMox-NFC, regardless of the freezing method, was mostly in the range of 3 μm to 7 μm. However, a pore wall thickness of 25 μm was observed in the aerogels prepared by unidirectional freezing . The samLyophilization also affects the external and internal structure caused by shrinkage . The aer2 drying [The mechanical behavior and shape recovery of the porous material depend on the geometry of the pores (shape and size) in the 3D architecture of the material ,37 and t2 drying ,15. Howe2 drying ,15. This2 drying . Thus, tO-acetylgalactoglucomannans (GGM) and NFC crosslinked with ammonium zirconium carbonate, showed that aerogels with a higher NFC content exhibited less shape recovery. A negative correlation was observed between compression shape recovery and the NFC content and compressive modulus. However, NFC showed positive effects on the GGM and NFC composite aerogels [The shape recovery (height recovery after compression) of the aerogels is presented in aerogels . During aerogels  for polyThe visual shape deformation of the cubical aerogel samples after compression testing is presented in Fusarium spp., which was produced recombinantly in Picihia pastoris and donated by Dr. Sybe Hartmans . Horseradish peroxidase  and catalase  were obtained from Sigma-Aldrich .Guar galactomannan (GM) obtained from Sigma-Aldrich  and tamarind seed xyloglucan (XG), a gift from Dainippon Sumitomo Pharma, Osaka, Japan, were used as aerogel-forming materials. NFC refined from birch pulp without chemical derivatization was purchased from UPM, Finland, and was used as a reinforcing agent. The NFC fiber was 10 nm to 50 nm wide and several micrometers long. The galactose oxidase (GaO) used was from GM and XG hydrogels were prepared using the previously explained method ,15. NFC For synchrotron radiation microtomography, the enzyme-inactivated hydrogels, from which air bubbles were removed by vacuum, were injected into polyether-ether-ketone (PEEK) capillaries  using a small syringe A,B and p3 in length, width, and height) from mechanically compressed (see Aerogel samples in the PEEK capillaries were glued on carbon fiber rods (diameter = 3 mm and length = 10 mm). All the samples were scanned at a height of 6 mm from the bottom to avoid any artifacts from the glue. Also, small samples  [Quantitative data analyses such as volume-weighted pore-size distribution and volume-weighted wall thickness distribution were calculated using the Local Thickness plugin to ImageJ software (ickness) . In thisickness) . The loc3) were compressed vertically for 6 mm at a rate of 1.3 mm/min. The unidirectional frozen aerogel samples were compressed in the freezing direction . The compressive modulus was determined from the stress-strain curve of the aerogels. Also, the sample recovery after compression was calculated by subtracting the height of the compressed sample from that of the uncompressed sample. The obtained value was then subtracted from the compression height (6 mm) to get the recovered height of the sample. Before the measurements, the aerogel samples were conditioned for 48 h in desiccators containing Mg(NO3)2 to set the relative humidity between 50% and 55%. All measurements were performed at 21 °C and 50% relative humidity (RH). The data were obtained from 10 replicates of each aerogel sample.The Instron 33R4465 universal testing machine  with a load cell of 100 N was used for the mechanical testing of aerogels. Cubical aerogel samples . NFC reinforcement exhibited an indubitable effect on the pore-size distributions, and this effect was more prominent when the unidirectional ice-templating method was used. NFC reinforcement also played its role in the rheological properties of the matrix polysaccharide (GMox/XGox) hydrogels. Pore-wall-thickness distributions were more or less independent of the processing conditions and the reinforcement agent. However, the mechanical properties such as shape recovery and compressive modulus were dependent on the pore-size distributions, which is the outcome of the nucleation and ice crystal growth kinetics of the ice-templating methods. This study provided very useful practical information that helps in the design of microporous materials of a desired morphology. Carefully controlling the nucleation and ice crystal growth results in the optimal properties of the porous material.


A meta-analysis comparing drug-eluting beads transarterial chemoembolization (DEB-TACE) with conventional transarterial chemoembolization (cTACE) has recently been published. On balance, no significant differences were found in terms of objective response and overall survival. The impact on healthcare costs had been studied in small series based on a hypothetical model and was in favor of DEB-TACE. We aimed to evaluate and compare health-care costs and effectiveness of both modalities in a cohort of patients from Nottingham, UK.Using a dedicated radiology database, we identified all patients who had undergone cTACE or DEB-TACE between 2006 and 2012 at a single tertiary referral center based in Nottingham. We collected clinical data, including treatment response, postprocedure complications and 30-day mortality. Costing models were constructed to present both our local hospital perspective as well as the national health service position.p=0.52). Adverse events were seen in 6/76 cTACE compared with 7/25 DEB-TACE group (p=0.16). Based on the predetermined standard pathway there was an unadjusted average cost difference of £3770.30  in favor of the DEB-TACE. Results from our costing models indicated a £2715.33 (95% CI £580.88–4849.77) cost difference in favor of the same procedure.During our study period, 101 procedures were performed on 43 patients (76 cTACE procedures on 26 patients and 25 DEB-TACE procedures on 17 patients). Overall, 11/26 in cTACE and 5/17 in DEB-TACE group had progressive disease (Even when the extra costs of DEB-TACE were considered, the overall treatment costs per patient were lower in relation to cTACE. Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and the second leading cause of all cancer-related mortality globally.p=0.015).7Response to TACE is commonly judged according to time-to-event, for example, progression as well as overall survival.TACE with drug-eluting beads (DEB-TACE) as opposed to conventional TACE (cTACE) involves the injection of beads into hepatic artery branches offering simultaneous embolization with sustained and controlled drug release. Consequently, DEB-TACE offers a favorable pharmacokinetic profile and lower peak plasma concentrations of chemotherapy.9Five meta-analyses comparing the safety and efficacy of DEB-TACE in comparison with cTACE have been published within the last 2 years.The study is registered by the Digestive Diseases and Thoracic Directorate of the Nottingham University Hospitals NHS Trust as an audit. Ethical approval and patient consent were not required for this retrospective study which was performed as a part of our service evaluation.We performed a retrospective analysis of patients who had cTACE or DEB-TACE between January 2006 and December 2012 for the treatment of HCC in a tertiary referral center. We identified the patients using a dedicated radiology database that is used to store and report procedural details as part of routine clinical practice. Clinical notes as well as electronic record information were used for data collection. We collected: 1) patient variables, including demographics, etiology of liver disease, Child–Pugh score and UK model for end-stage liver disease; 2) tumor variables, including size, location and number; 3) procedure variables, including number of TACE sessions required, length of hospital stay, postprocedure complications and 30-day mortality. Patients were excluded if they: 1) had not had follow-up in our unit; 2) had TACE for indications other than HCC; 3) had both cTACE and DEB-TACE on different occasions. Patients were followed up until discharge from clinic, including death or until the date of the last available cross-sectional imaging, if still under follow-up. Computerized tomography scans pre- and postprocedure were used to objectively assess tumor response using mRECIST. The images were reviewed by an expert radiologist as a part of the study. Cumulative tumor size of the 2 largest measurable lesions per liver was used where there was more than 1 lesion.5t-tests (parametric data) or Mann–Whitney U (non-parametric data) tests. A p-value <0.05 was considered statistically significant. Possible predictors of objective response were fitted into a proportional ordered logistic regression model. This analysis was performed on R statistical software using R packages mass and ggplot2 . TACE procedures were counted twice for the same patient if the interval between both procedures was more than 4 months and both were assessed by postprocedure cross-sectional imaging independently. Covariates were limited by data availability and included the following:Interval in weeks from TACE to cross-sectional imagingTumor size at baselineVascular invasion and/or extra-hepatic disease, including porta-hepatis lymph nodes.Differences between groups of continuous variables were assessed by unpaired In order to obtain per patient cumulative cost figures, a reference treatment pathway was constructed with the assumption that it would be followed by all patients during the study’s time. Follow-up procedures were modeled as shortened versions of the first procedure. Two different costing models were constructed in order to present both the local and the national perspectives. The local cost of the beads was added to both models since the national equivalent could not be identified. Details on the price weights can be found in To address the non-normality of distributions, costs were analyzed using generalized linear models (GLM) as described by Dunn et al.Fifty patients in total were identified. After exclusions, we analyzed 101 procedures  performed on 43 patients  as described in p=0.04). Despite this, the cumulative tumor size of all nodules per liver added together was not significantly different between both groups. DEB-TACE group required significantly less sessions (median=1) in comparison with cTACE (median=2.5) per patient (p<0.005).The majority of patients were male in both groups, with similar ages. Included patients had variable etiologies as outlined in One patient in the DEB-TACE group died from biliary sepsis on day 14 of the same admission. In the cTACE group, there was a single 30-day mortality from hepatic decompensation; on day 2, postprocedure, the patient was transferred to a specialist palliative care facility. Severe transaminitis  and hospital-acquired pneumonia occurred postprocedure in 1 patient in the cTACE group which prolonged admission in hospital by 5 days. One of the DEB-TACE cases had re-admission 6 days after the discharge from hospital with left hepatic artery dissection complicated by hospital-acquired pneumonia. The second admission had a duration of 7 days. There were no other notable severe complications otherwise, apart from limited transaminitis and postembolization syndrome .p=0.0004, OR=1.035, 95% CI 1.017–1.057).The method of TACE delivery (cTACE vs DEB-TACE) was not found to be independent predictor of objective response. Tumor size predicted objective response independent of all other variables as demonstrated in p=0.014).The analysis of the cost data took into account 40 out of the 43 patients for whom there was a complete dataset. The cTACE and DEB-TACE arms differed in the added cost of the beads for the DEB-TACE and the average number of follow-up procedures per patient. Using the aforementioned pathway, the 2 procedures had an unadjusted average cost difference of £3770.30  based on the NHS costing and of £3253.68  based on local costing, in favor of the DEB-TACE arm. Results are summarized in We have demonstrated that overall treatment costs of DEB-TACE are lower than that of cTACE for the treatment of HCC. Our analysis is based on a real-life scenario. In the UK, all patients with HCC are managed through a regional cancer network with treatment decisions made by multidisciplinary teams (MDTs) based on predetermined pathways. Our economic model was based on the predetermined standard pathway which included the patient’s journey from time of referral to the tertiary center until the time of discharge, death or last follow-up. It included the costs of outpatient appointments, MDT discussion, inpatient stay, blood tests, TACE procedure and cross-sectional imaging. This economic modeling has shown an unadjusted average cost benefit of £3770.30 for patients receiving DEB-TACE compared with those who received cTACE. Patients undergoing DEB-TACE have required less number of procedures over the observed study period. The data suggest that contribution to the cost comparison is in favor of DEB-TACE with potential benefits to healthcare costs. Although neither the OLS regression nor any GLM had a significant explanatory power, there is an indication that the DEB-TACE arm can be less costly in the medium term. This is consistent with recently published data by French and Italian groups.16Our limitations include the retrospective study design and possible selection bias, including the fact that patients treated by DEB-TACE had significantly fewer target liver lesions. The cost of beads reported in our study may lack generalizability as our analysis was based on our local purchase costs. However, the commissioning of health services is determined based on these estimates; so, these influence the design and delivery of services in the British NHS. The use of cTACE had been well established in our unit for many years. In contrast, DEB-TACE had been introduced as a relatively new technique. The choice of patients having DEB-TACE was mainly influenced by the time of introduction of the new service rather than specific clinical features. Similarly, classically, we will protocol cross-sectional imaging after 2 sessions of cTACE. However, as DEB-TACE was relatively new at the time of introduction, we protocoled cross-sectional imaging after each session of DEB-TACE in the majority of cases. Cross-sectional imaging after each session of cTACE may have led to different results. The study also highlights that, judging by objective response, we demonstrated that DEB-TACE is similar to cTACE. It also affirms the likelihood that its medico-economic consequences are more favorable in the medium term in comparison with cTACE.There is some controversy in the literature, however, about the effect of DEB-TACE on objective tumor shrinkage.Based on the recent meta-analyses, DEB-TACE has no statistically significant impact on survival or objectively evaluated treatment response according to RCTs. In contrast, the largest meta-analysis to date that included non-RCT prospective and retrospective studies, demonstrated that DEB-TACE was associated with survival benefit.19It is worth noting that there are several limitations of reviewing studies based on meta-analysis. First, the number of TACE sessions required and practically performed was not compared. The studies did not report if less sessions were required by DEB-TACE to achieve the same outcome. Second, the size of beads and dosage of chemotherapy was variable. Third, the timing and criteria used for radiological assessments were heterogeneous. Finally, the patient populations, for example, model for end-stage liver disease score and stage of fibrosis was not unified. Better efficacy and safety remain a theoretical possibility based on the aforementioned results.We conclude that DEB-TACE, a newer technique for the treatment of unresectable HCC, is an effective modality and our cost analysis shows it to have cost benefits when compared with cTACE. Prospective head-to-head trials on selected patient populations are needed to evaluate its efficacy as well as cost benefits to the healthcare system.Guru.Aithal@nuh.nhs.uk). Consent was not obtained from participants but the presented data are anonymized and risk of identification is low.The dataset is available from the corresponding author at (


In addition, the two-dimensional area covered by thrombi (%) in the capillary was analyzed every 3 minutes after sample applications. The AR10-AUC30 correlated weakly and negatively with plasma concentrations of DOACs, and the levels at On-anticoagulant were lower in all groups than at Off-anticoagulant. AR10-AUC30 levels at Off- and On-anticoagulant were identical among the groups. The thrombi areas in early phase were significantly larger in rivaroxaban and apixaban than warfarin and dabigatran groups. The findings suggested that visual analysis of the AR-chip can identify the differential inhibitory patterns of warfarin and DOACs on thrombus formation under flow condition.Direct oral anticoagulants (DOACs) have low risk of intracranial hemorrhage compared to warfarin. We sought to clarify the different mechanisms responsible for suppression of bleeding events using the Total Thrombus-formation Analysis System (T-TAS), a flow-microchip chamber with thrombogenic surfaces. Blood samples were obtained at Off- and On-anticoagulant (trough) from 120 consecutive patients with atrial fibrillation , which were used for T-TAS to compute the area under the curve (AUC) (AR Bleeding events are sometimes serious complications in AF patients on anticoagulation therapy. However, randomized control studies have shown the efficacy and safety of the DOACs in AF patients. For example, Chatterjee et al.5 reported that DOACs are associated with an overall low risk of intracranial hemorrhage (ICH). Another group reported that patients treated with rivaroxaban who develop ICH had relatively small hematoma, showed no signs of expansion of hematoma, and had favorable functional and vital outcomes compared to warfarin-associated ICH6.Direct oral anticoagulants (DOACs) are used to reduce the risk of stroke in patients with atrial fibrillation (AF), similar to warfarin8, there is currently no simple tool available to monitor the effects of DOACs9. Recently, the total thrombus-formation analysis system (T-TAS), a microchip-based flow chamber system designed to evaluate whole blood thrombogenicity, was developed as an easy-to-use system for quantitative analysis of thrombus formation. The T-TAS could be useful for monitoring the anticoagulant effects of DOACs and predicting periprocedural bleeding events13.The prothrombin time-international normalized ratio (PT-INR) is widely used to assess the anticoagulant effects of warfarin. Although it is possible to measure blood concentrations of DOACsThe aim of the present study was to determine differences in the anticoagulation patterns of warfarin and DOACs using the T-TAS in patients with AF who had undergone radiofrequency catheter ablation (RFCA).Figure Figure 10-AUC30 measured by T-TAS, we compared the parameters at the Off- and On-anticoagulants. At the Off-anticoagulants, there were no significant differences in PT-INR and AR10-AUC30 levels between the four groups . Figure 10-AUC30 levels and plasma concentrations in the three DOAC groups. There was almost no relation between AR10-AUC30 and plasma concentrations of rivaroxaban . On the other hand, there was a weak negative relation between AR10-AUC30 and dabigatran  and apixaban .To evaluate the utility of AR10-AUC30 levels at On-anticoagulants were identical among the four medications drug  the area of thrombi during the early phase of thrombus formation was significantly larger in patients treated with rivaroxaban and apixaban compared to those on warfarin and dabigatran, indicating that these differential patterns of thrombus formation might explain the mechanism of volume reduction of ICH. To the best of our knowledge, this is the first report that determined the differential mechanism of antithrombotic effects in patients treated with warfarin and DOACs by using T-TAS, a novel quantitative assessment of whole-blood thrombogenicity.The major findings of this study were as follows: (1) AR9. Several previous studies reported that dabigatran prolonged APTT in a concentration-dependent manner15, rivaroxaban prolonged PT in a concentration-dependent manner17, and apixaban prolonged PT and APTT, but their correlation was modest or weak19. In the present study, dabigatran, rivaroxaban, and apixaban significantly prolonged PT-INR and APTT, though their effects were not identical. Therefore, it could be difficult to set the optimal therapeutic range of using the PT-INR and APTT for the anticoagulant therapy with DOACs. However, AR10-AUC30 levels measured by T-TAS were uniformly lower under treatment by both DOACs and warfarin, indicating that the AR10-AUC30 level can reflect the anticoagulant effects on any anticoagulant therapy with an easy-to-use procedure.Monitoring of the anticoagulant effects of warfarin requires the measurement of PT-INR to determine the optimal dose, but there is no available tool for the monitoring of DOACs8. To investigate the relationship between AR10-AUC30 levels and plasma concentrations, we measured the latter employing coagulation assays. Analysis of the relationship between AR10-AUC30 levels and plasma concentrations showed almost no relation in patients treated with rivaroxaban, and weak relation in dabigatran and apixaban. The reason for this finding might be that the overall coagulability depends on not only the plasma concentration of DOAC, but also other coagulation factors, old age, and presence of pathological conditions . In a previous study from our laboratories, although the number of patients was relatively small, statistical analysis identified the AR10-AUC30 level measured by T-TAS as a potentially useful predictor of periprocedural bleeding events in AF patients undergoing RFCA12. Especially, in the evaluation of bleeding risk of patients treated with oral anticoagulants, assessment of total coagulability is probably more important than measurement of plasma concentrations of DOACs alone. Further prospective studies of large population are needed to establish the utility of AR10-AUC30 level for monitoring the anticoagulant effects and the risk of bleeding events in patients treated with warfarin or DOACs.Measurement of plasma concentrations is often used for monitoring DOACs, and several coagulation assays have been developed for monitoring of treatment with DOACs5. The authors hypothesized that the finding indicated different mechanisms of action for DOACs and warfarin, and that the unique physiology of the brain, such as tissue factor-rich basal membrane, provides protection against spontaneous intracerebral hematoma formation20. While these mechanisms were supported by the results of several animal studies22, there are no clinical studies that analyzed those mechanisms. In the present study, we used an AR-chip with a surface coated with collagen plus tissue factor, for analysis of thrombus formation under flow conditions, and examined the formation of platelet thrombi, which was calculated as the relative area of thrombi. Interestingly, the thrombi area during the early phase  was quite different among the four treatment groups, although there were no significant differences in the final AR10-AUC30 level measured by the T-TAS. The thrombi area at 3 or 6 min was significantly larger in rivaroxaban and/or apixaban (direct inhibitors of coagulation factor Xa) than dabigatran (direct inhibitor of thrombin) and warfarin . These findings suggest that thrombin inhibition or decrease seems to markedly reduce the thrombi area during the early phase of the measurement by the AR-chip, whereas factor Xa inhibition does not diminish it significantly, and could reflect the protective mechanism of factor Xa inhibitors against ICH clinically. Why was the thrombi area smaller in dabigatran than in factor Xa inhibitors (rivaroxaban and apixaban)? Against this observation, dabigatran and rivaroxaban have comparable bleeding risk in patients with AF even in real-world practice24. In addition, previous experimental studies showed that direct thrombin inhibitors, but not factor Xa inhibitors, enhance thrombin generation paradoxically via intrinsic pathway or inhibition of the protein C system26. These discrepancies might be explained by potential mechanism, such as the influence of CaCl2 and corn trypsin inhibitor in the collection tubes, and difference in the rivaroxaban dose used clinically in Japan and western countries. However, the clinical relevance of this observation is not clear and it should be evaluated in future clinical studies.One previous meta-analysis showed that the use of DOACs is associated with reduced risk of ICH compared to warfarin10-AUC30 and thrombi area) and clinical outcome. Thirds, the parameters of RFCA cannot be strictly regarded as ‘off-anticoagulation’ since bridging anticoagulation with unfractionated heparin is used before RFCA in some patients. Finally, the pharmacokinetics and pharmacodynamics need to be analyzed when considering the factors that can influence the effects of anticoagulation. These factors can include for example the activities of drug-metabolizing enzymes and any genetic polymorphism. Such confounding factors, which were not measured in the present study, might limit the significance of the present study due to the retrospective nature of the study design.The present study has several limitations. First, the results could be overestimated based on the small sample size in this single-center observational study. Second, the long-term outcome (thrombotic and bleeding events) was not evaluated in AF patients treated with anticoagulants. A large population study is needed to evaluate the relationship between T-TAS parameters , inflammatory disease (n = 1), use of antiplatelet drugs (n = 8), and refused participation (n = 15). The remaining 120 patients treated with anticoagulants were the subjects of this study. They were divided into four treatment groups according to type of anticoagulant used; warfarin (n = 29), dabigatran (n = 19), rivaroxaban (n = 47), and apixaban (n = 25) groups  were stopped in the morning the day before RFCA, and were bridged with unfractionated heparin until 6 hours before RFCA. Warfarin was interrupted 4 days before RFCA, and bridged with unfractionated heparin, similar to DOACs. All anticoagulants were restarted from the next morning after RFCA, but warfarin was bridged with unfractionated heparin from the next morning after RFCA until the target PT-INR of 1.6 to 2.6. During the bridging anticoagulation with unfractionated heparin, the heparin dose was adjusted so as to maintain the APTT level at about 1.5 to 2.5 times the baseline level.Details of the blood sampling points and the washout period of anticoagulants during RFCA were described previouslyWe analyzed blood samples obtained on the day of RFCA (Off-anticoagulants point), and 1 month after RFCA .10-AUC30 and plasma concentration of DOACs, we measured plasma concentration of DOACs at On-anticoagulants (at trough) using the commercially available standardized assay kits . For rivaroxaban and apixaban, the BIOPHEN DiXaI chromogenic assays were used, as well as the BIOPHEN HEMOCLOT Thrombin Inhibitors clotting assay for dabigatran, according to protocols recommended by the manufacturers.To investigate the relation between AR29. Briefly, the atheroma chip (AR-chip) of the T-TAS, which contains a single capillary channel , is coated with type I collagen plus tissue thromboplastin. Whole blood collected into 3.2% sodium citrate-containing tubes was blended with CaCl2 and corn trypsin inhibitor immediately before the assay. The 450-μL mixture was applied to the AR-chip at a flow rate of 10 μL/min, corresponding to an initial wall shear rate of 600 s−1. Both the collagen and tissue thromboplastin activate the platelets and the coagulation system simultaneously inside the AR-chip. The fibrin-rich platelet thrombus formation processes were assessed by continuous monitoring of the flow/pressure change resulting from capillary occlusion. The AUC was computed to evaluate platelet thrombogenicity in the AR-chip. AR10-AUC30 is defined as the AUC for the first 30 minutes for the AR-chip tested at flow rate of 10 μL/min. The absolute changes in the quantity of AR10-AUC30, PT-INR, and activated partial thromboplastin time (APTT) was calculated by the following formula:We used the T-TAS , which is a microchip-based flow chamber system equipped with a flow pressure sensor and a videomicroscope, for the analysis of thrombus formation under flow, by computing the area under the flow/pressure curve (AUC), as described previously28. The thrombus formation process inside the microchip was recorded at 3, 6, 9 minutes after application of the blood sample.In addition to the flow/pressure analysis, thrombus formation in the capillary was visually observed by the built-in light microscope Fig. , and theP value of <0.05 denoted the presence of a statistically significant difference. All statistical analyses were performed by using The Statistical Package for Social Sciences software version 23.0 .Data are expressed as mean ± SD or median. Differences between parameters were compared with the chi-square test, Fisher’s exact test, one-way ANOVA or Kruskal-Wallis test, as appropriate. We also used the Wilcoxon signed-rank test to assess the serial changes in AR10-AUC30, PT-INR, APTT, and the relative area of thrombi (%). A two-tailed


Antibodies play an important role in immunity to malaria. Recent studies show that antibodies to multiple antigens, as well as, the overall breadth of the response are associated with protection from malaria. Yet, the variability and reliability of antibody measurements against a combination of malarial antigens using multiplex assays have not been well characterized.A normalization procedure for reducing between-plate variation using replicates of pooled positive and negative controls was investigated. Sixty test samples , together with five pooled positive-controls and two pooled negative-controls, were screened for antibody levels to 9 malarial antigens, including merozoite antigens , sporozoite CSP, and pregnancy-associated VAR2CSA. The antibody levels were measured in triplicate on each of 3 plates, and the experiments were replicated on two different days by the same technician. The performance of the proposed normalization procedure was evaluated with the pooled controls for the test samples on both the linear and natural-log scales.Compared with data on the linear scale, the natural-log transformed data were less skewed and reduced the mean–variance relationship. The proposed normalization procedure using pooled controls on the natural-log scale significantly reduced between-plate variation.For malaria-related research that measure antibodies to multiple antigens with multiplex assays, the natural-log transformation is recommended for data analysis and use of the normalization procedure with multiple pooled controls can improve the precision of antibody measurements.The online version of this article (doi:10.1186/s12936-017-1933-6) contains supplementary material, which is available to authorized users. Plasmodium falciparum infections. Studies show that antibodies to a combination of antigens, not just a single antigen, are associated with protection from malaria [Antibodies play an important role in immunity against  malaria –5. Altho malaria , 7 and a malaria .Traditionally, antibodies to malarial antigens have been measured using an enzyme-linked immunosorbent assay (ELISA) that measures antibody to only one antigen at a time. However, recently, multiplex bead-based assays have been developed for measuring antibodies to multiple antigens simultaneously in the same well –12, therFor multiplex assays, measurement error occurs both within- and between-plates . Within-Several general approaches for data normalization have been reported. For example, logarithmic transformations have been used to reduce the skewness of the data and alleviate the association between the variance and mean of the responses, i.e., the mean–variance relationship , 17. A lThe objective of the study was to reduce between-plate variability associated with measuring antibodies to a combination of malarial antigens based on multiplex assays at the data analysis stage. Positive pooled controls were developed based on the average ranks of malaria antigens of interest, using archival plasma samples. A simple ANOVA-based normalization procedure was tested that uses between-plate replicates of pooled controls to normalize test samples across different plates on both the linear and natural-log scales. First the differences between the observed and the averaged values for the pooled controls across plates  were calculated, and then test samples were adjusted using the estimated plate effects. The performance of the proposed normalization procedure was then evaluated by comparing the between-plate variation with versus without the normalization.Escherichia coli, kindly provided by C. Long Malaria Vaccine Development Branch (MVDB), NIAID, NIH; MSP2 (Fc27 strain) expressed in yeast, kindly provided by R. Anders ; MSP3 (HB3) expressed in Escherichia coli, kindly provided by J. Rayner ; Erythrocyte binding antigen-175 RII (EBA-175), obtained from Science Applications International Corp., Frederick, MD; and full-length VAR2CSA (FV2) (FCR3) expressed in Baculovirus from A. Salanti . The extracellular domains of full length MSP11 and Pf41 (merozoite surface antigens) were made by gene synthesis and expressed as biotinylated proteins in MEK293E cells [A multiplex bead-based immunoassays was used to measure antibody levels to nine malarial antigens, including merozoite antigens, sporozoite CSP, and pregnancy-associated VAR2CSA. These nine antigens include recombinant proteins, gene expression proteins, and synthetic peptides. Recombinant proteins included: Apical merozoite protein 1 (AMA1) 3D7 strain expressed in yeast (molecular weight 83 kD), the c-terminal 42,000 portion of merozoite surface protein-1 (MSP1-42) expressed in 3E cells , provide2 fragment, Goat anti-human IgG Fc fragment specific, Jackson Immunoresearch, West Grove, PA, Cat # 109-116-170) diluted to 2 µg/ml in PBS-1% BSA was added to each well and incubated as described above in the dark for 1 h. Wells were washed, microspheres were re-suspended in 100 µl PBS-1% BSA and 85 µl of the microsphere suspension was analysed using a Liquichip M100 reader . The reader was programmed to read a minimum of 100 beads per spectral address, DD Gate 7500–15,000 and 35 s timeout. The results were expressed as median fluorescent intensity (MFI).The MAP assay was performed as previously described . RecombiIn a prior study, archival, coded plasma samples from 1377 pregnant women residing in Yaoundé, Cameroon, a malaria-endemic area, were screened for antibodies to 28 malarial antigens . AntibodAfter excluding samples included in the pooled controls, 30 malaria-positive (M+) and 30 malaria-negative (M−) plasma samples were randomly selected from the 1327 remaining samples for a total of 60 samples for testing. These 60 test samples were assayed in triplicate  with 20 samples (10 M+ and M−) on each of three different plates on a single day. Then, the same technician duplicated the process and reran three plates of the same samples on another day, with the same plate and well assignments as on the first day. On each of six plates, the same panel of seven pooled controls was also assayed in triplicate.The descriptive statistics of antibody levels (expressed as MFI) were summarized for each antigen on both the linear and natural-log scales. The proposed adjustment approach to reduce between-plate variation was built on the plate effect estimated using the same group of pooled controls assayed on all plates. The key idea was that the MFI for a sample  should remain constant across different plates for each antigen. The estimated plate effect for each antigen was then the average difference of the pooled controls on a given plate from the overall means of the corresponding controls across all plates. Four different sets of controls were considered to estimate the plate effect: five positive pooled controls only ; five PC plus the US-negative control ; five positive controls plus the pool of Cameroonian male control  ; and all 7 pooled controls (n = 7). For each antigen, the proposed analytical procedure for reducing between-plate variation included the following 6 steps: (1) Obtain the linear MFI value for pooled control samples and test samples; (2) Convert the MFI values to natural-log values; (3) For each pooled control, calculate the differences between the mean MFI averaged across the replicates for each plate and the overall mean MFI values across both replicates and plates; (4) Obtain the estimated plate effect for a specific plate by averaging the calculated differences across all pooled controls; (5) For test samples on a given plate, obtain the adjusted MFI values by subtracting the estimated plate effect from the unadjusted MFI. Steps 3–5 were conducted in both linear and natural-log scales.ijk be the MFI value for the ith pooled control on the jth plate and the kth replicate. Given a balanced design in which there are I controls, J plates, and K replicates, the overall mean for the ith pooled control was then Specifically, let yijk be the unadjusted MFI value for the ith test sample, jth plate, and kth replicate. The adjusted MFI value for the test sample, ijk to zijk to emphasize that test samples were not used in the estimation of the plate effects.Note that For each antigen, the between-plate variation was calculated by first averaging the three within-plate replicates. The between-plate variation for the unadjusted MFI values was then estimated by averaging the squared differences of the unadjusted MFI values between the two plates for the 60 test samples. Similarly, the adjusted between-plate variations were estimated by the average squared differences of the adjusted MFI values between the two plates for the sixty test samples. The impact of the adjustment on between-plate variation for each antigen was investigated on both the linear and natural-log scales. Paired t test was used to compare between the unadjusted and adjusted values in between-plate variations. Data were analysed using the SAS software version 9.4 . A two-sided test was used and a p < 0.05 was regarded as statistically significant.Table Table Figure This investigation demonstrated that the natural-log transformation of MFI data was more consistent than the linear MFI scale for malaria-related antibodies using the multiplex assay. The mean–variance relationship based on the natural-log transformed MFI values was reduced.The proposed normalization procedure using multiple pooled controls significantly reduced the between-plate variation on the natural-log scale. Compared with other normalization procedures, a major strength of the proposed ANOVA-based normalization procedure is its simple implementation. As long as a given laboratory has a well-defined set of controls, a multiplex assay can be easily adjusted by calculating the difference between the observed controls and the average values for the controls across plates . In addition, despite the replication built into this study, the proposed normalization procedure does not require within- or between-plate plate replicates of the test samples. Only the pooled controls are required across plates. Finally, in contrast to other normalization methods, the proposed normalization maintains a straightforward interpretation of final study results by back-transforming the adjusted values to the linear scale. However, there exists residual plate-effect after adjustment. This indicates that the normalization procedure is not-optimal. One assumption of the proposed normalization procedure is that the pooled controls possess the same plate effects as the test samples. However, the existence of a plate-by-sample interaction where the plates effects are different for pooled controls and test samples would have violated this assumption. Testing the plate-by-sample interaction on pooled controls is a potential approach to assess the validity of the normalization procedure. Any statistical approach for assessing the validity of the normalization approach could be improved by also incorporating information on antibody characteristics that could impact the between-plate adjustments.The selection of controls is critical for normalization. In the current study, five pooled positive controls were prepared based on the deciles of the average antibody response using pre-screened samples. The pre-screening of the pooled controls may also ensure that each control better covers the range of potential MFI values with variant ranks across antigens. By pooling different samples with different ranks of the antigens, it had hoped to achieve a range of expression levels to all antigens. The results for pooling samples as shown in Additional file Between-plate replication of test samples was included to evaluate the feasibility of the proposed normalization procedure. However, in practice, the proposed normalization procedure only requires between-plate replication of the pooled controls and not the test samples. This should help the investigation of responses to multiple malaria antigens across many subjects as between-plate replication of test samples may not be feasible due to limited amounts of biological specimen. Yet, since the proposed normalization procedure only relies on the pooled controls, residual plate-effects may still exist for the test samples after the proposed adjustment. Regardless, the current model should be useful in most situations.Overall, the proposed normalization procedure has significant advantages. The procedure does not assume that the between-plate distributions are rank invariant and can be easily implemented. The simplicity in the estimation of the plate effects improves the transparency of the final adjusted values of the test samples. Most importantly, the normalized MFI values result in lower between-plate variation on the natural-log scale. This should help reduce the overall variation and improve the estimation and statistical power for malaria-related research using multiplex assays.An analytical method for data produced by multiplex assays is important since future malaria studies will require the measurement of antibodies to multiple antigens, including large scale studies that require the evaluation of samples on multiple assay plates. Results from this study provide a method for analysis of data from multiplex assays. Further studies are needed to help scientists understand the consequences of failing to control for between-plate and, more generally, between-assay variability, on the accuracy of predicting outcome based on antibodies data.The natural-log transformation should be used for data analysis in using malaria antibody measurements. The normalization procedure using pooled controls could reduce the between-plate variability and improve the precision of malaria related research using multiplex assays.Additional file 1: Table S1. List of antigens used in the study.Additional file 2: Table S2. Summary of MFI values in linear scale for the pooled control samples across six plates.