The elevated iron supporting this bloom is likely of hydrothermal origin based on the recurrent position of the bloom relative to two active hydrothermal vent fields along the Australian Antarctic Ridge and the association of the elevated iron with a distinct water mass characteristic of a nonbuoyant hydrothermal vent plume.Primary production in the Southern Ocean (SO) is limited by iron availability. Hydrothermal vents have been identified as a potentially important source of iron to SO surface waters. Here we identify a recurring phytoplankton bloom in the high-nutrient, low-chlorophyll waters of the Antarctic Circumpolar Current in the Pacific sector of the SO, that we argue is fed by iron of hydrothermal origin. In January 2014 the bloom covered an area of ~266,000 km 2, but phytoplankton growth is limited by iron. Here the authors show that iron from hydrothermal vents fuels massive phytoplankton blooms in the Southern Ocean that have recurred in the same location for decades.Primary productivity in the Southern Ocean plays an important role in the drawdown of atmospheric CO Because net primary productivity (NPP) in the SO is limited by the availability of iron (Fe)4, understanding the distribution of Fe availability in the SO and its relationship to spatial patterns in NPP is essential to quantify the capacity of the SO to act as a carbon sink.Primary productivity in the Southern Ocean (SO) is critical in governing atmospheric CO5 proposed that Fe of hydrothermal origin is an essential part of the SO Fe budget and that the observed Fe distribution in the SO cannot be replicated without the inclusion of hydrothermal sources of Fe. Tagliabue et al.5 further proposed that the utilization of hydrothermal Fe by phytoplankton fuels increased NPP in the SO that results in 5–30% more carbon export. The first report of hydrothermal vent emissions fueling a large phytoplankton bloom was from the Indian sector of the SO around 40°E near the Southwest Indian Ridge (SWIR). Ardyna et al.6 used historical measurements of elevated δ3He to suggest that deep water concentrations of Fe in the vicinity of the bloom (observed by BGC-Argo floats) were elevated and of hydrothermal origin. They further demonstrated that enhanced eddy kinetic energy (EKE) resulting from the interaction of currents with bottom topography brought the hydrothermal vent emissions to the surface, stimulating phytoplankton growth.In 2010, Tagliabue et al.7. KR1, the southerly of the two vent systems (KR2 is further to the northwest), coincides with the positions of the southern boundary of the Antarctic Circumpolar Current (sbACC) and the southern ACC front (sACCf), which run very close together in this part of the ACC  and NPP  waters of the ACC. By combining field data and satellite imagery, we were able to shed light on the likely cause for such an intense phytoplankton bloom in a region of generally low NPP.a concentration from November through February exceeded 0.25 mg m−3 was ~266,000 km2.Satellite imagery showed that the position of the bloom, adjacent to the AAR and the sbACC Fig. , to the a concentration within the top 40 m of the water column  and only 0.33–0.97 mg m−3 outside the bloom (stations 119 and 140/149). The depth-integrated Chl a ranged from 56.7 mg m−2 at station 119 and 60.0 mg m−2 at station 140 outside of the bloom to 114.8 mg m−2 at station 135 on the edge of the bloom to 319.0 mg m−2 at station 130 and 255.0 mg m−2 at station 131, in the interior of the bloom. Five stations  had depth-integrated Chl a exceeding 140 mg m−2. Particulate organic carbon (POC) concentrations inside the bloom ranged from 705.4 to 833.9 mg m−3, with values of only 101.2–189.9 mg m−3 outside the bloom  depth-integrated NPP was only 0.40 and 0.35 g C m−2 d−1, respectively.Depth-integrated NPP inside the AAR bloom was around three times higher than outside of the bloom. At stations 130 and 150 where Chl 2 and nutrient drawdown by phytoplankton. Surface pCO2 outside the bloom ranged from 394 to 410 μatm while pCO2 inside the bloom was reduced to 196–228 μatm  ranged from 23 to 30 m, outside the bloom (stations 119 and 140/149) from 16 to 35 m, and on the margin of the bloom  from 17 to 41 m.The AAR bloom was also associated with significant COatm Fig. . Similaratm Fig. . The depPhaeocystis antarctica, which accounted for 90–100% (n = 9) of the phytoplankton Chl a inside the bloom. Outside the bloom, diatoms were dominant, comprising 61.6–90.4% (n = 11) of phytoplankton Chl a. The dominance of P. antarctica inside the bloom was also strongly indicated by the lack of silicate drawdown, despite high nitrate drawdown, with silicate concentrations inside the bloom (65.10–67.89 μM) roughly equivalent to those outside the bloom (60.15–71.88 μM).CHEMTAX analysis of HPLC pigments (confirmed by microscopy) showed that the AAR bloom was dominated by the haptophyte p < 0.0001) than DFe concentrations measured below the ferricline at stations outside the bloom (0.13–0.16 nM at 150 m). Deep (>250 m) DFe concentrations inside the bloom were also significantly elevated relative to those outside the bloom down to our deepest sampling depth of 2000 m (p < 0.0001). Vertical diffusivity  was significantly higher (p = 0.0002) in subsurface (90–250 m) waters beneath the bloom (mean = 2.8 × 10−3 m−2 s−1) than outside the bloom  was substantially depleted in surface waters both inside and outside the AAR bloom, with concentrations falling to 0.04–0.05 nM Fig. . The sub−3. At bloom stations, waters with a potential density anomaly between 27.6 and 27.8 kg m−3 were colder, fresher, and more oxygenated than at non-bloom stations.Additionally, profiles of DFe Fig. , potenti−1, which falls within the range of cellular Fe:C ratios reported for P. antarctica (2.3–8.6 μmol mol−1)13, the dominant phytoplankton inside the bloom. At stations outside of the bloom, the ratio of DFe removed to POC accumulated ranged from 13.0 to 21.2 μmol mol−1, which is consistent with the range of cellular Fe:C ratios for diatoms (10–20 μmol mol−1)13, the dominant phytoplankton group in these waters. These findings demonstrate that the amount of DFe removed from surface waters was sufficient to support the observed phytoplankton biomass accumulation both inside and outside of the bloom.To determine if the observed drawdown of DFe in surface waters of the AAR bloom was sufficient to support the accumulated phytoplankton biomass, we calculated the ratio of DFe removed from surface waters (assuming the depletion in DFe concentration between the surface and 150–200 m depth was due to phytoplankton uptake) to POC accumulated (the average POC concentration above 40 m). At stations within the bloom, the ratio of DFe removed to POC accumulated ranged from 4.0 to 5.2 μmol mol2 fixation  by phytoplankton measured in situ could be supported by the delivery of DFe across the ferricline, we compared the calculated vertical DFe flux to depth-integrated NPP for stations 130 and 150 inside the bloom and stations 119 and 140 outside of the bloom. The ratio of DFe delivered to CO2 fixed inside the bloom was 4.37–4.70 μmol:mol, consistent with the cellular Fe:POC range for P. antarctica13. Outside the bloom, the DFe delivery to CO2 fixation ratio was 2.82–3.14 μmol:mol, which is much lower than the cellular Fe:POC range for diatoms13, suggesting that the community of diatoms growing outside of the bloom would be extremely Fe-stressed. Presumably, P. antarctica was the dominant phytoplankton in the AAR bloom due to its favorable Fe requirements.In order to assess whether the rate of COa and vertical DFe flux using linear regression analysis. Depth-integrated Chl a was significantly correlated with vertical DFe flux such that higher DFe flux was associated with higher depth-integrated Chl a . Depth-integrated Chl a was not significantly correlated with either vertical diffusivity (p = 0.18) or the DFe concentration gradient across the ferricline (p = 0.14).We also examined the relationship between depth-integrated Chl 2 s−2 between 150°E and 160°E. NPP along the sACCf climbs steadily starting around 153°E, reaching a maximum near 160°E downstream of where the sACCf passes over KR1.To better understand the hydrologic conditions influencing the AAR bloom, we examined the NPP, deep EKE, isopycnal depth, and bathymetry of the waters in and around the bloom  were directed southeasterly during the month of January 2014 in the vicinity of the AAR bloom, decreasing in intensity from the northeast to the southwest across the bloom  exceeded the depth-integrated Chl a of the most intense ACC bloom thus far measured by BGC-Argo floats (98.1 mg Chl a m−2)6. It also eclipsed the large Fe-enriched blooms located downstream of the relatively shallow Kerguelen  and Crozet plateaus, which have a mean phytoplankton biomass of ~120 mg Chl a m−216 and 180–229 mg Chl a m−217, respectively. The maximum depth-integrated Chl a measured in our bloom is more than three times higher than that measured in the hydrothermally-driven SWIR blooms6.The AAR bloom that we sampled in the Pacific Sector of the SO was large, long-lived, and contained a substantial amount of phytoplankton biomass. Both climatological NPP Fig.  and annu−2 d−1) are 2.7-3.8 times higher than the satellite-based daily NPP estimates at the peak of the spring bloom in open SO waters (0.30–0.40 g C m−2 d−1)11. NPP in the AAR bloom was within the range of that measured over the Crozet Plateau (0.52–3.00 g C m−2 d−1 at stations where depth-integrated Chl a exceeded 50 mg m−2)17. The areal extent of the AAR bloom  is also within the range for the Crozet Plateau bloom , although it was substantially smaller than the Kerguelen Plateau bloom 18. The Kerguelen bloom typically persists from November through January and the Crozet bloom from October through December18, which is approximately equivalent to the length of the AAR bloom (December through February).The productivity rates in the AAR bloom were also very high compared with other blooms in the ACC. Our daily NPP measurements in the bloom  compared to that of non-bloom stations (0.12–0.17 nM) combined with a higher vertical diffusivity. While the vertical gradient of DFe measured at our bloom stations is only slightly higher than that reported for the Kerguelen Plateau20, the vertical DFe fluxes we calculated  across the ferricline (0.9–4.4 × 10−3 m−2 s−1) is an order of magnitude higher than the mean value generally reported for the SO of 10−4 to 10−5 m−2 s−122. Although our Kz values are relatively high, they still fall within the range of values reported previously in the SO. Kz values as high as 10−1 have been reported in the upper mixed layer of the SO during times of strong winds24, and values of 10−3 to 10−4 are typical of the seasonal pycnocline23. We observed that buoyancy frequency was slightly higher at bloom stations (though not significantly) at the same depths where elevated Kz was observed 31, upwelling of deep water32, and hydrothermal vent emissions that recently have been shown to be a significant source of Fe to deep waters in the SO33. We can rule out aeolian dust flux and iceberg melt, as estimates of dust deposition south of 60°S are vanishingly small (0.001–0.003 mmol Fe m−2 yr−1) 27 and the path of icebergs in the Pacific sector of the SO lies too far south to impact the AAR bloom28. While sea ice may account for some DFe input30, we can rule it out as the primary source of DFe since the bloom does not track the sea ice edge . Furthermore, the input of DFe from dust deposition, iceberg melt, and sea ice cannot account for the elevated DFe concentrations observed down to 2000 m. Advection from continental shelves is also not a strong candidate. While weak currents appear to move from the region of the shelf in the south into our study area, results from a Lagrangian model of horizontal DFe delivery shows almost no advected DFe from continental shelves making it to the vicinity of the AAR bloom31. Most notably, none of these potential Fe sources can explain why the bloom recurs in the same location every year. The remaining potential sources of DFe are upwelling of Upper Circumpolar Deep Water (UCDW) and hydrothermal vent emissions.The potential sources of new Fe in the SO that could fuel the AAR bloom are aeolian dust deposition10. The position of the AAR bloom is adjacent to the sbACC and sACCf, which flow very close together in this region. This means that the AAR bloom is positioned close to the steeply tilted and outcropping isopycnals associated with these fronts. In addition to the meridional shallowing of isopycnals southward across the sACCf and sbACC, there is also a zonal shallowing of isopycnals along the sACCf as the ACC flows over the AAR , nitrate concentrations in the density range associated with UCDW (27.35–27.75 kg m−3)36 was 31.1–36.6 μM, while DFe concentrations varied over a full order of magnitude from 0.07 to 0.70 nM  DFe concentrations at bloom stations , and will bear the signature of the water mass properties  at that depth41. The water mass properties in our depth profiles between 1800 and 2000 m, which we use as a proxy for the water mass at the same depth immediately adjacent to the vent, are colder and more oxygenated than the overlying UCDW  and a maximum CO2 flux of −131 mmol m−2 d−1 (−1.57 g C m−2 d−1). These values are much higher than the mean CO2 flux measured at the peak of the Kerguelen bloom in November (−17.9 mmol m−2 d−1)42 and the mean CO2 flux in the Ross Sea in January (−12.5 mmol m−2 d−1)43.We propose that the AAR bloom is a new type of natural Fe fertilization system that receives its DFe subsidy in a way that is much different from previously described systems on the Kerguelen and Crozet Plateaus. Instead of being supported by DFe from the advection of a shelf source, the AAR bloom is most likely supported by DFe from hydrothermal vent emissions. Systems receiving persistent natural Fe fertilization are marked by higher export efficiencies44 found high concentrations of both krill and cephalopods around the sbACC, including the area encompassing the AAR bloom, and suggested that the physical processes along the sbACC provide predictably productive foraging opportunities. Humpback whales tagged near Eden, off the southeast coast of Australia, in October of 200845, fed primarily downstream of KR1 and KR2 in the region of elevated Chl a associated with the AAR bloom 12 over the last several decades46. However, our understanding of the changes in ocean circulation that result from a positive SAM suggest that NPP in the AAR bloom should increase due to increased upwelling of nutrient-rich deep water47 instead of the decrease observed. Perhaps the decrease in NPP in the AAR bloom is due to light limitation of phytoplankton growth resulting from an increase in MLD48. Regardless, without understanding the mechanisms controlling production in the AAR bloom, we cannot predict how it will be impacted by future changes anticipated for the SO, and how those will affect the role of the AAR bloom as a CO2 sink and upper trophic level hotspot.The AAR bloom is the first recurring bloom that is likely to be stimulated by hydrothermal Fe to be identified in the SO. The importance of the AAR bloom to upper trophic levels and its potential role as a COa concentrations were unusually high. We sampled a total of nine stations, six of which extended to a depth of 400 m and three to 2000 m. Bottom depth at our sampling stations ranged from 2762 m at station 135 to 3126 m at station 140/149  were collected from the flow-through seawater system. Wind speed was measured using a Gill 1390-PK-062/R anemometer. At each station, vertical profiles of conductivity, temperature, and depth (CTD) were measured (24 Hz sampling rate) using a Sea Bird 911 plus CTD package and fluorescence was measured using a WET Labs ECO-HFL/Fl fluorometer. The depth of the upper mixed layer (MLD) was defined as depth of the maximum buoyancy frequency50.Underway measurements of temperature (Seabird SBE-38), salinity (Seabird SBE-45), oxygen (Oxygen Optode 3835), fluorescence (WET Lab AFL), and 33, and a conventional CTD-rosette package. For biological sampling, water was collected at depths of 10, 25, 50, 75, and 100 m, and an additional six to seven depths between 100 and 400 m. Water samples were collected for dissolved Fe (DFe), macronutrients , Chl a, particulate organic carbon (POC), phytoplankton pigments, and simulated in situ primary production (SIS). Additionally, for the three casts conducted down to 2000 m, we collected water every 200 m below 400 m with the TMC-CTD-rosette for DFe analysis.Discrete water samples were collected during each cast using both a trace-metal clean CTD-rosette package (TMC-CTD)51.DFe samples were collected and filtered  in a trace metal clean van. Handling of DFe samples prior to analysis was conducted on a laminar flow bench inside a positive pressure, TMC, plastic bubble. DFe samples were analyzed onboard using the automated flow injection analysis method52. Measurements were made for silicate, nitrate and nitrite together, and nitrite separately. All measurements were calibrated with standards diluted in low nutrient seawater, which was also used as rinse water between the samples.Samples for macronutrients were filtered through 0.2 μm Acrodisk filters. Separate samples were taken for silicate, which were stored at 4 °C, and for nitrate and nitrite, which were stored frozen. After the cruise, nutrient samples were analyzed colorimetrically on a Bran and Luebbe trAAcs 800 Autoanalyzer53.Samples for POC analysis were filtered onto precombusted (450 °C for 4 h) 25 mm Whatman GF/F filters. Filters were dried at 60 °C for analysis on a Costech Elemental Analyzer using acetanilide as a calibration standarda were collected and analyzed on board with a Turner Model 10 AU fluorometer using the acidification method54. Depth-integrated Chl a was calculated down to 100 m at all stations by linearly interpolating between discrete Chl a samples. An integration depth of 100 m was chosen to ensure that all phytoplankton biomass was accounted for and to enable direct comparison with depth-integrated Chl a values reported elsewhere in the SO. Below 50 m, Chl a concentrations were very low and contributed a negligible amount to depth-integrated values.Samples for Chl 53. Detection was based on retention time and diode array spectroscopy (Waters 996) at 436 nm53. The CHEMTAX analysis package (version 1.95)56 was used to assess phytoplankton class abundance57.Samples for HPLC analysis of phytoplankton pigments were filtered onto 25 mm Whatman GF/F filters, flash-frozen in liquid nitrogen, and stored at −80 °C until analysis within six months of collection. Filters for pigment analysis were extracted in 90% acetone (48 h at 4 °C) and then separated by HPLC 14C-based SIS production on-deck incubations58. Depth-integrated NPP was calculated to the depth of the 1% light level. The depth of light levels used for the integration  were calculated using Beer’s Law and a diffuse attenuation coefficient that was a function of the mean Chl a concentration in the mixed layer at each station59. The depth of the 1% light level at stations 119 (87 m) and 140 (107 m) outside of the bloom was much deeper than at bloom stations 130 (30 m) and 150 (35 m).Depth-integrated daily NPP was assessed at four different stations  using Kz) was calculated from vertical density profiles using Thorpe-scale analysis60 . The DFe concentration gradient for each station was calculated as the slope of the ferricline (defined as the difference between the minimum DFe concentration between 15 and 56 m and the maximum DFe concentration between 150 and 205 m) divided by the thickness of the ferricline . Mean vertical diffusivity was calculated as the mean Kz in the ferricline. Station 135 was excluded from this analysis because it had an anomalously high vertical diffusivity coefficient that was more than two standard deviations above the mean.The vertical flux of DFe across the ferricline for each station was calculated by multiplying the DFe concentration gradient by the mean vertical diffusivity. Vertical diffusivity  and the atmosphere (pCO2a = 400 µatm)61. Air-sea CO2 flux  was calculated as62:k is the gas transfer velocity and K0 is the solubility of CO2 in seawater63.Air-sea COa images were obtained daily during the cruise and were used to target areas of high Chl a. To obtain bloom information after the cruise, we used monthly Chl a concentrations from level 3 MODIS/Aqua data  from SeaWiFS (1997–2002) and MODIS/Aqua (2002–2019) were used to calculate images of mean seasonal Chl a . Satellite-based NPP was calculated using the algorithm of Arrigo et al.11  using IDL  and SeaDAS version 7.5.3 (NASA). Geostrophic currents derived from satellite altimetry were produced by Ssalto/Duacs and distributed by the European Copernicus Marine Environment Monitoring Services. This multi-satellite global product provides daily velocities with a 1/4° spatial resolution.MODIS/Aqua Chl 65. Lagrangian trajectories were derived by applying a Runge–Kutta fourth-order scheme with a time step of six hours to the velocity fields, which are linearly interpolated in both space and time. For each advection period, the model identified the most recent contact of each water parcel to the area at the surface directly above the hydrothermal vents. As in Sergi et al.65, hydrothermal vents were identified with disks of 50 km radii centered in the hydrothermal vents’ locations. For each phytoplankton growing season from 1997–2019, a map was produced every 15 days between 1 November and 30 April. The climatological signal  during the high NPP period (Nov–Apr)66. We used the 3° × 3° resolution gridded climatology data to look at eddy kinetic energy  in our region of interest.The Argo New Displacements Rannou and Ollitrault (ANDRO) dataset uses the displacement of Argo floats at their parking depth to map global ocean circulation67. The maps are based primarily on Argo CTD data, supplemented by shipboard and ice-tethered profiler CTD data. The optimal interpolated (0–1950 dbar) sigma-gridded data for January were used to plot the zonal change in the pressure associated with different isopycnals.The Monthly Isopycnal/Mixed-layer Ocean Climatology (MIMOC) provides global monthly ocean property maps at 0.5° resolutiont-test was used to compare DFe and Kz values at stations inside the bloom to those outside the bloom for different depth bins. A Shapiro–Wilk normality test was used to evaluate the normality of the variable distributions. Linear regression analysis was used to evaluate the relationship between vertical DFe flux, vertical DFe gradient, and vertical diffusivity and depth-integrated Chl a. Statistical analyses were considered significant for p values < 0.05. Statistical analysis was done using R version 3.6.2 .A two-sample Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The falling number was significantly (p < 0.05) lower for WWF compared to WF or BF, indicating higher alpha-amylase activity. Thus, bread made from WWF without BF substitution exhibited a higher glycemic response similar to the bread made from WF. When partly replaced with BF, the GR of the bread made with WWF or WF reduced significantly (p < 0.05) in healthy individuals.Whole foods are generally considered healthier choices compared to processed foods. For nutritional consideration, whole wheat bread is recommended over the white bread. However, it has a similarly high effect on glycemic response (GR) as the white bread. This study is aimed at assessing the microstructure of whole wheat flour (WWF), white flour (WF), chickpea flour (BF), their blends, and dough and the GR of the bread made thereof. Scanning electron microscope analysis showed clear distinctions in the microstructure of the three flours. WWF particle size distribution had the widest spread with a polydispersity index (PDI) of 1.0 (±0.0) and wider average diameter, with  Wheat bread is a basic staple in the diet of people around the world and a major protein source for the less affluent population. Therefore, majority of research has been dedicated to the improvement of its amino acid profile –4. RelatWheat bread is classified as a high glycemic index (GI) food. Rapid elevations in blood glucose after consuming high GI foods more frequently may cause metabolic stress, specifically associated with insulin resistance and type 2 diabetes . ComplexThe research to date defines GI of a carbohydrate food as a postprandial property determined by the rate of digestion and absorption. Nevertheless, the glycemic property of bread is a complex interaction of several preprandial factors, to name a few, the chemical structure of starch, the changes in the starch molecule during heating, the size of starch granules, particle size of flour, and the presence of interfering substances between starch and the hydrolytic enzymes. Hence, to develop a good quality wheat bread with low GR, it is imperative to understand the factors that influence the microstructure characteristics of wheat flour as well as the dough, which might be impacting the glycemic response of bread.α, 1-4 linked glucose chain with branching as α, 1-6 linkages, and the amylose α, 1-4 glucose polymers rendering the starch structure a combination of amorphous and crystalline areas. Starch molecules go through the process of gelatinization when heated in the presence of water. Water enters the amorphous spaces, causing the starch granule to swell, disrupting its crystalline structure with amylose leaching out into the surrounding area. The process of heating gelatinizes the starch and cooling recrystallizes it, a phenomenon known as retrogradation. The higher crystallinity that occurs in a starch molecule will lead to lower digestibility and, therefore, to a lower GR [Starch in the bread is present in two chemical forms: amylopectin, an lower GR .The shape or size of starch granule also affects its digestibility and GR. The larger starch granule has a bigger surface area, with greater gelatinization tendency and a better probability of enzymatic hydrolysis . The preLegumes, a source of good quality proteins and dietary fiber, are low in total starch yet high in amylose starch, which can convert to resistant starch during baking, rendering it to produce low GR. Supplementing chickpea flour (BF) to wheat flour can help in decreasing the GR of wheat bread. BF-supplemented composite wheat bread has been evaluated for enhancement of the nutritional quality by many researchers, but only a few studies exist on the GR of the bread with inconsistent results , 15.The present research is aimed at exploring the hypotheses that microstructure characteristics of whole wheat and refined wheat flour doughs differ and thus variably impact bread quality and its glycemic effect; also, wheat-chickpea composite blends will modulate dough attributes for the production of superior quality of bread exhibiting low glycemic effect. The objectives of the study were to assess the microstructure differences of the doughs made from WWF, WF, and their blends with chickpea flour (BF); test the preprandial differences of the doughs (if any) affecting the bread on the postprandial glycemic responses; and determine the best ratio of wheat-chickpea blend for producing a low-glycemic bread. To achieve these objectives, three experiments were conducted to measure (1) the microstructure properties of dough made from WWF and WF and (2) of the wheat-BF blends at various ratios and (3) the GR of WWB and WB alone or as composite blends with BF.Both WWF and WF were procured from Kuwait Flour Mills & Bakeries Company, Shuwaikh. Commercially available BF was obtained from the local market.D) of the dispersed particles and evaluates the hydrodynamic radius (rh) in terms of the Stokes-Einstein equation: rh = kT/6πη D, where η, k, and T are the viscosity of the medium, the Boltzman constant, and the absolute temperature, respectively. The observed z value (intensity-weighted average dia (in nm), polydispersity index (PDI)), i.e., the width of particle size distribution peak, and areas under the particle size distribution peaks have been reported.Particle size analysis was performed by the dynamic light scattering method  using dyThe microstructure of WWF and WF dough samples was evaluated using a SEM  at an accelerating voltage of 20 kV. Dough was prepared by using Farinograph Water Absorption (FWA) . The floα-amylase activity of wheat flour. The higher the FN value, the lower the α-amylase activity and vice versa.Falling number values of these blends were determined as per the standard AACC method 56-81B  with a FBF substituted WWF and WF in different percentages ; blends were prepared by mixing them thoroughly in dry condition. Moisture content in these flours, blends, and dough was determined by the standard AACC method 44-15A .Particle size analysis, SEM images, and FN measurements of the chickpea flour, flour blends prepared by BF substitution of WWF and WF, and their dough were determined by the same procedure as explained in the earlier section.Composite bread from both WWF and WF substituting BF at 0% (control), 20, 30, and 40% level was prepared using the formula given earlier , 18 unde2) of 22.8 ± 2.2, participated in the study. Subjects were recruited at the College of Life Sciences, Kuwait University, through flyers. A sample size (n = 15) was calculated based on power analyses from a similar study to detect significant differences among the breads with a level of α 0.05 and β 0.08 [Young healthy males and females, age 18–28 years (mean ± SD of 22.4 ± 2.4) with normal range of body mass index  was purchased for a reference from the local market (Kuwait Flour Mills and Bakeries Co.). The commercial bread was similar in nutritional composition to the experimental control bread. All the test breads were provided and consumed in an amount to supply 50 g of available carbohydrates. Blood glucose concentration was measured at baseline and then at 15, 30, 45, 60, and 90 min by a finger-prick method using a Monojector Lancet Device and a portable blood glucose monitoring system .p < 0.05 are presented.All the results for the microstructure of flour and dough are expressed on a 14% moisture level. Research data were analyzed for analysis of variance using SPSS version 17 for Windows. Wherever appropriate, the mean values ± standard deviations of these samples using a significance level of p < 0.05.Repeated measures analysis of variance was conducted on blood glucose concentration as a change from the baseline at each time point to assess treatment, time, and any time by treatment interaction over 90 min using two-way ANOVA, followed by Tukey's post hoc tests to identify significant mean differences among treatments at each time point of measurements. Area under the blood glucose concentration curves was analyzed using one-way ANOVA and Tukey's post hoc assessment. Data are presented as mean ± SD, and the significance level is set at The WWF, WF, and BF used in the study had moisture contents of 10.58, 10.54, and 7.61%, respectively.z value of WWF differed significantly (p < 0.05) from that of WF and BF. The higher intensity-weighted average diameter (in nm) for WWF may be due to the large particles of wheat bran [The distribution of the particle size of WF, WWF, and BF samples is presented in eat bran . Figure μm and small granules about 2-10 μm, whereas the BF shows small granules of about 10 μm, but a lot of protein bodies and protein wedge-like structures  for WWF than both the WF and BF, and no significant difference was observed between WF and BF. The FN values are affected by the flour granulation, type of sprout damage (sprouting increases α-amylase content 1000-fold), and extent of damaged starch content of the flour samples, and this ultimately affects the quality of finished bakery products [α-amylase enzyme, thus giving similar FN values to those of WF. Also, because finer particles of BF gelatinized much faster, giving higher viscosity in the FN apparatus, this leads to higher FN value  versus 658.9 (±160.4) for WF  for WF  has rend) for WF  suggestsα-amylases is a common practice for the standardization of flour for their antistaling and loaf volume enhancement effects in commercial bread making [Supplementation with d making . Beta-amd making , 31. A md making  is benefd making , 8, 9 miChickpea flour substitution became effective but only at the higher levels of 30-40% in both series of breads, similarly, compared to the control breads (without BF) after the 45- to 60-minute time period Figures . The micin vivo starch hydrolysis and the release of glucose into the blood circulation [The ample body of literature available on the postprandial glycemic responses of carbohydrate foods, affected by factors that modify the starch structure and its accessibility to digestive enzymes, is at the gut level. For example, the development of resistant starch through recrystallization of the starch molecule via the process of retrogradation, formation of amylose-lipid complex, presence of protein or fiber, etc., all limit culation , 13, 21.in vivo glycemic responses. The average larger particle size and the widest spread of WWF reflected in significantly lower falling number values, indicating high starch accessibility for its excessive α-amylase activity than either of the WF and BF. Thus, the higher hydrolytic products of starch from whole wheat, supposedly complex carbohydrate with higher proportion of fiber, converted the WWB into high-glycemic bread similar to the bread from white flour, a refined carbohydrate. The lower glycemic response of the higher BF : wheat flour composite breads (40 : 60 levels) may be attributable to the coating of starch granules by the presence of a high amount of protein and fiber in BF, as evidenced by the scanning electron microscope results.This study is the first to our knowledge that has compared the microstructure properties of whole wheat flour versus refined wheat flour doughs and their breads on the Health professionals recommending whole wheat bread for its high fiber and other nutrient content need caution in suggesting it to the people with diabetes, because patients with diabetes, considering WWB as a better option over the white bread, may consume it excessively and bring their blood glucose regulations at a halt. From these data, wheat-chickpea composite bread would be a better choice than plain whole wheat bread for controlling blood glucose levels within the normal range. Future research focused on the microstructural characteristic modulated by hydrolytic enzymes, natural or supplemented, and other additives to the wheat flour on the glycemic response of bread in people with or without diabetes is warranted, in order to succeed in developing high-quality staple pan bread with the low glycemic index.


Ex vivo prolonged lung preservation has resulted in successful transplantation of high-risk donor lungs. Normothermic MP of hearts and livers has displayed safe (heart) and superior (liver) preservation in randomized controlled trials (RCT). Normothermic kidney preservation for 24 h was recently established. Early clinical outcomes beyond the market entry trials indicate bioenergetics reconditioning, improved preservation of structures subject to IRI, and significant prolongation of the preservation time. The monitoring of perfusion parameters, the biochemical investigation of preservation fluids, and the assessment of tissue viability and bioenergetics function now offer a comprehensive assessment of organ quality and function ex situ. Gene and protein expression profiling, investigation of passenger leukocytes, and advanced imaging may further enhance the understanding of the condition of an organ during MP. In addition, MP offers a platform for organ reconditioning and regeneration and hence catalyzes the clinical realization of tissue engineering. Organ modification may include immunological modification and the generation of chimeric organs. While these ideas are not conceptually new, MP now offers a platform for clinical realization. Defatting of steatotic livers, modulation of inflammation during preservation in lungs, vasodilatation of livers, and hepatitis C elimination have been successfully demonstrated in experimental and clinical trials. Targeted treatment of lesions and surgical treatment or graft modification have been attempted. In this review, we address the current state of MP and advanced organ monitoring and speculate about logical future steps and how this evolution of a novel technology can result in a medial revolution.Organ transplantation is undergoing profound changes. Contraindications for donation have been revised in order to better meet the organ demand. The use of lower-quality organs and organs with greater preoperative damage, including those from donation after cardiac death (DCD), has become an established routine but increases the risk of graft malfunction. This risk is further aggravated by ischemia and reperfusion injury (IRI) in the process of transplantation. These circumstances demand a preservation technology that ameliorates IRI and allows for assessment of viability and function prior to transplantation. Oxygenated hypothermic and normothermic machine perfusion (MP) have emerged as valid novel modalities for advanced organ preservation and conditioning.  The velocity and significance of progress in transplantation started to decrease around the turn of the century. Up until that point, the number of transplantations performed was ever-increasing, and the outcomes continued to improve. The clinical benefit to the patient from the immediate and effective treatment of organ failure was defined by improved patient survival. Patient survival as an endpoint followed by graft survival as a surrogate and indicator of judicious organ attribution were the framework of significant momentum that succeeded in making transplantation a standard of care. This success was fueled, however, not only by the healthcare quality gain but also by a flourishing industry producing high-quality and expensive immunosuppressive medication—to be taken for life after transplantation –3.The honeymoon period came to an end when both donor rates and short-term results stagnated. The established endpoints for clinical trials are difficult to improve in the short run after transplantation, and the field was slow in anticipating and responding to the changing circumstances—specifically the shortage of standard criteria donors (SCD). The field of transplantation now finds itself in very difficult circumstances: 90%+ patient and graft survival rates leave little room for improvement but also little room for error. At the same time, the conditions and circumstances in organ donation are radically changing, with increasing donor age and comorbidities and donation from non-heart-beating donors. In countries spearheading the evolution, donation after cardiac death (DCD) and extended criteria donor (ECD) organs make up more than 50% of the organ pool. With the pressure to maintain and further improve the excellent short- and long-term results, but working with very different resources, the most prominent immediate challenge is to better define and preserve, if not improve, organ quality in transplantation.With this as a driving force, modalities to prolong and improve preservation together with the tools to assess organ quality and function are emerging as the new frontier. Beyond serving an immediate medical need, the technologies evolving herald a more fundamental potential for change in healthcare: with the extracorporeal long-term preservation and assessment of organs, the capability for human organ treatment and repair arises. Hence, 20 years after transplantation advanced to become a standard of care, the field has the potential to play a key role and add a chapter to healthcare once again –10.In this review, we aim to address the current state of the field. A clarification of the terminology and the definitions of marginal organs, including an overview and comparison between the definitions for each organ, shall help to provide a better picture of the actual status. Considering the changing circumstances, the actual impacts of a condition on organ quality and function need to be addressed not only from the viewpoint of an empirical correlation. The individual parameters need to be further addressed for their reversibility and treatability. Building on this concept, the various modalities of MP in their different stages of development and actual benefit require a critical reflection. While the assessment of organ quality and function during extracorporeal perfusion is in its infancy, the growing body of data may help to eventually develop a comprehensive picture of the quality of the components of organs as relevant to transplantation and beyond. An important focus in this context is the assessment, definition, and relevance of the immune system of organs if isolated from the human body. Immunomodulation and immunomasking seem more realistic if attempted under the conditions of an isolated and perfused organ. The vision of a realization and refinement of the repair, treatment, and modification of organs is starting to trigger a boost in interest both in academia and in industry.In the light of organ shortage, critical reflection on the expansion of the donor pool is essential. The definition of organs that are marginal for transplantation is often considered a well-defined entity. In reality, however, the definition is very different for each organ, and there is incongruence between the various definitions. It is clear and well-justifiable that different organs are considered to be extended criteria organs going by different criteria, since the relevance of one single factor may be very diverse. The assessment of the outcome after transplantation of extended criteria organs, however, needs to be seen and interpreted with reference to the diverse definitions used throughout the years.The determination of ECD evolved from the term “marginal donors,” and much of the literature focuses on criteria for living donors. Since this is not the focus of this article, we will restrict the research to deceased donors and deceased donor organs.It is well-known that a number of factors may impact the eventual outcome after transplantation. The definition of cadaveric kidney organs marginal for transplantation emerged from prioritization of and preference for factors that may outrank other contributors in magnitude and simplicity of assessment . In the Still, compared to other organ systems in SOT, a comparatively good definition of marginal or ECD has been established. Noteworthily, the term “marginal” should be avoided in favor of ECD, as “marginal” may be considered pejorative by the patients who receive them and also by the programs that transplant them .The ECD criteria most widely used in kidney transplantation are the OPTN-approved criteria, as described by Metzger et al. . They arA helpful tool that combines such parameters is the Kidney Donor Risk Index (KDRI), which was generated by weighting the following factors into a single number in order to predict the risk of post-transplant graft failure: age, weight, height, race, history of hypertension, history of diabetes, cause of death, hepatitis C status, serum creatinine, and DCD. The association of KDRI with the outcome in the Eurotransplant senior program (ESP) was evaluated by Schamberger et al. . Kidney In liver transplantation, the term “marginal” does not refer to a number of distinct internationally accepted criteria that could characterize a graft, and nor does “ECD.” Instead, both terms encompass a variety of factors that have been observed to limit graft quality, but the impact of each of these factors remains to be defined. Therefore, several models for the assessment of the sum of such risk factors have been introduced . In thisper se considered as ECD, Mihylov et al. suggested criteria for “ECD DCD” livers in order to subclassify DCD organs  started to become a clinical reality and routine. With the publication by Collins et al., in which the authors described a method for the transportation of kidneys on ice using a preservation solution with the result that the organs could be shipped in a small box and showed almost no damage after 30 h, the era of static cold storage (SCS) had begun . The impRecently, Ruiz et al. presented a series of 46 livers transplanted after NRP and concluded that their results, including 23% early allograft dysfunction, are superior to standard DCD organ procurements and comparable to donation after brain death (DBD) results . The CamIt is noteworthy that, only recently, a protocol for NRP was established even for DCD heart transplantation. Due to a restoration of function of the arrested heart, organ assessment via echocardiography, pressure-volume loops, and cardiac-output measurements could be implemented .2; central venous pressure <12 mm Hg; pulmonary capillary wedge pressure <12 mm Hg; left ventricular ejection fraction > 50% in transoesophageal echocardiography.Accepted criteria for subsequent heart transplantation after weaning from mechanical support are defined as: cardiac index >2.5 L/min/mNRP leads to a meaningful increment of survival benefits and helps to enlarge the available donor pool by utilizing marginal donor organs in a safe way , 46. FutNew England Journal of Medicine and demonstrated a significantly lowered DGF rate in recipients receiving a hypothermically perfused kidney compared to a renal graft stored on ice in the common way  has finished recruiting, and 1-year-follow-up results were presented during the American Transplant Congress 2019. This international RCT on oxygenated HMP of DCD kidneys included 197 kidney pairs, of which 106 were successfully hypothermically perfused and transplanted. Approximately 80% of the transplanted kidney pairs were eligible for the primary analysis and resulted in a similar eGFR at 1 year after transplant for oxygenated (HMPO) and standard HMP. Analysis of all-cause graft failure, however, showed a higher eGFR in HMPO than in HMP. Overall graft loss was significantly lower in HMPO, leading the authors to suggest that HMPO improves 1-year kidney graft function when accounting for the beneficial effect on allograft survival.Since 2009, dynamic cold storage—hypothermic machine perfusion (HMP)—has progressed to become clinical routine in several fields of SOT—above all, in kidney preservation. Moers et al. published their landmark paper in the mmon way . Applyinmmon way –52. Durimmon way . One of In contrast, in the field of pancreas transplantation, dynamic preservation technologies have not yet experienced a breakthrough. Neither hypothermic nor NMP technologies are well-established, and SCS remains the standard procedure. A recent review outlined nine studies on HMP and 10 studies on NMP. All of them were experimental; none of the human pancreatic grafts were transplanted. However, the common conclusion of all of the published articles considered was that IRI, thrombosis, and morbidity after whole organ pancreas transplantation might be reduced by both technologies –56.Liver transplantation can be regarded as the field in SOT that primarily focuses on the development of preservation techniques, currently. The most commonly used perfusion types in the daily routine are HOPE (hypothermic oxygenated perfusion), D-HOPE , and normothermic liver preservation. HOPE and D-HOPE studies have produced promising results, showing higher adenosine triphosphate (ATP) levels during preservation, less IRI, excellent graft survival after DCD liver transplantation, and a significantly reduced rate of bile duct injuries –60. The NMP has been shown to be beneficial in terms of immediate graft function in all organ types in which it has been explored clinically: this includes the kidney –65, liveWith a new technology, the accompanying terminology is often somewhat unspecific and unprecise in the beginning. This also applies to MP. It is paramount, however, to eventually clarify the terminology and define a uniformity for its correct use. Simply put, no other terminology than organ preservation would be justified at this point in the clinical application, since no targeted means for organ reconditioning  have been established. While this remains a glorious goal for the future, it also represents a significant legal and ethical challenge, since the framework for the treatment of human organs remains to be established. It remains entirely unclear whether extracorporal organ reconditioning and/or repair can be achieved. The hope and the hype are building on a growing body of experimental data that indicates the feasibility of success or organ-specific treatment. While this remains a most significant opportunity for the field, we herein aim to adequately define organ preservation, organ (re-)conditioning, and organ treatment.https://www.ncbi.nlm.nih.gov/pubmed/?term=organ+preservation). It seems to be a term where the definition appears so clear and easy that it does not need to be written down. Yet again, a uniform description of organ preservation is not straightforward since it is not necessarily defined by organ retrieval, temperature, metabolic state, or any other sole condition. The one possible common ground for a definition is the purpose. Putting an organ into a state that allows later reactivation and restitution of its original function with the aim of minimizing damage during the period of mal- and non-perfusion.It is interesting to note that no entry exists on organ preservation in Wikipedia , while a PubMed search yields 17189 entries  seems relatively well-defined at first glance, but the actual definitions leave room for interpretation. PNF does not indicate no organ function at all but rather a function insufficient to prevent/avoid longer-term organ replacement therapy.For example, in liver transplantation, according to Ploeg et al., PNF is defined by poor initial function, requiring re-transplantation or leading to death within 7 days after the primary procedure without any identifiable cause of graft failure . Such paThe original vision of organ preservation as formulated in 1813 was built on the continuation of circulation and blood supply . The subThe current status of organ preservation remains largely defined by SCS. The well-established and standardized procedures of organ storage in three sterile plastic bags filled with preservation solution (bag 1) or saline/ringer lactate/other (bags 2/3) is well-established, with a relatively good understanding of the risk accumulating with the duration of preservation. The major advantage of SCS is the safety of the procedure. Pretty much nothing can happen to an organ in plastic bags, on ice, in a plastic container. The major limitations of this technique relate to the fact that metabolism continues—even though at a very low rate—eventually resulting in the accumulation of succinate and reactive oxygen species (ROS) –81. The search “reconditioning,” September 2019). The way this can be read in reference to human organs is that no intervention aiming beyond the restoration of the condition of an organ prior to retrieval (a) or beyond the optimal (naïve) condition of a healthy human organ (b)—depending on how the term “original” is interpreted in this context—shall be included. These two possible views already point toward very different states of an organ. The way the term was used in the recent scientific transplant literature is in agreement with repair of the immediate injury prior to and during organ retrieval and storage. The key element of reconditioning/repair as clinically applied at this point relates to the prevention of an eventual ROS burst by mitochondria and subsequent disruption of ATP production, mitochondrial permeability transition pores, and danger-associated molecular pattern (DAMP) release upon reperfusion. An electron shift toward succinate and subsequent reverse electron transfer upon reperfusion is discussed as a mechanism of damage and target of therapy in liver HMP . While the common understanding of organ repair might be more mechanistic and that of organ regeneration more biological, the distinction between the terms seems vague.Human organ repair, apparently, is treated as an equivalent to organ regeneration  does not provide much guidance regarding this issue.Directive 2010/45/EU of the European Parliament and of the Council of 7 July 2010 on standards of quality and safety of human organs intended for transplantation  and compared them to the equal numbers of DCD liver transplants without HOPE or DBD liver transplantations. Five-year outcomes of HOPE-treated DCD liver transplants were similar to those of DBD primary transplants and superior to those of untreated DCD liver transplants (HOPE DCD 94% vs. SCS DCD 78%)  .Preserving an organ under physiological conditions is the ideal condition to prevent deleterious effects of ischemia as well as IRI following SOT. Similar to HMP, NMP has recently been implemented in clinical routine at a variety of centers. The transfer from the experimental stage to clinical application was first described by Ravikumar et al. in 2016. In this phase 1 (first-in-man), non-randomized prospective trial, short-term outcomes of 20 recipients of NMP-perfused donor livers were matched in a ratio of 1:2 to cold-stored livers. In the study group, livers were preserved under normothermic conditions over the entire preservation period (ranging from 3.5 to 18.5 h). Except for a significantly reduced peak serum AST in NMP-liver recipients, no differences regarding short-term recipient survival, PNF rate, or early allograft dysfunction were observed. Therefore, the safety and feasibility of this dynamic preservation method could be confirmed, opening the field for further clinical investigations with major implications on logistics .Feasibility and reproducibility following transplantation were further underlined in a U.S. phase I non-randomized pilot study . HoweverNasralla et al. were first to perform RCT to test the efficacy of NMP vs. SCS. After informed consent had been obtained from the recipient, the allocated liver was either randomized to SCS or NMP. NMP was performed over the entire preservation period. Outcomes of 121 NMP-graft recipients were compared to outcomes from 101 well-matched SCS-graft recipients. The key findings of this study were a significantly reduced peak serum AST, a significant reduction of post-reperfusion syndrome, and a 72% lower adjusted odds rate of developing early allograft dysfunction (EAD) in the NMP liver recipients compared to controls. Furthermore, NMP of the liver graft resulted in increased organ utilization with a 50% lower discard rate in this group .The additional positive effect of NMP is the “almost” physiological aspect of this method, which opens up new possibilities by means of organ assessment and organ selection. This fact was a matter of investigation in an observational study, where livers considered unsuitable for transplantation as well as highest risk liver were included. In total, 47 livers were biochemically assessed during the preservation, resulting in 39 liver transplants. Remarkably, two out of 19 livers deemed unsuitable were transplanted .In summary, both HMP/HOPE and NMP are accepted and clinically established preservation methods, particularly interesting in the preservation and preconditioning of high-risk organs. Especially in this organ category, including ECD and DCD livers, they have both been shown to be superior to SCS. Whether HMP/HOPE or HMP is superior to the other is still a matter of debate since the two methods have not been correlated with each other in clinical settings, and further RCTs are needed.The amount of clinical evidence for dynamic preservation of kidney grafts is rapidly increasing. Moers et al. were first to perform an international RCT on hypothermically perfused kidneys for transplantation. One kidney from 336 deceased donors was randomly assigned to HMP, and the other to SCS. A reduced risk of DGF and improved graft survival in favor of MP-preserved organs was shown . These pFurthermore, in another RCT, kidneys were either preserved by HMP or SCS. In accordance with Moers as well as Jochmans, the primary end-point was the occurrence of DGF. Secondary endpoints were primary non-function as well as graft survival. Both the risk of DGF and the incidence of non-function were significantly reduced by HMP in their cohort. One-year graft survival and function could be improved by dynamic cold graft preservation . DespiteThe optimal timepoint for, as well as the optimal duration of, HMP is still a matter of debate. HMP can be used either as a primary preservation method or in the end-ischemic phase as a reconditioning tool. Matos et al. were able to show a faster recovery of renal function if grafts were subjected to HMP prior to transplantation after a mean SCS period of 22 h. MP time was at least 6 h. However, this beneficial effect was limited to donors under the age of 50, who represent a rather selective group . FurtherNMP of the kidney may eventually resolve many issues related to damage induced by ischemic phases. The idea of preserving kidney grafts under almost physiological conditions with the possibility of real-life assessment of the graft prior to transplantation sounds intriguing. However, while the number of preclinical studies using animal models as well as discarded human kidneys is steadily increasing, evidence for NMP of the kidney in a clinical setting is rather poor. Hosgood et al. were first to transform preclinical experience into clinical reality by transplanting a 62-year-old ECD kidney into a 55-year old recipient. After 11 h of SCS, the graft was perfused with a plasma-free red cell-based solution at 33.9°C for 35 min prior to transplantation. Although a slow graft function was observed, the patient remained dialysis free with a serum creatinine level of 132 μmol/L at 3 months post-transplantation. In contrast, the recipient of the opposite kidney developed DGF . Based oTaken together, HMP of the kidney has been shown to have its place, especially in the preservation of marginal grafts, including grafts from DCD and ECD, with superior outcomes in the early phase compared to SCS. However, CIT is still the limiting factor influencing short- as well as long-term outcomes. In contrast, although the amount of preclinical data is rapidly increasing, NMP is still in its early phase. However, feasibility and non-inferiority to SCS have been described at this early stage, and multicenter RCTs are planned.In contrast to the dynamic preservation of liver and kidney, pancreatic MP is challenging. This is attributed to the physiologic as well as the anatomical characteristics of the pancreatic graft . MP of pIn contrast to the liver as well as the kidney, the pancreas is characterized by a low-flow and low-pressure environment, which impedes the direct translation of the existing perfusion machines for use in dynamic pancreas preservation . HoweverTherefore, assessment and evaluation of a pancreatic graft prior to transplantation would be of the utmost importance, since changes in donor demographics result in an increased need for the acceptance of higher-risk grafts at high-volume centers. However, MP of pancreatic grafts must still be considered experimental at this stage.ex vivo lung perfusion (EVLP)  . Needlesn (EVLP) .ex vivo prolonged lung preservation resulted in successful transplantation of high-risk donor lungs  Heart. At mild hypothermia (34°C), the system uses a combination of donor blood and a proprietary solution as perfusate for the heart. In 2014, heart NMP has led to the first distant procurement DCD heart transplantation. To date, over 100 DCD heart transplants have been performed using a cardiac perfusion system , and MP Currently, the decision as to whether organs are suitable for transplantation is determined on the basis of more or less subjective empirically established clinical parameters that have been shown to be associated with an increased rate of early allograft dysfunction or graft failure. Parameters such as donor past medical history, last known laboratory values, findings during procurement, and other procurement variables such as expected ischemia times primarily determine the acceptability of a graft. These parameters include prolonged warm ischemia time (WIT) >30 min during DCD, prolonged CIT, and parenchymal alterations within the graft . Viability testing and functional assessment prior to transplantation are likely to extend the utilization of suboptimal or marginal organs –111.HMP is an alternative method to standard SCS for the preservation of organs. For different types of kidney grafts, HMP offers superior preservation compared with SCS. In terms of graft quality assessment during HMP, some biochemical parameters of the released perfusate and hydrodynamic parameters are found to independently correlate with the outcome—a finding that may help clinicians in their decision making.Increased Glutathione S-Tranferase (GST), N-acetyl-beta-D-glucosaminidase (NAG), heart-type fatty acid-binding protein (H-FABP), or lactate dehydrogenase (LDH) concentrations during MP may serve as indicators for suboptimal graft quality. Furthermore, levels of redox-active iron measured in the perfusate have been correlated with DGF rates –114.Moreover, the measurement of vascular resistance during MP represents an additional and objective source of information that can assist clinicians in their decision-making process. High vascular resistance as a hydrodynamic parameter has been shown to be associated with an increased risk of DGF development and predictive for 1-year graft failure –117. TakConcerning kidney NMP, a scoring system has been developed by Hosgood and Nicholson. The decision on whether to transplant an NMP-kidney was based on macroscopic appearance, renal blood flow, and total urine output during NMP. The authors conclude that, currently, a high percentage of retrieved kidneys are being unnecessarily discarded , 118.Compared to the kidney, data on liver monitoring during HMP remain scarce, although recent publications suggest promising parameters. A correlation between the AST levels in the perfusate during HMP and the peak of AST after liver transplantation has been described. Furthermore, during HMP of porcine livers, a cumulative release of Fatty Acid- Binding Protein 1 (FABP) or AST may be represented by a linear or logarithmic equation, respectively. Each equation is characterized by a b-coefficient that is able to discriminate between livers likely to fail. Similarly, during HMP of human livers discarded for transplantation, AST release in the perfusate could discriminate between livers suitable for transplantation and unsuitable ones. More sophisticated methods to determine graft viability include evaluation of ATP content assessed by magnetic resonance imaging and spectroscopy , 119–121Furthermore, there is evidence that a decline in arterial flow in a pressure-controlled system of MP can be used as a marker for decreasing graft viability. However, in general, it cannot be advised to use flow values as an indicator of liver damage and viability during human liver MP , 91, 122Concerning NMP of liver grafts, this technology has been applied with promising results for utilization and outcomes , 123–126There is growing evidence that hepatic ATP content may serve as a viability marker. In clinical studies, hepatic ATP levels prior to organ retrieval or during preservation correlated with primary liver function after transplantation. Bruisma et al. showed that differences in mitochondrial respiration and restoration of cellular ATP contents are possible viability markers in the setting of NMP. Moreover, NMP offers the option to perform dynamic metabolomic analyses in both bile samples and sequential liver biopsies , 131.However, although these results seem impressive, none of the above-mentioned viability markers have been validated in larger studies, and the small numbers of patients included in the studies make it difficult to draw robust conclusions.Regarding a potential viability assessment of human pancreas grafts during HMP, flow indices and histological assessment of duodenal and pancreas-parenchyma biopsies throughout the perfusion duration have been proposed. Branchereau et al. described an absence of edema, a decrease in resistance indices, and normal staining for insulin, glucagon, and somatostatin as markers for good graft quality , 101. BaConcerning heart transplantation, organ assessment during NMP mainly relies on metabolic parameters and measurements of lactate levels in the aortic root and pulmonary artery. In the PROCEED II trial, three hearts were discarded due to rising total perfusate lactate concentrations. Histopathological examination showed myocardial injury  or left ventricular hypertrophy in those organs . CalculaIn EVLP, assessment of the organ relies primarily on functional and macroscopic parameters, as has been reviewed recently . AdequatInterestingly, given the observation that perfusate IL-1β and TNF-α at 30 min during EVLP were effective markers for differentiating between in-hospital survival and non-survival post-transplant, blocking the IL-1β pathway during EVLP might reduce endothelial activation and subsequent neutrophil adhesion on reperfusion .per se is a rather arbitrary one. The most commonly used is the dichotomy between standard criteria (SCD) and extended criteria donors (ECD), which is simply discriminated by age and three donor criteria for the DBD  kidney cohort  induces a pro-inflammatory environment, which could lead to an activation of innate and adaptive immune responses –141. AgiAn alteration of immunogenicity in aging organs could be one reason to focus on preservation techniques  such as ex vivo setting might provide not only a more physiological preservation method but also the ability to assess the organ prior to transplantation and to investigate its immunological characteristics in isolation  offers a previously non-existent link between basic and clinical science. The opportunity to treat an organ during preservation creates a new field of translational research. In this context, one of the main targets that can now be addressed is the modulation of the immunogenicity of a specific graft. In theory, such immunomodulation could allow the obviation of lifelong immunosuppression or even forge a path to the holy grail in transplantation—the achievement of tolerance. Importantly, MP offers a platform to apply immunomodulatory strategies that have been elucidated in In the existing literature, three major such strategies have been investigated, which are discussed herein. One of the most promising is the application of stem or progenitor cells, with the potential to suppress immunogenicity and help repair injured tissue. Another strategy is the administration of anti-inflammatory/immunomodulatory drugs and agents, and, finally, gene transduction by adenoviral vector gene delivery.per se seems not only to protect against preservation injury but also to downregulate the immune system and blunt the alloimmune response, as was shown in a rodent model of hypothermic oxygenated perfusion (HOPE)  . In partn (HOPE) –157. On n (HOPE) , 158–160ex vivo porcine organ perfusion models a suitable platform for translational transplant research. According to a recent review, in 2017, 22 articles discussed ex vivo porcine organ perfusion within the context of transplant preservation surgery , induced adult pluripotent stem cells, fetal stem cells from placenta, membranes, amniotic fluid, and umbilical cord, and hematopoietic cells . Among tin vitro  model. Twelve-hour NMP of DCD lungs with intravascular delivery of MSCs (150 × 106) resulted in reduced levels of interleukin-8 (IL-8), a pro-inflammatory cytokine associated with reperfusion injury, along with increased levels of vascular endothelial growth factor (VEGF)  . The facr (VEGF) .ex vivo normothermic perfusion. The authors used an exsanguinous metabolic support (EMS) tissue-engineering platform to study five pairs of kidney allografts from DCD donors. Whereas, one kidney of each pair solely underwent perfusion, the partner kidney was EMS perfused with MSC (1 × 10). Indeed, a reduced inflammatory response was observed along with increased synthesis of adenosine triphosphate and growth factors and a normalization of the cytoskeleton and mitosis , and galactosidase and increased IL-10 levels .+ and CD8+ T cells, and increases endothelial cell nitric oxide (ex vivo lung perfusion (EVLP). Indeed, EVLP with targeted A2AR agonist treatment could attenuate IRI after transplantation of DCD donor lungs subjected to prolonged 12-h cold preservation in a preclinical porcine model  promotor to porcine donor hearts during NMP and prior to heterotopic implantation. Along with a high copy number of vector genomic DNA in transplanted hearts, there was no evidence of vector DNA in either the recipient's native heart or liver, substantiating the applicability and safety of the protocol.Only recently has a normothermic These findings make it likely that this technology could be feasible for other organ systems as well. A wide variety of interesting genes could be targeted. As has been reviewed lately , promisiOutstandingly, Figueiredo et al. recently showed that antigen silencing is feasible during NMP. In a porcine model of lung NMP, short hairpin RNAs were delivered by lentiviral vectors, successfully reducing the immunogenicity of the lung by silencing MHC expression on the endothelium. The authors concluded that the decrease in immunogenicity carries the potential to generate immunologically invisible organs to counteract the burden of rejection and immunosuppression .Recent experimental data primarily focusing on marginal livers furthermore suggested that NMP offers a platform for organ reconditioning and repair.Impressive data from Birmingham, UK, showed that livers discarded due to pronounced steatosis could be effectively treated with defatting agents during 6 h of NMP. Tissue triglycerides were lowered by 38% and macrovesicular steatosis by 40%, which was associated with a down-regulation of inflammatory marker expression relevant for oxidative injury and activation of immune cells , combined with reduced inflammatory cytokines in the perfusate  . In addiIn summary, immunomodulation in the context of MP seems to offer a plethora of novel strategies. A series of porcine and human studies distinctively underline its effectiveness and feasibility. However, as formulated in a recent review , 192, thMuch speculation about the changes in transplantation is currently energizing the field. The conceptual approach of extracorporal organ preservation and monitoring begs for speculation about organ reconditioning, repair, and treatment. While these are all valid considerations and perfectly reasonable hopes, the implementation of machine perfusion is the first step. Immediate and widespread adoption of HMP and NMP is unlikely since this technology does not immediately improve the outcome with regard to the primary endpoints in transplantation: patient and graft survival. Authority approval and market entry are largely based on secondary endpoints such as delayed graft function  or primary poor function , which are considered to serve as surrogates for improved long-term results. The conceptual limitation with surrogates is, however, that the predictive value is not uniformly robust or formally established and immediately reproducible. The DGF rate, for example, in kidneys from DCD donors is high, but the predictive value of DFG in these cases is low . The defFor clinical implementation, several regional and center factors play an important role. First, the retrieval team may be different from the team eventually performing the transplantation. Even if the retrieval team is familiar with the technology, the preparation of the graft prior to NMP (more than HMP) is more substantial and more definitive for the fate of the graft. Hence, in a system with longer-distance organ exchange, additional steps and a new routine would need to be established. The technology and the shifting of the work of the backtable procedure to the retrieving team need to be considered. The alternative approach where the organ is transferred to the recipient center and is then machine perfused is currently being pursued, but data indicating the actual advantage of this approach are lacking . For nowThe consideration of longer-term organ preservation under normothermic conditions further carries the challenge of defining the responsibilities for managing and monitoring the organs. Eventually, the working place description of health care workers involved in this will require adoption and formal training. Standard operating procedures (SOP) and safety parameters need to be established for a wider spread use of the technology. It is likely that the advanced technological requirements and the need for 24-h availability of knowhow and personnel would favor the establishment of regional hubs for MP.Despite the hype in the field, the clinical adoption of MP is slow. In addition to the above-mentioned factors, the lack of reimbursement is a stringent limitation in many regions. For the technology to be reimbursed, the authorities will require hard facts. Hence, the path to more widespread clinical use might be long, and the focus should remain on the demonstration of superiority regarding the most relevant clinical endpoints.One major important step toward collective advancement in this field would be the definition of data points during and after MP and the establishment of registries for coordinated data collection. Such data points should include the definition and terminology of the various time points and actions such as cold flush and storage prior to MP, temperature and flow during MP, parameters indicating metabolic function and bile/urine production during MP, second flush, second cold ischemia time, and others. One of the hurdles in improving NMP is the large number of variables added to preservation. Identifying the relevance of individual parameters will require attention to the details of the procedure and adequate data collection and handling. To the knowledge of the authors, no routine data collection and no consensus toward data points have been established at present.The decision-making process in MP is relatively arbitrary since the data collected during MP are suggestive but not formally established as quality-defining parameters. Since the decision to transplant an organ or not is extremely meaningful, great care needs to be applied in the process, and detailed documentation of the reasoning for decision making should be carried out. A greater effort toward orchestrated data collection could help to streamline and eventually enhance the robustness of the decision-making process.The preference for MP to be used for the preservation of marginal organs results from the greater need for organ assessment and the greater potential benefit of better preservation. This might be particularly true and relevant for organs from uncontrolled DCDs, where the circumstances and the accumulated damage to the organs might be less clear. The value of an additional assessment under these conditions is not only meaningful with respect to the number of additional organs for transplantation but also for preventing the transplantation of organs that are severely damaged. While the assessment of this subject would be highly valuable and is much needed, the behavior of the investigators in the NMP trial by Nasralla et al. indicates the limitations and conflicts observed with this approach . Since tThe fantasies building on the realization of extracorporal organ preservation under physiological conditions are currently fuelling hopes that this technology could facilitate tissue regeneration, organ repair, immunomodulation, xenograft humanification, and many other things. MP as a platform may impact medicine far beyond transplantation and delivers a unique chance to alter the treatment of organ failure and organ disease. Between the imagination and the realization of medical advancement stands a mountain of work and an as yet unknown but probably large number of technical and methodological challenges. An important initial goal is the expansion of the duration of MP and the establishment of an equilibrium of the condition of the organ. Preservation of organs for several days will likely require additional modifications from the currently existing technologies. Preservation of the acid-base equilibrium, nutrition organ weight-induced pressure, electrolyte shift, hemolysis, and many other challenges may require closer attention. A tissue- and/or cell-specific and targeted treatment is a realistic consideration, and proof of concept trials indicate the feasibility of this approach , 197. ThTR and SS conceived and designed the study and wrote the manuscript. BC, RO, AW, JD, CK, and CB wrote sections of the manuscript. DO and MG helped to revise the manuscript, tables and figures. All the authors contributed to manuscript revision, read, and approved the submitted version.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.


To treat small-for-size syndrome (SFSS) after living donor liver transplantation (LDLT), many procedures were described for portal flow modulation before, during, or after transplantation. The selection of the procedure as well as the best timing remains controversial.A 43-year-old female with end-stage liver disease underwent LDLT with extended left with caudate lobe graft from her donor who was her 41-year-old brother (graft volume/standard liver volume (GV/SLV), 35.7%; graft to recipient weight ratio (GRWR), 0.67%). During the surgery, splenectomy could not be performed owing to severe peri-splenic adhesions to avoid the ruined bleedings. The splenic artery ligation was not also completely done because it was dorsal to the pancreas and difficult to be approached. Finally, adequate portal vein (PV) inflow was confirmed after portal venous thrombectomy. As having post-transplant optional procedures that are accessible for PV flow modulation, any other procedures for PV modulation during LDLT were not done until the postoperative assessment of the graft function and PV flow for possible postoperative modulation of the portal flow accordingly. Postoperative PV flow kept as high as 30 cm/s. By the end of the 1st week, there was a progressive deterioration of the total bilirubin profile (peak as 19.4 mg/dL) and ascitic fluid amount exceeded 1000 mL/day. Therefore, splenic artery embolization was done effectively and safely on the 10th postoperative day (POD) to reverse early allograft dysfunction as PV flow significantly decreased to keep within 20 cm/s and serum total bilirubin levels gradually declined with decreased amounts of ascites below 500 mL on POD 11 and thereafter. The patient was discharged on POD 28 with good condition.SFSS can be prevented or reversed by the portal inflow modulation, even by post-transplant procedure. This case emphasizes that keeping accessible angiographic treatment options for PV modulation, such as splenic artery embolization, after LDLT is quite feasible. Living donor liver transplantation (LDLT) is a commonly used treatment option for patients with end-stage liver disease, particularly in eastern countries , 2. SmalHerein, we report a case of LDLT recipient who developed early graft dysfunction after LDLT and SFSS with evident high portal flow. Postoperative splenic artery embolization (SAE) was done as a post-transplant portal inflow modulation, and SFSS was successfully treated.2. Her blood type was O Rh (+). Preoperative assessment reveals the Child-Pugh score was grade C as 13 pts and the model for end-stage liver disease score was high as 19. Radiological evaluation revealed partial PV thrombosis in the main trunk. She underwent partial SAE 8 years ago and umbilical hernia repair. Her donor was her 41-year-old brother who weighted 79 kg and body mass index was 26.7 kg/m2. His blood type was B Rh (+). Preoperative 3-dimensional volumetry revealed that the extended left with caudate lobe graft volume was 555 mL, which was 49.2% of the recipient standard liver volume (SLV). The donor and recipient have incompatible blood types, so the recipient was subjected to preoperative rituximab protocol.The patient was a 43-year-old female with end-stage liver disease secondary to HCV hepatitis. Her weight was 60 kg and her body mass index was 24.3 kg/mThe patient underwent LDLT using extended left with caudate lobe graft. The actual graft weighted 467 g (402 g after UW reperfusion) of which GV/SLV was 35.7% and GRWR was 0.67%. It had middle and left hepatic veins, left hepatic artery, left PV, and left hepatic duct. Upon laparotomy, there was 10,500 mL of ascites as well as perihepatic adhesions and periumbilical adhesions. On laparotomy, portal venous pressure (PVP) monitoring was 22 mmHg. The splenic artery was dorsal to the pancreas and difficult to be approached, and finally, the upper pole branch of the splenic artery was identified and ligated at the level of the distal pancreas, by which only the upper pole of the spleen turned pale. Splenectomy could not be performed due to severe peri-splenic adhesions to avoid the ruined bleedings. After PV thrombectomy was done, adequate PV inflow was not confirmed until left gastric vessel ligation was performed. After graft reperfusion, PVP was 20 mmHg but PV flow was relatively low, 520 mL/min. At this time, we choose not to ligate the other branches of the splenic artery nor the lienorenal shunt until postoperative assessment of the graft function and the PV flow for possible postoperative modulation and boost of the portal flow accordingly. The point is that safe angiographic procedures for portal flow modulation are accessible postoperatively, such as splenic artery embolization for portal decompression and balloon-occluded retrograde transvenous obliteration (BRTO) for boosting portal flow.3/μL) by POD 8 and INR levels kept below 1.5 along the whole clinical course. Considering this relatively insufficient graft volume along with the clinical and laboratory parameters suggestive of SFSS, then we decided to do splenic artery embolization (SAE) for portal decompression. The patient underwent arterial splenic angiography on POD 10 via percutaneous trans-femoral Seldinger’s technique. After angiographic assessment, partial SAE occluding the main branch of the splenic artery was achieved by trans-catheter coil embolization , widely vary , 49. In r et al.  reportedGiving post-transplantation portal modulation dilemma , 32, 50,We report here the case of a patient who recovered from SFSS by the postoperative portal flow modulation by splenic artery embolization. The present case suggests that keeping the accessible angiographic treatment options for PV modulation after LDLT is quite feasible.