The fluid balance associated with a better outcome following emergency surgery is unknown. The aim of this study was to explore the association of the perioperative fluid balance and postoperative complications during emergency gastrointestinal surgery.We retrospectively included patients undergoing emergency surgery for gastrointestinal obstruction or perforation. A perioperative fluid balance of 2.5 L divided the cohort in a conservative and liberal group. Outcome was Clavien-Dindo graded complications registered 90 days postoperatively. We used logistic regression adjusted for age, sex, American Society of Anesthesiologists’ classification, use of epidural analgesia, use of vasopressor, type of surgery, intraabdominal pathology, and hospital. Predicted risk of complications was demonstrated on a continuous scale of the fluid balance.p < 0.001, and cardiopulmonary complications 3.2 (95% CI 1.9 to 5.7), p < 0.001, were increased in the liberal group. A perioperative fluid balance of 0–2 L was associated with minimal risk of cardiopulmonary complications compared to 1.5–3.5 L for renal complications.We included 342 patients operated between July 2014 and July 2015 from three centers. The perioperative fluid balance was 1.6 L IQR [1.0 to 2.0] in the conservative vs. 3.6 L IQR [3.0 to 5.3] in the liberal group. Odds ratio of overall 2.6 (95% CI 1.5 to 4.4), We found a perioperative fluid balance above 2.5 L to be associated with an increased risk of overall and cardiopulmonary complications following emergency surgery for gastrointestinal obstruction or perforation. A perioperative fluid balance of 0–2 L was associated with the lowest risk of cardiopulmonary complications and 1.5–3.5 L for renal complications.The online version contains supplementary material available at 10.1186/s13741-021-00235-y. We aimed to study the effect of a perioperative fluid balance above 2.5 L on postoperative complications following emergency gastrointestinal surgery.We found that a perioperative fluid balance above 2.5 L was significantly associated with an increased risk of overall- and cardiopulmonary complications and that the predicted risk of cardiopulmonary complications was at a minimum at a perioperative fluid balance between 0 and 2 L compared to 1.5–3.5 L for renal complications.Our results, from this multicenter observational study, imply a clinical potential of an optimized perioperative fluid strategy in patients undergoing emergency gastrointestinal surgery.Worldwide, more than 310 million patients undergo major surgery each year  and the Danish Data Protection Agency (REG-149-2016) prior to data extraction. Ethical approval for this study (J.nr. 16-000014) was provided by the Ethical Committee, Zealand Region, Denmark, on 14 December 2016. The requirement for written informed consent was waived by the committee. We retrospectively collected data on patients admitted between 1 July 2014 and 31 July 2015 at three teaching hospitals in the Region of Zealand, Denmark. The study sites offer treatment free of charge for a population of approximately 800,000 citizens. Local guidelines for intraoperative fluid administration during emergency gastrointestinal surgery were not present during the study period. The Strengthening the Reporting of Observational studies in Epidemiology (STROBE) statement was used in drafting this manuscript , pregnant women, patients receiving regular dialysis, or patients with a traumatic or iatrogenic perforation. If eligible for inclusion, more than once patients were included only at the first procedure. We excluded patients who had had intraabdominal surgery 30 days prior to eligibility or patients without data on the intra- and postoperative fluid therapy. The Danish Civil Registration System provides uniform identification of every citizen through a personal identification number used to access all electronically stored medical and anesthetic records. It offers complete information on death for all Danish residents  for up to 24 h. Fluid administration included crystalloids, glucose-containing fluids, colloids, intravenous drugs, packed blood products, and per oral intake. Fluid loss included diuresis, aspiration, emptied ascites, blood loss, and perspiration calculated as 0.5 mL kgThe primary outcome was complications until postoperative day 90. The Clavien-Dindo classification (CDC) . Renal complications included the need for dialysis or other renal complications (nephritis or hydronephrosis treated with a nephrostomy catheter). Infectious complications included superficial wound infection, pneumonia, urinary tract infection, or cutaneous infection. A clinical doctor set the diagnosis and initiated medical treatment.The three participating hospitals used identical software and uniform registration of variables. We screened the booking system for patients undergoing abdominal surgery. All emergency procedures meeting the inclusion criteria and unclassified cases were further explored. We accessed the medical and anesthetic records on each patient eligible for inclusion. The data collected preoperatively were physiological status, co-morbidities, sepsis-2 score, and American Society of Anesthesiologists’ (ASA) classification. Intraoperatively, we registered the fluid administration and loss as specified above, vasopressor use and dose, hypotensive episodes defined as mean arterial pressure < 50 mm Hg at any time intra- and postoperatively, and the use of epidural analgesia.Case report forms were used for data collection by our medically trained team. All team members were trained in the use of the Clavien-Dindo classification. AAA and AWV collected the anesthetic data, fluid administration, and losses. Two independent team members assessed each patient file and registered data on complications in two separate case report forms. Regular audit by the project leader (AWV) corrected irregularities. The senior advisor (BB) was consulted in case of incongruity. Database entry was conducted twice and inconsistencies were corrected by revisiting the case report form.p-value < 0.01. We presented the predicted risk of complications depending on the fluid balance on a continuous scale. A generalized additive model with smoothing splines and four degrees of freedom was used. The statistical plan was approved by the authors before commencing the analysis of data. The statistical software was R version 3.5.0 GUI 1.70 El Capitan©R, 2016 and RStudio version 1.1.453.Data were tested for normality and parametric or non-parametric statistics was used as appropriate. The primary outcome was analyzed with multiple logistic regression. Confounders included were settled between the authors and a statistician based on a priori knowledge of variables known to be associated with the fluid administration by the physician and the postoperative complications  patients had complications. The overall risk of complications was significantly associated with the liberal fluid group with an adjusted OR of 2.6 (95% CI 1.5 to 4.4), 01 Table . No dataThe association between the predicted risk of complications and the perioperative fluid balance on a continuous scale is presented in Figs. p = 0.077)  patients developed a major complication (CDC ≥ 3). The risk of a major complication was not significantly associated with the liberal group (OR 1.6 (95% CI 1.0 to 2.7), 2.5% patiWe analyzed our data after excluding the 51 patients with a preoperative sepsis score of 3–4 and three patients of which data were missing. The risk of complications remained largely unchanged (Supplementary Table SOur study of patients undergoing emergency surgery for gastrointestinal obstruction or perforation showed a perioperative fluid balance of 3.6 L [IQR 3.0 to 5.3] compared with 1.6 L [IQR 1.0 to 2.0] to be significantly associated with a higher risk of postoperative complications, especially cardiopulmonary complications. The correlation remained robust after the exclusion of patients with preoperative severe sepsis or patients directly admitted at the ICU following surgery. The predicted risk of cardiopulmonary and major complications were at a minimum at a perioperative fluid balance of 0–2 L, whereas the predicted risk of renal complications were at a minimum at a fluid balance of 1.5–3.5 L.A little is known about the influence of the perioperative fluid therapy on postoperative complications in patients undergoing emergency gastrointestinal surgery. One pilot study randomized 29 patients undergoing emergency abdominal surgery to two different fluid strategies (Harten et al., We divided the patients into two groups, a liberal and a restrictive, after the intravenous fluid volume given; however, the as discussed below, the terms are not well defined in the literature.We found more cardiopulmonary complications in the patients given a liberal fluid therapy. The group also received more vasopressors postoperatively. The dominating drug given was norepinephrine, which for most parts was given in the intensive care unit. Even so, our result remained robust in the sensitivity analysis when excluding patients directly admitted to the intensive care unit. This indicates that cardiopulmonary complications are not related to the greater use of vasoactive drugs in the liberal group in our study.We demonstrated a U-shaped correlation between the fluid balance and postoperative complications. This has previously been suggested in meta-analysis of studies comparing restrictive vs. liberal fluid strategies during elective abdominal surgery (Bundgaard-Nielsen et al., −1 h−1. In similarity, we found a more favorable outcome of a perioperative fluid balance of 1.6 L comparable to a fluid administration of 5.9 mL kg−1 h−1 for overall and cardiopulmonary complications. Our data suggest that renal function might benefit from a greater fluid administration, and are supported by the study including the largest number of elective surgical patients randomized to a liberal versus restricted fluid strategy: more patients with renal failure were found in the restricted group. Noteworthy, the protocol for that trial did not recommend fluid administration to patients with postoperative oliguria (Myles et al., Our results suggest that the risk of cardiopulmonary and renal complications is differently associated with the perioperative fluid balance. Findings were in agreement with a registry study of patients admitted for elective non-cardiac surgery. Shin and colleagues included 92,000 patients in the study and divided the group in quintiles according to the fluid administration. They found a perioperative fluid administration of > 2.7 L to be significantly associated with an increased risk of respiratory complications, acute kidney injury, and mortality at 30 days (Shin et al., The limitations of our study lay within the retrospective design. The baseline data suggest a possible bias by indication: more patients in the liberal group had gastrointestinal perforation with sepsis and a high ASA score. We chose to adjust for the ASA score. Severe sepsis and co-morbidities are both inherent in the ASA score and as such dependent variables. In addition, more patients in the liberal group had hypotensive episodes treated with IV-fluid and/or vasopressors. We accommodated this by adjusting for the use of vasopressors in the regression model. However, we did not distinguish between different vasoactive drugs, nor a single- versus continuous administration. Blood loss, hypotension, and sepsis are likely to prompt fluid administration but are also linked with increase in morbidity which challenge interpretation of study results (Vincent et al., The strengths of our study are the detailed prospectively registered record-data of perioperative fluid administration. Our data included fluid given as iv-medicine which is often omitted in other studies. Further, double registration of the fluid data and complications was performed to ensure the completeness of available data and avoid misclassification of complications. We adjusted for known confounders influencing the fluid administration and the postoperative complications, further strengthening our findings. The multicenter design strengthens external validity of the study results. Yet, the design has inherent limitations and causal relations are for future trials to explore.With reservations to the inherent limitations in the study design, we found a perioperative fluid balance above 2.5 L to be significantly associated with an increased risk of overall and cardiopulmonary complications following emergency surgery for gastrointestinal obstruction or perforation. The predicted risk of complications demonstrates a U-shaped correlation with the perioperative fluid balance. A perioperative fluid balance of 0–2 L was associated with the fewest cardiopulmonary complications. The equivalent estimate was 1.5–3.5 L for renal complications. Our findings support our thesis that avoiding fluid overload in patients undergoing emergency gastrointestinal surgery may reduce the risk of complications.Additional file 1:. Supplementary Fig. S1.Additional file 2:. Supplementary Fig. S2.Additional file 3:. Supplementary Fig. S3.Additional file 4:. Supplementary Table S1.Additional file 5:. Supplementary Table S2.


Passive micromixers are miniaturized instruments that are used to mix fluids in microfluidic systems. In microchannels, combination of laminar flows and small diffusion constants of mixing liquids produce a difficult mixing environment. In particular, in very low Reynolds number flows, e.g., Re < 10, diffusive mixing cannot be promoted unless a large interfacial area is formed between the fluids to be mixed. Therefore, the mixing distance increases substantially due to a slow diffusion process that governs fluid mixing. In this article, a novel 3-D passive micromixer design is developed to improve fluid mixing over a short distance. Computational Fluid Dynamics (CFD) simulations are used to investigate the performance of the micromixer numerically. The circular-shaped fluid overlapping (CSFO) micromixer design proposed is examined in several fluid flow, diffusivity, and injection conditions. The outcomes show that the CSFO geometry develops a large interfacial area between the fluid bodies. Thus, fluid mixing is accelerated in vertical and/or horizontal directions depending on the injection type applied. For the smallest molecular diffusion constant tested, the CSFO micromixer design provides more than 90% mixing efficiency in a distance between 260 and 470 µm. The maximum pressure drop in the micromixer is found to be less than 1.4 kPa in the highest flow conditioned examined. Over the past few decades, improvements in microfabrication technology  and succ−9–10−11 m2/s [In passive micromixers, fluid mixing arises as a challenging work due to advection-dominant transport developed in microchannels. Strictly laminar fluid flow that is usually Reynolds (Re) number << 100 ,17 and v−11 m2/s ) fundame−11 m2/s ,19,20,21−11 m2/s ). IncreaTo date, several passive micromixer geometries have been devised to improve the degree of fluid mixing over a short distance. Although majority of these efforts enhance fluid mixing by developing secondary flows when Re > 10–20, usually a considerable mixing length increase is observed at very low flow conditions. For instance, Gidde et al.  studied −7 m2/s. Goullet et al. [In several other studies, researchers focused on improving fluid mixing specifically at very low Re numbers. Fang et al.  studied t et al.  surveyedAs discussed above, mixing performance can be boosted in virtue of complex flow patterns which are mostly generated at relatively high Re numbers. When, however, very low Re conditions are examined, a substantial decrease in mixing performance is seen because of yielding a small contact surface between fluids. This is mainly caused by ineffective flow manipulations in microchannels. Although some injection and design strategies help to improve mixing at low Re numbers, overall micromixer length can still rise to the centimeter level which is not desired as noted earlier. In the present paper, we propose a novel fluid overlapping passive micromixer design to surpass the small interfacial area restrictions at very low fluid flow conditions. Unlike the conventional micromixer configurations, where the effective use of advection process is prioritized to enlarge contact surface, the novel design proposed enables forming a predefined interfacial area between fluid bodies in a compact geometry. Therefore, a rapid inter-diffusion between fluids is ensured, and mixing distance is decreased significantly.µ is the viscosity (Pa∙s), p is pressure (Pa), ρ is the density (kg/m3), u =  is the velocity vector (m/s), c is scalar concentration, and D is molecular diffusion coefficient (m2/s).Computational Fluid Dynamics (CFD) simulations are performed to numerically describe the fluid flow and scalar transport in the micromixer. In all scenarios tested, it is assumed that the system is isothermal and gravitational effects are negligible. Fluids to be mixed are of constant density and viscosity, miscible, and non-reactive. Based on the assumptions above, flow field is simulated using incompressible Navier-Stokes (NS) and continuity equations as presented in Equations (1) and (2), respectively. Advection–diffusion (AD) equation is employed to resolve the scalar transport domain in the micromixer as given in Equation (3).simpleFOAM and scalarTransportFOAM solvers are employed to simulate steady-state incompressible fluid flow and passive scalar transport, respectively, a modified form icoFOAM solver is exploited in time-dependent solutions of coupled flow and scalar transport equations. Solvers are arranged to treat the advection and diffusion terms in the governing equations with second-order accurate upwind [simpleFOAM solver, SIMPLEC (semi-implicit method for pressure linked equations-consistent) [−6 threshold as typically practiced in several numerical micromixer studies [Equations (1)–(3) are solved using OpenFOAM    algorithsistent)  is used  studies ,32,33. IA is area, u is velocity, σ and σ2 are variance and the maximum variance, respectively, c is concentration, and MP and FP denote the parameter values, obtained from the numerical solutions of a certain mesh density and the finest mesh, respectively. ΔM-FD shows the difference, as a percentage, between a mesh level and the finest mesh with respect to the parameter employed. In this research, pressure drop (Δp) and mixing efficiency (MI) parameters are used to assess numerical errors in fluid flow and scalar transport simulations, respectively.Dimensionless Reynolds (Re) and Peclet (Pe) numbers are used to determine the characteristics of fluid flow  and scalar transport  in the micromixer, respectively. Re and Pe numbers are computed in the exit channel of the micromixer as defined in Reference . In the il and el) and cross-section area (cA) of 200 µm and 2 × 104 µm2, respectively. In the CSFO design, five identical mixing units are used to observe the effect of fluid overlapping approach in a wide range of flow conditions. The height (uh) and radius (ur) of a single mixing unit are 60 µm and 300 µm, respectively. Each mixing unit is divided equally in the z-direction with a solid, impermeable, and thin-plate disk element which is coaxial with the mixing unit and has a radius (dr) of 270 µm. It is assumed that the existence of physical joining parts between a disk element and mixing unit will affect the overlapping flow pattern trivially. Therefore, for the sake of designing convenience in the present study, these parts are excluded in the CSFO geometry. In physical applications of the CSFO design, the disk elements can be attached to mixing units from various points as indicated by the line arrows I, II, and III in ch) is 10 µm and radius (cr) is equal to that of inlet and outlet channels. Nonetheless, it should be noted that the purpose of including these connection parts is only to be able to measure mixing performance at the outlet of the mixing chambers. In a physical design, these extensions can be omitted since the short residence time of fluids in these sections will contribute to diffusive mixing negligibly. The dimensions of the CSFO micromixer were selected consistent with the 3-D passive micromixer designs studied in the literature. The 3-D CSFO passive micromixer proposed can be fabricated using the current microfabrication technology. Multi-layer fabrication methods [In this study, a 3-D circular-shaped fluid overlapping (CSFO) passive micromixer is developed. As shown in  methods ,36 can b methods ,37,38,39In the CSFO micromixer, nested-type inlets are used to create overlapping flow profile throughout the disk surfaces in mixing units. The core and outer segments of inlet surfaces are used to inject fluids as depicted in 1 = 3.0 × 10−10  and viscosity (µ) of 103 kg/m3 and 10−3 Pa·s, respectively. To solve the governing flow equations, the following boundary conditions are prescribed in numerical simulations: a uniform velocity profile at the inlets, zero-gauge pressure at the outlet, and zero fluid velocity, i.e., no-slip condition, on solid surfaces. In scalar transport simulations, the gradient of scalar concentration is set to zero at the outlet and solid boundaries to prevent the scalar undergo diffusion over these surfaces. To investigate fluid mixing in the micromixer, relative scalar concentrations, 0 and 1, are imposed on the inlet surface as described schematically in Micromixer performance is examined extensively establishing several molecular diffusion constants, i.e., Din water ), D2 = 1in water ) and D3 of water ), in a bof water ,44 with 6, 2.45 × 106, 1.58 × 106, and 1.05 × 106, respectively. Numerical simulations are carried out for the highest Pe number scenario examined in the CSFO–B micromixer configuration . Mesh study outcomes are presented in Numerical solution of advection-dominant transport systems is prone to create high amount of numerical  diffusion errors ,47. This4). Hence, much smaller mesh elements are required to approximate sharp scalar gradients accurately. Accordingly, mesh study outcomes need to be evaluated in refence to scalar transport simulations to employ a suitable mesh density in the simulations. For the MI parameter given in 6 elements, between L1–L2 mesh levels, L2 mesh level is determined to conduct numerical simulations. Furthermore, this selection is also validated by estimating an effective diffusivity coefficient from the scalar transport solution of L2 mesh level as proposed in [posed in . The ratAt small Re numbers, ineffective manipulation of fluid bodies cause yielding a small contact area between fluid bodies, which in turn limits mixing by diffusion. To overcome this problem and enlarge the interfacial area between mixing fluids, a typical approach is to create several laminations in microchannels . In thisAs shown in 2 and D3 mixing scenarios, more than 94% mixing efficiency is obtained at the exit of the first mixing unit of the CSFO–A micromixer. As can be pursued from the change of the trendlines in increasing Re numbers, reducing contact time between fluid bodies suppresses the inter–diffusion continually. Hence more mixing units are required to enhance the degree of mixing in the micromixer. Notably, the combination of low contact times with small diffusion coefficients develops the most challenging mixing conditions in the micromixer. At Re = 1, while D2 and D3 diffusivities can still be tolerated against the fluid residence time reduced, the use of the smallest diffusion constant becomes difficult. In that case, the mixing distance increases to 400 µm (E3) and 470 µm (E4) to obtain nearly 86% and 93% MI, respectively. In all mixing scenarios tested, the lowest mixing efficiencies are obtained for the two highest flow conditions of the D1 case as expected. The MI values at the exit of the micromixers are found to be nearly 65% and 54% for Re = 5 and 10, respectively. As mentioned previously, although the contact surface area is increased substantially in CSFO geometry, the development of mixing by diffusion is prohibited by rising flowrates. Nonetheless, the MI values are still promising for the higher molecular diffusion constants, D2 and D3, as shown in 2 scenario provides more than 86% mixing efficiency at the exit of the third mixing unit (E3), only two units are required to reach a MI value of nearly 91% (E2) in D3 case. For the same diffusivity scenarios, D2 and D3, the highest flow condition, Re = 10, yields 84% and 92% fluid mixing at E5 and E4 exits, respectively. For all mixing conditions examined, the distribution of scalar concentrations on the outlets of CSFO–A and CSFO–B micromixers are presented in 1, the development fluid mixing on different cross-sections along the CSFO–A configuration can be seen from When the mixing performance of micromixers are measured, the outcomes evidently show that diffusive mixing—in the vertical direction—is activated across the large interfacial areas formed. 1 and Re = 1, 5, and 10). The evolution of mixing efficiencies is observed with respect to time at the exit of each mixing unit. The results are plotted in As discussed above, when the CSFO micromixer is operated with constant fluid injection, large contact surfaces are yielded between fluid bodies in the horizontal directions. However, in the CSFO design, the overall interfacial area can be enhanced further if the fluids are injected sequentially over the core and outer inlet segments as described in At Re = 5, as constant fluid injection (f = 0 Hz) can only offer a MI value around 63% (t = 280 ms) at E5 location, more than 85% MI (t = 120 ms) is obtained at the exit of the second mixing unit (E2) by the use of sequential fluid injection in the CSFO geometry (f = 25 Hz). For higher injection frequencies, f = 50 and 100 Hz, the degree of mixing rises to 95% (t = 160 ms) and 98% (t = 180 ms) levels at the same location (E2), respectively. Unlike the Re = 1 flow condition, the effect of injection frequency is more visible at Re = 5. While f = 25 Hz case provides nearly 65% (t = 140 ms) mixing efficiency at the exit of the first mixing box (E1), the MI values reach 81% (t = 120 ms) and 90% (t = 100 ms) levels in f = 50 and 100 Hz scenarios, respectively. In the highest flow scenario, Re = 10, while nearly 53% MI (t = 140 ms) can be measured at the last exit location (E5) of the CSFO–A micromixer, CSFO–C configuration provides more than 61% MI (t = 70 ms) at the exit of the first mixing unit (E1) for the lowest injection frequency tested (f = 50 Hz). When the injection frequency is set to f = 100 and 250 Hz, the MI value reaches 77% (t = 80 ms) and 88% (t = 100 ms) on the same exit location (E1), respectively. As the best-case scenarios at Re = 5 (f = 25 Hz) and Re = 10 (f = 50 Hz), CSFO–C configuration develops approximately 85% (t = 120 ms) and 83% (t = 70 ms) mixing efficiencies in a distance less than 330 and 400 µm, respectively. When these mixing figures are compared with the outputs of the CSFO–A configuration, mixing conditions are improved significantly in terms of efficiency, distance, and time. Notably, such an improvement could be achieved by means of the extra contact areas formed between consecutive fluid segments during the sequential injection.The CSFO micromixer and nested-type inlets developed in this study offer a novel design approach to mix fluids at microscales. Unlike the conventional micromixer designs, where the enhancement of interfacial area strongly depends on the effective manipulation of fluid flow in microchannels, the CSFO geometry inherently develops a large contact area without requiring a complex flow formation in the micromixer domain. Therefore, better operating conditions are obtained. As can be seen from It should also be noted that the use of nested-type inlets is not only limited to the CSFO micromixer, but also can be used in any type of active or passive micromixer designs. Concentric flows that are developed in nested-type inlets basically provide two main advantages. First, when the fluids are injected concentrically, the deformation of fluid bodies in the micromixer becomes relatively much easy compared to the conventional fluid injections in separate channels. For instance, in split-and-recombination (SAR) micromixers ,61,62, sThe CSFO micromixer can also function without employing the nested-type inlets when the fluid injection is sequential. In such a case, the entire inlet surface is used to feed the micromixer with different fluids sequentially. However, in this condition, the interfacial area on the horizontal plane is not formed and the overall contact surface is developed by the wave pattern as described schematically in In addition to the circular micromixer design, the fluid overlapping mixing approach can also applied in rectangular or polygonal  geometries. However, when a rectangular geometry is used, a non-uniform velocity distribution can develop on the rectangular plane that divides mixing box volume equally. As displayed in Consequently, considering the plain design structure and high mixing performance, the CSFO passive micromixer can be integrated with microfluidic systems or used as a stand-alone device to mix fluids at microscales. In this research, fluid overlapping mixing approach and nested-type inlets are introduced for passive micromixers. A 3-D circular-shaped passive micromixer design is developed to enhance fluid mixing particularly at low flow conditions that is Re < 10. The mixing performance of the CSFO micromixer is examined numerically in various fluid flow and molecular diffusion conditions. The effects of alternative design configurations and injection strategies are tested. Numerical simulation results indicate that the CSFO design creates a large contact surface between mixing fluids in both upper and lower volumes of each mixing unit. In the case of constant fluid injection, the overlapping fluid pattern develops an interfacial area throughout the disk elements on the horizontal plane. However, when the fluids are injected sequentially, additional contact areas are formed between consecutive fluids. While symmetrical and alternating fluid feeding types provide almost identical results in the constant injection scenarios, the mixing effect of injection frequency is increased with rising Re numbers in the sequential injection cases. In both injection conditions, high mixing efficiency values could be achieved with a reasonable pressure drop in the CSFO micromixer. The maximum pressure drop is found to be less than 1.4 kPa at Re = 10. For the smallest diffusion coefficient and constant fluid injection, more than 90% mixing efficiency is quantified in a distance of 260, 400, and 470 µm for Re = 0.1, 0.5, and 1 flow scenarios, respectively. The mixing distances are reduced further even in high flow conditions when fluids are injected sequentially. When the mixing outcomes are compared with that of reported in the literature, the CSFO design offers a high amount of fluid mixing over a very short distance. Therefore, the CSFO micromixer can be employed in next generation microfluidic systems, where short mixing distances will be required, to mix fluids at microscales.


Surgical resection followed by chemo-radiation postpones glioblastoma (GBM) progression and extends patient survival, but these tumours eventually recur. Multimodal treatment plans combining intraoperative techniques that maximise tumour excision with therapies aiming to remodel the immunologically cold GBM microenvironment could improve patients’ outcomes. Herein, we report that targeted photoimmunotherapy (PIT) not only helps to define tumour location and margins but additionally promotes activation of anti-GBM T cell response.EGFR:03115) was conjugated to IR700. The response to ZEGFR:03115-IR700-PIT was investigated in vitro and in vivo in GBM cell lines and xenograft model. To determine the tumour-specific immune response post-PIT, a syngeneic GBM model was used.EGFR-specific affibody molecule (ZEGFR:03115-IR700 to produce reactive oxygen species upon light irradiation. ZEGFR:03115-IR700-PIT promoted immunogenic cell death that triggered the release of damage-associated molecular patterns (DAMPs)  into the medium, leading to dendritic cell maturation. In vivo, therapeutic response to light-activated conjugate was observed in brain tumours as early as 1 h post-irradiation. Staining of the brain sections showed reduced cell proliferation, tumour necrosis, and microhaemorrhage within PIT-treated tumours that corroborated MRI T2*w acquisitions. Additionally, enhanced immunological response post-PIT resulted in the attraction and activation of T cells in mice bearing murine GBM brain tumours.In vitro findings confirmed the ability of ZEGFR:03115-IR700 to accurately visualise EGFR-positive brain tumours and to destroy tumour cells post-conjugate irradiation turning an immunosuppressive tumour environment into an immune-vulnerable one.Our data underline the potential of ZThe online version contains supplementary material available at 10.1186/s12916-021-02213-z. Glioblastoma (GBM) is the most common primary malignant brain tumour in adults and is associated with an extremely aggressive clinical course and poor prognosis . The medThe current neurosurgical management of GBM aims for maximal resection while avoiding additional neurological damage. Numerous methods have been developed to facilitate surgery, including 5-aminolevulinic acid (5-ALA) fluorescence-guided surgery, intraoperative neuro-navigation, and neurophysiological monitoring , 5. HoweApproximately 57% of GBMs contain a mutation, rearrangement, splicing alteration, and/or amplification of the epidermal growth factor receptor (EGFR). The most common EGFR variant is a deletion of exons 2–7, EGFRvIII, which often co-occurs with focal EGFR amplification, which together are associated with a more aggressive, immuno-evasive tumour phenotype and worse prognosis . Despite Interestingly, recent studies have shown that inhibiting EGFR signalling may reduce tumour cell-intrinsic EGFR-induced programmed death-ligand 1 (PD-L1) upregulation, as well as extrinsic IFNγ-induced signals associated with CD8+ T cell infiltration into the tumour microenvironment (TME) , 10. ThiOne way to activate the TME immunologically would be through the use of photoimmunotherapy (PIT) and conventional photodynamic therapy (PDT).NCT03769506) [PIT is a light-mediated therapeutic approach, where a photosensitiser (PS) is conjugated to a highly specific monoclonal antibody (mAb), antibody fragment, or affibody molecule that has the ability to engage the selected target of interest. Near-infrared (NIR) light irradiation of the conjugate lead to ligand release reaction of IR700 and under normoxic conditions to the production of heat and reactive oxygen species (ROS) that, consequently, initiate target-selective cell death and stimulate inflammation, followed by vascular shutdown and tissue ischaemia –16. For 3769506)  and was 3769506) .  Furthe3769506) .In view of the high expression rate and oncogenic nature of EGFR, we have postulated that PIT targeting this receptor could promote CD8+ T cell attraction and activation and overcome the immunologically “cold” status of GBM.EGFR:03115-IR700), aiming for more effective tumour penetration, faster delivery, and clearance from non-targeted tissues [EGFR:03115-IR700 cell uptake enables imaging of EGFR expression in an orthotopic brain tumour model (U87-MGvIII), our proof-of-concept in vivo PIT study also showed the conjugate’s therapeutic efficacy in subcutaneous glioma xenografts [As an alternative to full-size antibodies, we have previously investigated the smaller, IR700-labelled EGFR-specific affibody molecule  maturation in vitro. In addition, when applied in a syngeneic mouse model, the treatment induces T cell responses that might overcome the “immunologically cold” status of GBM. Therefore, we believe that this therapeutic approach, following complete or cytoreductive resection of GBM, could lead to (i) elimination of residual or surgically inaccessible EGFR+ve cancer cells and (ii) subsequent stimulation of anti-tumour immunity.In the current study, we report that ZEGFR:03115-Cys affibody molecules  is described in detail in the supporting information  to the ZMycoplasma contamination .Human GBM cell line DKMG and murine GBM cell line GL261 were purchased from the Celther Polska (Poland) and the German Collection of Microorganisms and Cell Cultures , respectively. U87-MG and U87-MGvIII were kindly provided by Dr. Frank Furnari  . The pri1O2) production was determined using the Singlet Oxygen Sensor Green reagent  according to the protocol provided by the manufacturer. More details about the assay are described in the supporting information.Singlet oxygen (EGFR:03115-IR700 (30 nM) for 1 h at 4 °C, and samples were analysed using flow cytometry (BD™ LSRII). To test the targeting specificity and internalisation of the conjugate, cells were plated on confocal glass-bottomed dishes  in complete medium with ZEGFR:03115-IR700 (1 μM) for 1 h at 4 °C or 1, 3, and 6 h at 37 °C and analysed using a Zeiss LSM700 confocal microscope . A detailed description of the procedures is given in the supporting information.Human and murine GBM cells were harvested and incubated in a medium with ZEGFR:03115-IR700 (0.1 to 1 μM) for 1, 3, or 6 h at 37 °C. The media were then changed for phenol red-free DMEM medium and cells irradiated (8 or 16 J/cm2) using a LED light source . Cell viability was determined using the CellTiter-Glo®  luminescent assay 24 h post-light exposure. To assess ROS production, 5 μM 2′,7′-dichlorofluorescein diacetate  was added to phenol-red free medium during irradiation. The cell death at 1, 4, and 24 h post-irradiation was assessed using the Annexin V/Dead Cell Apoptosis Kit  according to the manufacturer’s instruction. To determine the post-PIT ATP and HMGB1 release, the ENLITEN® ATP assay  and an HMGB1 ELISA kit  were used. Calreticulin exposure on the membrane was measured by flow cytometry (BD™ LSRII). All the methods are described in detail in the supporting information.U87-MGvIII cells were seeded on petri dishes 24 h before experiments. Afterwards, cells were incubated with ZThe experimental details about co-culturing the immature dendritic cells (iDCs) with PIT-treated U87-MGvIII or DKMG cells are given in the supporting information.Western blotting was performed as previously described . ProteinEGFR:03115 and its radiolabelling with the 18F-Al complex was performed as previously described [The preparation of NOTA-Zescribed .All experiments were performed in compliance with licences issued under the UK Animals (Scientific Procedures) Act of 1986 and following local ethical review. Studies were compliant with the UK National Cancer Research Institute Guidelines for Animal Welfare in Cancer Research  and the 3, mice were randomly distributed into the experimental groups.The detailed methods are described in the supporting information. Briefly, NCr athymic female mice (5–6 weeks) were bred in-house. C57BL/6J female mice (6–7 weeks) used for the syngeneic model, were purchased from Charles River, UK. The orthotopic GBM U87-MGvIII or BL6-NPE-GFP-Luc mouse models were established as previously described , 24. For2) and (ii) 18 μg ZEGFR:03115-IR700 with light exposure . For immunocompetent mice bearing intracranial tumours, 50 J/cm2 light dose was used. The tumours were irradiated with a LED light source  1 h post-conjugate i.v. injection. More details are provided in the supporting information.For PIT treatment studies, subcutaneous and intracranial GBM U87-MGvIII xenografts were randomised into the following treatment groups: (i) light exposure only . The imaging protocols are described in the supporting information.To monitor the orthotopic tumour growth, mice were imaged using the 1 T M3™ MRI system  with a Tn = 5) with MRI-confirmed brain tumours received an i.v. injection of 18F-AlF-NOTA-ZEGFR:03115 , and PET/CT scans were acquired 1, 3, and 5 h post-injection of the radiotracer using an Albira PET/SPECT/CT imaging system. The detailed imaging and data analysis protocols are given in the supporting information.Mice  and snap-frozen in liquid nitrogen.Further experimental details are given in the supporting information.In vivo and ex vivo fluorescence images were acquired as stated in the supporting information using an IVIS Spectrum/CT system .Formalin-fixed brain and tumour tissues were embedded in paraffin, sectioned (5-μm-thick slices), and mounted on microscope slides. Frozen embedded tissues were sectioned into 10-μm-thick slices and mounted on microscope slides before being fixed in ice-cold acetone. The detailed staining procedures with the various antibodies are described in the supporting information.Tumour and surrounding brain tissue were harvested and dissociated via enzymatic digestion . Single-cell suspension was prepared by straining the digested tissue through a 70-μm mesh. Further experimental details are given in the supporting information.The serum was separated from the whole blood collected from the mice at the 24 h endpoint, snap-frozen, and stored at − 80 °C until further analysis. Concentrations of various cytokines were analysed using a Mouse Cytokine Proinflammatory Focused 10-plex Array .Unless otherwise stated, data were expressed as the mean ± SD. Statistical significance, sample size calculations, and correlation analysis are described in detail in the supporting information.EGFR:03115), which recognises the murine and human extracellular epitope of EGFR, was conjugated to IR700. Specific and receptor expression-dependent ZEGFR:03115-IR700 binding , as measured by flow cytometry, was in line with the total EGFR level assessed via Western blot  in a panel of human and mouse GBM cell lines. In order to confirm that ZEGFR:03115-IR700 PIT induces target-specific cell death, U87-MGvIII (EGFR high), DKMG (EGFR high), WSz57 (EGFR medium), and U87-MG (EGFR low) cells were incubated with increasing concentrations of the conjugate  and exposed to dose of NIR light selected based on our previous studies [2 , both cell lines demonstrated a dramatic loss in cell viability . A small but significant reduction in cell viability  was observed in U87-MG cells when the highest concentration of the conjugate was tested (0.5 μM). The patient-derived WSz57 cell line was less sensitive to the treatment, most likely due to highly heterogeneous EGFR expression . A longer incubation (6 h) of U87-MG, DKMG, and WSz57 cells with the ZEGFR:03115-IR700  led to an enhanced PIT-mediated cell death that was in line with an increased internalisation of the conjugate as confirmed via confocal microscopy .The affibody molecule (Z studies . A signi2), there were three distinct cell populations: viable , apoptotic , and necrotic . Importantly, by 24 h post-irradiation, the population of viable cells was 17.5% and 4%  compared to the control groups (91%)  after NIR light activation was initially measured in cell-free conditions. The studies confirmed a significant light dose-dependent SOSG fluorescence enhancement post-irradiation . Moreover, U87-MGvIII cells subjected to ZEGFR:03115-IR700-based PIT  showed a prominent increase of intracellular ROS production that was significantly suppressed by the ROS scavenger NAC  to lead to DAMPs  release in U87-MGvIII cells. As shown in Fig. S2A, there was a transient but significant increase in CRT expression level as early as 5 min post-NIR irradiation. We also observed a rapid (5 min) secretion of ATP into the cell culture media post-irradiation  and most likely high sensitivity of these cells to oxidative stress. No other DAMP upregulation was detected in any of the control groups  (n = 3) [50 = 5.14 ± 1.17)  as early as 1 h post-injection in mice bearing MRI-confirmed tumours .The capability of Z n = 3) . The ima . The imEGFR:03115-IR700-PIT shows an anti-tumour effect against GBM in vivo, subcutaneous and intracranial U87-MGvIII tumours were established in nude mice. In contrast to our previous studies using three ZEGFR:03115-IR700-PIT doses over three consecutive days [2. A significant delay in tumour growth was observed during the initial 7 days in the group receiving ZEGFR:03115-IR700-PIT compared to the control (light only 100 J/cm2) . However, due to the re-growth of measurable tumours at this point, a second PIT dose was delivered. Unfortunately, no further growth inhibition was observed . Next, we studied the early anti-tumour response to PIT using the orthotopic U87-MGvIII model by MRI. Following tumour establishment, T2- and T2*-weighted images were acquired at baseline and 1 or 4 h post-ZEGFR:03115-IR700-PIT . T2*-weighted imaging was selected because of its sensitivity to the presence of paramagnetic species such as deoxyhaemoglobin and its capability to detect cerebral and intratumoural micro-haemorrhage under pathologic conditions. T2*-weighted images showed an intratumoural signal decrease corresponding to haemorrhage and hemosiderin deposition 1 h post-NIR irradiation in mice treated with ZEGFR:03115-IR700-PIT as compared to the control group  for the control group vs 11.3% (n = 3) and 41.1% (n = 2) for 1 h and 4 h groups post-PIT, respectively), which effectively confirmed an acute tumour response to PIT. Hypointense cores highlighted after PIT are compliant with early necrosis. These changes were consistent with H&E staining of the brain sections showing tumour necrosis and micro-haemorrhage patterns associated with pyknosis and apoptotic bodies on the margins of the PIT-treated tumours  Fig. SB. Howeve2) Fig. SB. Howeve2) Fig. SB. Howeve2) Fig. SB. Howeve2) Fig. SB. HoweveEGFR:03115-IR700 in the brain setting  showed higher levels of CD4+ and CD8+ immune cells in tumours exposed to PIT compared to the control groups . Subsequently, we detected a significant decrease of PD-L1 in U87-MGvIII cells in response to ZEGFR:03115-IR700-PIT with and without IFN-γ stimulation compared to the controls in vitro . These results were next corroborated by in vivo findings showing a downregulation of PD-L1 also on the surface of BL6-NPE-GFP-Luc cells post-PIT  leads to higher tumour:background contrast and enhanced tumour necrosis when compared with full-size mAb-based IRDye700DX conjugate . Along tEGFR:03115-IR700-PIT can trigger a local immune response in the brain tumour microenvironment. The conjugate binding to EGFR on the membrane of GBM cells induced receptor expression-dependent cell death upon NIR light exposure which was, in part, due to ROS production. Interestingly, Kato et al. have recently provided a theoretical mechanism by which photoactivated hydrolysis reaction following irradiation of mAb-based IR700 conjugates cause changes in the silicon-oxygen bond and silanol formation, which converts the dye from very hydrophilic to very hydrophobic [EGFR:03115-IR700 will need to be investigated. Additionally, the efficacy of ZEGFR:03115-IR700 in vitro increased in a conjugate concentration-dependent manner, and significant phototoxicity was observed within 1 h post-light exposure of conjugate-treated cells.Herein, we further demonstrated that Zrophobic . WhetherEGFR:03115-IR700 will result in the release of these danger signals. We observed high-level cell surface CRT exposure, rapid ATP secretion, and HMGB1 release only in PIT-treated cells, indicative of ICD. However, in cells treated with either ZEGFR:03115-IR700 or light alone, these signals were not enhanced compared to controls. Furthermore, significant release of DAMPs by PIT-treated GBM cells subsequently activated and promoted maturation of antigen-presenting iDCs, as indicated by a marked expression of CD86 and HLA-DR.Of importance, several studies, including ours, provide evidence that PIT can induce mobilisation of DAMPs involved in ICD , 33, 37.EGFR:03115-IR700-PIT.Thereafter, in order to determine whether the conjugate is capable of inducing selective tumour cell death in vivo, we treated mice bearing subcutaneous U87-MGvIII xenografts with ZEGFR:03115) with high specificity recognise EGFR in vivo. For example, the U87-MGvIII-bearing mice injected with ZEGFR:03115-IR700 displayed a strong fluorescent signal as compared to ZTAQ-IR700 (a non-specific affibody molecule). The tumour fluorescent intensity of ZEGFR:03115-IR700 was 6-fold higher than ZTAQ-IR700 already 1 h post-injection [EGFR:03115 was radiolabelled with zirconium-89, only very low accumulation of the radioconjugate was found in tumours with low EGFR expression levels [Burley et al. have recently reported that EGFR targeting affibody molecule , which validated the model and procedure we employed.Apart from a targeting vector, also the light dose delivered and the method by which it is delivered are crucial to the success of PIT. However, physical dosimetry during PIT is a complex process due to the nature of dynamic interactions between light, conjugate, oxygen, and biological response of different tissues, which clearly depends on the concentration of cytotoxic photoproducts and on the intrinsic photosensitivity. In the murine models of GBM, the explicit dosimetry to map the distribution of light delivery and direct measurement of the light fluence are technically challenging. Therefore, for the purposes of this manuscript, we individually selected the intensity of light for U87-MGvIII and BL6-NPE-GFP-Luc models based on the initial validation experiments. For the xenograft model, the therapeutic light fluence was chosen to be 100 J/cmEGFR:03115-IR700, we initially used the radiolabelled conjugate 18F-AlF-NOTA-ZEGFR:03115 to assess the efficacy of the affibody molecule in targeting EGFR-positive tumours in the brain setting. The acquired PET/CT images showed discrete focal accumulation of the radiotracer in the brain lesions already 1 h post-injection. Considering the small difference in size between the two conjugates, we expected ZEGFR:03115-IR700 to exhibit similar in vivo behaviour to 18F-AlF-NOTA-ZEGFR:03115.Encouraged by this potent anticancer activity in vitro and in vivo, we further evaluated this approach in the brain setting. It is well known that GBM progression leads to blood-brain barrier (BBB) structural changes including neuronal death, astrocyte endfeet displacement, and heterogeneous pericyte and astrocyte subpopulations, all of which can reduce the barrier functions through the formation of fenestrations and disruption of tight junctions . Even thEGFR:03115-IR700 administration clearly indicated accumulation of the conjugate in the tumour and provided insights into its delivery. Despite a relatively equal distribution of ZEGFR:03115-IR700 in the tumours, we observed some variability in the response to PIT between the mice. This could be linked to a non-uniform irradiation through the burr hole in the mouse skull resulting in uneven NIR-light delivery and light-induced photochemical production of ROS. In spite of these issues, hypointense signals were depicted on T2*w images of U87-MGvIII tumours within 1 h post-PIT that corresponded to microhaemorrhagic lesions. Moreover, histopathological examination of the brain sections revealed high levels of necrosis induced by irradiation of ZEGFR:03115-IR700 24 h post-treatment. Of importance, necrosis has been previously reported to be the characteristic form of cellular death post-PIT [EGFR:03115-IR700-PIT, as confirmed by IHC staining of tumour sections. As published earlier, the translocation of HSP70 depends on the NIR light dose and is related to either mitochondrial or direct surface stress disruption [EGFR:03115-IR700 PIT. Excitingly, we identified enhanced immunological response after conjugate irradiation which resulted in the attraction and activation of CD4+ and CD8+ T cells in PIT-treated tumours compared to the control group. Furthermore, the expression of both IL-1β and IL-6, which have the ability to enhance the immune response against tumours by activating CD8+ T cells was also markedly increased. Interestingly, we also observed that ZEGFR:03115-IR700-PIT reduced the level of compensatory immunosuppressive PD-L1 in U87-MGvIII and BL6-NPE-GFP-Luc cells in vitro. We speculate that the remaining PD-L1+ cells could still suppress the anti-tumour immune response and allow the tumour cells to survive immunologic cytotoxicity. Of note, Kleinovink et al. have recently shown in tumour models of colon carcinoma that the addition of CTLA-4 blockade prior to bremachlorin-PDT leads to a significant reduction in tumour burden compared to either treatment alone [Indeed, fluorescence images of the entire brain captured ex vivo post-Zpost-PIT , 42. Fursruption , 44. Morsruption . For exasruption . In addisruption . Finallynt alone .EGFR:03115-IR700 fluorescence could guide resection of the tumour mass, and ZEGFR:03115-IR700-PIT lead to the eradication of residual tumour GBM cells simultaneously turning an immunosuppressive TME into an immune-vulnerable one.In conclusion, the surgical options for GBM patients have not changed significantly over the last three decades and performing a complete tumour excision often presents an insuperable challenge. Residual tumour cells located in close proximity to critical functional areas are often left in the margins of the resection, leading to disease relapse. The possibility of enhanced surgical precision together with intra-operative adjuvant treatment could improve the outcome of GBM patients. In fact, it has been recently shown in patients with recurrent high-grade glioma that a combination of 5-ALA fluorescence-guided resection and open PDT after tumour removal is a promising strategy for local tumour control and targeting non-resectable, visibly fluorescent tumours . ConsistEGFR:03115–IR700-PIT holds a tremendous potential as a novel therapeutic approach against this aggressive type of brain tumour.Overall, more work is needed to fully unlock the potential of PIT as an effective treatment for GBM, especially concerning local and systemic immune responses and synergies with adjuvant treatments. In addition, there are practical aspects in the procedure that need further investigations including the assessment of light intensity, light delivery protocols, and dosimetry. In addition, as bleeding into the tumour may potentially result in oedema of brain parenchyma clinical application of PIT should be considered for GBM remnants within tumour resection cavity or for patients with small and deeply located tumours where stereotactic PIT could be attempted. Nevertheless, ZAdditional file 1: Figure S1. Characterisation of ZEGFR:03115-IR700 binding capacity on EGFR-positive GBM cells. Figure S2. Post-PIT DAMPs release. Figure S3. Ability of 18F-AlF-NOTA-ZEGFR:03115 to accumulate in orthotopic U87-MGvIII tumours. Figure S4.In vivo EGFR-targeted PIT in U87-MGvIII subcutaneous tumours. Figure S5. Immune response to PIT.


Fluid resuscitation is frequently required for cardiac surgical patients admitted to the intensive care unit. The ideal fluid of choice in regard to efficacy and safety remains uncertain. Compared with crystalloid fluid, colloid fluid may result in less positive fluid balance. However, some synthetic colloids are associated with increased risk of acute kidney injury (AKI). This study compared the effects of succinylated gelatin (4%) (GEL) with compound sodium lactate (CSL) on urinary AKI biomarkers in patients after cardiac surgery.2-isoprostanes concentrations. Differences in change of biomarker concentration between the two groups over time were compared with mixed-effects regression models. Statistical significance was set at P < 0.05.Cardiac surgical patients who required an intravenous fluid bolus of at least 500 mL postoperatively were randomly allocated to receive GEL or CSL as the resuscitation fluid of choice for the subsequent 24 h. Primary outcomes were serial urinary neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C concentrations measured at baseline, 1 h, 5 h and 24 h after enrolment, with higher concentrations indicating greater kidney injury. Secondary biomarker outcomes included urinary clusterin, α1-microglobulin and Fn = 20 per group) with similar baseline characteristics were included. There was no significant difference in the median volume of fluid boluses administered over 24 h between the GEL  and CSL group  (P = 0.42). There was a significantly greater increase in urinary cystatin C (P < 0.001), clusterin (P < 0.001), α1-microglobulin (P < 0.001) and F2-isoprostanes (P = 0.020) concentrations over time in the GEL group, compared to the CSL group. Change in urinary NGAL concentration (P = 0.68) over time was not significantly different between the groups. The results were not modified by adjustment for either urinary osmolality or EuroSCORE II predicted risk of mortality.Forty cardiac surgical patients  for fluid resuscitation after cardiac surgery was associated with increased biomarker concentrations of renal tubular injury and dysfunction, compared to crystalloid fluid. These results generate concern that use of intravenous gelatin fluid may contribute to clinically relevant postoperative AKI.Trial registration ANZCTR.org.au, ACTRN12617001461381. Registered on 16th October, 2017, http://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=373619&isReview=true.The online version contains supplementary material available at 10.1186/s40635-021-00412-9. Optimising cardiac preload with intravenous fluid boluses is frequently required in cardiac surgical patients admitted to the intensive care unit (ICU). However, positive fluid balance and fluid overload are associated with adverse patient outcomes , 2, and Patients undergoing cardiac surgery are at high risk of postoperative AKI , a major2-isoprostanes concentrations.In this randomised controlled trial, we compared the effects of succinylated gelatin (4%) (GEL)  on a range of urinary AKI biomarkers, compared to a balanced crystalloid solution, compound sodium lactate (CSL), in patients who had undergone major cardiac surgery. We hypothesised that fluid bolus therapy with CSL would be associated with a lesser increase in concentrations of urinary NGAL and cystatin C over time, compared to GEL. Secondary outcomes included additional urinary biomarkers of AKI; clusterin, α1-microglobulin and FThe GELATIne Fluid and Acute Kidney Injury in Critical Illness (GELATI) Trial was an investigator-initiated, single-centre, randomised, open-label, parallel-arm designed clinical trial. Approval was obtained from the South Metropolitan Health Service Human Research Ethics Committee , and registration with the Australian and New Zealand Clinical Trials Registry  before commencement. After obtaining written informed consent prior to surgery, 40 participants (20 in each group) were enrolled and randomly allocated to receive either GEL or CSL for fluid boluses after cardiac surgery in the 30-bed ICU of the Fiona Stanley Hospital in Perth, Western Australia.−1 prior to surgery; end-stage renal failure requiring dialysis; urine output < 10 mL h−1 over the previous 4 h; severe AKI requiring renal replacement therapy (RRT), or likely to require RRT within the next 24 h; known allergy to GEL; received GEL in the previous 48 h; admitted to ICU for longer than 96 h; previously enrolled in the study; a limitation of care order had been placed; death was perceived to be imminent; or the treating clinician perceived that participation in the study was not in the patient’s best interests. The serum creatinine concentration limit of 265 µmol L−1, or 3 mg dL−1, corresponded with an estimated creatinine clearance of < 30 mL/min for the anticipated demographic, consistent with severe chronic renal insufficiency [After postoperative admission to the ICU, consented patients were screened again by a study investigator, in conjunction with the ICU clinician. Eligible patients were those for whom the treating ICU clinician intended to administer an intravenous fluid bolus of 500 mL or more within 60 min, and in whom they believed there was equipoise in using either GEL or CSL. The exclusion criteria were: < 18 years old; serum creatinine > 265 µmol Lficiency .Intraoperative management was conducted according to local standard practice. All participants received routine monitoring including 5-lead electrocardiogram, systemic arterial and central venous pressure monitoring. Pulmonary artery catheterisation was performed at the treating anaesthetist’s discretion. For participants receiving cardiopulmonary bypass (CPB), 2 g of tranexamic acid was administered as an intravenous infusion, commencing after induction, and heparin was dosed to achieve an activated clotting time (ACT) of > 480 s. CPB circuits were primed with 1600 mL of balanced isotonic crystalloid fluid . Following CPB, heparin was reversed with protamine targeting an ACT of < 130 s. For participants not receiving CPB, administration of drugs was similar except that target ACT was > 300 s during heparinisation and < 150 s post-reversal. Fluid management in the operating theatre consisted of blood products and balanced isotonic crystalloid, as required, but no synthetic colloid fluid.Patients were randomly allocated to receive intravenous fluid bolus therapy with either GEL or CSL. Randomisation was performed via block design of 4 blocks of 10 participants each (5 in each treatment arm) generated by an online random number generator −1 or 3000 mL, or 24 h after randomisation, whichever occurred first, the choice of further fluid therapy was at the discretion of the treating clinician. The use of more than 50 mL kg−1 or 3000 mL of GEL was strongly discouraged. Use of other fluids during the intraoperative and postoperative period, including crystalloid for maintenance fluid requirements or drug administration, and blood products including human albumin solution, was at the discretion of the treating clinicians.Participants were randomised to receive an initial study fluid bolus of at least 500 mL. Fluid delivery started as soon as a baseline urine sample had been collected. Further boluses of the assigned study fluid were administered if considered clinically indicated by the treating clinician. After administering a total study fluid volume of 50 mL kg2-isoprostanes concentrations. Urine collection occurred immediately after randomisation (T0), then at 1 (T1), 5 (T5) and 24 (T24) hours after the start of the first fluid bolus. Details of the sample collection methods are provided in Additional file 2-isoprostanes concentration, which indicates induced oxidative stress, was measured by gas chromatography–mass spectrometry (Agilent 6890N gas chromatograph coupled to an Agilent 5975B mass spectrometer) using electron capture negative ionisation and selective ion monitoring. Urine osmolality was measured by freezing point depression . Urine creatinine concentration was measured using a commercial analyser .The primary outcome was urinary NGAL and cystatin C concentrations over time up to 24 h post-randomisation. Secondary outcomes included other biomarkers of AKI: urinary clusterin, α1-microglobulin and FThe following clinical outcomes were also recorded in this study: maximum AKI stage within the first 7 days, RRT-free days within the first 28 days, number of blood product units transfused during the ICU stay, ICU-free days within 28 days, and 60-day all-cause mortality. Clinical data collection details are described in Additional file α = 0.05) to detect a twofold difference in NGAL. A conservative sample size of 20 per arm  was planned to reduce the risk of type II error, which would detect a mean difference in NGAL > 55 ng.mL−1 between the two groups with a power of 80%.Sample size calculation was based on the clinically important difference in NGAL that has been reported to be associated with AKI in a critically ill population. Patients with burn injury and early AKI had an approximately twofold increase in urine NGAL concentration within 48 h after admission, compared to those without AKI . ExtrapoP values for biomarkers that remained slightly skewed following log transformation. P values are presented for the interaction between treatment and time in the unadjusted model. Biomarker concentrations were summarised as predicted mean  estimated by the unadjusted models. A Bonferroni-corrected P value significance cut-off of < 0.01 for each linear mixed-effects model was further applied in order to reduce Type I error created by multiple biomarker analyses. The effects of urine concentration, as osmolality, and illness severity, as estimated by EuroSCORE II mortality prediction [Normality of data was assessed by visual inspection of histograms and Q–Q plots. Data are presented in median and first to third quartiles (Q1–Q3) or counts and percentages, as appropriate. Inferential tests were not applied to baseline characteristics, in line with the 2010 CONSORT statement . Total sediction , were alClinical outcomes were compared using Fisher’s exact test for categorical variables or Wilcoxon rank sum test for non-normal continuous data. Ventilation time was compared using negative binomial regression due to the nature of its distribution. No adjustment was applied to account for multiple comparisons in the exploratory analyses of clinical outcomes.P < 0.05, except for Bonferroni correction where stated.Analyses were performed using Stata 14  and SAS  with significance set at two-sided n = 20 per study arm) were enrolled and randomised  , clusterin (P < 0.001), α1-microglobulin (P < 0.001) and F2-isoprostanes (P = 0.020) concentrations in the GEL group, compared to the CSL group . The overall result for each biomarker was not modified by adjustment for urine osmolality and there was no association detected between biomarker concentrations and EuroSCORE II predicted risks of mortality. Results of the linear mixed-effects model analysis for the primary outcomes, both unadjusted and adjusted for osmolality, are provided in Additional file Of the five urinary biomarkers analysed, there was a significantly greater increase over time for cystatin C (P = 0.73) (Table P = 0.27). However, there were more participants in the GEL group that received packed red blood cell transfusions, compared to the CSL group  (P = 0.047). The other clinical outcomes were not significantly different between the two groups   for fluid resuscitation after cardiac surgery was associated with an increase in some biomarkers of renal tubular injury and dysfunction within 24 h, compared to crystalloid fluid. These results support the concern that use of gelatin fluid may be associated with increasing the risk of post-operative AKI. There was a signal, albeit still limited, that succinylated gelatin (4%) may affect bleeding tendency. Given that succinylated gelatin (4%) is more expensive than crystalloid fluid, an adequately powered randomised controlled trial focused on clinical outcomes is needed to confirm its safety in cardiac surgical patients.Additional file 1. Sample and data collection of cardiac surgical patients who were randomised to receive either succinylated gelatin (4%) or compound sodium lactate as resuscitation fluid therapy after cardiac surgery.Additional file 2. Linear mixed-effects model analysis of the associations between serial change in urinary neutrophil gelatinase-associated lipocalin and cystatin C concentrations and treatment.Additional file 3. Urine data  from cardiac surgical patients who were randomised to receive succinylated gelatin (4%) (GEL) or compound sodium lactate (CSL) as resuscitation fluid therapy after cardiac surgery. Urine was sampled before the intervention (T0), then 1 h (T1), 5 h (T5) and 24 h (T24) later. There were missing data for urine output at T0 , T1  and T24 .Additional file 4. Urinary biomarker concentrations  NGAL, cystatin C, clusterin, α1-microglobulin and F2-isoprostanes of cardiac surgical patients who developed acute kidney injury (AKI) (closed squares) or did not develop AKI (open squares). AKI was defined as a maximum KDIGO score > 0 within 7 days of randomisation. Urine was sampled before the intervention (0 h), then 1, 5 and 24 h later. Abbreviations: AKI; acute kidney disease; KDIGO, Kidney Disease Outcomes Kidney Disease: Improving Global Outcomes; NGAL, neutrophil gelatinase-associated lipocalin.


Whether fluid overload is associated with vascular stiffness parameters in hemodialysis (HD) patients has not been fully elucidated. We hypothesized that interdialytic fluid accumulation increases vascular stiffness parameters, which improves with intradialytic ultrafiltration.Fluid overload and vascular stiffness parameters were assessed in 39 HD patients (20 with and 19 without fluid overload) and compared to 26 healthy controls. Fluid status was assessed 15 minutes before the mid-week HD session by bio-impedance spectroscopy. Following this, ambulatory pulse wave velocity (PWV) and augmentation index (AIx) were measured for 24 hours before another mid-week HD session and then for 5 hours starting 30 minutes before and ending 30 minutes after the session.HD patients had significant fluid overload compared to healthy controls  and baseline PWV was higher . There was no significant difference between PWV and AIx in fluid overloaded and non-fluid overloaded HD patients prior to, or during the HD session. AIx of non-fluid overloaded HD patients improved after the HD session (P = 0.04). Average 24-hour AIx was higher in fluid overloaded HD patients (P<0.001).Inter- and intradialytic changes in fluid volume were only weakly related to vascular stiffness parameters in HD patients. Although there was a modest reduction in AIx in non-fluid overloaded HD patients after the dialysis session, fluid removal did not improve vascular stiffness parameters during the HD session. We speculate that the effect of fluid overload correction on vascular stiffness parameters requires long-term adjustments in the vasculature. Patients with end-stage kidney disease treated with hemodialysis (HD) have a high prevalence of fluid overload , 2 and hVascular stiffness in HD patients is increased by structural and functional changes. Structural changes, such as atherosclerosis and vascular calcification, presenting as media sclerosis, increase PWV and AIx and increase vascular stiffness , 11. FunAltogether, whether fluid overload affects vascular stiffness in HD patients remains unclear. We hypothesized that interdialytic fluid accumulation increases vascular stiffness parameters, which improves with intradialytic ultrafiltration. We aimed to confirm that vascular stiffness parameters are higher in HD patients compared to healthy controls and to investigate the effect of inter- and intradialytic fluid changes on PWV and AIx measurements in fluid overloaded and non-fluid overloaded HD patients.We recruited 39 prevalent HD outpatients from the University of Alberta Hospital, Edmonton Alberta, Canada. Inclusion criteria were 18 years of age or above and dialysis-dependency for more than 6 weeks, with a three times per week frequency and a HD duration of 3.5 to 4 hours per session. Exclusion criteria were acute illnesses that included cardiovascular events and infection, surgery within 6 weeks of the study, pregnancy, nocturnal dialysis, kidney transplantation during the study, implanted pacemakers, defibrillators, pins, metallic stent, artificial joints, and skin lesions at the site where bioimpedance electrodes should be positioned. As far as applicable, the same criteria were used for the healthy subjects. A health questionnaire was obtained from the healthy subjects. Written informed consent was obtained from all participants. The study was approved by the Human Research Ethics Board at the University of Alberta Hospital.® Fresenius Medical Care, Bad Hamburg, Germany) [th and 90th percentiles of the healthy distribution to define the normovolemia range which yielded to -1.1L and +1.1L, respectively. Also, fluid overload normally varies from -1.1 L below normal ECFV after HD run to + 1.1 L above normal ECFV before the next HD run with a 75 mmol/d sodium intake [Extracellular fluid volume, intracellular fluid volume and total body fluid volume of all study participants were assessed using a validated, portable bio-impedance spectroscopy device  , 15. TheGermany) . For eacGermany) , 18. TheGermany) . The 1.1m intake .TM, TensioMed, Budapest, Hungary) [We used an oscillometric device to measure aortic PWV, brachial AIx, peripheral systolic and diastolic blood pressure, mean arterial pressure, and pulse pressure using an upper arm cuff using the non-fistula arm for HD patients (Arteriograph24Hungary) . This deDialysis vintage, vascular access, ultrafiltration, and current target weight were obtained from the patients’ record. Cardiovascular events like coronary artery diseases and other illnesses were obtained from patient’s medical history reported in the electronic medical record. Interdialytic weight gain was calculated by subtracting the post-dialysis weight of previous HD session from the pre-dialysis weight of the HD session. Patients were considered diabetic if it was documented in the chart or they were prescribed anti-diabetic medications. Antihypertensive medications were obtained from the electronic medical record.Continuous data are expressed as mean ± standard deviation if normally distributed or as median 25–75 percentile if not normally distributed. Categorical variables are expressed as percentage of total. Shapiro-Wilk test was used to test normal distribution of variables. Chi-square test was used to compare frequencies. Mann-Whitney U test was performed to compare nonparametric parameters between HD group versus healthy controls and between fluid overloaded HD versus non-fluid overloaded HD groups. Two-way repeated measurement ANOVA was used to analyze the variance of vascular stiffness parameters during the HD session, the changes of variables in pre-and post-HD session, and the 24 hours measurements. Tukey multiple comparison test was applied as post-hoc test. The missing data and outliers were imputed by performing a regression analysis using the entire data set of each parameter per subject. Univariate and multivariate linear regression analysis were performed to determine the factors predicting pre-HD PWV. Correlation between non-parametric data were analyzed with Spearman’s test and Pearson’s test was used for parametric data. Graphpad Prism version 8  and SPSS version 25  were used for data analysis. P<0.05 was considered statistically significant.Table 1.HD patients were significantly older than healthy controls and were fluid overloaded. Fluid overloaded HD had higher dialysis vintage than non-fluid overloaded HD patients. Fifty-nine percent of HD patients were on anti-hypertensive medications; beta-blockers were prescribed most frequently. Anti-hypertensive medications were prescribed more commonly in fluid overloaded HD compared to non-fluid overloaded HD patients. Both fluid overloaded HD and non-fluid overloaded HD patients had similar interdialytic weight gain, prevalence of cardiovascular disease  and hypertension, and causes of end stage renal disease. Demographic and clinical characteristics, the baseline measurements before the 5 hours measurements of all study participants as well as the mean 24h measurements of HD patients are shown in Fig 1A–1C). After the HD session, fluid overload remained significantly higher in fluid overloaded HD compared to non-fluid overloaded HD patients. Over the 5 hours of measurements, no significant changes in PWV and AIx were found in both HD groups and that was similar to healthy controls . In healthy individuals over 49 years of age, PWV and AIx were significantly higher than in younger individuals S1 Table. Multivariate analysis showed that advanced age was an independent predictor of high PWV in healthy individuals S2 Table. After univariate linear regression analysis of baseline PWV predictors, extracellular fluid volume and total body fluid volume were included in the multivariate analysis. In the multivariate analysis, neither extracellular fluid volume nor total body fluid volume were independent predictors for baseline PWV in HD patients S3 Table.HD patients had significantly higher systolic blood pressure, mean arterial pressure, pulse pressure, and PWV values compared to healthy controls as assessed from the measurements immediately prior to the HD session for HD patients or at the start of the 5 hours measurements in the healthy controls. Pulse pressure was higher in fluid overloaded HD than non-fluid overloaded HD patients, however, systolic blood pressure, diastolic blood pressure, and mean arterial pressure were not different. The PWV and AIx were also not different between fluid overloaded HD and non-fluid overloaded HD patients as well .Average 24-hour systolic blood pressure, mean arterial pressure and pulse pressure were significantly higher in fluid overloaded HD patients than non-fluid overloaded HD patients. AIx was higher in fluid overloaded versus non-fluid overloaded HD patients, yet average PWV was not different .Univariate linear regression analysis regarding average interdialytic AIx revealed mean arterial pressure, fluid overload, and fluid overload/extracellular fluid volume, extracellular fluid volume /total body fluid volume, and extracellular fluid volume /intracellular fluid volume ratios as potential predictors. In the multivariate analysis, mean arterial pressure was the only significant predictor for average interdialytic AIx We investigated inter- and intradialytic fluid changes and vascular stiffness parameters in HD patients. First, we confirmed that HD patients have higher vascular stiffness parameters than healthy controls, which is consistent with other publications , 21, 22.Very few studies have evaluated vascular stiffness parameters in HD patients with varying levels of fluid overload , 24. Oneintradialytic fluid removal would acutely improve vascular stiffness parameters in HD patients. We found that PWV and AIx did not change during the HD run when all patients were considered. A previous study observed a significant decline in intradialytic PWV only at 135 and 210 minutes into the HD run, whereas AIx remained unchanged [We then investigated whether nchanged . The latnchanged , 24, 26.nchanged , 26, 27.nchanged . Yet, thThere are several physiological responses to ultrafiltration during the HD session which could obscure changes in vascular stiffness parameters. Options are activation of the sympathetic nervous system and adjustments in vascular resistance by blood flow autoregulation , 30. Aftinterdialytic fluid accumulation could lead to an increase in vascular stiffness parameters in HD patients. Our findings could not confirm a gradual change in PWV and AIx during the 24-hours of fluid accumulation in HD patients. However, since changes in vascular stiffness parameters could be more pronounced in fluid overload versus non-fluid overloaded HD patients, we also compared these two groups. Two previous reports are available about interdialytic changes in vascular stiffness parameters in HD patients both using 48 hours measurements, yet, using two different technologies [We hypothesized that nologies , 32. Thenologies . Also, tnologies . Second,nologies . From a nologies . The bloAlthough the current study provides a full description of vascular stiffness parameters changes during inter-and intradialytic fluid volume changes, there are limitations. Our small sample size could potentially be perceived as limitation; however, the study was adequately powered to demonstrate a biologically relevant change in vascular stiffness parameters. Yet, our findings are consistent with the existing literature , 25, 31.Another possibility is that fluid overload affects vascular function via mechanisms other than classical hemodynamics, such as by a direct influence of salt on vascular function. The endothelial glycocalyx can become functionally and structurally impaired by high sodium . In addiIn conclusion, our study was unable to demonstrate that interdialytic fluid accumulation and intradialytic fluid removal affects vascular stiffness parameters in HD patients. The post-HD AIx reduction in non-fluid overloaded HD patients suggests that an adequate ultrafiltration volume could improve the wave reflections but not PWV. These and previous observations call for studies on chronic adjustments in fluid volume status and vascular stiffness parameters, including studies directed towards the direct effects of salt on vascular function.S1 TableP value < 0.05 is considered significant; FO, fluid overload; PWV, pulse wave velocity; AIx, augmentation index.(DOCX)Click here for additional data file.S2 TableP value < 0.05 is considered significant; ESRD, end stage renal disease; FO, fluid overload; ECFV, extracellular fluid volume; ICFV, intracellular fluid volume; TBFV, total body fluid volume; PWV, pulse wave velocity; AIx, augmentation index.(DOCX)Click here for additional data file.S3 TableP value < 0.05 is considered significant; IDWG, intradialytic weight gain; FO, fluid overload; ECFV, extracellular fluid volume; ICFV, intracellular fluid volume; TBFV, total body fluid volume.(DOCX)Click here for additional data file.S4 TableP value < 0.05 is considered significant; FO, Fluid overload; PWV, pulse wave velocity; AIx, augmentation index.(DOCX)Click here for additional data file.S5 TableP value < 0.05 is considered significant; FO, Fluid overload; PWV, pulse wave velocity; AIx, augmentation index; net ultrafiltration, is the pre- and post- HD weight difference; delta calculated as the difference between the post- and pre-HD measurement.(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


The monitoring of antibiotic use in animals is a crucial element to ensure food safety. The main goal of this study was to analyse the distribution of selected antibiotics to porcine oral fluid, as well as to demonstrate that an oral fluid is an alternative to other biological matrices used in the control of antibacterials. Therefore, an animal study with pigs treated using seven different antibiotics was performed. Sulfadoxine (SDX) with trimethoprim (TRMP), lincomycin (LIN), tiamulin (TIAM), tylosin (TYL), amoxicillin (AMX) and penicillin G (PEN G) were injected intramuscularly to pigs, and concentrations of these analytes in the oral fluid were assessed. Ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) was used to quantify the analytes. On the first day of medication, the highest concentrations for SDX and TRMP at the level of 22,300 µg/kg and 14,100 µg/kg were found, respectively. The concentrations of LIN  and TIAM (7600 µg/kg) were also relatively high. The peak of TYL was recorded on the second day of drug administration. Most of the analytes were present in oral fluid for 30 days, apart from TYL, which was detected for 27 days. It was found that AMX and PEN G were quantified only for 5 and 8 days, respectively, at very low concentrations. It was found that oral fluid can be used for the verification of antibiotics on pig farms. Antimicrobial residues are of food safety concern. The residues of drugs may result in many biological adverse effects and allergic reactions in consumers, as well as the spread of drug-resistant bacteria and bacterial resistance acquisition . ConsoliIt is possible to determine drugs level in blood plasma; however collection of this type of material is associated with exposure of animals to stress, as well as considerable inconvenience for the person taking the samples. The pig’s safety, stress reduction, time-saving, and material collection convenience, determine different sampling techniques. Even though urine and milk samples can be successfully utilised as matrices for the detection of drugs in animals, the first material in question is used solely for the analysis of banned compounds, whereas usage of the latter is limited to cows only . In the There are many benefits of using oral fluid in the control of antimicrobial residues compared to matrices used so far. Collecting oral fluid is animal-friendly and noninvasive, devoid of any needles or snout loop . The tecOral fluid was demonstrated as a matrix in the evaluation of some metabolic diseases in humans by detecting a variety of analytes in the olden days . After tIn this paper, the transfer and concentrations of seven different antibiotics in swine oral fluid, commonly used in pigs, were studied for the first time. In six separated experimental groups, animals were therapeutically treated with a veterinary product containing sulfadoxine (SDX) with trimethoprim (TRMP), lincomycin (LIN), tiamulin (TIAM), tylosin (TYL), amoxicillin (AMX), and penicillin G (PEN G), one drug per group. The aim of this study was to develop and validate a multiresidue method for the simultaneous determination of seven antibiotics as well as demonstrate an oral fluid as an alternative to other biological matrices used so far in the control of antibacterials.2 > 0.997) for all analytes. The coefficients of variation  for repeatability, were lower than 10% and below 15% for within-laboratory reproducibility. The selectivity testing allowed us to verify that no peaks from endogenous compounds were detected in the retention time corresponding to each analyte or internal standard. The average recoveries were in the range of 87.6–107%, depending on the analyte. The method was satisfactorily sensitive with limits of quantification (LOQs) established in the range of 2–10 µg/L, whereas limits of detection (LODs) were in the range of 1–5 µg/L. The results are shown in The UHPLC-MS/MS method used in this research for the determination of seven different compounds in oral fluid was fully validated. All matrix-matched calibration curves showed good linearity  and TIAM (7600 µg/L) were also relatively high on the first day of medication. The peak of TYL was recorded on the second day of drug administration, but concentrations in the oral fluid were much lower from the abovelisted compounds. It was found that AMX and PEN G were poorly passed to oral fluid, and the level of these analytes was determined as 11.3 µg/L for AMX and 93 µg/L for PEN G. The concentrations of antimicrobials at each time point are listed in The antimicrobials overused in domestic animals can lead to residues in tissues and other animal products intended for human consumption. In some cases, the lack or inadequate cleaning of water supply systems or feed dispensers after previous treatment can also give positive results. Apart from the risk to public health, the presence of antibiotics in tissues often leads to the removal and destruction of a significant quantity of meat, which contributes to a serious economic consequence. Therefore, a new method for the control of antibacterials during rearing is strongly needed. The fast and simple sampling of oral fluid with the cotton rope is an effective tool for the monitoring of reasonable treatment. In case of positive oral fluid analysis results, appropriate measures can be taken to prevent the slaughtering of positive animals intended for sale in order to avoid huge costs connected with the disposal and utilization of contaminated meat.For the analysis of antibacterials in oral fluid, a UHPLC-MS/MS analytical method was developed. The extraction used in the presented method allows for simultaneous analyses of seven different compounds from six various chemical groups. Additionally, the simple and fast sample preparation, as well as a cleanup step by filtration, give an opportunity to test a lot of samples in a short time. The paper described a biochip array-based immunoassay method for the detection of six different antimicrobial drugs, tetracyclines were very poorly recovered in dirty samples (5%), and for other drugs , recovery values were much higher than 100% . This meIn the described work, oral fluid was tested for the detection of some antibacterials for food safety applications. To demonstrate the validity of this matrix, seven drugs from various groups of antibacterials with different chemical and therapeutic properties were administered by intramuscular injection. The compounds tested in this experiment were selected based on a review of the use of antimicrobial drugs used in swine . The rouOral fluid is a mixture of saliva, oral mucosal transudate and gingival crevicular fluid . AntimicThe pharmacokinetics properties determine, to some extent, the distribution of drugs in many structures of the organism. The results of our research demonstrate the strong relationship between the pharmacokinetics and penetration to oral fluid for most of the analysed substances. Concentrations of most compounds presented in the experiment were high and reached great levels in oral fluid. Most of the analysed antibiotics are basic compounds with relatively high volumes of distribution. LIN is a basic compound with pKa values of about 7.6. It has high lipid solubility and consequently a large apparent volume of distribution. LIN is widely distributed in many fluids and tissues. This antibiotic diffuses across the placenta in many species, and the apparent volume of distribution is >1 L/kg . For TIAPEN G, for which concentrations in the oral fluid were very low, is an organic acid with pKa 2.7. The group of penicillins have relatively small apparent volumes of distribution (0.2–0.3 L/kg) and short half-lives (0.5–1.2 h) in all species of domestic animals . Acid hyThe transport of some drugs to the oral fluid can also be determined by the degree of protein binding. It was observed that compounds with high protein binding show a greater ability for diffusion to saliva. Sulfonamides and diaminopyrimidines, which are generally highly bound to proteins in serum (60–90%), as well as lincosamides, in which serum protein binding is 72%, reached high concentrations in oral fluid ,29. SubsAll animals were born in the sow farm (8000 DanBred sows) located in Northern Poland, after crossing ♀(♂Landrace × ♀Yorskshire) × ♂Duroc. Piglets were weaned after 4 weeks of lactation with an average weight of 6 kg and then moved to the weaner stable . After the next 7 weeks, animals were transported to the 9000-places fattening farm with an average body weight of 28 kg, where the trial was performed. All farms in the aforementioned three-phase model used weekly batches and an all-in all-out system. Specialist veterinary and production management care were provided in each location.For the purpose of the study, 240 immunologically castrated boars were divided into 8 groups, 30 animals in each pen. For the control group, 30 pigs were used in separate pens. Their initial body weight was between 40 and 60 kg. Pigs were housed at 20–25 °C. Fatteners were fed ad libitum with antimicrobial-free water and feed provided by feeders throughout the study. Before the experiment, water and feed were checked for antimicrobials, and no antimicrobial treatments were administered to these pigs before. All animals in one pen were treated due to the same health issue using disposable needles and the same drug and dosage. Pens were separated from each other with concrete walls. The drugs and dosage administered to each experimental group with an indication of withdrawal period for tissues are listed in The research material was one pooled sample of oral fluid from 10 pigs, in triplicate, per pen. Ropes were removed immediately after collection. The first oral fluid sample was collected 24 h after the first injection. The collection of each subsequent sample took place at the same time (11:30 A.M.) for 35 consecutive days. Additionally, oral fluid samples were collected from pigs not treated with any antibiotics. Oral fluid from each pen was pooled into one 50-mL Falcon plastic tube and centrifuged at 9447× rcf for 15 min. Centrifuged material was transferred to a 15 mL plastic tube and frozen at −20 °C until analysis.3 (LIN-d3), amoxicillin-d4 (AMX-d4), sulfaphenazole (SFF) and azithromycin (AZT) were used as internal standards. All standards were manufactured and obtained from LGC Standards . Formic acid was from Sigma Aldrich . Acetonitrile was obtained from J.T. Baker . Heptafluorobutyric acid was from Fluka . Syringe 0.22 μm Hydrophilic Polyvinylidene Fluoride (PVDF) Membrane Filters were from Restek .All reagents used were of analytical grade. Reference standards of SDX, TRMP, LIN, TIAM, TYL, AMX and PEN G were used for the analysis and quantification of the analytes. Lincomycin-dIndividual stock standard solutions (1000 µg/mL) for AMX, AMX-d4, and PEN G were prepared in ultrapure water and stored in polypropylene vessels. LIN, LIN-d3, TYL, TIAM, AZT, SDX, TRMP and SFF stock standard solutions (1000 µg/mL) were dissolved in methanol. All individual stock standard solutions were stable for at least 6 months when retained in a dark place at −18 °C. A working solution for each analyte was prepared from stock solutions by dilution in ultrapure water and stored at 4–8 °C for 1 month.Before the analysis, the oral fluid samples were centrifuged at 9447× rcf. Next, an aliquot (1 mL) of oral fluid was placed into a 2 mL polypropylene centrifuge tube, and the 30 µL of the internal standard at the concentration of 2 µg/mL was added. For the extraction of analysed antibiotics, 600 µL of 0.5% formic acid was added, vortexed for 30 s and centrifuged for 10 min at 9447× rcf. Then, the supernatant was filtered through a 0.22 mm PVDF filter into an amber glass vial before chromatographic analysis.2)—20; nebulizer gas (N2)—60; collision gas (N2)—medium; auxiliary gas—65; ion spray voltage—4500 V. Mass spectrometric conditions are shown in The analysis of SDX, TRMP, LIN, TIAM, TYL, AMX and PEN G was conducted by an UHPLC Shimadzu Nexera X2  system connected to the SCIEX 4500 triple quadrupole mass spectrometer  controlled by Analyst 1.6.2 software . The mass spectrometry detection was operated in the positive ESI mode with multiple reaction monitoring (MRM). The operating parameters were set as follows: temperature—450 °C, curtain gas  column  with an octadecyl guard column (2 × 4 mm) maintained at 35 °C. The mobile phase consisted of 0.025% heptafluorobutyric acid (A) and acetonitrile (B) at a flow rate of 0.6 mL/min with an injection volume of 5 µL. Gradient elution was conducted as follows: 0–4 min 90% A, 4–5.3 min 20% A and finally from 5.31 to 7 min back to 90% A. The total run time was set as 7 min.n = 20) and checked for potential interferences with endogenous substances. The precision for each analyte was determined by the repeated analysis (n = 6) of oral fluid samples spiked with SDX, TRMP, TYL, TIAM, LIN, AMX and PEN G at three concentrations corresponding to 0.5, 1.0, 1.5× VL. The precision was calculated and expressed as the percentage coefficients of variation . For repeatability, samples were analysed on the same day by the same operator. For within laboratory reproducibility, another two sets of fortified samples at the same concentration levels as for the repeatability were analysed on two different days with different operators. The extraction recovery experiment was carried out at the three concentration levels, in the same experiment as precision, by comparing the average area obtained for each analyte with the concentrations of analytes in spiked samples. Additionally, LOQ as the lowest point of the matrix-matched calibration curve, where the least abundant diagnostic ion for quantitative analysis can be detected was determined. The LOD was estimated as the signal to noise ratio (S/N) of 3, and LOQ was determined as the lowest validated concentration that produced a signal to noise ratio > 10.The method was validated according to the Commission Decision 2002/657/EC . The folThe results of conducted experiment illustrate that oral fluid can be used to monitor most antibacterial substances administered in pig farms. This paper shows the usefulness of oral fluid as a biological material for the non-invasive detection of drugs administered on pig farms. An analytical UHPLC-MS/MS method for the simultaneous determination of seven antibacterials in the oral fluid has been developed and validated. The implemented method is suitable for the simultaneous rapid detection and quantitation of SDX, TRMP, TYL, TIAM, LIN, AMX and PEN G in porcine oral fluid. The concentrations of these compounds found in this work showed high distribution to oral fluid for SDX, TRMP, TYL, TIAM and LIN, except for AMX and PEN G, where low penetration was observed.Analysis of antibiotics in oral fluid offers a cost-effective approach for the screening of an individual animal or populations of pigs. The results of this study demonstrate the oral fluid analysis as an important “security tool” for the rapid verification of the declaration of treatment on pig farms.