Pro-inflammatory IL-1β gene expression measured in synovial fluid was significantly higher (p = 0.0156) than systemic/blood expression. Senescent-associated factor MMP-3 and regenerative factor TIMP-2 were significantly higher in synovial fluid compared to blood serum. Senescent-associated factor MMP-9 and regenerative factor TGFβ-2 were significantly elevated in serum compared to synovial fluid. Correlation analysis revealed that C12FDG++/CD87++ senescent cells in synovial fluid positively correlated with age-related growth-regulated-oncogene , IFNγ , IL-8 , and gene marker p16 . Conclusions: There is an abundance of senescent cells locally and systemically after an acute ACL tear without a significant difference between those present in peripheral blood compared to synovial fluid. This preliminary data may have a role in identifying strategies to modify the acute environment within the synovial fluid, either at the time of acute ligament injury or reconstruction surgery.Background: Senescence, a characteristic of cellular aging and inflammation, has been linked to the acceleration of osteoarthritis. The purpose of this study is to prospectively identify, measure, and compare senescent profiles in synovial fluid and peripheral blood in patients with an acute knee injury within 48 h. Methods: Seven subjects, aged 18–60 years, with an acute ACL tear with effusion were prospectively enrolled. Synovial fluid and peripheral blood samples were collected and analyzed by flow cytometry, using senescent markers C12FDG and CD87. The senescent versus pro-regenerative phenotype was probed at a gene and protein level using qRT-PCR and multiplex immunoassays. Results: C Post-traumatic osteoarthritis (PTOA) frequently develops after anterior cruciate ligament (ACL) and concomitant meniscal or other ligament pathology injury ,2,3. TheSynovial fluid profiles, which have shown cartilage extracellular matrix degradation and upregulation of inflammatory markers in the setting of intraarticular ligament injury similar to those found in chronic osteoarthritis (OA), may play a significant role the development of PTOA, fibrosis and graft incorporation ,12,13,14Few human studies exist examining synovial fluid biomarkers at the time of both ACL reconstruction and meniscal repair/debridement surgery and, to our knowledge, there are no studies examining senescence in synovial fluid in the knee at the time of acute injury ,22,23,24p16 and p21, have all been seen in chronic OA, and have been detected in cartilage, subchondral bone, and synovium in animal studies [Senescence, a characteristic of cellular aging and growth arrest in damaged or stressed tissue, and a critical component in controlled apoptosis for tumor suppression, has also been associated with OA ,26,27,28 studies ,32. Addi studies ,33. TargThe purpose of this study is to prospectively identify, measure, and compare senescent profiles in synovial fluid and peripheral blood in patients that have sustained an acute ACL tear within 48 h. Secondarily, we aim to evaluate the senescent phenotype  at the local versus systemic level. We hypothesized that senescent cells would be detectable in synovial fluid and in peripheral blood after an acute ACL tear, with no difference in the senescent phenotype. Additionally, the study aims to provide preliminary data as the first to detect and characterize senescent profiles in synovial fluid and peripheral blood after an acute knee injury.This study was approved by our Institutional Review Board (protocol 2020-49). Patients between 18 and 60 years of age, who presented within 0–48 h of a primary, acute knee injury with significant intraarticular effusion and confirmed ACL tear on magnetic resonance imaging (MRI), were prospectively enrolled in the study between 8 February 2021 and 30 June 2021 . DemograSynovial fluid samples were obtained at the time of injury (0–48 h) by performing an arthrocentesis procedure. The patient was positioned in the supine position on an exam table, and the lateral knee was marked at the level of the superior pole of the patella. A sterile prep was performed with chloraprep, and the skin was anesthetized with up to 5 mL of 1% lidocaine. After the superficial layers were anesthetized, additional chloraprep was applied to the knee, and an 18- or 22-gauge needle was inserted into the intraarticular space using a patellofemoral approach. Then, up to 30 mL of synovial fluid was aspirated into a sterile syringe with 3 mL of anticoagulant citrate dextrose, formula A (ACD-A) to prevent coagulation. The synovial fluid was then transferred to a 50 mL conical tube. A compression bandage was placed over the knee after the aspiration was performed.Venous peripheral blood was collected on each enrolled subject at the time of the arthrocentesis. Using a standard venipuncture or intravenous blood collection procedure, approximately 15 mL of peripheral blood was drawn in a syringe prefilled with 3 mL of ACD-A, and an additional 5 mL of peripheral blood was drawn into a red-top tube without any anticoagulant for serum isolation.Synovial Fluid: The acellular and cellular layers of the synovial fluid were separated by centrifuging the sample at 1500× g and 20 °C for 10 min. If necessary, additional ACD-A was added prior to processing, to prevent clotting and cell clumping. A total of 300–1500 µL of supernatant was aliquoted into three sterile microcentrifuge tubes, and subsequently snap frozen at −80 °C for protein analysis using multiplex immunoassays. If red blood cells were visible in the cell pellet, the cellular layer was processed either using a red blood cell lysing buffer  or a SepMateTM tube  with a LymphoprepTM  density gradient separation system according to the manufacturer’s protocol, until red blood cells were no longer visible. Cells were then counted and aliquoted into cryovials with TRIzol®  for RNA isolation or CryoStor®  with 1% anticoagulant for flow cytometry. Samples for RNA isolation were snap frozen at −80 °C, while cells for flow cytometry were frozen at the controlled rate of 1–2 °C/min. A minimum volume of 2 mL of synovial fluid was necessary for the sample preparation methods described, which did not allow for processing of small amounts of synovial fluid or diluted samples from contralateral normal knees.Peripheral Blood: Peripheral blood mononuclear cells (PBMC) were isolated using the SepMateTM tube with LymphoprepTM density gradient separation system, according to the manufacturer’s protocol. Cells were counted and aliquoted for RNA isolation and flow cytometry following the same protocols detailed for synovial fluid.12FDG and the monoclonal antibody for human CD87 . C12FDG is a compound that, when hydrolyzed by β-galactosidase, an enzyme upregulated during senescence, fluoresces at a wavelength of 514 nm [We have recently developed flow cytometry-based protocols to detect senescent cells using the fluorescent compound Cf 514 nm ,35, wherf 514 nm ,37,38.5 cells per 1 mL culture media . Bafilomycin  was added, and cells were incubated on a shaker (150 rpm) in 5% CO2 at 37 °C for 1 h. Half of each sample was then separated and stained with C12FDG , and the other half remained an unstained control. All samples were then diluted (2:1) with FACS buffer, centrifuged, washed, and resuspended in 100 µL PBS. Zombie aqua viability stain  was added per the manufacturer’s instruction, and samples were incubated at 4 °C for 15 min. Cells were then diluted with 1 mL FACS buffer, washed, and resuspended in 100 µL FACS buffer. 5 µL Human Seroblock  was added to all samples for 10 min at room temperature. Samples were transferred to ice, stained with CD87 , and incubated at 4 °C for 30 min. Cells were then washed at 500× g for 5 min and resuspended in FACS buffer. A Cytek® Northern Lights  flow cytometer was used to quantify the number of live/dead, CD87 positive, and C12FDG positive cells present in each sample in triplicate. Gating strategies were developed in accordance with established methods [® software .Synovial fluid and PBMC samples were rapidly thawed in a water bath and immediately diluted (4:1) in fluorescent activated cell sorting (FACS) buffer , 5% FBS, and 5 mM EDTA Bio-Rad Cat #: 1610729). Cells were centrifuged, and counted to ensure a minimum of 2.0 × 10 methods ,35,37,39g for 10 min to remove debris. Approximately 300 µL of each sample were transferred to microcentrifuge tubes for multiplex immunoassays on the Luminex® 100/200TM System . Analytes were measured according to the manufacturer’s protocols using magnetic bead kits  described in TM Immunoassay Curve Fitting Software System .Plasma, serum, and the acellular synovial fluid fractions were placed in a warm bead bath until just thawed, then centrifuged at 1000× ® SYBR® Green FastMix  on an Applied Biosystems StepOnePlus RT-PCR thermocycler . Primers were designed using PRIMER-Blast (NCBI), and primer sequences are listed in Total RNA was isolated and purified from synovial fluid cells and PBMCs using TRIzol™, and transcribed into complementary DNA using the reverse transcription cDNA synthesis kit qScript™ , according to the manufacturer’s protocol. Expression of inflammatory and senescence markers was detected by quantitative real-time PCR (qRT-PCR) using PerfeCTap16 gene expression, a widely recognized marker of senescence, and C12FDG++/CD87++ senescent synovial fluid cells. The post hoc power analysis revealed that the study achieved a power of 0.84 using a one-tailed test with a significance level of 0.05 and a sample size of 7 subjects. Based on these results, the study had a sufficient statistical power and sample size to assess the presence of senescent cells in synovial fluid, as confirmed by the expression of a known senescence-related gene. Power analysis calculations were performed using G*Power statistical software (version 3.1.9.6).Power Analysis: Due to the exploratory nature of the study, a post hoc power analysis was conducted to evaluate the achieved power of the study based on the observed data, including the sample size, effect size, and significance level. To address our primary hypothesis to detect and measure senescence in synovial fluid and/or peripheral blood, we examined the Spearman correlation coefficient (ρ = 0.83) between 12FDG or CD87. Samples were measured in triplicate, and triplicates were averaged. Percentages of synovial fluid and PBMCs were measured positive for senescence using dim (+), bright (++), and total (dim + bright) senescence markers C12FDG and CD87. The combination of both senescence markers was used to measure the percentages of double positives for both C12FDG and CD87 in cellular synovial fluid and PBMC samples. Results were analyzed for normality, and normally distributed samples were tested with a ratio paired t-test (α = 0.05). Samples that did not fit a normal distribution pattern were analyzed with a Wilcoxon’s matched-pairs signed rank rest (α = 0.05). Statistical significance was defined as p < 0.05 using GraphPad Prism® software .Flow Cytometry Analysis: Synovial fluid and PBMC results were paired by patient, and analyzed for senescence markers, defined as the percentage of live cells positive for C−ΔCT, Wilcoxon signed rank testing was performed on 2−ΔCT, and normalized gene expression is presented as log(2−ΔCT). All significance testing was performed with paired, Wilcoxon rank sum testing on non-transformed data using GraphPad Prism® software .qRT-PCR Analysis: Expression of genes relative to housekeeping gene GAPDH was determined using 2® software .Biomarker Analysis: For the multiplex analysis, a non-parametric mixed-effects ANOVA test was used due to the non-normally distributed data that contained values that were undetectable or below the threshold, to test the overall null hypothesis of equivalency among the synovial fluid, plasma, and serum supernatant samples . Only anCorrelation Analysis: A spearman rank-based correlation analysis was performed between senescent cell percentages, SASP, and senescent-associated and inflammatory gene expression analytes separately for both PBMCs and synovial fluid. Sample size allowed for detection of correlation coefficient magnitudes ≥ 0.8 with 80% statistical power. Correlation calculations and graphical figures were performed in RStudio statistical software  .2) was 26.0 ± 2.8 (range 21.3–28.2). No patient used tobacco products. Patient demographic and injury details are summarized in Twelve consecutive patients with acute ACL injuries confirmed on MRI and presented within 48 h of injury were evaluated; one was excluded due to age, one due to presence of osteoarthritis, and three refused knee aspiration. A total of seven subjects with confirmed ACL injury were enrolled in the study, with a mean age of 42.0 ± 15.3 years (range 21.4–60.7). Mean BMI . The majority of senescent cells detected were double positive for dim C12FDG and dim CD87 in both samples  and bright (++) expression for both Cand CD87 . No stats white, H–I.IL-1β, IL-8, IL-6) and the senescence-associated genes  were measured in the cellular component of the synovial fluid and PBMCs. All data were log transformed to achieve normal distribution. IL-1β gene expression in synovial cells was significantly higher than PBMCs (p = 0.0156). Relative expression of IL-8 and IL-6 was not significantly different in synovial fluid and peripheral blood. Similarly, relative expression of senescence markers p53, p16, and p21 were not significantly different.  and trending higher in MCP-1 and IP-10. MMP-9 was the only senescence-associated analyte that was higher in serum than synovial fluid (p = 0.0313). Similarly, consistent trends were not observed for the regenerative factors at the local versus systemic level; pro-regenerative MMP inhibitors TIMP-1 and TIMP-2, as well as the interleukin-1 receptor antagonist IL-1RA, were higher in synovial fluid than serum ; however, the angiogenic factor PDGF-AA (p = 0.0625) and growth factors TGFβ-1 (p = 0.0625) and TGFβ-2 (p = 0.0313) were higher in serum compared to synovial fluid , IFNγ , and IL-8  . Additionally, a significant positive correlation was observed between non-senescent C12FDG−/CD87− synovial fluid cells and the regenerative factor IL-1RA  in acellular synovial fluid (p = 0.0724) and TGFβ-1 .We next aimed to understand which factors most strongly correlated with in the senescent cell classification determined by flow cytometry, to determine which biomarkers most strongly drove the double bright C 0.0374) A. The exal fluid B. Strong12FDG++/CD87++ PBMCs, but no significant relationships were found (12FDG++/CD87+ PBMCs positively correlated with the expression of inflammatory marker IL-1β (ρ = 0.60), plasma MCP-1 (ρ = 0.60), serum MCP-1 (ρ = 0.54), and serum MMP-1 (ρ = 0.43) (12FDG++/CD87++ PBMCs and SASP/inflammatory biomarkers: plasma TNF-α (ρ = −0.80), plasma MMP-3 (ρ = −0.70), plasma IFNγ (ρ = −0.70), serum MMP-12 (ρ = −0.54), serum GRO (ρ = −0.50), and gene expression of inflammatory marker IL-8 (ρ = −0.60) (12FDG−/CD87− PBMCs and the regenerative factor IL-1RA (ρ = 0.70) , we were able to identify unique staining patterns and suggest that the combination of these two markers may help to categorize cells in various states of senescence. The composition of total senescence was driven (~54%) by a population of dim senescent cells (C12FDG+/CD87+), compared to the less frequent double positive C12FDG++/CD87++ cells . While the science supporting senescent cell detection is rapidly expanding, the functional consequence of these different populations remains unclear. We propose that the C12FDG++/CD87++ cells may represent a subset of highly senescent cells, whereas the C12FDG+/CD87+ may correlate with a pre-senescent state. We speculate that the pre-senescent state may less deleterious or possibly associated with an acute injury response.By using two different senescent detection modalities  following acute knee injury, and will form foundational data from which to build on in future investigation. Importantly, our data suggests no statistically significant difference in the quantification of senescent cells from the blood compartment compared to synovial fluid, which may increase the feasibility of conducting future longitudinal studies using blood only. Further investigation is warranted to evaluate temporal changes in synovial senescence and biomarkers at serial time points after ACL injury or after surgical treatment, and will assist in better understanding the complex synovial environment after injury and in development of senolytic treatment to potentially prevent PTOA.We recognize this study is not without limitations, which includes low patient enrollment, particularly due to the logistical difficulty of capturing patients within 48 h of an acute intraarticular knee injury. Larger cohorts could increase the power and sensitivity. We were unable to establish a control group to evaluate synovial fluid senescent cells in normal knees, as the low aspiration volume attainable in a physiologically normal knee was too low for detection with current technology, and dilution of the samples was not comparable in pilot testing. Further exploration of differences between the bright versus dim senescent cell populations are needed to determine if they are functionally similar or different in the setting of acute knee injury and development of OA/PTOA. Additionally, we acknowledge senescence and SASP are complex, with both reparative and catabolic functions that will require further investigation to delineate the clinical implications of senescent cells.There is an abundance of senescent cells locally and systemically after an acute ACL tear, without a significant difference between those present in peripheral blood compared to synovial fluid. This preliminary data may have a role in identifying strategies to modify the acute environment within the synovial fluid either at the time of acute ligament injury or reconstruction surgery.


Intraventricular hemorrhage is characterized by blood leaking into the cerebral ventricles and mixing with cerebrospinal fluid. A standard treatment method involves inserting a passive drainage catheter, known as an external ventricular drain (EVD), into the ventricle. EVDs have common adverse complications, including the occlusion of the catheter, that may lead to permanent neural damage or even mortality. In order to prevent such complications, a novel dual-lumen catheter (IRRAflow®) utilizing an active fluid exchange mechanism has been recently developed. However, the fluid dynamics of the exchange system have not been investigated. In this study, convective flow in a three-dimensional cerebral lateral ventricle with an inserted catheter is evaluated using an in-house lattice-Boltzmann-based fluid–solid interaction solver. Different treatment conditions are simulated, including injection temperature and patient position. Thermal and gravitational effects on medication distribution are studied using a dye simulator based on a recently-introduced (pseudo)spectral convection–diffusion equation solver. The effects of injection temperature and patient position on catheter performance are presented and discussed in terms of hematoma irrigation, vortical structures, mixing, and medication volume distribution. Results suggest that cold-temperature injections can increase catheter efficacy in terms of dye distribution and irrigation potential, both of which can be further guided by patient positioning.The online version contains supplementary material available at 10.1007/s10439-022-03130-9. However, this procedure lacks a well-defined clinical protocol and must be performed at the physician’s discretion.19The majority of hemorrhagic strokes happen due to intracerebral (ICH) or subarachnoid hemorrhages (SAH).active fluid exchange). Several clinical studies have illustrated the ability of this dual-lumen catheter system to deliver tissue Plasminogen Activator  in a continuous manner while enabling quicker evacuation of blood in the ventricles while negating catheter occlusions41 (increasing EVD efficiency and decreasing complications52).In order to take advantage of irrigation while maintaining sterility, a novel dual-lumen self-irrigating catheter system has been recently developed . This dual-lumen catheter is connected to an intelligent digital cassette  and powered by a control unit with a user interface. The catheter is designed to prevent the issues observed with conventional EVD usage by delivering periodic irrigation with drainage and continuous pressure monitoring , this study investigates the fluid exchange system within the lateral cerebral ventricle at different fluid injection temperature and patient body position. The results of this study might provide insights into treatment procedures of active-irrigation catheters and, further, may contribute to improved patient care by elucidating optimized drug delivery patterns.Although case studies with this catheter system (IRRAflow®) have documented the effectiveness of such self-irrigating catheters in shortening hospitalization times with no catheter-related complications,12 This approach has been utilized for various fluid dynamics problems including those related to turbulent,14 multiphase,40 thermal,39 and bio-fluid46 flows.In order to simulate fluid flow, we used a lattice Boltzmann method (LBM) that combines discrete kinetic theory and modified molecular dynamics. Unlike conventional continuum-based methods, LBM is a mesoscopic method that behaves like a conservation equation solver for the corresponding macroscopic variables while providing a viable way for the inclusion of microscopic dynamics at the interfaces.47 In the present work, our model has two sets of distributions 9 with 19 discrete velocity vectors (D3Q19), given byIn LBM, a particle distribution function ensures that fluid particles move synchronously on a regular lattice. The function is chosen to satisfy conservation equations, where the fluid is Galilean invariant and isotropic.39; pressure 46). The buoyancy force density 31 asHere, 6 Three-dimensional (3D) rendered images of this geometry is acquired from a repository43 and subsequently separated using SOLIDWORKS®  to obtain the final form. MeshLab13 is used for smoothing the geometry and generating three-node triangular mesh elements .22 IBM regulates the force term 35 The Lagrangian-interface force exerted on the wall by the fluid 22 given by20 Such a procedure can be summarized by the following steps, where steps one through five are iterated until the boundary conditions are satisfied:Step 0: Step 1: Step 2: Step 3: Step 4: Step 5: The fluid and solid domains are coupled via a summation of all intermediate values of such forces over all iterations, i.e., 30 to those concerning biological flow dynamics.46 A particular advantage of using this immersed-boundary lattice Boltzmann solver for FSI simulations lies in the ability of LBM to easily incorporate other physical effects as right-hand-side forcing terms of Eq.  with one drainage and six injection ports  whose locations corresponds to those found in the IRRAflow® catheter. The tip is inserted 18 mm into the ventricular domain in order to ensure all ports are completely inside the ventricle.37 Ideally, the injected and drained volumes should be equal in order to prevent collapsing or possible bursting of the ventricle.48 The average velocity is calculated based on the injection volume, and spatially-uniform velocity profiles are prescribed at the injection and drainage sites during their corresponding operating time intervals. The onsets of these velocities are regularized-in-time by partition-of-unity-based functions to obtain appropriately smooth and physically-relevant simulations.We have employed the standard duty cycle of IRRAFlow® in all simulations: 20 s with three distinct time intervals corresponding to injection (1 s), observation (9 s), and drainage (10 s). The considered injection volume of 44 The brain cells can tolerate low temperatures 44 However, they start to develop ischemia at temperatures that are slightly elevated above normal body temperature 7 Therefore, injections can only be performed at temperatures colder than the normal body temperature To the best of our knowledge, there is no prescribed injection temperature for this procedure, although it should be within a realistic range so as to avoid neural injury.46 This scheme is applied to the outer boundaries of the fluid domain as well as to the catheter boundaries , whereas the fixed temperature condition 31Fixed temperature boundary conditions and a bounce-back scheme are applied for the fluid flow simulations. The bounce-back method assures no-slip and no-penetration conditions by reversing the outgoing velocity distribution functions.11 with a thickness corresponding to Simulations are performed in non-dimensional lattice units, where the governing equations are non-dimensionalized by four reference parameters: a density 32 For the case of an injection temperature of All simulations are performed using a uniform mesh with a lattice spacing of 4 i.e.,32 and where The evolution of a dye concentration At all other boundaries, including rigid walls 4 We note4 based on a Fourier continuation (FC) methodology5 for accurate Fourier series expansions of non-periodic functions. For a dimension Solution of Eqs. –17 is fa of Eqs. –17 can t4The resulting function k in Eq.  can be c4 Such time marching incurs linearly-scaling Courant-Friedrichs-Lewy (CFL) constraints on the timestep.5 Together with the FC-based spatial scheme, the overall solver enables high-order accuracy and a faithful preservation of the dispersion/diffusion characteristics of the underlying continuous operator . The latter property is particularly important for avoiding non-physical  diffusion in simulated dye concentrations.A high-order explicit time integration scheme is utilized to complete the numerical treatment of the advection–diffusion system.fluid blending in the ventricle using Eq.  and prone (lying on the stomach with the face downwards). Other body positions are not considered since they have been demonstrated to be clinically impractical.Simulated snapshots, at various times, of the velocity and vorticity fields inside the ventricle are presented in Figs. Every velocity magnitude image in Fig. mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimSnapshots of the time evolution of the vortex structures corresponding to each configuration is presented in Fig. fluid blending caused by the injected fluid via the space-averaged vorticity for each configuration. We observe that vorticity curves produced by all injections are similar at their onset. However, midway through each injection, the subsequent evolution of the average vorticity begins to differ drastically as a function of temperature. We note that the coldest injection results in up to twice as much blending than at room temperature. Following the end of injection  and drug delivery performance (via consideration of vortex structures and dye evolution) under different injection temperatures and patient positions.This work studies the thermal and postural effects on irrigation patterns and injected fluid mixing of a newly-developed self-irrigating catheter to utilize an active fluid exchange mechanism for treating intraventricular hemorrhages. Through robust numerical simulations, we have investigated clot irrigation potential  instead of face-down (prone) for better irrigation . Additionally, we have not considered chemical reaction between injected tPA solution and hematomas in our simulations. Our results only elucidate the path of diffusion and not the actual activated interaction between tPA and hematomas . The addition of such a reaction term to the governing model Eq.  is a subSome additional limitations should be mentioned. To the best of our knowledge, the properties of an intraventricular hematoma  are not well established and there is a dearth of literature. As such, multi-phase fluid models are not feasible. Hence, we have employed a single-phase model by assuming the hematoma is a uniform mixture of blood and cerebrospinal fluid, where medication immediately mixes with the hematoma at the injection port. Further research into the properties of a hematoma may one day enable consideration of fluid–fluid interactions; however, conclusions drawn from our study would still be reasonably accurate since increased thermal forces (lower injection temperatures) enhance irrigation and drug distribution in any case.via patient positioning. Our unique modeling approach may help facilitate rapid assessment of drug delivery performance before treatment. Indeed, the first two days are most critical for IVH patients, and this work has been motivated by the overarching goal of faithfully producing treatment-specific simulations towards enabling a priori catheter performance estimation within a few hours.Overall, this work demonstrates that both temperature and patient position are important factors in catheter efficacy. Results suggest that treatment administered at a high temperature difference Supplementary file2 (MP4 642 kb)Supplementary file3 (MP4 642 kb)Supplementary file4 (MP4 1620 kb)Supplementary file5 (MP4 1350 kb)Supplementary file6 (MP4 1946 kb)Supplementary file7 (MP4 1418 kb)Supplementary file8 (MP4 2563 kb)Below is the link to the electronic supplementary material.


Generally, intracellular pH and biological species such as various gases, metal ions, and anions constitute the foundation of cells and participate in the basic physiological processes, whose abnormal level can lead to poisoning, cardiovascular disease, and cancer in living organisms. Therefore, monitoring of their quantity plays an essential role in understanding the status of organisms and preventing, diagnosing, and treating diseases. In the last decades, remarkable progress has been made in developing ICT probes for the detection of biological elements. In this review, we highlight the recent ICT probes focusing primarily on the detection of intracellular pH, various gases , metal ions , and anions . In addition, we discuss the issues and limitations of ICT-based fluorescent probes for in vivo detection and explore the clinical translational potential and challenges of these materials, providing valuable guidance and insights for the design of fluorescent materials.Fluorescent probes, typically based on the intramolecular charge transfer (ICT) mechanism, have received considerable research attention in cell detection due to their non-invasiveness, fast response, easy regulation, high sensitivity, and low damage tolerance for  It can be overexpressed in many cancer cells, and colon cancer cells can produce a large amount of H2S through excessive expression of cystathionine β-synthase (CBS). Therefore, high-sensitivity detection of small molecules and biomacromolecules in cells is of great significance for the understanding of the molecular-level cellular functions and the mechanism underlying disease development. Herein, we reviewed the recent developments of ICT probes focusing primarily on the detection of intracellular pH, various gases , metal ions , and anions . Additionally, the issues and limitations of ICT-based fluorescent probes for in vivo detection were discussed, and the clinical translational potential was explored.Cells are the basic units that constitute living organisms and carry out life activities. They rely on intracellular pH and biological species such as various gases, metal ions, and anions to function normally in a highly complex and networked spatial environment in order to maintain normal cellular activities . When thThe intracellular pH (pHi) plays an irreplaceable role in maintaining various metabolic processes in cells. Enzyme activity, signal transduction, and other cellular functions are closely related to pHi. Studies have shown that various diseases, including cancer, Alzheimer’s disease, and Down syndrome, are closely associated with abnormal pHi in cells. Therefore, real-time, accurate, and sensitive monitoring of intracellular pHi changes reveals important information for studying the pathological and physiological processes related to cellular pHi. Small-molecule fluorescent probes used for measuring the pH of specific compartments within cells usually require the following characteristics: 1) the probe must have the ability to target the specific compartment; 2) the linear response range of the probe must include the pH value of the compartment, and its pKa should be as close as possible to the pH value of the compartment to achieve maximum sensitivity; 3) the probe should have a strong anti-interference ability against various cations, amino acids, and other biomolecules present in the cell; 4) reversibility is necessary for the real-time monitoring of pH fluctuations using the probe; and 5) the probe should have low toxicity to cells, excellent membrane permeability, and leak-proof ability. For instance, a ratio-type fluorescent probe, benzo dithiophene (BTDB), with a large Stokes shift was prepared based on the ICT luminescence mechanism . BTDB em2S, CO, H2O2, NO, and SO2 always lead to serious diseases, including ischemia–reperfusion injury, vasodilation, cell death, angiogenesis, neural regulation, inflammation therapy, insulin signaling, and gas stress response  to H2O2 upon ICT effect in HepG2 cells  detection in vitro and in vivo.Normally, abnormal concentrations of cellular gases such as Hresponse . Niu et rescence . Furthershown in . NO partshown in . In addi2+, Hg2+, Pb2+, Zn2+, and Al3+, can cause chronic poisoning when they accumulate to a certain concentration  and rhodamine B linked by ethylenediamine. Then, the boronate ester was grafted to the compound to react with HClO at 1.36 nM  interaction between the donor phenolate anion on IS-NR-O and the acceptor acetonitrile, thereby achieving a dual colorimetric and fluorescent response to fluoride ions with excellent selectivity. Furthermore, the probe exhibits a good linear relationship with fluoride ions over a wide concentration range of 0.38–6.84 ppm and a low detection limit of 0.09 ppm (Anions in cells such as ClOsduction . They ex 1.36 nM . Next, awith CN− . In addiG2 cells . Last bu0.09 ppm .Recently, various fluorescent probes based on ICT have been designed and gained for intracellular pH and biological species detection, which was believed to promote the developments in applications of biomedicine. In summary, through the design and modification of ICT fluorescent probes, biological targets can be detected accurately. Ratio-type fluorescent probes with a large Stokes shift can dynamically monitor changes in intracellular pH in real time. By designing the D-A structure, different types of ICT fluorescent probes can be used for the rapid detection of various gases, heavy metal ions, and anions in living organisms at the nM level.However, there are still several aspects that need to be further explored. These include the following: 1. High sensitivity. Reducing the effective concentration threshold of the fluorescent probe improves its sensitivity, which was at lower concentrations of reactive groups, resulting in better detection results. 2. Specificity and selectivity. Fluorescent probes should be able to recognize and bind to target molecules or pathological changes while not interacting non-specifically with other molecules or cells. It is challenging to improve the specific detection of fluorescent probes and reduce non-specific signal interference to obtain better imaging results. It is also promising to cleverly design and synthesize probes to achieve the detection of multiple biological activities with a single probe, thereby achieving multiple detection biological species. 3. Biocompatibility. It is a basic requirement to ensure the safety to the human body and compatibility with biological tissues in the conversion process of fluorescent probes. Fluorescent probes must have low toxicity, low immunogenicity, and good biocompatibility in order to be safely used in clinical applications. The metabolic pathways and toxicity of synthesized molecular compounds must be clarified clearly in organisms, especially for probes that are bound to high-molecular-weight polymers. The metabolic pathways of degradation products and inflammatory reactions also need to be investigated in organs such as the heart, liver, spleen, lungs, and kidneys. 4. Cell permeability. Fluorescent probes should own the ability to pass through the cell membrane and reach target cells or tissues. This requires fluorescent probes to have appropriate chemical properties and size in order to effectively penetrate and distribute in the organism. 5. Integrated diagnosis and therapy. It is a promising solution to integrate the detection of fluorescent probes with treatment by combining them with drugs or nanomedicine delivery systems, to achieve integrated diagnosis and therapy.in vivo environment, unaffected by factors such as light, acidity or alkalinity, and temperature. This is crucial for achieving reliable fluorescent signals in clinical applications. In addition, fluorescent probes must have good optical properties such as high fluorescence quantum yield, long fluorescence lifetime, and high photostability. This can enhance the intensity and stability of fluorescent signals, making them more suitable for medical imaging or diagnostics. Effectively labeling fluorescent probes onto suitable carriers or nanoparticles and delivering them to target cells or tissues is also a challenging task. More importantly, converting fluorescent probes into clinical applications requires conducting clinical trials to validate their safety and efficacy. Additionally, they need to comply with relevant regulatory requirements and regulations. These challenges require continuous research and development to drive the translation of fluorescent probes from the laboratory to the clinic. Interdisciplinary collaboration and technological innovation will play a crucial role in overcoming these challenges.More importantly, how to translate laboratory findings into clinical applications and successfully achieve the clinical translation of fluorescent probes is a challenging task that needs to be urgently addressed. Moreover, fluorescent probes need to maintain satisfying stability in the


Humoral immune responses to respiratory syncytial virus (RSV) infection in hematopoietic cell transplant (HCT) recipients have not been well characterized. We measured RSV pre-fusion F antibody (RSVpreF) titers in HCT recipients in 3 groups: upper respiratory tract infections (URTI) only, lower respiratory tract infections (LRTI) only, and those who progressed from URTI to LRTIs (progressors).10 RSVpreF titers among groups using linear mixed effect models with restricted cubic splines.We evaluated adult and pediatric allogeneic HCT recipients who acquired RSV from 12/2011 to 12/2019. Prospective and leftover clinical serum specimens were collected from the Fred Hutchinson Cancer Center. Samples included baseline prior to infection, at RSV URTI or LRTI diagnosis, and after infection (both 4-6 and >8 weeks). Quantitative RSVpreF titers were determined using the MesoScale Discovery electrochemiluminescence assay. We compared longitudinal logA total of 129 samples from 39 patients were analyzed; 24 with URTI only, 8 progressors, and 7 with LRTI only. The median age at infection was 45 years, but HCT recipients with LRTI only had an overall higher median age of 54 (range 23-63) (Table 1). Most had acute leukemia  and received peripheral blood stem cells  as their cell source (Table 1). Median RSVpreF values were similar across the 3 different groups at each timepoint (Table 2). Trajectories of RSVpreF titers over time for each patient trended towards an increase following RSV diagnosis in all groups . Predicted curves in RSVpreF titers over time did not differ significantly by group .(A) RSVpreF titers for allogeneic HCT recipients with RSV infection. Each line represents a patient. The top panel shows patients with RSV URTI only, the middle panel shows patients who progressed from URTI to LRTI, and the bottom panel shows patients who presented with RSV LRTI. Open circles represent time points prior to RSV diagnosis, filled circles represent time points just prior to, on or after URTI, and asterisks represent time points just prior to, on, or after LRTI. (B) RSVpreF titers plotted by days from RSV diagnosis. Open circles represent observed values and lines represent model predicted trajectories. Black circles and lines correspond to patients with RSV URTI only, blue corresponds to patients who progressed from RSV URTI to LRTI, and dark orange represents patient who presented with RSV LRTI at diagnosis. An outlying observation at day -53 was excluded.Among allogeneic HCT recipients with RSV infection, RSVpreF titers increased in response to RSV infection, but did not differ among patients with URTI only, progressors, or those with LRTI only across pre and post infection time points. Our novel immunoassay results are similar to those from previously described RSV neutralization assays, but extended the observation period beyond 4 weeks after infection, revealing a modest increase in RSVpreF titers. Further characterization of RSV humoral immunity, including mucosal immunity, is needed.chikara Ogimi, MD, bioMerieux Japan Ltd.: Honoraria|Horiba: Honoraria|Pfizer: Honoraria Janet A. Englund, MD, Ark Biopharma: Advisor/Consultant|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant Emily T. Martin, PhD, MPH, Merck: Grant/Research Support Michael J. Boeckh, MD PhD, Allovir: Advisor/Consultant|Amazon: Grant/Research Support|Ansun: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Symbio: Advisor/Consultant Alpana Waghmare, MD, Allovir: Grant/Research Support|Amazon: Grant/Research Support|Ansun Biopharma: Grant/Research Support|GlaxoKlineSmith/Vir: Grant/Research Support|Kyorin Pharmaceuticals: Advisor/Consultant|Pfizer: Grant/Research Support|Vir Biotechnology: Advisor/Consultant


Pneumonia is the most common indication for antibiotics in the ICU. Better diagnostic tools are needed to target therapy. We evaluated the impact of the BIOFIRE Pneumonia Panel (PNP) coupled with aggressive stewardship interventions in the ICU.The PNP was implemented 5/2020 with interpretation guidance, provider education, and intermittent stewardship feedback. From 2/21 to 7/21 (Intervention) stewardship personnel reviewed all ICU PNP and provided structured feedback. We compared this group to patients with a respiratory tract culture in the ICU 9/19-2/20 (Control). We evaluated only first PNP/culture and excluded age < 19 years and expired < 24 hours after PNP/culture. Antibiotic use for 7 days after PNP/culture was compared between groups with time to de-escalation as the primary outcome. Times were measured from PNP/culture collection.Table 1). PNP results were available 4 hours after collection and positive in 56.9% (culture positive 38.3%) with the most common pathogens detected being S. aureus , H. influnezae (32), respiratory viruses (30), and P. aeruginosa (24). Use of urine antigens and respiratory pathogen panel testing was less common in the Intervention group (Table 1) while stewardship interventions were more common (82% vs. 13%) and occurred 23 hours earlier (Table 2). Intervention period antibiotic de-escalation occurred 18 hours earlier (P< 0.0001) and time to stopping anti-MRSA and anti-Pseudomonal therapy was shorter . Median antibiotic days were decreased  and days of vancomycin, metronidazole, azithromycin, and Anti-Pseudomonal therapy significantly decreased during the intervention.A total of 313 intervention patients and 315 controls were compared with differences noted in ICU location and LOS, COVID detection, specimen type, and mortality (PNP detected more pathogens and when coupled with an aggressive stewardship intervention resulted in faster pathogen detection, decreased urine antigen testing, faster de-escalation of antibiotics, and less antibiotic use. Clinical characteristics and outcomes were different between the groups which may be due to differences in the populations including the presence of COVID.Trevor C. Van Schooneveld, MD, FSHEA, FACP, AN2 Therapeutics: Grant/Research Support|Biomeriuex: Advisor/Consultant|Biomeriuex: Grant/Research Support|Insmed: Grant/Research Support|Thermo-Fischer: Honoraria Scott J. Bergman, PharmD, bioMerieux, Inc.: Honoraria Erica J. Stohs, MD, MPH, bioMerieux: Grant/Research Support|Merck: Grant/Research Support


Varicella-zoster virus (VZV) vasculopathy is a granulomatous vasculitis that has a wide variety of clinical presentations. It is most common in patients with HIV not on anti-retroviral therapy (ART) with low cluster of differentiation (CD)4 cell counts. This disease affects the central nervous system and can cause small intracranial bleeds. Our patient presented with stroke-like symptoms in the setting of recent VZV reactivation in the ophthalmic distribution with HIV on ART. Her MRI scan showed a small punctate bleed and the CSF workup was consistent with VZV vasculitis. She was treated with 14 days of acyclovir and five days of high-dose steroids with clinical improvement to baseline. Varicella-zoster virus (VZV) has been known to cause chickenpox and in some cases resurface as shingles. Shingles occur due to the reactivation of the virus within the ganglion of a dorsal root, cranial nerve, or autonomic nervous system with spread to the corresponding dermatome or neural tissue. This typically starts with pain, tingling, or itching preceding the development of the unilateral rash. Shingles can be associated with complications including post-herpetic neuralgia, ophthalmologic disease, motor dysfunction , transverse myelitis, and vasculitis ,2.Varicella-zoster virus vasculopathy, which has previously been called granulomatous angiitis, post-varicella arteriopathy, and varicella zoster vasculitis, is a granulomatous vasculitis that has a wide variety of clinical presentations. This occurs due to the reactivation of the virus in a cranial nerve ganglion followed by the spread and infection of the blood vessels. It is characterized by vessel wall damage, usually transmural inflammation, with multinucleated giant cells and with or without epithelioid macrophages. Central nervous system (CNS) small or medium vasculopathy is most common in immunocompromised individuals, including those with HIV infection and organ transplant recipients. However, HIV vasculitis is uncommon and is not typically at the top of the differential for patients presenting with acute neurological symptoms. There are lumbar puncture studies that will suggest the diagnosis in patients in whom the presence of VZV is suspected. Heterogenous presentations can occur considering that VZV can also precipitate arterial dissection, intracranial aneurysms, and venous sinus thrombosis . Among HA 70-year-old African-American female with a past medical history significant for chronic kidney disease (CKD) IV, coronary artery disease status post-myocardial infarction, HIV , and distant history of seizures presented to the ED via ambulance for altered mental status. History was obtained from a family member who states the patient was watching television when she "blanked out and stopped responding." The patient was unable to recall this event but later reported that two to three weeks prior to admission she was diagnosed with shingles in the left ophthalmic distribution with a persistent burning sensation in the same area. She did not receive treatment for shingles at that time.When she presented to the ED, she had a left-sided facial droop which improved back to baseline prior to the initial stroke exam performed in the ED. Her initial exam showed no focal extremity weakness, no cranial nerve deficits, normal speech pattern, no sensation deficits, and no coordination deficits. Initial vitals were temperature 36.4 degrees celsius, heart rate 64 beats per minute, respiratory rate 18 breaths per minute, blood pressure 123/74 millimeters of mercury, and pulse oximetry 98%. Initial labs were significant for iron deficiency anemia with hemoglobin 7.4 g/dL , hematocrit 24.2% , iron 29 ug/dL , total iron binding capacity 247 ug/dL , and iron saturation 11% . Acute kidney injury (AKI) with a creatinine of 2.76 mg/dL  was detected on her basic metabolic panel. Serum glucose on arrival was 160 mg/dL . Her coagulation profile including international normalized ratio (INR), prothrombin time (PT), and activated partial thromboplastin time (aPTT) were within normal limits. Erythrocyte sedimentation rate and c-reactive protein were not collected during this encounter. Non-contrast head CT . Due to her history of HIV, recent shingles infection, and small hemorrhagic stroke, there was high suspicion for VZV vasculopathy and she was started on IV acyclovir. Her AKI was treated with IV fluids which were continued for renal protection while on acyclovir. She received 1 unit packed red blood cells, IV iron supplementation, and oral iron supplementation on hospital day (HD) 1 for hemoglobin <7, which improved her anemia while hemoglobin and hematocrit remained stable throughout her hospital stay. Cerebral spinal fluid findings were consistent with viral etiology with glucose 44, WBC 89 (97% mononuclear), and protein 72. Clinically, her presentation was not consistent with meningitis/encephalitis. The MRI/MRA of the head and neck demonstrated a left periventricular lesion suggestive of a small focus of subacute hemorrhage Figures . The MRAClinical manifestations of VZV vasculopathy can vary widely due to infection of both large and small arteries. When it affects the CNS, it can result in cerebral ischemia or hemorrhage. The classical presentation is ophthalmic-distribution zoster followed by acute contralateral hemiplegia, but it also can occur with headache, mental status change, focal weakness or sensory loss, aphasia, ataxia, hemianopia, or vision loss. Ophthalmic distribution typically leads to unifocal vasculopathy, especially in children and the elderly, while multifocal vasculopathy most commonly occurs in immunocompromised individuals. Some may also present with encephalitis followed by focal deficits. Up to two-thirds of these patients have a history of either zoster infection or varicella rash. The time from rash to neurologic symptoms is sometimes simultaneous but averages about four months apart . Based oIntracerebral VZV vasculopathy occurs with VZV infection of intra and extracranial arteries. After infection, these vessels undergo pathological changes ranging from thrombosis, necrosis, dissection, or aneurysm formation. It has been described as having similar histological changes to giant cell arteritis and granulomatous aortitis. It tends to affect both large and small arteries in the cerebral vasculature. Patients with symptoms consistent with stroke are typically imaged according to local stroke protocol. Brain imaging usually shows ischemic or hemorrhagic infarction. These lesions tend to be bland and multifocal, but single lesions are also reported. An MRI shows lesions in the superficial and deep-seated in both gray and white matter, especially at the gray-white matter junction. After VZV infection there is an increased stroke risk of 2% to 9% in the first year after infection, regardless of the immunological status .A VZV vasculopathy diagnosis involves CSF testing. The CSF analysis usually has a modest pleocytosis (<100 cells/microL) with mononuclear cells. Protein is elevated with normal glucose and oligoclonal bands. Detection of VZV DNA and/or the presence of anti-VZV IgG can help establish the diagnosis. The sensitivity of CSF VZV IgG antibody has been found to be higher than VZV DNA detection by PCR 93% vs 30%) . The abs3% vs 30%The first-line treatment for VZV vasculopathy is intravenous acyclovir 10 mg/kg to 15 mg/kg every eight hours for seven to 14 days.  In VZV CThis case highlights the most important clinical aspects regarding clinical presentation, diagnosis, treatment, and complications of VZV vasculopathy in HIV patients. Varicella-zoster virus vasculopathy should be considered in patients with clinical presentation of stroke in the setting of HIV and recent VZV ophthalmopathy. This should be on the differential even in patients with adequate CD4 count, undetectable HIV DNA, and good highly active antiretroviral therapy (HAART) medication compliance. The use of brain imaging and CSF studies are key in the diagnosis. The treatment for this condition is acyclovir administration with or without high-dose steroids and has a high mortality rate if it goes untreated.


Glomus tumour is an uncommon soft tissue tumour which commonly occurs in the distal extremities, particularly the subungual region of the finger. Due to its rarity, there is a paucity of literature concerning glomus tumour. Therefore, this paper aims to report a case series based on our institution’s experience.A retrospective cross sectional study was performed in a single tertiary institution in Singapore. All patients diagnosed with glomus tumour confirmed on histology from January 2019 to October 2022 were included in the study. Patient demographics and clinical information  were retrieved from existing medical records.A total of 31 cases of glomus tumour were diagnosed from January 2019 to October 2022, and the relevant demographics and clinical presentation were reported. Majority of glomus tumours occurred in the finger (61.3%). Pain was present in almost all the cases (96.8%), while a lump was visible in less than half (48.4%). An average of 44.0 months elapsed before patients were properly diagnosed and treated. There were no cases of recurrence despite involved margins in three cases.Glomus tumour can be easily missed if clinicians do not have an index of suspicion for it, resulting in delayed treatment. Once diagnosed, glomus tumour can be treated with complete excision with good outcomes. Glomus tumours are uncommon neoplasms arising from the glomus body, which is part of the thermoregulation pathway in the dermis , 2. FirsThe classical symptoms of glomus tumour, as described by Carroll et al. , are paiHistologically, glomus tumours are usually well-circumscribed nodules comprised of uniform round cells with centrally located nuclei and well-defined cell borders Fig. A, B. TheGenerally considered to be a benign neoplasm, there have been cases of malignant transformation , 13. SomDespite being described more than a century ago, most of the available literature on glomus tumours is limited to case reports and a few case series. In this study, we present a series of 31 cases of glomus tumours in our institution, including a case report of a subungual glomus tumour of the toe, which to the authors’ knowledge, is one of the larger studies available. In doing so, we hope to further shed light on the clinical characteristics of this uncommon neoplasm.A retrospective cross sectional study was performed in a single tertiary institution in Singapore. All patients diagnosed with glomus tumour confirmed on histology by independent pathologists in our institution from January 2019 to October 2022 were included in the study.The cases were identified through our institution’s histopathology record database. Relevant clinical information of included patients was retrieved from existing medical records. The following information was collected: Demographics , presenting signs and symptoms , tumour parameters based on surgical or histological reports  and presence of recurrence on last clinical follow-up.Continuous variables were presented as mean and standard deviation, while categorical variables were presented in terms of frequency and percentage of the whole sample.This study was approved by our institution’s ethics committee .A total of 31 cases were included in the study. The mean age of presentation was 48.8 years, and there was equal distribution between genders. Most had a histological diagnosis of glomus tumour (93.5%), with only two cases diagnosed as glomangioma.Majority of glomus tumours was found in the extremities, with the fingers being the most common location 61.3%), followed by around the knee region (16.1%). One case had a glomus tumour located in the left 11th rib, while another was found in the stomach. Pain was the defining presenting symptom in almost all cases (96.8%), except for the tumour located in the stomach. A visible lump was only present in less than half of the cases (48.4%). The presence of preceding trauma was found in only 16.1% of the cases in this study. Symptoms were present for an average of 44.0 months before initial consultation. Table .3%, follAll the patients underwent an excisional biopsy, of which two were reported to have involved margins. The two cases with involved margins are as follows: Glomus tumour of the right knee followed-up for 785 days and glomus tumour of the 11th rib followed-up for 638 days. Overall, after an average follow-up period of 174 days across the 31 cases, there was no case of recurrence. All the cases were histologically benign, except for the 11th rib glomus tumour which had atypical features . No adjuvant treatment was performed.Glomus tumour is often encountered by hand or orthopaedic surgeons, owing to its usual occurrence in the extremities, especially the digits of the upper extremities. Majority of the cases in this study were found in the fingertips, which is similar to current literature , 8, wherGlomus tumours are reported to occur more commonly in women, and around the age of 20 to 40 years old . InteresThe clinical presentation of the patients in our study is comparable with that of the literature , 15, witRadiological investigations may be used to supplement clinical suspicion. Although a soft tissue tumour, glomus tumour may sometimes result in scalloping of adjacent bone which can be picked up on plain radiographs , 11, 14.Complete excision is considered curative, often with good outcomes . Almost The key strength of this study is the number of patients, representing one of the largest in the current literature. The clinical characteristics and outcomes presented will hopefully shed further light on this condition and aid clinicians in managing patients with this uncommon neoplasm.Glomus tumour is an uncommon soft tissue neoplasm that commonly occurs in the extremities. As it is often clinically unapparent aside from the presence of significant pain, it may be easily dismissed by clinicians, resulting in a prolonged period before the right diagnosis is made. While it is usually benign with a seemingly low recurrence rate, clinicians should have a high index of suspicion for those which have atypical features.


Amniotic fluid is a complex biological medium that offers protection to the fetus and plays a key role in normal fetal nutrition, organogenesis, and potentially fetal programming. Amniotic fluid is also critically involved in longitudinally shaping the in utero milieu during pregnancy. Yet, the molecular mechanism(s) of action by which amniotic fluid regulates fetal development is ill-defined partly due to an incomplete understanding of the evolving composition of the amniotic fluid proteome. Prior research consisting of cross-sectional studies suggests that the amniotic fluid proteome changes as pregnancy advances, yet longitudinal alterations have not been confirmed because repeated sampling is prohibitive in humans. We therefore performed serial amniocenteses at early, mid, and late gestational time-points within the same pregnancies in a rhesus macaque model. Longitudinally-collected rhesus amniotic fluid samples were paired with gestational-age matched cross-sectional human samples. Utilizing LC–MS/MS isobaric labeling quantitative proteomics, we demonstrate considerable cross-species similarity between the amniotic fluid proteomes and large scale gestational-age associated changes in protein content throughout pregnancy. This is the first study to compare human and rhesus amniotic fluid proteomic profiles across gestation and establishes a reference amniotic fluid proteome. The non-human primate model holds promise as a translational platform for amniotic fluid studies. It is a complex biological medium that surrounds the fetus, offers mechanical protection, provides nutrition, is critical for normal fetal growth and organogenesis, and can potentially influence fetal programming6. Amniotic fluid is swallowed by the fetus as early as 16 weeks of human gestation and plays a vital role in the development and maturation of the fetal gastrointestinal tract, lungs, and immune system9. In vitro studies have shown that amniotic fluid has intestinal epithelial cell trophic effects equivalent to breast milk and prior research has demonstrated the benefit of simulated amniotic fluid in reducing feeding intolerance in preterm human infants10.The in utero environment plays a significant role in fetal development and is vulnerable to adverse maternal environmental factors including chronic stress, environmental exposure, substance use, infection, and metabolic disease. A critical entity that influences fetal development in utero is amniotic fluid12. It is rich in proteins, lipids, oligosaccharides, ribonucleic acids (RNAs), and metabolites that potentially influence fetal development through bioactivities12. Amniotic fluid proteins and metabolites from human cross-sectional samples appear to evolve over the course of pregnancy14. However, alterations between different trimesters have not been confirmed within the same pregnancy because serial amniotic fluid sampling is not typically performed in human pregnancies.Despite its importance, the biology of amniotic fluid has been understudied. The existing literature suggests that the composition of amniotic fluid is dynamic and changes across gestation16.The knowledge deficit regarding the biology of amniotic fluid is partly because of the challenges in obtaining amniotic fluid samples from healthy human pregnancies, but also because of the limited translational strength of most animal models for amniotic fluid research. Human amniotic fluid studies typically examine samples collected at 16–20 weeks of gestation when amniocentesis is clinically performed for genetic or infectious testing. In addition, usually only one sample of amniotic fluid is collected before viability (approximately less than 23 weeks gestation) during a human pregnancy. This limits studies to a cross-sectional design, which does not allow insight into the potential longitudinal evolution of amniotic fluid composition within individual pregnancies. Consequently, detailed understanding of how amniotic fluid composition changes over gestation is limited, as is our understanding of the influence of the maternal environment on amniotic fluid composition, including the proteomeNon-human primate (NHP) models are an ideal proxy for studying human amniotic fluid biology because of their similar genome and developmental ontogeny. In addition, NHP models allows a high degree of experimental and environmental control and the ability to obtain serially collected amniotic fluid samples from the same pregnancy, which is not ethical or feasible to perform in humans. To address this research gap and overcome these obstacles, we employed a rhesus macaque NHP model to collect longitudinal amniotic fluid samples within the same healthy pregnancies at three time points across gestation. Tandem mass tag (TMT) isobaric labeling quantitative proteomic analyses were then performed with gestational age-matched human amniotic fluid samples collected cross-sectionally. Our objectives were to determine the translational strength of the rhesus macaque model for human amniotic fluid studies and to establish a reference proteomic profile across gestation to identify adversely affected pregnancies in future work. This is an important first step that will establish a pipeline for future studies and ultimately provide insights into the role of amniotic fluid protein composition on fetal development and the in utero environment, which define later-life offspring outcomes.Characteristics of the subjects that served as the source of amniotic fluid are shown in Table In this study, 24 human and 21 rhesus amniotic fluid samples were multiplexed using isobaric labeling and randomly distributed across 3 separate multi-dimensional LC–MS/MS runs. Common sample pools in all 3 plexes were used to perform internal reference scaling so that reporter ion intensities from all 45 samples were on a common protein abundance measurement scale. Species-specific FASTA searches were performed in parallel and yielded 1269 quantifiable proteins from rhesus amniotic fluid samples and 1381 quantifiable proteins in human amniotic fluid samples. The median inter-sample coefficient of variation was 24.4% in human and 29.8% in rhesus. After removing immunoglobulin family proteins, there were 1204 quantifiable rhesus proteins, of which 1148 (95.5%) overlapped with human proteins Fig. .Figure 1; Fig. Principal component analysis of the amniotic fluid proteome in either human or rhesus revealed clear separation of samples by gestational age. The first principal component (PC1) explained 21% and 27% of variability in human and rhesus datasets, respectively, and PC1 was strongly associated with rhesus equivalent gestational age within each dataset Fig. a. Out ofWe next compared functional terms overrepresented among the amniotic fluid proteins associated with gestational age across species and by direction of effect. The top 5 unique terms per category and subgroup are presented in Fig. 17. Analysis of all quantifiable proteins in amniotic fluid identified enrichment of liver, placenta, esophagus, cervix, gallbladder, and lung signatures  as a referenceres Fig. a. Among res Fig. b. Uniqueres Fig.  were mosres Fig.  were mosres Fig. c. Uniqueres Fig. . Proteinres Fig. .Figure 618. We identified 165 of the 320 previously observed amniotic fluid proteins in human amniotic fluid samples from our study, and 75 of the 165 were consistently differentially expressed with gestational age . The coefficient of variation for both rhesus and human amniotic fluid proteomes was small, likely reflecting both the stability of amniotic fluid across diagnoses and its importance as a developmental medium. Taken together, our findings establish the NHP as a potentially ideal translational model for the study of human amniotic fluid biology.A barrier to amniotic fluid research has been the lack of a suitable research model for the study of amniotic fluid longitudinally. The study of amniotic fluid using a NHP model offers several advantages over other model systems due to their similarity to humans, including placentation18. This study utilized amniotic fluid samples collected longitudinally from rhesus macaques at ~ G85, G110, and G135  and relative gestational age-matched human amniotic fluid samples collected cross-sectionally. These data reported herein are consistent with previous reports that utilized methods with less sensitivity or reduced gestational time points18. When comparing the recent report of Bhatti et al.18, of 320 differentially expressed human amniotic fluid proteins to the 440 differentially expressed human amniotic fluid proteins reported in this study, we found that approximately 50% of proteins were identified in both datasets, with half of those overlapping proteins varying consistently in both studies. There were differences between Bhatti et al.18 and our study in the timing of sample collection  and proteomics methods used (protein array versus mass spectroscopy-based), however both studies indicate a reorganization of amniotic fluid composition with the progression of fetal development. The proteomic methods used herein have several strengths that improve proteome data quality. Isobaric labeling of tryptic peptides allows for a combination of both protein identification and quantification with high sensitivity, there is little missing data, and synchronous precursor selection mass spectroscopy (SPS-MS3) acquisition improves the dynamic range of the reporter ion measurements .The complexity of the amniotic fluid proteome is further amplified by the evolution of the proteome over the course of gestation in healthy pregnancies, which has to date only been sparsely reported and limited to cross-sectional samples36. These results also suggest that the rhesus model may be used for the study of amniotic fluid biology, and to discover and validate biomarkers of pregnancy complications relevant to human health. For example, in a previous study of amniotic fluid from spontaneous preterm birth associated with infection/inflammation, lipocalin-1 (LCN1) was among several biomarkers identified and was decreased in cases versus controls37. In this study, we observed a ~ 26-fold increase in LCN1 in human amniotic fluid collected at G85 to G135 rhesus equivalent windows, in support of an important role of this protein in normal fetal development. A separate study of amniotic fluid proteins using mass spectrometry and validation by ELISA identified lumican (LUM), apolipoprotein (APOA4), and kininogen-1 (KNG1) as biomarkers of preterm birth38. Lumican is a major proteoglycan expressed in the cornea and skin, as well as the placenta, where it is hypothesized to play a role in preeclampsia39. In our study, LUM was the 9th most abundant protein in human amniotic fluid and the 22nd most abundant in rhesus amniotic fluid, and was downregulated in both species with increasing gestational age. The clinical significance of LUM concentration in amniotic fluid is unknown, but previous studies have observed association with preterm delivery as well as intra-amniotic inflammation and/or microbial invasion of the amniotic cavity in women with early preterm labor40.Importantly, the evolution of the human amniotic fluid proteome across gestation is replicated in the NHP model with longitudinally-collected samples, suggesting that these changes in amniotic fluid protein composition across gestation may have shared biological relevance among primate species. Only in the early third-trimester did we observe differences in the trends of some human and NHP amniotic fluid proteins Fig. . Specifi41 through innate immune defense. Similarly PIP, which is involved in regulation of water transport in lung airways, salivary glands, sweat glands, and apocrine glands including the mammary gland42, also increases in abundance across gestation. Interestingly, PIP is an aspartyl protease that cleaves fibronectin, thus its increasing abundance in amniotic fluid throughout gestation may have a relationship to the biological processes leading to delivery, and also may play a role in immune regulation43. Although we anticipated an increase of fetal gastrointestinal proteins with advancing gestational age to prepare for milk digestion after delivery, we observed an increase in esophageal proteins that may serve that purpose.It is possible that the diverse array of proteins, including bioactive proteins, and the large-scale alteration of composition and abundance across pregnancy is a by-product of fetal development and the maternal–fetal interaction over the course of gestation. However, the sheer complexity of the amniotic fluid protein composition and its cross-species reproducibility hint at the potential for amniotic fluid to directly influence fetal development through its rich and importantly, dynamic array of bioactive molecules. Use of GO, KEGG, and tissue enrichment analysis of the human and rhesus amniotic fluid data indicated increases and decreases in families of proteins associated with a diverse array of cellular functions and tissues. Specifically, we observed an increasing abundance of proteins expressed by respiratory tissues  and decreasing abundance of proteins overrepresented in placental and gastrointestinal tissues. The increasing presence of proteins associated with lung, such as surfactant proteins SFTPB, C, and D, biologically align with preparation of the fetal lung for breathing and gas exchange after birth through production and regulation of surfactant. In addition to its role in surfactant homeostasis, SFTPD is a collectin that may function in amniotic fluid to prevent intraamniotic infection44. The tear fluid proteome is enriched for proteins involved in immune function, which may also explain the biological abundance of these proteins in amniotic fluid45. Interestingly, several antimicrobial proteins differentially expressed in nasal mucosa from patients with chronic rhinosinusitis 46 were upregulated in amniotic fluid with increasing gestational age. Amongst those,  function as antimicrobial proteins46 and are related to immune and inflammatory processes, which may help prime the fetus for the process of labor and the postnatal environment47. The biological relevance of amniotic fluid proteome composition and dynamics and the potential role(s) of amniotic fluid in fetal development remain to be defined. However, continued efforts to characterize the composition of amniotic fluid over the course of gestation, by leveraging the NHP model and longitudinal sampling, may provide additional insight.Further investigation is needed to better understand these observed profiles across gestation. Interestingly, 8 of the top 10 most upregulated proteins in human amniotic fluid , have been described as biomarkers for tear fluid and proteins involved in ocular diseasesThis study leverages the translational rhesus macaque model to address the critical knowledge gaps in our understanding of normal amniotic fluid proteome composition across gestation. Strengths of this model include similar physiology, genetic, and developmental properties with humans and the ability to perform serial biological sampling and experimental manipulation that are not feasible or ethical in humans.49, despite differences in the underlying etiology for performing amniocenteses, and we observed striking similarity across species in protein abundance and dynamics.We employed isobaric labeling quantitative proteomics because of the high fraction of identifiable proteins that can be quantified with little missing data. Increased multiplexing capacity and advanced data acquisition methods that improve reporter ion signal quality are also isobaric labeling strengths. We quantified 1269 proteins in rhesus amniotic fluid (21 samples) and 1381 in human amniotic fluid (24 samples) with 1090 uniquely mapping orthologs. The coefficient of variation for the human and rhesus proteomes at each gestational age ranged between 24 and 30%, lower than inter- and intra-subject variability reported in other biofluids18, and the data presented here: differences in amniotic fluid gestational age time-points and methodological differences in proteomics platforms that limit comparison between the two reports. While our methods detected ~ 1300 amniotic fluid proteins after albumin depletion, larger numbers of reported human amniotic fluid proteins have been reported after top 14 protein depletion26, and future work may allow for a deeper comparison of the amniotic fluid proteome across gestation, as well as between the human and rhesus models. The size of our NHP cohort was relatively small (n = 7), however this limitation is balanced by our longitudinal, repeated-measures design, which minimizes inter-sample variability. Due to the small sample size, we were not able to stratify our analysis by fetal sex, which should be a focus of future investigations on amniotic fluid composition. Additional strengths of this study are that all rhesus amniotic fluid samples were collected by a single board-certified Maternal Fetal Medicine specialist under ultrasound guidance, and that NHPs are selected with similar age, size, and housing conditions, which eliminates many of the confounders of human cross-sectional study designs.There are several differences between the recent cross-sectional report of the human amniotic fluid proteome by Bhatti et al.This is the first study to perform parallel analyses of rhesus and human amniotic fluid proteomes across gestation. Overall, we demonstrated high homology across these two species in amniotic fluid proteome composition and dynamics with gestational age, with large-scale evolution of protein identity and abundance. Importantly, our study suggests that the rhesus macaque model can be used for future investigations into the biological role of the amniotic fluid proteome in normal pregnancy, fetal development, and may also be relevant in developing biomarkers of obstetrical or fetal complications.https://arriveguidelines.org)50. This study used indoor-housed, reproductive age female rhesus macaques (n = 7) maintained on a standard chow diet . Human amniotic fluid samples were obtained from the OHSU Knight Diagnostic Laboratory where informed consent was previously achieved to utilize the samples for research purposes.All methods were carried out in accordance with relevant guidelines and regulations. Protocols were all approved by the Oregon Health & Science University (OHSU) Institutional Review Board (IRB) for human amniotic fluid (#20623), and the Oregon National Primate Research Center (ONPRC) Institutional Animal Care and Use Committee for rhesus amniotic fluid (IP0001389). Non-human primate methods are reported in accordance with the ARRIVE guidelines . All animals (n = 7) underwent ultrasound-guided amniotic fluid sampling on or near gestational days 85 (G85), G110, and G135 (term is ~ 168 days)  prior to intubation and maintained on 1.5% isoflurane gas with continuous monitoring of vital signs. Animals were in a dorsal recumbent position, and image-directed pulsed and color Doppler equipment with a 5- to 9-MHz sector probe (GE Voluson 730) was used to measure the amniotic fluid index, placental location, and confirmation of complete chorion and amnion fusion prior to sterile preparation of the skin. A 22-gauge needle was introduced transcutaneously under direct ultrasound guidance into the amniotic cavity, avoiding the placenta, and a 10 mL fluid sample aspirated, centrifuged at 1500×g for 10 min to remove amniocytes. Samples were aliquoted and stored at − 80 °C.Standard clinical amniocentesis was performed by a board-certified OHSU Maternal Fetal Medicine physician. Patients were unsedated and in a supine position. 2% chlorhexidine gluconate solution was used to sterilize the abdomen and under direct ultrasound guidance, a 20-gauge needle was introduced transcutaneously into the amniotic cavity while avoiding the placenta. A total of 30 mL of fluid was aspirated, and sent to OHSU Knight Diagnostic Laboratory, where the samples were centrifuged at 2000×The rhesus macaque amniotic fluid sample group comprised of 7 animals longitudinally sampled at 3 gestational ages (in days): G85, G110, and G135 . These gestational time points were matched by percent completed gestation with 7 human samples at each time point . 3 additional human samples at the G110-equivalent human gestational age were included for a total of 24 human samples. The comparable human gestational age time point for G85 is approximately 20 weeks (sample range 19 5/7 weeks to 20 4/7 weeks), for G110 approximately 25.5 weeks (sample range 24 1/7 to 26 2/7 weeks), and for G135, approximately 31.5 weeks (sample range 30 3/7 to 31 3/7 weeks) . Samples were depleted of albumin using an albumin depletion spin cartridge . Samples were reduced and alkylated and digested with trypsin using the EasyPep Mini digestion kit . Amniotic fluid peptides were labeled with tandem mass tag (TMTpro) 18-plex reagent kits . TMTpro labeled peptides were combined and analyzed by LC–MS on a Dionex Ultimate HPLC operating in 2-D mode coupled to an Orbitrap Fusion Tribrid mass spectrometer  using the SPS MS3 scan mode for TMT quantitation.51 using the Comet search engine (version 2016.03)52 in parallel searches of Uniprot proteomes UP000006718  with canonical FASTA sequences  and UP000005640  for identification and quantifications of proteins from each species. Statistical analysis was performed using the Bioconductor package edgeR53 within Jupyter notebooks.Data was processed using the PAW pipelineThe clinical characteristics of subjects included in this study were summarized in Table 54. The coefficient of variation (CV) for each species and gestational window was calculated by dividing the standard deviation of each protein by the mean, and is reported as the median percent across all proteins. The data was then loaded into an edgeR DGEList object and normalized using TMM53, library scaling, and log transformation. The logcpm matrix for each species was used for principal component analysis using the stats package in R (version 4.1.3). The rhesus and human logcpm matrices were merged based on ortholog matching to generate a scatterplot of mean protein abundance in rhesus versus human using ggplot2 and a heatmap of log intensity for all samples and orthologs using ComplexHeatmap.For each species, Internal Reference Scaling (IRS) was performed separately as previously describedlimma with gestational age in days in the design variable followed by eBayes moderation of standard errors. For rhesus, we also added a blocking argument and correlation value to account for within-subject variance. The resulting coefficients (logFC) from the linear models therefore describe the change in protein abundance for each day of gestation. The “AveExpr” represents the average log intensity for all samples per protein. We considered an FDR adjusted p-value < 0.05 as significant. We used the VennDiagram package to visualize the overlap of gestational-age associated proteins between species and to calculate enrichment of overlap using the hypergeometric test.For each species, we fit separate linear models for each protein using clusterProfiler and enrichplot packages in R version 4.2.2 to test for and visualize enrichment of GO and KEGG pathways. Proteins differentially expressed with gestational age (FDR p < 0.05) were mapped to human Entrez gene identifiers for each dataset (rhesus and human). Enrichment analyses were performed using the compareCluster function separately with enrichGO and enrichKEGG with the default background gene list of all genes in the database. Redundant terms were simplified using a correlation cutoff of 0.7. An adjusted p-value < 0.05 was considered significant.We used the TissueEnrich R package55. We defined tissue enrichment based on RNA-seq data across 35 human tissues in the Human Protein Atlas dataset17. Genes with an expression level greater than 1 (TPM or FPKM) that also have at least five-fold higher expression levels compared to all other tissues were classified as enriched for a particular tissue. This package uses the hypergeometric test to calculate tissue-specific gene enrichment where, N is the total number of genes, K is the total number of tissue-specific genes for a tissue, n is the number of genes in the input gene set, and k is the number of tissue-specific genes in the input gene set. An adjusted p-value of 0.05 was considered significant to identify enriched tissues. To visualize the overall longitudinal pattern of expression in a given enriched tissue, we normalized expression for each protein by subtracting the mean and dividing by the standard deviation calculated from the reference group . We then take the mean across proteins enriched for a given tissue to obtain tissue-specific Z-scores and plot longitudinally relative to rhesus GA equivalent.To determine potential tissue sources of proteins differentially expressed with gestational age, we used the 18. We used the VennDiagram package to visualize the overlap of gestational-age-associated proteins between studies and to calculate enrichment of overlap using the hypergeometric test. For proteins identified in both studies, we compared the effect sizes using a scatterplot and Pearson correlation between logFC with gestational age.We compared gestational age associated protein changes in human amniotic fluid from this study with results from a previous cross-sectional study measured using a high-throughput aptamer-based platformSupplementary Information.Supplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Table 6.


P < 0.001). The f-UAS should be brought closer to the stone in RIRS. And more stone fragments can be taken out of the body by the effect of irrigation fluid.This study aims to explore the optimal location of flexible ureteral access sheath (f-UAS) in retrograde intrarenal lithotripsy (RIRS). RIRS model was built by AutoCAD 2011 software, and imported COMSOL 5.6 software to computer simulation. An RIRS model was constructed in vitro to analyze the distribution pattern of stone fragments and compare the weight of stone fragments carried out by the irrigation fluid when the f-UAS is in different positions. Computer simulation showed that the highest flow of irrigation fluid was in the channel of flexible ureteroscopy (f-URS) and in the lumen of f-UAS. From the f-URS to the renal collection system and then to the f-UAS, the velocity of irrigation fluid changes gradually from high-flow to low-flow and then to high-flow. When the f-URS and the f-UAS are at the same level, the irrigation fluid is always at a state of high flow during the process from f-URS to f-UAS. When the f-URS and the f-UAS are at the same level, it can increase the local intrarenal pressure (IRP) at the front of f-URS. The stone fragments are mainly sediment in the low-flow region of irrigation fluid. More stone fragments could follow the irrigation fluid out of the body when the tip of f-URS and the tip of f-UAS are at the same level ( The ureteral access sheath (UAS) facilitates flexible ureteroscopy (f-URS) exercise, reduces operative time, lowers intrarenal pressure (IRP), and improves stone-free rate (SFR) in retrograde intrarenal surgery (RIRS) –4. ThereA computerized model of RIRS was established to analyze the distribution pattern of irrigation fluid in RIRS. And vitro RIRS model was established to analyze the distribution pattern of stone fragments under the action of irrigation fluid. Compare the weight of stone fragments carried out by the irrigation fluid. We inferred the optimal placement of f-UAS in RIRS.The RIRS model was created using AutoCAD 2011 software and then imported into COMSOL 5.6 software for computer simulation. The irrigation fluid was set at 80 ml/min. A two-dimensional model was generated to analyze the distribution pattern of irrigation fluid when the f-URS position was stationary in renal calyces, and the f-UAS was in the upper ureter or renal pelvis.An RIRS model was established using a tube (10 ml), f-UAS , and f-URS . Stone fragments  were placed at the bottom of the tube. The irrigation fluid was set at 80 ml/min. We analyzed the distribution pattern of stone fragments under the action of the irrigation fluid when the f-UAS was in different positions.Flexible ureteral access sheath (f-UAS): the f-UAS is a new type of UAS that has a tip of 10 cm tube that can be passively bent with bending of flexible ureteroscope (f-URS), and this degree of bend is about 145º. The f-UAS is still able to ensure a positive circular shape of tube lumen when bending, and it can be connected to a vacuum suction device .A group: the f-UAS and the f-URS are basically at the same level, and both move synchronously and gradually approach the stone fragments. B group: the f-URS was ≥ 2 cm away from the f-UAS, the f-URS was in constant motion, and the f-UAS was fixed . The experiment was conducted for 10 min in both groups and the weight of stone fragments carried out by irrigation fluid was compared between the two groups. Repeat 20 times each in two groups.t test was applied to continuous data. Statistical significance was set at P < 0.05.Student’s Computer simulation showed that the highest flow of irrigation fluid is in the channel of f-URS and in the lumen of the f-UAS, which is a region with the relatively highest flow. A region with a relatively low flow of irrigation fluid has been reported elsewhere. During the process in which the irrigation fluid from the f-URS to the renal collection system and then to the f-UAS, the velocity of the irrigation fluid changes gradually from high-flow to low-flow and then to high-flow.When the tip of the f-URS and the tip of the f-UAS are at the same level, the irrigation fluid is always in a state of high flow during the process from f-URS to f-UAS, and there is almost no low-flow state. Comparing the two situations where the f-UAS is in different positions, it was found that a localized higher IRP is generated at the tip of the f-URS when the tip of the f-URS and the tip of the f-UAS are at the same level Fig. .Fig. 1CoP < 0.001) .Stone fragments are constantly moving under the action of irrigation fluid, but at the same time, the stone fragments are also subjected to the action of gravity, which eventually leads to sedimentation. Observed in conjunction with Fig. When the f-UAS and f-URS are at the same level, only a localized high pressure is generated at the front of the f-URS, rather than an increase in intrarenal pressure throughout the renal collecting system. In our opinion, one of the reasons for high intrarenal pressure is the presence of some kinetic energy in the irrigation fluid. In addition, in the computer model, the f-URS is equivalent to being placed in the calyx, and a certain thickness of the sheath exists, which indirectly reduces the outlet of the calyx when the f-UAS is also placed in the calyx, i.e., reduces the outlet of the irrigation fluid. This eventually leads to an increase in IRP. The IRP can be reduced by connecting a vacuum device; an f-URS and a UAS at the same level and connected to a vacuum device might better improve the stone free rate and reduce intrarenal pressure –16.FD) in fluid dynamics. FD is one of the forces on the sediment particles by the current [FD equation: CD: drag and lift coefficients; A: frontal area exposed to the flow; ρ: densities of fluid and sediment) [FD is proportional to FD increases exponentially with the increase in current velocity, which causes the stone fragments to move more easily with the irrigation fluid. The irrigation fluid is always at a high-flow rate from f-URS to f-UAS when f-UAS is at the same level as f-URS, so more stone fragments are taken out of the body by the action of irrigation fluid. The velocity of irrigation fluid cannot be increased indefinitely in RIRS, but bringing the high-flow region closer to the position of stone fragments can indirectly increase the action of irrigation fluid on stone fragments. However, the irrigation fluid flow rate is influenced by several factors [To understand the effect of irrigation fluid on stones, we introduced the concept of the drag force ( current . FD equaediment) . FD is p factors . A vacuu factors –21. Our Powdered and granular stones are inevitably produced during RIRS , 22, 23.The experiments showed that more stone fragments could be carried out of the body under the action of irrigation fluid when the UAS is at the same level as the f-URS and both move synchronously. In reality, it is difficult for the non-flexible UAS to follow the f-URS into the renal pelvis or calyces. For non-flexible UAS, perhaps the optimal location in ureteral stones and bladder stones may also be close to the stone. However, this study was only an in vitro experiment and computer simulation without the influence of devices such as a laser; this does not represent the true in vivo situation, which requires further clinical validation.During the process in which the irrigation fluid from the f-URS to the renal collection system and then to the f-UAS, the velocity of irrigation fluid changes gradually from high-flow to low-flow and then to high-flow. Stone fragments were mainly sediment in the low-flow region of irrigation fluid. More stone fragments could follow the irrigation fluid out of the body when the f-UAS is at the same level as the f-URS and both move synchronously and progressively closer to the stone fragments.