After the nitroglycerin challenge, patients with shock had an increase in MFI , TVD , PPV , PVD , venular RBC velocity , and a decrease in MHI . Compared to the control group, patients with shock had a higher microcirculation heterogeneity index (MHI 0.33 vs. 0.12,  Traditionally shock is categorized by macrocirculatory etiologies including hypovolemic, distributive, obstructive, and cardiogenic shock, but downstream effects on functional capillary density and blood flow are heterogeneous3. A number of mechanisms have been identified for reduced microcirculatory blood flow during shock states, including inflammatory-mediated vascular endothelial injury, microthrombosis, and an inadequate balance between vasoconstrictive and vasodilating molecules which can lead to a global or heterogeneous reduction in capillary blood flow5. Unfortunately, many of the often used therapies to reverse hemodynamic derangements do not reverse microcirculatory impairment8. Interventions during the optimization and stabilization phases of shock resuscitation are limited, and often guided by surrogate markers of tissue perfusion2.Circulatory shock is a life-threatening, generalized form of acute circulatory failure where there is an imbalance between oxygen demand, supply, or utilization9. Vascular endothelial injury and impaired endothelial NO production have been implicated as a mechanism of shock severity. Nitroglycerin (NTG) is an endothelium-independent nitric oxide donor and vasodilator that has been shown to increase capillary density and blood flow in patients with vasopressor-dependent septic shock and acute heart failure11. Initiation of vasodilatory therapy in patients with vasoactive-dependent shock may be counterintuitive and might only benefit a select group of patients. In this case, a strategy that identifies nitroglycerin-responsive patients prior to systemic administration would be ideal.Nitric oxide (NO) impacts many of the key pathologic changes observed in shock, including regulation of vascular endothelial function, microvascular permeability, leukocyte adherence, and platelet activation at the capillary level12. A microdose is defined as being below the dose calculated to yield a pharmacological effect and less than 100 µg of the active drug13. In healthy adults, low-dose topical nitroglycerin has been used to measure microcirculatory reserve using sublingual incident dark-field (IDF) microscopy, but this approach could also be used in patients with shock to test for a physiologic response without exposing patients to undesired effects that may be experienced with systemic administration14. Our primary aim was to determine if a microdose of topical nitroglycerin could improve microcirculatory density and capillary blood flow in critically ill patients with vasoactive-dependent circulatory shock. A secondary aim was to identify nitroglycerin responders using a patient-specific pharmacodynamic response threshold15.Microdosing novel therapeutics in conjunction with local tissue imaging has gained popularity in fields outside of critical care to test for important physiologic responses to treatmentForty-four patients were consented for enrollment in the study. Twenty-four patients with post-operative shock received the topical nitroglycerin challenge and 20 preoperative patients served as a control group. Control patients had common chronic medical conditions for patients with cardiovascular disease but did not have any pre-operative end-organ injury such as chronic kidney disease, reduced left ventricular ejection fraction, chronic liver disease. Two patients were excluded from the experimental arm prior to receiving the study drug . Twenty-two total subjects received the study drug to assess for microcirculation responsiveness. Two additional subjects were excluded after enrollment for having an inadequate number of quality videos to meet consensus standards. The CONSORT flow diagram is included in Supplemental Data. Patient information and demographics are in Table Compared to the control group, patients with postoperative shock had reduced microvascular flow index (MFI), heterogeneity (MHI), total vessel density (TVD), proportion of perfused vessels (PPV), and perfused vessel density (PVD) Table .Table 3Three hundred and twenty-six video clips of the sublingual microcirculation were obtained, stabilized, and graded for quality prior to inclusion for final analysis. Eighty-six videos were excluded from the NTG challenge group for having a Massey score > 10, leaving 240 acceptable videos for final analysis. The analyzed video clips were of good quality with a mean Massey score of 0.3 ± 0.4 . Bland–Altman bias was 0.021 (95% limit of agreement − 0.11 to 0.15).2, p = 0.19), proportion of perfused vessels , perfused vessel density , microvascular flow index , and heterogeneity  between control subjects and patients with shock after the NTG challenge. All microcirculation parameters returned to baseline values 30 min after the NTG challenge.Nitroglycerin administration reduced flow heterogeneity, increased microcirculatory density, and increased venular RBC velocity (RBCv) within 3 min of nitroglycerin microdosing Table . There w2 compared to baseline using a pooled baseline SD of 3.03. Baseline microcirculation characteristics of NTG responders and non-responders are listed in Table Pharmacodynamic (PD) response to nitroglycerin was defined as an increase in PVD > 5.46 mm/mmThere was no significant change in mean arterial blood pressure, central venous pressure, cardiac index, or systemic vascular resistance index during the 30-min challenge Fig. . PatientBased on the results of this study, we were able to show that (i) sublingual capillary density and blood flow is reduced during postoperative circulatory shock compared to a preoperative control; (ii) reduced microcirculation density and flow could be increased using a topical endothelium-independent nitric oxide donor without any effect on systemic hemodynamics; and (iii) we could identify nitroglycerin responsive patients using a pharmacodynamic statistical method used in early phase 0 trials.2. Compared to our control group, patients with shock had lower capillary density and blood flow indicating reduced tissue perfusion. Current resuscitation strategies to reverse shock include augmenting intravascular volume and vasoactive therapies to normalize systemic hemodynamics, but unfortunately, these interventions do not always lead to improvements in capillary blood flow17. In this study, we found that topical nitroglycerin increased regional PVD and reduced heterogeneity in patients with shock which have been associated with poor clinical outcomes7. The MFI and RBCv of our shock cohort also increased after the NTG challenge, but both of these values appear to be close to values that may be considered within the normal range based on consensus definitions—specifically MFI18. Large studies to define human reference ranges have yet to be completed. It is unclear if targeting higher values would have any clinical or physiologic impact. The small vessel abnormalities identified with IDF are likely the result of a number of common post-cardiopulmonary bypass pathologies including a SIRS-related vasoplegia, low cardiac output, vascular endothelial injury, and microvascular shunting19. Nitric oxide donors are of particular interest as they have been found to be a mediator of endothelial ischemia–reperfusion injury and can provide a number of other tissue-protective effects including reduction of small vessel resistance, leukocyte adherence, platelet aggregation, and vascular endothelial cell permeability21.Circulatory shock is defined as an imbalance between oxygen demand, supply, or utilization22. A significant reduction in venular pressure would increase the pressure differential across the capillary network to improve PVD and flow homogeneity identified with IDF. It is not possible to determine if the topical solution primarily acted on the pre- or post-capillary resistance vessels within the microvascular unit using IDF microscopy. As expected, the duration of the pharmacodynamic response was transient, lasting less than 30 min, which is consistent with well-studied nitroglycerin pharmacokinetics23.The rapid change in perfused vessel density and flow heterogeneity suggests that an endothelium-independent nitric oxide donor can reverse early post-operative derangements in capillary blood flow. The time-course of the observed change in microcirculatory flow was consistent with the known pharmacokinetics of nitroglycerin. Although nitroglycerin does provide a vasodilatory effect on both arteries and veins, evidence suggest the most significant effect is on the venous system24. We chose our dose of NTG based on the previous findings of Hilty et al., who also identified an increase in microcirculatory blood flow without a change in hemodynamics in healthy volunteers14. By incorporating handheld video microscopy (HVM) imaging, we were able to identify a pharmacodynamic effect of nitroglycerin using a solution that delivered less than 2% of the standard dose of sublingual nitroglycerin without causing a change in hemodynamics. Our method appears to be a low-risk test for identifying nitroglycerin responsiveness in patients with shock.Administration of a vasodilatory medication to patients with vasopressor-dependent hypotension is a significant departure from conventional treatment strategies and could result in significant hypotension. Topical nitroglycerin has been shown to improve local microcirculation without disturbing hemodynamics in hemorrhagic shock and in healthy volunteers25. Our measurements of postoperative microcirculation parameters are similar to other reports in the literature when using consensus standard analysis methods26.When evaluating our NTG response groups for potential confounders, both NTG responder and non-responder groups were similar. There was no difference in vasoactive medications, vasoactive dose, mean arterial pressure, central venous pressure, or other hemodynamic measures between NTG responders and non-responders. Nitroglycerin responders had a longer cardiopulmonary bypass and cross clamp time, which has been associated with more pronounced vascular endothelial injury and decreased NO productionIn our study, 65% of patients increased their PVD > 1.8 SD from baseline, indicating that NO donor responsiveness is likely individualized. In fact, NTG responders had lower baseline perfused vessel density, largely due to a reduced TVD. There was no difference in baseline MHI between groups, yet 15/20 subjects who received the NTG challenge resulted in an MHI that approached 0 indicating minimal flow heterogeneity and essentially uniform blood flow through most capillaries.27. It is possible that previous studies included patients that were either not responsive to nitroglycerin therapy or the dose administered was inadequate to stimulate regional blood flow in the vascular beds being imaged. The hesitancy to administer vasodilators in patients requiring vasopressor therapy is understandable, because current small molecule therapeutics cannot limit their effects to sites of microvascular injury. The development of novel NO-donor or NO-preserving therapeutics using nanocarrier technology, designed to target injured vascular endothelial cells, may be able to improve capillary blood flow in disturbed microvascular beds while minimizing undesirable side effects that can occur with current small molecule therapeutics30.Previous research in vasoactive-dependent septic shock utilized a fixed-dose nitroglycerin infusion (30 mcg/min) which improved peripheral perfusion but did not improve sublingual microcirculatory function or survival compared to placeboThere are a few limitations to our study that should be identified. We did not perform a stepwise increase in nitroglycerin dosing, therefore it is possible that patients labeled as non-responders did not receive a high enough dose of nitroglycerin to improve capillary blood flow. While incorporating HVM to identify a real-time response to nitroglycerin can identify a physiologic response, significant time-constraints for the analysis of video sequences are a barrier to use in routine clinical practice. Development of software capable of providing real-time automated analysis would reduce the time required to interpret changes in PVD, allowing for more timely, individualized therapeutic interventions. Finally, there may be natural variation in sublingual microcirculation over time, which could account for a portion of the change we observed after the nitroglycerin challenge. While we did not perform an analysis to evaluate for regression to the mean, our findings overall reflected the anticipated change in microcirculatory function based on established nitroglycerin pharmacology. Future research that examines natural variation in microcirculatory flow over time on a larger dataset would be a valuable addition to the literature.In conclusion, we were able to demonstrate that abnormalities in functional capillary density, heterogeneity, and flow during circulatory shock can be reversed using an endothelium-independent nitric oxide donor. The effect appears to be most pronounced in patients with abnormal perfused vessel density. Individual responsiveness can be safely tested with the use of a low-dose topical nitroglycerin solution in conjunction with HVM.32.Adult patients (age > 18 years) receiving elective cardiac surgery requiring cardiopulmonary bypass were screened for eligibility from August to October 2021. Written informed consent was obtained from eligible patients prior to surgery. Patients were excluded if they had a nitroglycerin allergy, were taking oral phosphodiesterase inhibitors, were unable to tolerate microcirculatory flow image acquisition or did not have a pulmonary artery catheter for continuous cardiac output monitoring after surgery. Patients only received the study drug if they met the clinical criteria for circulatory shock, defined as having postoperative vasopressor-dependent hypotension or low cardiac output requiring inotropic support, with signs of end-organ injury . A second cohort of patients were enrolled to serve as a preoperative control, which were matched to the experimental group for age, co-morbidities, and planned operation. Study data were collected and recorded using a well-established clinical database tool  hosted at the University of Pennsylvania−2 μmol per drop) were applied to the sublingual space after obtaining baseline post-operative microcirculation images. A microdose  was administered to avoid any systemic effects.This investigator-initiated, open-label study was conducted at the Hospital of the University of Pennsylvania, an urban quaternary academic medical center. The study was approved by the University of Pennsylvania’s institutional review board, registered with ClinicalTrials.gov (NCT05102734), and conducted in accordance with the principles of the Declaration of Helsinki. Sample size was determined based on an anticipated 30% increase in TVD, and setting a one-sided α of 0.1, and ß of 0.8. A 1% topical nitroglycerin solution was prepared immediately prior to enrollment by reconstituting 400 mcg of nitroglycerin  in 4 mL of sterile water within a dropper bottle that would administer the solution in 0.05 mL aliquots. Two drops (0.1 mL) of the 1% nitroglycerin solution  duration were taken at each time point with attention to quality factors, especially the absence of pressure artifact, excess saliva, and proper location in accordance with the accepted consensus for microcirculation analysis. Image quality was assessed using the 6-factor Massey quality score, which scores each video for appropriate illumination, duration, focus, content, stability, and pressure. Images were only included in the final analysis if the Massey quality score was < 1034. Acceptable clips were deidentified and coded for analysis after enrollment was complete.Sublingual microcirculation imaging was performed using HVM  in the preoperative staging area (for controls) or within 2 hours of arriving to the intensive care unit after surgery by a trained member of the investigative team (JCG or FMT)Microcirculation videos were exported using commercial software  and manually analyzed using the validated Automated Vascular Analysis software . Video analysis was performed by trained investigators (JCG and FMT) who were blinded to the conditions of the subject. Only microvessels ≤ 20 μm in diameter were included in the calculation of TVD and PVD.18. Microcirculatory function was quantified according to the current best practice guidelines for microcirculation imaging18. Blood flow within each microvessel was graded using a semiquantitative scale, based on the vascular flow pattern ranging from 0 to 3 . Vessel perfusion was dichotomized as nonperfused (no flow or intermittent flow) or perfused (continuous or sluggish) for the calculation of PPV and PVD.Red blood cell flow velocity was calculated within appropriate venules 20–30 μm in diameter by manually measuring the slope of individual RBC movement within software generated space–time diagrams15.An exploratory analysis was planned to identify the incidence of a patient specific pharmacodynamic response to the nitroglycerin challenge. A PD response threshold was set as a post-administration PVD > 1.8 standard deviations from the baseline, which was calculated by averaging the intra-patient variance between the three baseline PVD video sequences, which was then pooled across the entire cohort. This 1.8SD threshold was set to achieve 90 percent confidence interval . This method has been used in previous Phase 0 trials to provide a statistically rigorous threshold for individualized PD responses to novel therapeutic interventions2) were continuously monitored by pulmonary artery catheter . Arterial blood pressure was measured continuously using a standard invasive arterial line. Systemic hemodynamic data, vasopressor infusion doses, and mixed venous oxygen saturation were recorded at baseline then every 3 min for 30 min after application of the topical nitroglycerin solution35. The vasopressor-inotrope score (VIS) was calculated to summarize the degree of post-operative vasoactive support was required at the time of the study36. Norepinephrine equivalents were calculated for each patient at each time point37.Subject demographics, pre-operative risk score (euroSCORE II) and medical history were collected during initial screening. Cardiac index (CI), central venous pressure (CVP), pulmonary artery pressure (PAP), and mixed venous oxygen saturation . Statistical significance was assumed at This study was approved by the University of Pennsylvania’s institutional review board (IRB # 829765) and informed consent was obtained prior to enrollment. All consent forms were copied in triplicate, one given to the subject, the second placed in the official medical record, the third kept in a secured location within the PI’s office.Supplementary Information.


The incidence of cardiovascular diseases is continuously increasing around the world. Therefore, the study of new methods for diagnosing cardiovascular diseases is very important. Early diagnosis and evaluation of the effectiveness of treatments are among the most important tasks. In this work, we study changes in vascular compliance and vascular tone of the lower extremities in a patient diagnosed with an early stage of varicose veins. The study is based on recording the bioimpedance signals of the lower extremities and their parts using the Rheo-32 multichannel device. Registration in the monitoring system takes place in two stages: the first in a state of relaxation, and the second after applying a local massage on one of the legs for five minutes. The results indicate a change in the type of vascular tone of the lower extremities after the massage, while the type of vascular tone changes and shifts on average towards the normotonic type. The method proposed in this study makes it possible to quantitatively and qualitatively assess changes in the tone of the vessels of the extremities. Cardiovascular diseases are the leading cause of death worldwide and among the most common diseases in the general population . The neeImpedance plethysmography is used to measure the blood volume changes associated with changes in electrical impedance. This technique can be used to measure blood flow in a limb or finger .Electrical impedance plethysmography  records the impedance of the lower extremity as the blood volume varies. IPG has been proven to be safe, inexpensive, painless, reliable, and effective in monitoring and diagnosing vascular diseases ,5,6.Bioimpedance technology has been used in many studies that aimed to monitor changes in blood flow in the lower extremities under unstable conditions, and quantitative results were obtained using this method . The venOne of the very common diseases in adults that affects the quality of life is varicose veins in the lower extremities. Empirically, local massage has been proven to be very effective in treating the symptoms of this disease and relieving pain .In a methodical and structured manner, a specialized therapist performs with the hands a series of manual manipulations during the massage aimed at mobilizing the soft tissues. The activation of the blood circulation resulting from the massage is temporary and has a role in relieving pain or comforting the patient for a short period .Considering that the patient actually feels comfortable after the massage, as has been proven by experimental studies, in this work we ask whether it is possible to measure the effectiveness of this process.In order to regulate blood flow to living tissues, soft tissues, and organs, peripheral vascular resistance is regulated by vascular tone .For assessment of the type of vascular tone using IPG signals, the ratio of the amplitudes of the diastolic and systolic waves is studied. A small amplitude of the diastolic wave relative to the systolic (or direct) wave corresponds to a hypertonic tone. If the amplitude of the diastolic wave approaches half the amplitude of the direct wave, then the vascular tone can be considered normotonic.Points C, I, and D express the blood volume change for all vessels for a specific part of the body. This means that changes in the amplitude and time of these points are related to changes in blood volume ,13. TherIt has been shown that IPG is sensitive to changes in blood volume in arterial branches up to the sixth branch. In contrast, PWV is primarily used to assess vascular tone in large or major arteries.Therefore, in our study, we used both IPG and PWV to investigate the evaluation of vascular tone changes in all blood vessels in the studied segment. By combining these two techniques, we were able to assess vascular tone in both small arterial branches and large arteries, providing a more comprehensive understanding of vascular function in the studied extremities ,7,14,15.Among markers of arterial disease, arterial stiffness has emerged as an important parameter in estimating cardiovascular risk. Of the various methods used to assess arterial stiffness, the carotid-to-femoral pulse wave velocity (PWV) is relatively easy to determine, has been shown to be reliable, and is the most important. As a matter of fact, it has emerged as the gold standard method as there is a wealth of evidence to support it. It has an association with cardiovascular disease occurring in different populations, and is independent of traditional risk factors . The useThe main objective of this work is to investigate and verify methods for monitoring vascular tone assessment in lower extremities using bioimpedance. In addition, we are interested in monitoring the local femoral pulse wave velocity changes measured through two channels of bioimpedance.In this study, monitoring was performed using electrical bioimpedance with a multichannel device. This method allows for the monitoring of blood volume changes in the studied section and for several cardiac cycles, in addition to the possibility of obtaining information about the phase of breathing without the use of additional equipment. By using appropriate electrode configurations and recording the signals before and after leg massage, we determined the resulting differences in the signals and analyzed them.2), with a genetic predisposition in the family to the occurrence of varicose veins. For four years, the subject had annual examinations to detect varicose veins. The measurements were made after confirming the varicose vein in the right leg in the early stages, and the second measurement was made after a five-minute local massage of the right leg.The subject was a female nonsmoker, 27 years old, BMI = 23.1 , with a minimal contribution from reactance (the imaginary component). This is important when studying changes in blood volume using bioimpedance, as the value of bioimpedance should mainly reflect changes in resistance. Using a frequency of 100 kHz has been shown to allow for the determination of up to 90% of the resistance changes associated with a change in blood volume, while minimizing the influence of reactance on the measurement ,21. As aCh1—transthoracic channel;Ch2—right thigh (RLU);Ch3—right leg (RL);Ch4—lower part of the right leg (RLD).Each electrode was connected to the contact point of the limb symmetrically from two sides. Thus, eight electrodes per channel were required. CERACARTA Italy, Top Trace ECG Electrodes (50 mm) with Ag/AgCl sensors were used for the experiments. In addition to the ECG signal, the signals were recorded from three major segments for each leg; the following channels were for the right leg:The same was true for the left leg.Measurement points for these signals are shown in For the first step, the subject was lying down and fully relaxed. Recording of the bioimpedance signals began after 20 min of complete relaxation. The signals were recorded for 3 min with free breathing without interruptions. After that, a local massage was performed on the right leg by pressing in an annular manner from the bottom of the foot to the head of the thigh, massaging the muscle areas for a period of 5 min, in order to push the blood stuck in the veins upwards and stimulate blood circulation. Then signals were recorded for 2 min. The experimental stages are presented in First of all, the signals had to be smoothed. Many methods can be used for smoothing the bioimpedance signal ; in thisECG processing and R-peak detection were performed using the Heartpy library based on .In order to determine the cycles that follow inhalation and exhalation, the transthoracic bioimpedance signal was used ,25. At lThe legend of the signal colors is presented in When a pressure wave passes through a blood vessel, it expands, and this expansion is expressed by vascular tone . To idenAC, AI, and AD are the amplitudes of the systolic wave peak, incisura, and diastolic wave peak, respectively. The vascular tone type can be determined based on the DKI–DCI phase plane. This tone has three main types: normotonic, hypotonic, and hypertonic . DetaileE is Young’s modulus in the circumferential direction, h is the wall thickness.Pulse wave velocity (PWV) is an important parameter in the analysis of arterial tree behavior and a significant marker of cardiovascular risk. It is considered the gold standard for assessment of arterial elastance and is directly related to arterial stiffness (as described in Equation (3)). Measuring PWV is relatively simple, but results can vary depending on the method used. While the desired value can be obtained from either the transit time between two points of the flow or diameter wave  or the pressure wave over a given distance, it is technically easier to measure pressure than flow or diameter. Most previous studies have only considered the propagation velocity of the invasively measured pressure wave ,30.(3)PPWV, and found an average speed for 20 s of about 7.7 m/s. The measurement was made using traditional methods, where the velocity of the pulse transmission between two points is calculated with a known distance between them.Kusche et al.  used a mIn Kusche’s study, the time between the two peaks of the diastolic wave was used  is the closest to the theory, considering that it expresses the beginning of the change in the volume of blood and thus the beginning of the change in pressure, while the rest of the points are affected by pressure and other factors. For example, we studied the PWV in the arteries, while the bioimpedance signal expresses changes in blood volume not only in the arteries, but in the studied section as a whole, including veins and capillaries as well. Therefore, even horizontal and small electrode combinations will not lead to an accurate calculation of the velocity because, in any case, this impedance will pass through the capillaries and be affected by them. However, with regard to the use of time (PTTf-f), it was not taken into account in this study because in real signals, this point is unstable and the error rate will be very high.PTT on PWV is given by Equation (4) [l is the distance between two measurement points (see The dependency of tion (4) :(4)PWV=lints see .The recording information shows that the subject was in a state of complete rest during the first recording before the massage and the second after the massage, as the heart rate and breathing rate did not change significantly during the experiment and were in the normal range.To monitor vascular tone changes, we used two methods:First, all the recording cycles are shown where they are located within  points, and we could determine the type of vascular tone during the entire recording. Showing points for all cardiac cycles can confuse the display of information due to the large number of cycles, so we show only the points that represent the center of gravity of the points  for each phase of respiration.Variations in vascular tone for each segment are shown before massage (1) and after massage (2), for the right leg (massaged) in We notice from As for the thigh, the type of vascular tone in general is located in the normotonic area, but on both limbs it is very close in the first recording (before the massage) of the hypertonic area. This indicates the presence of several heart cycles where the vascular tone is within the hypertonic area and, therefore, we used the second method to display changes in vascular tone in the thigh area over time (or successive cardiac cycles).In The figures show no pattern of vascular tone changes in either thigh prior to massage. The tone moves from hypertonic to normotonic and then back without a specific rhythm. However, it can be clearly observed that the number of cycles in which the vascular tone is hypertonic in the pre-massage stage is much smaller than after the massage, but in a varying proportion, as the transition to normotonic appears in the massaged thigh.Many of the cycles following the hypertonic before the massage occurred during the end of the exhalation (black color); we noticed the absence of cycles that followed the end of the exhalation in the recording after the massage.PWV and for the studied subject it is equal to The velocity of the pulse wave measured in this study is the velocity of the wave in the thigh region. The distance between the first measuring electrode in the thigh and the first measuring electrode in the lower leg is the dimension used to calculate the 0 cm see . The velBefore the massage, the average speed was 9.2 m/s in the left leg and 8 m/s in the right leg. The measured velocity decreased slightly in both legs after the massage, to 8.4 m/s in the left leg and 7.4 m/s in the right leg. The results show a decrease in the range of the left leg (massaged) after the massage: before the massage it had the ratio [The results of changes in the type of vascular tone before and after the massage show the approach of the type of vascular tone after massage to the normotonic area in all studied segments, even if the points were completely located in the hypertonic region (whole leg and lower leg), which means an increase in the wave amplitude in the diastolic stage to the amplitude of the systolic wave.Studying the temporal changes in the type of vascular tone not only gives information about the general changes in the type of vascular tone, but also allows us to know the extent to which the type of vascular tone has improved by studying the number of cycles in which the vascular tone was hypertonic before and after the massage. As shown in The results of this study are consistent with , in whicThe transmission of the change in the type of vascular tone to the opposite end gives an indication that this transmission is due to the neural regulation of the vascular tone , but thePWV in this study are close to the values of PWV from previous studies [PWV between the recordings before and after the massage indicates a decrease in the arterial tension in the studied area. These results are consistent with the nature of the experiment, as massage leads to an increase in the effectiveness of blood circulation and temporarily relieves vascular tension. Therefore, the use of this technique enables us to evaluate the nature of the procedures used in the temporary or permanent treatment of vascular diseases associated with pulse wave velocity.The results of  studies ,35. The The methods used in this study depend on the amplitude of the recorded signals for each cardiac cycle, but do not study changes in the overall signal shape. That is, the crest time or width of the systole or diastole wave is not taken into account, and monitoring these indicators requires independent studies.PWV using PTTf-f, but it remains to achieve this frequency for 32 combined channels as the device is designed to be technically difficult.The capabilities of the device used, especially for the sampling time with 500 Hz, do not allow us to obtain very accurate results regarding the pulse wave velocity. If a similar device is used with a sampling time equal to 1 kHz or more, the results will be more accurate and we can calculate the This study relates to a special case only and its results cannot be generalized in terms of diagnosis. Rather, it can be considered confirmation of the possibility of using the bioimpedance monitoring technique to study local changes in circulatory activity and hemodynamics.Based on the results of the current study, our subsequent studies will focus on testing the technique on a larger number of healthy subjects and patients, with the aim of obtaining a criterion that differentiates normal changes in the type of vascular tone from pathological changes.PWV during the different phases of breathing in healthy subjects.Future studies will focus on the changes in the type of vascular tone and the The changes in the type of vascular tone were transmitted to the other limb (non-massaged), but, due to the nature of the experiment, we could not determine the time at which this transmission began, so a study must be carried out that records the signals during massage in one limb only and monitors the changes in vascular tone in the opposite limb. This may explain the nature of the transmission caused by different kinds of vascular tone regulation.In this work, an evaluation of the information that can be obtained by tracking specific vascular tone changes using bioimpedance signals was made. The study was applied to a subject who was diagnosed with varicose veins in the lower extremities in the early stages. The signals from the lower extremities were recorded before and after applying a local massage to one of the extremities for five minutes. The obtained results confirm that the use of bioimpedance signals can provide quantitative and qualitative information about the amount of improvement in blood circulation and the type of vascular tone before and after applying a massage. We recommend using this method for monitoring in the event of applying other types of treatment, whether physical or pharmacological.


Hypercalcemic hyperparathyroidism has been associated with poor outcomes after kidney transplantation (KTx). However, the clinical implications of normocalcemic hyperparathyroidism after KTx are unclear. This retrospective cohort study attempted to identify these implications.Normocalcemic recipients who underwent KTx between 2000 and 2016 without a history of parathyroidectomy were included in the study. Those who lost their graft within 1 year posttransplant were excluded. Normocalcemia was defined as total serum calcium levels of 8.5–10.5 mg/dL, while hyperparathyroidism was defined as when intact parathyroid hormone levels exceeded 80 pg/mL. The patients were divided into two groups based on the presence of hyperparathyroidism 1 year after KTx. The primary outcome was the risk of graft loss.P < 0.001). Cox hazard analysis revealed that normocalcemic hyperparathyroidism was an independent risk factor for graft loss .Among the 892 consecutive patients, 493 did not have hyperparathyroidism (HPT-free group), and 399 had normocalcemic hyperparathyroidism (NC-HPT group). Ninety-five patients lost their grafts. Death-censored graft survival after KTx was significantly lower in the NC-HPT group than in the HPT-free group (96.7% vs. 99.6% after 5 years, respectively, Normocalcemic hyperparathyroidism 1 year after KTx was an independent risk factor for death-censored graft loss. Early intervention of elevated parathyroid hormone levels may lead to better graft outcomes, even without overt hypercalcemia.The online version contains supplementary material available at 10.1186/s12882-022-02840-5. Secondary hyperparathyroidism is a frequent complication of chronic kidney disease (CKD) and increases the risk of mortality and various other complications . AlthougConsecutive patients who underwent KTx between January 2000 and December 2016 at the Japanese Red Cross Nagoya Daini Hospital  were included in the study. Data were collected on December 31, 2020. Our patient-exclusion criteria were as follows: those for whom data were lacking; those who had hypercalcemia or hypocalcemia within 1 year of KTx; those who were undergoing treatment with calcimimetics; those who had lost their kidney graft within 1 year of KTx; or those who were under 16 years of age at KTx. Furthermore, those who underwent parathyroidectomy (PTx) were also excluded because their PTH values were affected by the autografted parathyroid function. Hypercalcemia was defined as total serum Ca levels > 10.5 mg/dL. Hypocalcemia was defined as total serum Ca levels < 8.5 mg/dL. Normocalcemic hyperparathyroidism was defined as intact PTH levels of > 80 pg/mL without hypo/hypercalcemia 1 year after KTx. Intact PTH level has been reported to be ≤80 pg/mL in over 98% of healthy individuals irrespective of vitamin D status in both PTH assays used in this study .Patients who met the inclusion criteria were divided into two groups based on the presence (or absence) of hyperparathyroidism: the HPT-free group, comprising patients without hyperparathyroidism and the NC-HPT group, comprising patients with normocalcemic hyperparathyroidism 1 year after successful KTx. Each patients’ sex, age, body mass index (BMI), dialysis vintage, number of HLA mismatches, positivity of donor-specific HLA antibody (DSA), laboratory data, mean blood pressure (MBP) 1 year after KTx, and graft survival were documented. The primary outcome was the risk of death-censored graft loss. Blood sample analyses were performed on all patients every month for 1 year following transplantation and every second month thereafter. The values of serum Ca and intact PTH obtained from blood samples 1 year after KTx were used for patient enrollment and classification. This study is reported according to Strengthening The Reporting of Observational studies in Epidemiology guidelines.www.srl-group.co.jp, reference range [10–65 pg/mL]), or an enzyme immunoassay . When serum albumin values were < 4.0 g/dL, all serum Ca values were corrected with serum albumin values as follows [Serum Ca and phosphorus (P) levels were measured using standard methods. Intact PTH levels were measured using second-generation immunoassays: an electrochemical luminescence immunoassay , mycophenolic acid, mizoribine, everolimus, and glucocorticoids. Basiliximab was used as induction therapy. Furthermore, rituximab administration or splenectomy was used as induction therapy in anti-donor antibody-positive patients with KTx, except in those with low antibody titers.P < 0.05.Pearson’s chi-squared test was used to analyze nominal variables, and the Mann–Whitney U test was used for continuous variables. All results are presented as median (interquartile range) because of their non-normal distribution, confirmed by the Kolmogorov–Smirnov test. Spearman’s rank correlation coefficient was used to evaluate the correlations among the variables . KaplanP = 0.591). There was a weak correlation between intact PTH and age, dialysis vintage, BMI, serum Ca, serum P, and eGFR, with an absolute correlation coefficient of less than 0.2 . Of the 892 patients, 493 were assigned to the HPT-free group, and 399 were assigned to the NC-HPT group Fig. . PatientP = 0.022) despite the shorter follow-up period in the NC-HPT group  (Table P < 0.001)   , BMI , diabetes mellitus , DSA positivity , serum P 1 year post-KTx , eGFR 1 year post-KTx , and normocalcemic hyperparathyroidism    and > 100 pg/mL  (Fig. The univariate Cox proportional hazards revealed that the independent risk factors for death-censored graft loss were donor age (The multivariate analyses in this study demonstrated that BMI, DSA, serum P, and eGFR 1 year post-KTx were risk factors for graft loss, which is consistent with previous reports , 21, 22.Since GFR reduction is a direct cause of PTH elevation, it is difficult to determine whether high PTH after KTx is mainly due to low GFR or to parathyroid gland hyperplasia . In addiThe optimal management of normocalcemic hyperparathyroidism in kidney transplant patients is controversial due to a lack of consensus. However, aggressive treatment for elevated PTH after KTx may improve clinical outcomes, as several reports have demonstrated that therapeutic intervention for normocalcemic hyperparathyroidism in non-CKD patients can reduce complication risks, including bone lesions , 46, nepAlthough the “Kidney Disease: Improving Global Outcomes” guidelines recommend active vitamin D and bisphosphonates as medical treatments for hyperparathyroidism in the first year after KTx, these treatments are often difficult to implement because of concerns about hypercalcemia, insufficient kidney function, and/or low bone turnover . TherefoThis study has several limitations, most of which are due to its retrospective nature, its confinement to a single center, the inherent possibility of unmeasured confounders, selection bias, and absence of data regarding endogenic vitamin D levels, FGF23, other bone biomarkers, and renal lithiasis. Hence, further studies with larger sample sizes are necessary to validate the findings of this study.Hyperparathyroidism 1 year after KTx was found to be an independent risk factor for death-censored graft loss, even without overt hypercalcemia. Early management and intervention of elevated PTH levels may contribute to better graft outcomes after kidney transplantation.Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.


Endoscopic procedures are rarely performed in children with congenital heart disease (CHD); therefore, the associated complications are unknown. We report an abrupt change in circulatory and respiratory condition during endoscopic injection sclerotherapy for esophageal varices.A 9-year-old boy with a history of total anomalous pulmonary venous connection (TAPVC) repair and Fontan procedure for asplenia and a single ventricle with TAPVC underwent endoscopic injection sclerotherapy under general anesthesia for esophageal varices. Systolic blood pressure decreased from 70 to 50 mmHg following a sclerosant injection; a second injection reduced his peripheral oxygen saturation from 93 to 79% secondary to ventilation difficulty. Although we suspected anaphylaxis intraoperatively, postoperative imaging suggested that balloon dilation performed to prevent sclerosing agent leakage caused compression of the pulmonary venous chamber and trachea owing to the anomalous intrathoracic organ anatomy.Thorough understanding of the complex anatomy is important before performing endoscopic procedures in children with CHD to preoperatively anticipate possible intraoperative complications and select the optimal therapeutic approach and anesthesia management. In recent years, surgical outcomes for patients with congenital heart disease (CHD) have improved, and non-cardiac surgery for postoperative children with CHD have become popular. However, endoscopic therapy is rare for children with CHD, and it is unknown what complication would be happened during procedure. We report a rare case who had severe hypotension and respiratory failure during endoscopic injection sclerotherapy (EIS) for esophageal varices (EV) in a patient with CHD.A 9-year-old boy  with asplenia, dextrocardia, single atrium, single ventricle, common atrioventricular valve, and total anomalous pulmonary venous connection (TAPVC) had undergone a TAPVC repair and a Fontan surgery with fenestration by 3-year-old, in addition, multiple coil embolizations for major aortopulmonary collateral arteries. He had developed protein losing enteropathy and plastic bronchitis after Fontan surgery. Furthermore, he experienced endoscopic esophageal variceal ligation (EVL) for EV twice at 7-year-old without any adverse events.This time, he was rushed to the emergency room at another hospital for massive hematemesis. His condition was stabilized by conservative treatment such as a blood transfusion, and he was transferred to our hospital. An emergency gastroscopy revealed moniliform moderate varices with redness extending from the gastric cardia to the upper esophagus; however, there was no active bleeding. Subsequent cardiac angiography revealed the central venous pressure of 14 mmHg, and there was no indication for a catheterization procedure. Thus, EIS under general anesthesia was scheduled for the treatment of EV.2) was 96% at 1 L/min of oxygen through a nasal cannula. Preoperative electrocardiogram showed sinus rhythm, and chest X-ray indicated a cardiothoracic ratio of 44.5%. Transthoracic echocardiography revealed ejection fraction of 56% with mild common atrioventricular valve regurgitation, and blood tests showed mildly decreased hemoglobin (12.5 g/dL) and elevated hepatobiliary enzymes .His preoperative blood pressure (BP) was 84/46 mmHg, heart rate (HR) was 91 beat per minute, and peripheral oxygen saturation  0.47. He was monitored via electrocardiogram, SpO2, continuous arterial BP, and capnometry.After the induction of general anesthesia with midazolam 3 mg, fentanyl 50 μg, remifentanil 0.3 μg /kg/min, and rocuronium 20 mg, he was intubated, and we maintained general anesthesia with sevoflurane 1.5–2% and remifentanil 0.05–0.15 μg/kg/min. The ventilator mode was set to pressure-controlled ventilation, with driving pressure of 10–13 cmHWe used an OLYMPUS GIF TYPE Q260J endoscope with a diameter of 9.9 mm and 5% ethanolamine oleate with iopamidol as the sclerosing agent (SA) for EIS. We attached an EV balloon  near the tip of the endoscope and dilated the balloon with 20 ml of air just before injecting the SA to prevent it from leaking out. The SA was injected into the varicose vein with a 25G puncture needle under X-ray fluoroscopy.2 decreased from 93 to 79%, end-tidal CO2 (EtCO2) increased from 36 to 51 mmHg, and HR increased from 88 to 98/min , followed by a percutaneous fenestration re-creation on the 26th POD, and he was transferred to another hospital for rehabilitation on the 55th POD.Regarding gastroesophageal varices after CHD surgery, Kiesewetter et al. reported that 7 of 12 patients in the distant stage after Fontan surgery had congestive cirrhosis, and 4 of them had gastroesophageal varices , there aWe compared the preoperative CT images, intraoperative fluoroscopic images, and esophageal balloon size to find the cause of hypotension and ventilatory failure in this case. The position of the puncture needle and balloon during injection of the SA is supposed to be as shown in Fig. 2 and an increase in EtCO2, and direct stimulation of the trachea caused an increase in BP and HR (Fig. We assessed that cardiac output was dropped by balloon compressing of the common pulmonary venous cavity during the first injection, which resulted in decreased BP Fig. A. In theThere have been no reports of these same phenomena occurring in children without CHD. We thought that the reason was the anatomical difference between this patient and normal children without anatomic deformities. In this patient, the common pulmonary venous cavity after TAPVC repair was shorter in anteroposterior diameter than the left atrium of normal children Fig. A. TherefIO2 in advance. If we increase the inspiratory force against a decrease in VT due to airway obstruction, the high intrathoracic pressure by check valve would induce a BP decrease, especially in Fontan circulation. Therefore, if airway obstruction is predicted, it may be better to only prevent a significant SpO2 drop by raising FIO2 in advance and tolerate temporary hypoventilation for a while, which may reduce the risk of secondary complications. However, in cases with pulmonary hypertension, it may be critical due to increased pulmonary vascular resistance by hypoventilation-induced hypercapnia, so we think that it is necessary to discuss the treatment strategy with the surgeon.In order to properly respond to such complications of respiratory and circulatory changes, the following points should be mentioned. First, it is important to understand the complex anatomy preoperatively and anticipate possible complications. In addition, it is necessary to monitor an invasive arterial BP, and good communication should be maintained with the surgeon to understand the timing and duration of balloon inflation, since lack of information about balloon manipulation during this procedure was one of the factors that prevented us from identifying the causes. They would permit to take countermeasures against hypotension as in pattern of Fig. In conclusion, understanding each anatomy in CHD and anticipating possible intraoperative complications preoperatively may allow for appropriate treatment selection and anesthetic management.


Molecular therapies, including anti-IgE, biologicals and small molecules are increasingly used for treatment of asthma. The effectiveness of these therapies may be increased with biomarkers. Aim of this study was to assess the value of measuring cumulative IgE levels specific for respiratory allergens to increase the efficacy of anti-IgE therapy for severe bronchial asthma. One hundred and thirty seven patients with severe asthma were recruited from 2016 to 2022. Standard empirical allergy diagnosis , blood eosinophil counting, measurement of total IgE and of cumulative IgE-specific for respiratory allergens by Phadiatop™ were performed. Thirty four patients with severe allergic asthma, for whom all three diagnostic methods were performed, were then used to analyze the efficacy of anti-IgE treatment in patients stratified in two groups according to cumulative IgE levels specific for respiratory allergens determined by Phadiatop™. Group #1 patients (n = 8) had cumulative specific IgE values ≥ 0.35 and < 1.53 PAU/l while in group #2 patients (n = 26) they were ≥ 1.53 PAU/l. Treatment with Omalizumab was performed for at least 12 months. The level of asthma control (ACT questionnaire), the number of asthma exacerbations, the quality of life (AQLQ questionnaire), the need for systemic corticosteroids, and the respiratory function (FEV1) was determined by “before-after” analysis for each group, followed by a comparison of the dynamics between groups. In group 2 patients with an initial allergen-specific IgE level ≥ 1.53 kUA/L, the efficacy of Omalizumab treatment was better regarding asthma control, number of exacerbations, and quality of life than in group 1 patients. Our study provides evidence that measuring cumulative levels of IgE specific for respiratory allergens could be a useful screening method for detecting an allergic phenotype of severe asthma and may serve as biomarker to enhance the success of IgE-targeted therapy. Skin tests were assessed retrospectively if patients had earlier positive skin test reactions specific for allergen sources  with positive (histamine) and negative  controls .Dermatophagoides pteronyssinus D1, Dermatophagoides farinae D2), epithelium of a cat (Cat Dander-Epithelium E1), house dust (Greer H1), mold (Penicillum notatum M1), birch pollen (Birch T3), a mixture of grass allergens  GP1), mugwort pollen (Artemisia vulgaris W6). A result was considered positive when specific IgE levels were ≥ 0.35 kUA/l. Phadiatop™ was performed using the ImmunoCAP technique . Phadiatop™ level ≥ 0.35 PAU/l was reported as positive result, and < 0.35 PAU/l as negative result.Determination of total IgE was performed using a chemiluminescent method . Total IgE was considered as elevated at a level ≥ 100 IU/ml. Specific IgE was determined by the ImmunoCAP method   to dog dandruff allergens (Dog dander e5), house dust mite allergens . Patients with mixed bronchial asthma (n = 4) were not included in the analysis. Patients with the diagnosis of severe allergic asthma were selected for the treatment with Omalizumab  for at least 12 months according to the instructions of the manufacturer, taking into account the patient’s body weight and the level of total IgE (the corresponding amount of mg of the drug 1 or 2 times a month). Each patient had evaluation visits before starting the treatment with Omalizumab (baseline), at 4 months of therapy, and at 12 months of therapy. At each of these visits, patients completed the ACT (Asthma Control Test), AQLQ  questionnaires, spirometry (=respiratory function) was performed, and data on exacerbations were collected. Spirometry was done according to ATS/ERS recommendations 2005 and GINA guidelines . Exacerbhttps://www.ncss.com/). Quantitative variables were assessed for normality using the Shapiro-Wilk test or the Kolmogorov-Smirnov test. Quantitative variables following a normal distribution were described using mean (M) and standard deviation (SD), 95% confidence interval (95% CI) for the mean were estimated. Quantitative variables following the non-normal distribution were described using median (Me) and lower and upper quartiles (Q1 – Q3). Student’s t-test or Mann-Whitney-U test were used for comparisons between two groups following the normal or non-normal distribution of the data, respectively. Comparisons of three or more groups were done using the Kruskal-Wallis test and Dunn’s criterion with Holm correction as a post-hoc method. P-value under the set up value of α = 0.05 (two-sided) was considered as statistically significant. ROC analysis was used to assess the diagnostic performance of quantitative variables in predicting a categorical outcome. The optimal cut-off value was estimated using the Youden’s index. Comparison of frequencies in the analysis of 2 by 2 contingency tables was performed using Pearson’s chi-square test , and Fisher’s exact test . One-way repeated measures analysis of variance was used to compare three or more matched samples in regard to normally distributed quantitative variables. Statistical significance of dependent variable changes over time was assessed using the Pillai’s Trace and paired Student’s t-test with Holm correction as post hoc methods. When comparing three or more matched samples regarding to non-normally distributed quantitative variables Friedman test was used along with ConoverIman test with Holm correction as a post hoc method.Statistical analysis was performed using StatTech v. 2.1.0 , NCSS Statistical Software 2021  treated with Omalizumab for at least 12 months for whom all three diagnostic methods had been performed. The patients were divided into two groups. Group #1 (n = 8) included patients with cumulative IgE levels to respiratory allergens ≥ 0.35 and < 1.53 PAU/l while group #2 (n = 26) included patients with specific IgE levels ≥ 1.53 PAU/l. The efficacy of Omalizumab treatment was assessed by the level of asthma control (ACT questionnaire), the number of asthma exacerbations within one year before and one year after the initiation of therapy, the quality of life (AQLQ questionnaire), the need for systemic corticosteroids, and the respiratory function (FEV1). Patients were examined before the start of therapy, and after 4 and 12 months of therapy. “Before-after” analysis was performed for each group, followed by a comparison of the dynamics between groups.The analysis of Omalizumab therapy efficacy in group #1 (Phadiatop™ test < 1.53 PAU/l) and group #2 (Phadiatop™ test ≥ 1.53 PAU/l) revealed a significant decrease in the proportion of uncontrolled bronchial asthma in both groups   to 17 (Q1-Q3: 13-20) points in group #1 and from 10 (Q1-Q3: 8 -12) to 19 (Q1-Q3: 17-22) points in group #2   in group #2 while in group #1 there was no significant reduction in the number of asthma exacerbations per year per patient of Omalizumab therapy (p = 0.277)  to 59.1% (95% CI 56.0 – 62.8) in month 12 (p = 0.311). Group #2 also had an increase from 69% (95% CI 48.0 – 77.1) at baseline to 78% (95% CI 56.0 – 94.0) after 12 months (p = 0.0233). Regarding FEV1 there were no statistically significant differences between the groups (data not shown).Molecular testing using defined recombinant allergen molecules has revolutionized the understanding and diagnosis of allergy , 53. Serpost-hoc analysis of poly- versus monosensitized subjects did not provide unequivocal results regarding sensitization status  and the result of skin tests (p = 0.1), which has been also reported by other researchers , 27, 31.et al., 2018  . Due to the low sensitivity and high specificity, if the result is less than 1.53 PAU/l in the patient it can be predicted that the patient will not have clinically relevant sensitization. The cut-off level found by us is higher than that of Zeng c 97.5%)  which isc 97.5%) . AccordiComparison of the efficacy parameters of the Phadiatop™ test when used as isolated biomarker with the collection of allergic anamnesis, skin tests and the determination of specific IgE does not allow us to conclude that the Phadiatop™ test has a clear advantage for asthma phenotyping. However, the characteristics of the combination of the Phadiatop™ test with allergic anamnesis were superior to both allergic anamnesis and standard methods separate and in combination. Thus, Phadiatop™, in combination with the collection of an allergic anamnesis, can be used instead of cumber-some standard skin testing and hypothesis-driven determination of specific IgE.Indeed, response prediction to Omalizumab is challenging. This is true also in the setting of chronic spontaneous urticaria, where low total IgE is a strong predictor of poor response , 65, butIt has been suggested that typing of asthma according to underlying trigger factors may be useful for guiding newly emerging forms of molecular and personalized treatment of patients suffering from asthma. The limitations of our study are associated with the retrospective analysis and the absence of a control group comprisig patients with non-allergic severe asthma treated with Omalizumab. Furthermore, we were able to analyze only a relatively small number of patients with severe asthma for whom all diagnostic methods were performed and who were only treated with Omalizumab. However, it must be acknowledged that severe asthma patients account only for approximately 5% to 10% of patients with asthma, which makes it very difficult to find them in real clinical practice . NeverthThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the Ural State Medical University. The patients/participants provided their written informed consent to participate in this study.Conceptualization, VN, EB, DK, AK and RV. Methodology, VN, DK. Formal analysis, VN, EB, DK, and RV. Investigation, VN, EB, DK, and RV. Resources, VN, EB, DK, and RV. Data curation, VN, EB, DK, and RV. Writing — original draft preparation, VN, EB, DK. Writing — review and editing, KN, PE, and RV. Visualization, VN, EB, DK, KN. All authors have read and agreed to the published version of the manuscript.RV is a recipient of a Megagrant of the Government of the Russian Federation, grant number 14.W03.31.0024. This work was partially supported by the “Danube Allergy Research Cluster” Program of the Lower Austria (330950005).RV has received research grants from HVD Life-Sciences, Vienna, Austria, and WORG Pharmaceuticals, Hangzhou, China and from Viravaxx AG, Vienna, Austria. Furthermore, he serves as consultant for WORG and Viravaxx AG.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Objective: Renal cell carcinoma (RCC) is the most common malignancy of the kidney. However, there is no reliable biomarker with high sensitivity and specificity for diagnosis and differential diagnosis. This study aims to analyze serum metabolite profile of patients with RCC and screen for potential diagnostic biomarkers.Methods: Forty-five healthy controls (HC), 40 patients with benign kidney tumor (BKT) and 46 patients with RCC were enrolled in this study. Serum metabolites were detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and then subjected to multivariate statistical analysis, metabolic pathway analysis and diagnostic performance evaluation.Results: The changes of glycerophospholipid metabolism, phosphatidylinositol signaling system, glycerolipid metabolism, d-glutamine and d-glutamate metabolism, galactose metabolism, and folate biosynthesis were observed in RCC group. Two hundred and forty differential metabolites were screened between RCC and HC groups, and 64 differential metabolites were screened between RCC and BKT groups. Among them, 4 differential metabolites, including 3-β-D-Galactosyl-sn-glycerol, 7,8-Dihydroneopterin, lysophosphatidylcholine (LPC) 19:2, and γ-Aminobutyryl-lysine (an amino acid metabolite), were of high clinical value not only in the diagnosis of RCC , but also in the differential diagnosis of benign and malignant kidney tumors .Conclusion: The occurrence of RCC may involve changes in multiple metabolic pathways. The 3-β-D-Galactosyl-sn-glycerol, 7,8-Dihydroneopterin, LPC 19:2 and γ-Aminobutyryl-lysine may be potential biomarkers for the diagnosis or differential diagnosis of RCC. Kidney carcinoma is one of the most common malignancies of the urinary system, and renal cell carcinoma (RCC) accounted for more than 85% of kidney carcinoma . AccordiThe commonly used kidney function indicators in clinic, such as urea, creatinine (Cr), uric acid (UA), cystatin C (CysC), complement C1q (C1q), neutrophil gelatinase-associated lipocalin (NGAL), and estimated glomerular filtration rate (eGFR), can evaluate the kidney function of patients with RCC, but cannot indicate kidney tumors. To date, few reliable tumor-specific markers are available for clinical use in kidney carcinoma. Although kidney biopsy is the "gold standard” for the diagnosis and identification of kidney tumors, its clinical application is often limited because of its invasive nature and the possible risk of needle tract implantation and metastasis.in vivo  age ≥18 years; 2) All patients were diagnosed as RCC or BKT by pathological biopsy after finding abnormalities in computed tomography (CT) or magnetic resonance imaging (MRI) examination. Exclusion criteria: 1) patients with failure of blood sample collection; 2) women in pregnancy or lactation; 3) patients complicated with other metabolic diseases and tumors; 4) patients with RCC who underwent treatment with radiotherapy or immunotherapy before enrollment; 5) patients who had undergone nephrectomy. In addition, all diagnoses were confirmed by senior clinicians according to clinical diagnostic criteria.Forty-five healthy subjects who underwent physical examination during the same period were enrolled as healthy controls (HC). Inclusion criteria: 1) age≥18 years; 2) healthy volunteers with normal examination indexes during the same period; 3) subjects who did not take drugs that may affect kidney function within the last month. Exclusion criteria: 1) patients with failure of blood sample collection; 2) women in pregnancy or lactation.Detailed clinical information of the subjects is shown in g for 10 min, the serum was collected and equally divided into two parts. One part was tested for kidney function and lipid indicators within 2 h. The other part was stored at −80°C for metabolomics analysis.After fasting overnight, all study subjects were subjected to blood collection (approximately 5.0 ml of venous blood each) from 8:00–10:00 a.m. The SSTTM II Advance vacuum blood collection tube  containing separation gel and coagulant was used. After centrifugation at 3,000 The indicators for kidney function and lipid indicators were measured on LST008 automatic biochemical analyzer . The C1q kit was provided by Shanghai Beika Biochemical Reagent Co., Ltd. . The kits for other indicators were provided by Sichuan Maker Biotechnology Co., Ltd. .g for 15 min at 4°C to obtain the supernatant. The supernatant was filtered by 0.22 µm microporous membrane. Finally, 3 µl of the filtrate-solution was transferred by an autosampler and injected into the UPLC-MS/MS system for metabolomic analysis. In addition, 10 µl of serum from each sample was mixed as a quality control (QC) sample. QC samples were processed in the same way as the study samples. Serum metabolomics analysis was performed with Agilent® 1290 Infinity II UPLC system  and AB Sciex® Triple TOF 5600+ mass spectrometer system . UPLC-MS/MS analytical conditions used previous methods of our lab  and 800 µl of methanol-acetonitrile (v/v = 1:1) solution was sonicated at 4°C for 10 min, then the mixture was incubated at –20°C for 1 h, followed by centrifugation at 13,000  our lab . In addi our lab .http://www.5omics.com) provided by Dalian Dashuo Information Technology Co. . 2) quality analysis of the data based on the stability of the QC samples. The percentage of relative standard deviation (RSD) of metabolic mass spectrometry features in QC samples that were less than 50% should exceed 80%. Calibration of QC was performed using the statTarget analysis (http://metlin.scripps.edu/), Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.kegg.jp/kegg/pathway.html), LipidMaps (https://www.lipidmaps.org/), Human Metabolome Database (HMDB) (https://hmdb.ca/), MassBank (https://massbank.eu/), and PubChem Database (https://pubchem.ncbi.nlm.nih.gov/), parameters setting: Δm/z (MS1)≤0.01000Da; Δm/z (MS2)≤0.05000Da; MS2 Score Method = Forward; Reference Noise of Unknown MS2 to remove = 1.000000; Reference Noie of Standard MS2 to remove = 200.000000; the number of near fragments at least to merge peaks cluster = 2; investigate the maximum number of fragments = 2. According to the formal definition of metabolite annotation and identification specified by the Chemical Analysis Working Group of the Metabolomics Standards Initiative (MSI), the metabolites determined in this study would be considered as putative identification (levels 2) (http://www.genome.jp/kegg) and MetaboAnalyst database exploration .Raw data were collected using Analyst TF 1.7 software . The metabolomics data were subjected to a series of processing workflow, including peak picking, quality assessment, missing value imputation, normalization, transformation and scaling. The details were as follows: 1) the XCMS algorithm was applied to peak extraction based on the One-MAP platform  the evels 2) . Untargep < 0.05 indicated that the difference was statistically significant.Statistical analysis was performed using SPSS 26.0 software . Normally distributed data were expressed as mean ± standard deviation, and compared with one-way ANOVA followed by LSD test. Non-normally distributed data were expressed as median (interquartile range), and analyzed with Kruskal–Wallis H test. Count data were compared using chi-square test. Spearman correlation was used for correlation analysis. PCA and PLS-DA were performed using SIMCA 14.1 software . The validity of the PLS-DA model was examined using a random permutation test (100 times). Receiver operating characteristic curve (ROC) analysis was used to evaluate the diagnostic performance of the differential metabolites. When AUC = 0.50–0.59, 0.60–0.69, 0.70–0.79, 0.80–0.89, or ≥0.90, it means that the diagnostic performance is fail, poor, fair, good, or excellent, respectively . p < 0.0p < 0.05). Pairwise comparison analysis showed that compared with the HC group, NGAL , Cr , CysC , TG , LDL-C  and Apo-B  were significantly increased in the RCC group, whereas eGFR  was significantly decreased in the RCC group. NGAL , TG , LDL-C  and Apo-B  were significantly increased in the BKT group than in the HC group. Compared with the BKT group, Cr  and CysC  were significantly increased in the RCC group, while eGFR  was significantly decreased in the RCC group. These results showed that kidney function and lipid indicators were abnormal in patients with RCC to different degrees.As shown in The 131 serum samples and 14 QC samples were analyzed by UPLC-MS/MS in positive ion (ESI+) and negative ion (ESI-) modes. The results were analyzed by PCA analysis. The PCA score plots, total ion chromatogram (TIC) and base peak intensity (BPI) diagram of QC samples indicated that the metabolomics dataset of this study had good stability and reproducibility , while tp < 0.05 , CysC  and NCAL , while positively correlated with eGFR . 7,8-Dihydroneopterin was negatively correlated with Cr , CysC  and NGAL , whereas positively correlated with eGFR . LPC 19:2 had negative correlation with urea , Cr , CysC  and TG (r = -0.183 p = 0.040), but positively correlation with eGFR . Although the correlation coefficient is weak , either directly or indirectly by conversion to glutamic acid, but also a carbon source for amino acid and fatty acid synthesis in cancer cells . AdditioIn this study, 3-β-D-Galactosyl-sn-glycerol, γ-Aminobutyryl-lysine and 7,8-Dihydroneopterin were identified to be possible potential biomarkers to distinguish patients with RCC from patients with BKT and healthy subjects. The chosen kidney function and lipid indicators for comparative analysis in this study are commonly used in clinical diagnosis of kidney function damage or to judge whether the blood lipid level is normal. They are intermediate or end products of substance metabolism in the body. Because of their high level in blood, they can be accurately detected by conventional chemical analysis methods. In contrast, the new biomarkers screened in this study were obtained by untargeted metabolomics technology, which can only perform qualitative and semi-quantitative analysis on the analyzed substances . However, several potential biomarkers for diagnosis or differential diagnosis of RCC, which were not previously noticed, were found through the analysis of serum samples of subjects using untargeted metabolomics technology in this study. Many studies have also confirmed that untargeted metabolomics can comprehensively and systematically analyze small molecular substances in biological samples , and it p-values. Therefore, these compounds may also be worthy of attention and further research.In this study, serum levels of 6-Keto-prostaglandin F1α, 17α,21-Dihydroxypregnenolone and γ-Glutamylphenylalanine in RCC patients were found to be elevated which may be potential biomarkers to distinguish RCC patients from healthy individuals. However, the diagnostic performances of these three biomarkers for distinguishing RCC and BKT were not good . 6-Keto-prostaglandin F1α belongs to the prostaglandin and related compounds. Prostaglandin is unsaturated fatty acids produced by the cyclooxygenase-catalyzed arachidonic acid metabolism and is widely present in various vital tissues and body fluids in humans. It is reported that cyclooxygenase-2 overexpression in RCC may be associated with metastasis of tumor cells, tumor invasion and angiogenesis . Therefod-glutamine and d-glutamate metabolism pathway. Although these substances were discovered by screening, the final results only focused on 4 biomarkers, indicating that the changes of 1-2 compounds may not affect the changes of the whole metabolic pathway. In addition, the changes in their quantity (peak intensity and area of MS) of these substances are not large enough among the different study groups. Therefore, they were not finally screened as differential biomarkers.In the screening process of biomarkers, it was found that some compounds involved two or more metabolic pathways, such as phosphatidic acid (PA) 23:2 and phosphatidylinositol (PI) 18:4, and were associated with glycerophospholipid, phosphatidylinositol and glycerolipid pathways. Since these biomarkers are provided by database comparison, are they the intersections of metabolic pathways or subset of other pathways? Unclear. Fortunately, these substances have little significance for this study, so they are only cared about very little. Although these metabolites are not as important as those screened in this study, they actually involve some metabolic pathways. Perhaps these metabolites could be related to a certain disease in a particular way, but they cannot be observed in this study. Similarly, in the process of pathway analysis, it is found that some metabolic pathways only involve the changes of 1-2 markers, for example, only PA 23:2 and PI 18:4 were hit to the phosphatidylinositol metabolism pathway and only glutamic acid was hit to the Based on the above theories and research findings, we propose a hypothesis: there are some changes in metabolic pathways of substances in the body of RCC patients, which at least include lipid metabolism, amino acid metabolism, galactose metabolism and folate biosynthesis. Studying the signal pathways involved in these metabolic pathways may find a new way to explore the pathogenesis of RCC. This study only provides a new way for screening biomarkers for diagnosis and differential diagnosis of RCC. In the follow-up study, we will verify and evaluate the diagnostic performance of these potential biomarkers of RCC found in this study through targeted metabolomic analysis technology based on the research results, so as to explore the possibility of these biomarkers in clinical practice.d-glutamine and d-glutamate metabolism pathway, and the glycerolipid metabolism pathway in patients with RCC. These results suggest that the occurrence of RCC may be associated with changes in lipid metabolism, amino acid metabolism, galactose metabolism, and folate biosynthesis. These four metabolites may become markers for the diagnosis or differential diagnosis of RCC. However, this study is limited in the small sample size and the lack of targeted metabolomics validation. Therefore, follow-up studies with larger sample size and validation with other omics such as genomics and proteomics are warranted to clarify the metabolic mechanism of RCC and to further identify tumor markers for clinical application.In summary, this study analyzed serum metabolic profiles of healthy subjects, patients with BKT and patients with RCC using UPLC-MS/MS. Four potential biomarkers for the diagnosis of RCC were screened by multivariate statistical analysis and ROC analysis, namely LPC 19:2 involved in the glycerophospholipid metabolism pathway, 3-β-D-Galactosyl-sn-glycerol involved in the galactose metabolism, 7,8-Dihydroneopterin involved in the folate biosynthesis and γ-Aminobutyryl-lysine, an amino acid metabolite. In addition to the above metabolic pathways, there may also be changes in the phosphatidylinositol signaling system, the The original contributions presented in the study are included in the article/


Chronic kidney disease (CKD) affects about 10–15% of the global population, according to different estimates, and is a leading cause of morbidity and mortality worldwide [In this issue of the Journal, Schultz and colleagues report on the discrepancy between nephrology referral criteria suggested by different guidelines and real-life practice . Their sWhile one major issue, the evident need to align guidelines, is a topic that is beyond the scope of this editorial, considering several of the other points that emerge from the study could stimulate constructive debate.2 or in the presence of significant proteinuria, sustained unexplained microhematuria, progression of CKD, potassium alterations, a family history of kidney disease, recurrent nephrolithiasis, with a quality of evidence graded “moderate” [2 becomes a two-faced Janus: it is a clear indication that a patient must be referred to a nephrologist, but it is also an incongruent threshold in the attempt to halt, or even only slow disease progression, at least according to the recent KDOQI guidelines on nutritional management in kidney diseases [First of all is the question concerning what role guidelines play in regard to nephrology referral. Let us consider the KDIGO guidelines on CKD, now a decade old, according to which referral to a nephrologist is advised when the glomerular filtration rate (GFR) has fallen below 30 ml/min/1.72 mdiseases . The firTaking advantage of this new reimbursement system, our group analyzed the need for nephrology care and proposed a simple way to estimate it in keeping with the local situation . While tThese guidelines hold that all CKD patients, regardless of stage, should undergo regular assessment of nutritional status, and that starting in stage 3, in the absence of contraindications and without age restrictions, they should be managed with a low-protein diet to slow the progression of kidney disease and improve uremic symptoms and metabolic control; this intervention  is associated with better survival and lower mortality . The intFurthermore, how can we be sure that earlier CKD stages should be denied referral to a specialist?CKD has a heterogeneous progression towards end-stage kidney disease (ESKD) which depends on primary disease, concomitant risk factors and comorbidities, sex and age . MoreoveConsidering the relationship between renal function and histological correlates, due to the large kidney function reserve, a rise in serum creatinine only becomes evident when more than 50% of the nephron mass has been lost. Thus, if we have a chance to modify the natural course of CKD, it is most likely that this will be in its initial stages, with targeted interventions when kidney tissue is still relatively preserved.Finally, there is a huge difference in clinical practice between high- and low-healthcare resource countries, and universal guidelines developed in high-income settings may not be consistently applied worldwide but rather adapted to the local situation. A study from the United Kingdom showed that implementing eGFR for specialist referral led to a 265% increase in referrals in the first months since the program started but, later, it decreased to a steady rate of 40%, questioning the role of overwhelmed nephrology units . GeograpFor the reasons set forth above, ideally all patients with kidney alterations should be seen by a specialist at the first sign of disease, at least in a first phase, to determine whether to continue nephrology follow-up. Difficulties in predicting who will progress and who will not can explain the low rate of discharge of patients observed in Schultz’s study and can be the reason for the retention of patients in nephrology clinics: a prudent attitude of common sense in the light of our patients’ interest. The need to find a compromise between clinical needs and medical desertification, within healthcare systems already stressed by increasing pressure, should not influence guideline recommendations if we want to prevent ESKD.In conclusion, concerning referral of CKD patients to a nephrologist, rather than focusing on why physicians deviate from guidelines, should we not reconsider the guidelines, so that they do more to take patients’ needs into account?