Although placement of an intra-cerebral catheter remains the gold standard method for measuring intracranial pressure (ICP), several non-invasive techniques can provide useful estimates. The aim of this study was to compare the accuracy of four non-invasive methods to assess intracranial hypertension.We reviewed prospectively collected data on adult intensive care unit (ICU) patients with traumatic brain injury (TBI), subarachnoid hemorrhage (SAH), or intracerebral hemorrhage (ICH) in whom invasive ICP monitoring had been initiated and estimates had been simultaneously collected from the following non-invasive indices: optic nerve sheath diameter (ONSD), pulsatility index (PI), estimated ICP (eICP) using transcranial Doppler, and the neurological pupil index (NPI) measured using automated pupillometry. Intracranial hypertension was defined as an invasively measured ICP > 20 mmHg.r = 0.54; PI, r = 0.50; eICP, r = 0.61; NPI, r = − 0.41—p < 0.001 for all). The area under the curve (AUC) to estimate intracranial hypertension was 0.78 [CIs = 0.68–0.88] for ONSD, 0.85 [95% CIs 0.77–0.93] for PI, 0.86 [95% CIs 0.77–0.93] for eICP, and 0.71 [95% CIs 0.60–0.82] for NPI. When the various techniques were combined, the highest AUC (0.91 [0.84–0.97]) was obtained with the combination of ONSD with eICP.We studied 100 patients  with a median age of 52 years. The median invasively measured ICP was 17 [12–25] mmHg and intracranial hypertension was present in 37 patients. Median values from the non-invasive techniques were ONSD 5.2 [4.8–5.8] mm, PI 1.1 [0.9–1.4], eICP 21 [14–29] mmHg, and NPI 4.2 [3.8–4.6]. There was a significant correlation between all the non-invasive techniques and invasive ICP (ONSD, Non-invasive techniques are correlated with ICP and have an acceptable accuracy to estimate intracranial hypertension. The multimodal combination of ONSD and eICP may increase the accuracy to estimate the occurrence of intracranial hypertension. Intracranial hypertension is a common and severe complication after acute brain injury , 2. AlthAt present, the gold standard for ICP assessment is the placement of invasive devices, including external ventricular drains, which also enable drainage of the cerebrospinal fluid, or intraparenchymal micro-transducers . The lacA non-invasive, accurate, and safe tool to assess ICP would therefore be useful in this context. Various methods have been proposed, with different advantages and limitations, although none of them has been shown to be sufficiently accurate to replace invasive ICP measurement –13. TranThe aim of this study was therefore to compare the accuracy of different non-invasive methods to estimate ICP and to estimate the occurrence of intracranial hypertension in a heterogeneous cohort of brain-injured patients. The following non-invasive methods were used: (a) ONSD measurement using ultrasound, (b) PI and the estimated ICP (eICP)  using TCThis study was performed according to the STROBE reporting guidelines for observational studies.All consecutive adult (> 18 years) patients admitted to the Department of Intensive Care of the Hôpital Erasme (Université Libre de Bruxelles) over a period of 20 months (January 2017 to September 2018) were eligible if (a) they underwent invasive ICP monitoring for risk of intracranial hypertension; (b) they had a TBI, subarachnoid hemorrhage (SAH), or intracerebral hemorrhage (ICH); and (c) they had a “stable ICP” value (see below). Patients with other reasons for ICP monitoring, with known pupillary abnormalities , multiple sclerosis, ocular surgery, or severe peri-orbital edema limiting pupillary assessment were excluded. Patients without a temporal window for TCD assessment and without an arterial catheter for continuous blood pressure measurement were also excluded.The decision to insert invasive ICP monitoring was made by the attending ICU physician in collaboration with the neurosurgeons. The decision was based on the latest version of the Brain Trauma Foundation guidelines  for TBI Within the first 72 h after ICP insertion, the same experienced operator (FST) performed TCD, ONSD, and automated pupillometry  to help to better understand brain hemodynamics and compliance during ICP monitoring. In some patients with uncontrolled intracranial hypertension, the same tests were performed to evaluate the compromise of cerebral hemodynamics. These tests are all routinely used to monitor brain-injured patients in the ICU. The measurements were recorded bilaterally as, although an asymmetry between the right and left side of each variable may exist, ICP is considered as the result of the transmission of the pressure in the whole cranial cavity and the average of bilateral measurements could mirror more precisely brain pathophysiology, as previously suggested . These d2 monitoring, and pulse oximetry (SpO2). The local Ethics Committee approved the study (P2019/308), but waived informed consent.Invasive ICP data were collected simultaneously and, if variability exceeded this value, the non-invasive results were excluded. Intracranial hypertension was defined as ICP > 20 mmHg. EVD was closed during the measurement for correct ICP measurement. Systemic hemodynamic monitoring consisted of invasive arterial blood pressure from the radial artery, continuous electrocardiography, end-tidal COQuantitative automated pupillary light reactivity was measured in both eyes using the NeurOptics NPi-200  pupillometer, which uses a calibrated light stimulation of fixed intensity (1000 Lux) and duration (3.2 s) enabling rapid and precise measurement (0.05 mm limit) of pupil size, quantitative pupillary light reflex (PLR), constriction velocity, and latency . Using aONSD was assessed using a 7.5-MHz linear ultrasound probe  and the lowest acoustic power able to measure the ONSD. With the patient in the supine position and with head elevated to 30°, the probe was oriented perpendicularly in the vertical plane and at around 30° in the horizontal plane on the closed eyelids of both eyes without exerting pressure. Measurements were made in the axial and sagittal planes on the ultrasound images, 3 mm behind the retina in both eyes using an electronic caliper, and the final ONSD value was calculated by averaging four measured values as previously described . AbnormaTCD was performed by the same investigator using the temporal window on both sides and an echo-color Doppler device with a 2-MHz transducer . The TCD measurements were performed bilaterally on the middle cerebral artery (MCA); the mean flow velocity in the MCA was recorded 1 cm after the internal carotid artery bifurcation, and PI was automatically calculated using the formula: PI = [(systolic velocity − diastolic velocity)/mean velocity]. The final flow velocities were calculated by averaging the two measured values. PI was considered abnormal if > 1.2. eICP and estimated cerebral perfusion pressure (eCPP) were calculated using a validated formula , which iAll data were collected in an ICU patient data monitoring system . Demographics, main comorbidities, and clinical characteristics on admission and on the day of ICP assessment were collected. Use of drugs that might interfere with pupillary constriction  and of neuromuscular blocking agents (NMBAs) was also noted. The initial severity of the brain injury was assessed using the Marshall score , the Fisr) was considered as “strong” , “moderate” , “weak” (0.25–0.50) or “poor” . The agreement between ICP and eICP was assessed using the Bland-Altman method; the mean bias and their limits of agreement (LoA) (as 2.2 times SD of the bias) were computed. The discriminative ability of NPI, ONSD, and PI to identify intracranial hypertension was evaluated using receiver operating characteristic (ROC) curves with the corresponding area under the curve (AUC) and related sensitivity and specificity. The estimated AUCs with their 95% CI for the combinations of the non-invasive tools were constructed using the logistic regression model with intracranial hypertension as the dependent variable and the combined non-invasive tools as the independent variables. Youden’s index was computed for each non-invasive method to identify the cut-off value with the best sensitivity and specificity to estimate intracranial hypertension. The comparison of each ROC curve was performed using the non-parametric DeLong method. A p < 0.05 was considered as statistically significant. Data were analyzed using IBM® SPSS® Statistics software, version 22 for Windows .Data are expressed as mean ± standard deviation, median [interquartile range] or count (percentage), as appropriate. For continuous variables, normality assumption checking was performed by inspection of residual and normal plots. Differences between groups  were assessed using a Fisher’s exact test for categorical variables and a Wilcoxon rank test for continuous variables and one-way ANOVA for group comparisons. Each ICP value was correlated with PI (mean of both sides), NPI (mean of both sides), and ONSD (mean of both sides). Correlation between continuous variables was evaluated using the Spearman correlation, with 95% confidence intervals, as appropriate. The Spearman index of correlation (n = 7), hydrocephalus (n = 6), or monitoring after at risk surgery for brain tumors (n = 11) or infection of ventriculo-peritoneal shunt (n = 11). Of 140 eligible patients, we excluded 40 patients . Thus, 100 patients were included in the analysis, with TBI (n = 30), SAH (n = 47), or ICH (n = 23). The median Glasgow Coma Score on admission was 8 [5–12] and ICP assessment was performed 2  days after ICU admission. Other characteristics of the study cohort are shown in Table A total of 195 patients underwent invasive ICP monitoring during the study period; 20 patients were younger than 18 years and 35 were excluded because ICP monitoring was inserted for ischemic stroke , PI , and ONSD  and weak for NPI , PI (p < 0.001), and eICP (p < 0.001) were significantly higher than that of NPI. ONSD > 5.3 mm had 70% sensitivity and 75% specificity to assess intracranial hypertension; PI > 0.97 had 81% sensitivity and 78% specificity; NPI < 4.1 had 65% sensitivity and 70% specificity; and an eICP > 20 mmHg had 73% sensitivity and 84% specificity. The AUCs for different combinations of the non-invasive tools to evaluate intracranial hypertension are shown in Table Median invasive ICP values were 17 12–25] mmHg and intracranial hypertension was present in 37 patients. Median values from the different non-invasive techniques were ONSD 5.2 [4.8–5.8] mm, PI 1.1 [0.9–1.4], eICP 21 [14–29] mmHg, and NPI 4.2 [3.8–4.6]. The number of patients with abnormal ONSD, PI, eCIP, and NPI is reported in the –25 mmHg The main differences among TBI, SAH, and ICH patients are shown in Table p = 0.005), PI (p = 0.004), and eICP (p < 0.001) to estimate intracranial hypertension in TBI patients were significantly higher than the AUC for NPI. Similarly, the AUCs for ONSD (p = 0.001), PI (p < 0.001), and eICP (p < 0.001) to assess intracranial hypertension in SAH and ICH patients were significantly higher than the AUC for NPI.There were no significant differences in ICP, ONSD, PI, eICP, or NPI among the three groups Table . Correlap = 0.002), NPI (p = 0.02), and eICP (p < 0.001); however, the correlation was weak for all . PI was significantly correlated with NPI (p = 0.006) and eICP (p < 0.001); however, the correlation was strong for eICP (r = 0.79) and moderate for NPI (r = − 0.27). eICP was significantly correlated with NPI (p = 0.003); the correlation was moderate (r = − 0.29).ONSD was significantly correlated with PI  will provide additional data to solve this conundrum. Our results suggest a relevant role for such an approach to assess ICP; among the studied non-invasive indices, eICP had the highest correlation and accuracy with invasive ICP in the total population and in the different subpopulations of brain injury.Increasing values of ICP cause specific changes in the waveform analysis of TCD, with the diastolic flow velocity being primarily affected. Czosnyka et al. proposed a formula based on the diastolic flow velocity changes for non-invasive assessment of CPP (and therefore ICP), which showed an estimated error between estimated and measured CPP less than 10 mmHg in 71% of the examinations, and in 84% of the examinations, the error was less than 15 mmHg . This foThe ultrasonographic measurement of ONSD is gaining popularity for non-invasive ICP estimation and has been investigated in different clinical scenarios showing a good correlation with invasive ICP values and good inter- and intra-observer variability , 42. TheRecently, NPI has been suggested as a valuable technique for early recognition of increased ICP . The autTaken together, important disagreement exists in the literature on the role of non-invasive ICP methods. The observed conflicting results may be related to several factors including the heterogeneity of brain injuries included in the different studies, different methodologies, different expertise of the operators, and inter-observer variability. In our cohort, results from subgroups of different types of brain injury suggest that each method has different accuracy depending on the type of brain injury. For example, we found that PI had good correlation with ICP in TBI and ICH and a weak correlation in SAH; NPI had a good correlation with ICP in SAH, but a weak correlation in TBI; by contrast, ONSD and eICP had good accuracy in all three types of brain injury. The discrepancies across different types of brain injury may be related to the pathophysiological effect of ICP on brain function in the different conditions, and potentially to different critical ICP thresholds to worsen brain damage in different conditions. In particular, as each of the non-invasive methods relies on different pathophysiological mechanisms underlying intracranial hypertension , it is plausible that only their combination would improve the estimation of altered brain compliance and increased ICP. This hypothesis is in agreement with a recent study  comparin2, imaging, body temperature, or cumulative doses of sedatives, were difficult to assess, but can potentially have an important effect, especially on TCD-derived measurements. Also, timing from ICU admission to assessment was variable between patients. Third, the vast majority of measurements were obtained in patients with relatively well-controlled ICP, and we cannot extrapolate these findings with highly variable ICP values. Fourth, all measurements were performed by the same operator, which can increase the reliability of the observations, but also prevents assessment of interobserver variability. Fifth, averaging measurement from both sides, as for ONSD, could be questionable and one may argue that ONSD of a specific side  would be more adequate. However, most studies also used average data on both cerebral sides and comparison of the two approaches  have not been adequately addressed in other reports.This study has several limitations. First, the number of patients included was relatively small for each subgroup of patients, and only one measurement per patient was obtained; more measurements for each patient over time would have strengthened our findings and permitted to assess also estimation of ICP changes. Moreover, some confounding factors, such as the effects of PaCOIn this study, non-invasive methods to estimate ICP based on TCD, pupillometry, or optic nerve ultrasonography were significantly correlated with invasive ICP values, although the correlation was moderate to weak. The best accuracy was found for eICP method. The multimodal combination of such indices may increase the ability to identify intracranial hypertension.Additional file 1 Supplemental Table 1. Number of abnormal values for each monitoring tool, according to the different forms of brain injury. Supplemental Figure 1. Correlations and Bland-Altman graphs for ICP and estimated ICP (eCIP). The continuous line shows the mean difference (bias) and the dotted lines show the limits of agreement (2.2 ± SD of the bias). Supplemental Figure 2. Correlations and Bland-Altman graphs for ICP and estimated ICP (eCIP). The continuous line shows the mean difference (bias) and the dotted lines show the limits of agreement (2.2 ± SD of the bias). The mean bias between ICP and eICP were: − 1.37 mmHg (LoA − 21.78 to 19.05 mmHg) for TBI; − 0.68 mmHg (LoA − 19.67 to 18.30 mmHg) for SAH; − 3.91 mmHg (LoA − 21.36 to 13.54 mmHg) for ICH.


The aim of this study was to evaluate renal disease progression in ESRD and CKD according to DR severity in patients with type 2 diabetes. (2) Methods: We included 1329 patients and divided the cohort into two end-points. The first was to trace the incidence of ESRD in all enrolled participants and the other was to follow their progression to CKD. (3) Results: Significantly higher crude hazard ratios (HRs) of ESRD incidence in all enrolled participants were noted, and this ratio increased in a stepwise fashion. However, after adjustment, DR severity was not associated with ESRD events. Therefore, a subgroup of 841 patients without CKD was enrolled to track their progression to CKD. Compared with no diabetic retinopathy, the progression of CKD increased in a stepwise fashion, from mild nonproliferative diabetic retinopathy (NPDR) to moderate NPDR, to severe NPDR and to proliferative diabetic retinopathy (PDR), both in the crude and adjusted models. (4) Conclusions: The severity of retinopathy appeared to be associated with renal lesions and the development of CKD. Our findings suggest that the severity of DR is a risk factor for progression to CKD. Therefore, diabetic retinopathy is useful for prognosticating the clinical course of diabetic kidney disease. The major microvascular complications of diabetes mellitus (DM) include retinopathy, nephropathy, and neuropathy. Diabetic retinopathy progresses stepwise from milder stages of nonproliferative diabetic retinopathy (NPDR) to more advanced vision-threatening levels, including proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME). Diabetic retinopathy (DR) and diabetic nephropathy (DN) share common risk factors, such as poor glycemic control and systolic hypertension. Both have similar pathological abnormalities within the glomerular and retinal vessels ,2,3.Longitudinal studies have investigated whether the presence of retinopathy is associated with chronic kidney disease (CKD) progression among all patients with type 2 diabetes and chronic kidney disease ,5,6,7. HWe postulated that DR and DN share similar mechanisms, resulting in the concurrent progression of both complications. The aim of this study was to evaluate the renal disease progression (ESRD and CKD) in patients with type 2 diabetes and with/without diabetic kidney disease according to DR severity .The Tri-Service General Hospital, Taipei, Taiwan, provided their research data from 12 October 2012 to 11 September 2018. Research ethics approval was given by the institutional review board to collect data without individual consent (IRB NO 1-107-05-047). Any patients with diabetes and more than one fundus color photograph session were included in this study. The start of follow-up was defined as the first fundus color photograph session. There were 5974 potential cases included in this study, but we excluded the cases without type 2 diabetes. The definition of diabetes was one of the following conditions: (1) At least two International Classification of Diseases (ICDs) of type 2 diabetes (ICD9: 250 or ICD10: E11) from half a year ago to the start time and received oral hypoglycemic agents or insulin treatments, (2) at least 2 records of more than or equal to 126 mg/dL of fasting glucose from half a year ago to the start time, and/or (3) at least 2 records of more than or equal to 6.5% of HbA1C from half a year ago to the start time. Moreover, patients were excluded if they had one of the following conditions: (1) At least one ICD of end-stage renal disease (ESRD), which is ICD9:585.6 or ICD10:N18.6, (2) lack of serum creatinine measurement within 3 days of the start time, and/or (3) at least 2 records of serum creatinine measurements after the start time. Finally, there were 1329 patients included in this study . We diviDR was defined according to the following retinal microvascular lesions: Microaneurysms, hard exudates, intraretinal hemorrhages, venous beading, or prominent intraretinal microvascular abnormality, retinal or optic disk neovascularization, vitreous hemorrhage, or preretinal hemorrhage. DR was graded on the International Clinical Diabetic Retinopathy Disease Severity Scale  as follo2 and the presence of any kidney damage; stage 2, eGFR = 60–89 mL/min/1.73 m2 with any kidney damage; stage 3, eGFR = 30–59 mL/min/1.73 m2; stage 4, eGFR = 15–29 mL/min/1.73 m2; stage 5, eGFR < 15 mL/min/1.73 m2 [The stages of CKD were determined according to the Kidney Disease Outcomes Quality Initiative guidelines using the eGFR as calculated by the Chronic Kidney Disease Epidemiology Collaboration equation . The var/1.73 m2 .2 was defined as progression to CKD.We collected other laboratory records within 3 days: Total cholesterol, low-density lipoprotein (LDL), High-density lipoprotein (HDL), triglyceride, alanine aminotransferase (ALT), creatinine, hemoglobin, and white blood cell (WBC) and platelet counts. The missing rate of the above variables in this study was less than 30%. We used the multiple imputations method to impute the missing values. The other demographic characteristics and comorbidities were collected from electronic health records. The basic characteristics included sex, age, height, weight, systolic blood pressure (SBP), and diastolic blood pressure (DBP). The definition of comorbidities was based on the corresponding International Classification of Disease Ninth Revision and Tenth Revision. The comorbidities of hypertension, ischemic heart disease, stroke, and diabetic neuropathy were included in this study. An ESKD event was defined as the initiation of any hemodialysis, peritoneal dialysis, renal transplantation, or death from uremia. An eGFR decline to less than 60 mL/min/1.73 m1 = 0.9 and β2 = 0.999) and a batch size of 32 for optimization; (2) learning rate was set at 0.001; and (3) a weight decay of 10−4 [The evaluations for grading diabetic retinopathy were conducted by the artificial algorithm that we developed previously and based on convolutional neural network. The fundus color photograph session were provided by the Kaggle coding website , which c of 10−4 . The 100The final DR severity was confirmed by ophthalmologist and was determined by the result of the more severely affected eye.p < 0.05 throughout the analysis. The statistical analysis was carried out using the software environment R version 3.4.3.We presented the characteristics as the means and standard deviations, numbers of patients, or percentages, where appropriate. They were compared using either analysis of variance or chi-square tests, as appropriate. We used a significance level of The primary analysis was to evaluate the effects of different grades and the progression of diabetic retinopathy. We used Kaplan–Meier curves to present the progression difference between participants with HbA1c and each initial grade. All variables were evaluated for their association with diabetic retinopathy progression using univariate Cox proportional hazard models. A multivariable Cox proportional hazard model was used to adjust the potential confounding factors, and the selection of the adjusted variables was based on the significance of the univariate analysis.The secondary analyses were performed by stratified analysis. We only presented the analysis of the most significant type of initial grade of diabetic retinopathy progression. Because the initial conditions of different patients were variable in real clinical practice, we tried to use the initial grade of diabetic retinopathy and the baseline HbA1c as the stratified variables. The interaction analysis was based on a Cox proportional hazard model, and the adjusted variables were the same as the primary analysis.A total of 1329 patients were included in this study. We divided them into two end-points. The first was for the incidence of ESRD in all enrolled participants. The other was for people without CKD at the time of enrollment, 841 people in total, followed for their progression to CKD. A significantly higher crude HR of ESRD incidence in all enrolled participants was noted. The disease progressed in a stepwise fashion, from mild NPDR to moderate NPDR, to severe NPDR, to PDR. The crude HR of progression to ESRD events were 2.35 (1.01–5.43) in mild NPDR, 5.96 (2.99–11.89) in moderate NPDR, 2.79 (1.16–6.75) in severe NPDR, and 6.98 (2.22–21.94) in PDR compared to the non-DR group. However, after adjustment , DR severity was not associated with ESRD events. 2. However, only four had proteinuria. Therefore, 841 patients without CKD were enrolled for an evaluation of their progression to CKD. A total of 845 patients’ eGFR was greater than 60 mL/min/1.73 mCompared with no diabetic retinopathy, the HR for the progression of CKD increased in a stepwise fashion, from mild NPDR to moderate NPDR, to severe NPDR, to PDR, both in the crude and adjusted model . The crude HRs of progression to CKD events were 3.46 (95% CI 0.92 to 12.98) for patients with mild NPDR, 8.75 (95% CI 2.72 to 27.92) for patients with moderate NPDR, 5.73 (95% CI 1.63 to 20.04) for patients with severe NPDR, and 14.21 (95% CI 1.55 to 130.67) for patients with PDR. The adjusted HRs of progression to CKD events were 3.38 (95% CI 0.90 to 12.71) for patients with mild NPDR, 7.88 (95% CI 2.43 to 25.57) for patients with moderate NPDR, 5.08 (95% CI 1.35 to 19.04) for patients with severe NPDR, and 11.43 (95% CI 1.22 to 107.12) for patients with PDR. p < 0.001; p < 0.001). The Kaplan–Meier survival curve in each subgroup was showed in As shown in In patients with diabetes and without CKD initially, we found the progression of CKD proceeded in a stepwise fashion, from mild NPDR to moderate NPDR, to severe NPDR, to PDR, both in the crude and adjusted model . This finding suggested that the DR severity is an independent factor related to renal functional decline in non-CKD patients with diabetes.Previous studies demonstrated that the presence of DR is associated with an increased risk of end-stage renal disease , albuminThis study revealed DR severity might be associated with a faster renal functional decline. PDR was associated with DN progression. NPDR was associated with DN progression in some but not all studies. In some studies, the renal outcome  was significantly higher among subjects with either PDR or severe NPDR relative to mild NPDR ,19,20. TThus, in this Taiwan population-based study, we evaluated the risk for ESKD and CKD progression according to the severity of diabetic retinopathy from mild NPDR to moderate NPDR, to severe NPDR, to PDR. In our study, we found DR severity was independently associated with progression to CKD. The ESRD events among patients with diabetes were significantly associated with DR severity in a stepwise fashion. However, the association was not maintained in the adjusted model. Our study cohort included all CKD stages. A previous study found that DR is significantly correlated with CKD progression in later CKD stages . This maThere were significant differences in DR severity, age, uric acid, albumin and more hypertension events than in other studies ,23,24. POur study had a higher prevalence of NPDR but less PDR compared to previous studies  or angiotensin receptor blockers (ARBs), dietary protein restriction, and lipid-lowering therapy contribute to diminishing the risk of diabetic nephropathy ,26,27. CThe strengths of our study are the use of a longitudinal design rather than a cross-sectional design, including all CKD stages, and good validation of the grading of diabetic retinopathy. Our study has several limitations. First, the data in this cohort study were collected retrospectively from a single center. Sampling bias and selection bias is inevitable. The results could not be used to establish a cause-effect relationship. However, patients who were newly diagnosed with type 2 DM were routinely transferred to receive a fundus examination in our hospital. This study was sponsored by the national health insurance, and nearly 99.9% of the Taiwanese population is under National Health Insurance coverage . Second,In conclusion, DR severity was a prognostic factor for progression to CKD in patients with type 2 DM. Therefore, clinicians must evaluate the DR severity at the first visit. DR severity may be a powerful tool in predicting the clinical course of diabetic kidney disease in patients with type 2 diabetes. Routine follow-up and management of retinal disorders in all patients with type 2 diabetes would be important. Early initiation of renoprotective therapy in patients with DR may also be a future direction in the management of these patients.


Results: NGAL was significantly higher in patients with high total PV versus patients with low total PV. The NGAL amplitude for the prediction of high total PV was significantly higher when compared with hs-CRP. A high predictive value could also be observed for patients without established CVD. Conclusion: NGAL seems to be a promising biomarker for the identification of asymptomatic patients with atherosclerotic disease.Background/Objectives: Inflammation represents a cornerstone in the development of atherosclerosis and early detection is essential to avoid cardiovascular events. Biomarkers like interleukin-1 beta, interleukin-6, or high sensitivity CRP (hs-CRP) have been investigated intensively in this field. Since they have several limitations, additional biomarkers are needed for cardiovascular risk stratification. The acute phase protein, neutrophil gelatinase-associated lipocalin (NGAL), modulates inflammation and is elevated in cardiovascular disease (CVD). Moreover, it contributes to plaque destabilization. Methods: In this prospective, single-center study, we included 323 asymptomatic patients with at least one cardiovascular risk factor or established CVD. NGAL levels were measured in plasma samples using a commercially available ELISA. Carotid, femoral, and total atherosclerotic plaque volumes (PV) were measured using a 3D ultrasound system (Philips iU22). Patients were separated into a low ( Cardiovascular disorders represent the major cause of death in the industrialized world. Arterial hypertension, hypercholesterolemia, diabetes mellitus, and nicotine consumption have been identified as strong risk factors for the development of atherosclerotic lesions. Recent data suggest that atherosclerosis is driven by chronic vascular inflammation and that ongoing inflammation within plaques is associated with plaque vulnerability and disruption ,2. ElevaTo identify patients at high risk for cardiovascular events, the European Society of Cardiology recommends the use of risk scores . What thNeutrophil gelatinase-associated lipocalin (NGAL) is a 25 kilo Dalton (kDA) acute phase protein belonging to the lipocalin superfamily and was initially described as a biomarker for acute and chronic renal failure . These fExperimental data revealed that NGAL forms a complex with matrix metalloproteinase-9 (MMP-9), thereby prolonging the proteolytic activity of MMP-9 and contributing to plaque vulnerability. This could lead to plaque rupture and subsequently, to a cardiovascular event. In addition, vascular stability could be modified by NGAL. Animal experimental data show that mice are less prone to aneurysm formation after NGAL knockout .Recently, it has been postulated that adipose tissue is a reservoir of inflammation in obese patients. In accordance with these data, a significantly increased expression of NGAL could be detected in the visceral fatty tissue of obese patients. Congruently, a correlation of serum NGAL level with insulin resistance could be shown . These dAccording to the abovementioned characteristics of NGAL, we hypothesized that this biomarker may be altered also in patients with peripheral atherosclerotic disease and therefore, may be a valuable biomarker to guide patient management in preventive medicine.Patients were recruited from the prospective observational single-center cohort study “Correlation of Atherosclerotic Plaque Volume and Intima Media Thickness With Soluble P-selectin trial” . The baseline results of this trial have been reported [Briefly, individuals aged 30 to 85 years with established CVD (coronary artery disease (CAD), cerebrovascular disease, peripheral arterial occlusive disease (PAD) or at least one traditional cardiovascular risk factor  were included. A sonographic examination with automated measurement of the intima media thickness (IMT) with a linear L9-3 probe and quantification of the atherosclerotic plaque volume with a VL13-5 3D probe were performed using the Philips iU22 ultrasound system . A detailed description of the ultrasound examination is given below. Plaques were defined according to the Mannheim consensus . In addig and the plasma was stored at −80°. The level of NGAL was measured using a commercially available ELISA .Laboratory analysis included total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, hs-CRP, fasting glucose, hemoglobin A1c, creatinine, and estimated glomerular filtration rate (eGFR). For 342 patients, an additional EDTA blood sample was centrifuged at 1730× The study protocol has been approved by the Ethics Committee of the Medical University of Innsbruck and complies with the Declaration of Helsinki. All patients gave written informed consent before inclusion into the study. Due to missing data and withdrawn consent of some patients, 323 participants were included in the final analysis see .2. For diabetes, a fasting glucose level ≥ 126 mg/dL or the use of diabetes medication was considered relevant. Arterial hypertension was defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg and/or the use of antihypertensive therapy. Hyperlipidemia was defined as a low-density lipoprotein value ≥ 160 mg/dL and/or triglycerides ≥ 150 mg/dL and/or use of lipid lowering drugs.Family history of CVD was defined according to currently practiced recommendations as the occurrence of a premature cardiovascular event  in a first-degree relative . The eGFR was calculated using the Modification of Diet in Renal Disease (MDRD) formula and chronic kidney disease (CKD) was defined as estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 mEach participant underwent a routine sonographic examination measuring the flow velocity of the common carotid artery, the internal carotid artery, the external carotid artery, and the vertebrate artery, as well as the common femoral artery, the proximal superficial femoral artery, and the deep femoral artery. In addition, we performed measurements of IMT according to the recommendations of the Mannheim Consensus in the far wall of the distal carotid artery, as well as the proximal superficial femoral artery 1 cm distal to the flow divider along a 10 mm segment free of plaques . For IMTTo assess method reliability, interobserver variability of 3 different observers was calculated and showed a very good agreement between the raters with an intraclass correlation coefficient of 0.95 .To test for normal distribution, the Kolmogorov–Smirnov test was used. The baseline data for continuous variables are shown as mean ± standard deviation (SD) for normally distributed parameters or as median and corresponding interquartile range (IQR) for parameters not following a normal distribution. Categorical variables are displayed as absolute numbers and percentages. Subjects were analyzed according to total PV and separated into two groups: <75th percentile  and ≥75th percentile . A two-tailed, independent samples t-test or the Mann–Whitney U test were used to assess differences between continuous variables, as appropriate. The χ2 test was used to evaluate variations in categorical variables. Total PV was calculated adding the sum of carotid and femoral PV. To assess the linear correlations for selected variables, Pearson or Spearman test were used as appropriate.To calculate the predictive value of NGAL for high total PV, receiver operating characteristics (ROC) were performed to determine the area under the curve (AUC) and the optimal cut-off values with the highest sensitivity and specificity . AUC valp < 0.05 in univariate analysis were considered for multivariate regression.Univariate and multivariate linear regression were used to analyze the relationship between the dependent variables  and the predictor variables . Only variables with Interobserver variability comparing 10 vessels for plaque volume quantification was determined by an intraclass correlation coefficient (ICC).p-value of <0.05 was considered as significant. Statistical analyses were conducted with SPSS Statistic Version 24.0  and MedCalc Version 15 .For all statistical tests, a two-tailed In total, 323 patients , with a mean age of 64.2 ± 9.0 years, were included in the analysis and underwent 3D plaque volumetry of carotid and femoral arteries.Detailed demographic, clinical, and laboratory characteristics of the study population are summarized in About one-third of the study participants suffered from established CVD, but overall, the study group belonged to an intermediate risk population with a median of two cardiovascular risk factors.Hyperlipidemia (87.6%) and arterial hypertension (65.0%) were the most common CVRFs. One quarter of the study population had a family history of CVD (25.4%) and around 20% were smokers, while only a minority presented with diabetes (13%). A total of 81% of diabetics were treated with antidiabetics, with most of them receiving oral medication (83% of treated patients). The mean HbA1c of the diabetic patients was 7.2%.p < 0.001), less frequently females (p < 0.001), and suffered, not unexpectedly, more often from established CVD (p < 0.001).The study population was divided according to the plaque burden in low (<75th percentile) and high total PV (≥75th percentile). Patients with high total PV were significantly older  and lipid-lowering drugs (55.1%), with moderate and high intensity statins being taken more frequently by patients in the high PV group.p < 0.001) and femoral (p < 0.001) PV. In addition, the median pulse wave velocity, as well as mean carotid IMT and ankle brachial index, are shown.Median values for total PV, carotid PV, and femoral PV are displayed in ABI is known as strong indicator of generalized atherosclerosis and CV risk. An ABI ≤ 0.90 is associated, on average, with two to three times higher risk of total death and CV events ,23. In ap < 0.001). Interestingly, in the diabetic cohort, a better correlation was found .As shown in Moreover, patients in the high PV group showed increased pulse wave velocity compared to the low PV group. Similar to the ABI, pulse wave velocity is a marker of atherosclerosis and a predictor for cardiovascular events. Both the association with low ABI and high pulse wave velocity confirm that the patients in the high PV group represent a collective at higher cardiovascular risk.p < 0.001, p < 0.001), as well as carotid PV . Interestingly, the association between the biomarker hs-CRP and PV was not significant .Patients with high PV showed significantly higher median NGAL values compared to patients with low PV  with the optimal cut-off value of 81.18 µg/L revealed 52% sensitivity and 81% specificity in the prediction of higher PV. The AUC of hs-CRP  was significantly lower compared to the AUC of NGAL . A high predictive value for NGAL could also be observed for patients without established CVD . Conversely, the AUC of hs-CRP was not a significant predictor for high PV in these two subgroups.The area under the curve (AUC) of NGAL  than patients with lower NGAL values , NGAL , and established CVD  were the strongest predictors for total PV.In multivariate analysis, all included parameters showed a significant contribution. However, comparing regression coefficients, age (In this prospective, single-center study, we investigated the role of the acute phase protein NGAL on atherosclerosis in outpatients with at least one established CVRF or a manifestation of CVD.Recent data strengthen the hypothesis that thrombus formation and the development of atherosclerosis is fueled by immune cells through the activation of coagulation and release of chemokines . The cliIn a small study, Eilenberg and colleagues were able to detect NGAL in carotid plaques of patients undergoing carotid endarterectomy . In theiThe role of hs-CRP has been demonstrated in previous publications ,26. InteInterestingly, in our study, patients in the high PV group had lower LDL cholesterol levels. We know that NGAL levels are influenced by statins . AlthougSince Eilenberg et al. also reported an influence of metformin on NGAL levels, we also analyzed metformin intake in our study population. We did not find statistically significant different NGAL levels in diabetic patients with high or low PV in our study. Moreover, metformin intake was balanced between the low and high plaque volume group. In our study, NGAL levels were comparable between patients receiving metformin and those who were metformin-naïve . HoweverInterestingly, the high predictive value of NGAL for plaque burden was also observed for patients without established CVD, indicating that this protein might be a promising marker for atherosclerosis in patients with lower cardiovascular risk. The multivariate analysis identified age and NGAL as the strongest predictors for total PV, again, demonstrating that NGAL represents a selective biomarker for this disease.NGAL is also secreted from renal tubular cells and is a biomarker for acute kidney injury . TherefoIn some patients, we evaluated levels of IL-1beta and IL-6 . HoweverIn this publication, we present the baseline data of our study. A follow-up over five years with 3D ultrasound and NGAL measurement is ongoing. Therefore, we hope that we can provide data on the association of NGAL with plaque progression in the near future. To reliably document plaque progression, we decided to perform sonographic 3D plaque volumetry, which is a practicable and new approach for the exact determination of plaque volume in peripheral and carotid arteries ,29. ReceTo our knowledge, this is the first study demonstrating a correlation of NGAL levels with carotid and femoral plaque volume measured by 3D ultrasound. Considering the current literature and our data, we think that NGAL appears to be an interesting biomarker for risk assessment in patients with established CVD as well as in patients with subclinical atherosclerosis. The combination of classical risk scores with biomarkers like NGAL might be an innovative approach in preventive medicine.The design of our study includes several limitations. Although more than 300 patients were included, which is more than in several previous investigations, the sample size is still relatively small. Additionally, the study was conducted at one center and we do not provide a control group. To confirm ours and former findings, a multicenter study with a matched control group without manifestation of atherosclerosis would be desirable. Finally, our population belonged to a low to intermediate risk population. Therefore, at the moment the results are probably not transferable to a high risk group.


The plasma cfDNA fraction was measured using a targeted, single nucleotide polymorphism (SNP)-based assay. Pathological diagnosis was made according to the 2015 Banff Kidney Rejection Classification. The area under the ROC curve (AUC-ROC) was determined using the bootstrapping method to estimate median and 95% confidence interval (95% CI). The sensitivity, specificity and Youden index, positive predictive value (PPV), and negative predictive value (NPV) were calculated for specific cfDNA fractions.In this prospective single center observational study, we enrolled kidney allograft recipients between November, 2016 and September, 2017 at the First Affiliated Hospital of Sun Yat-sen University. Kidney allograft recipients with ABMR, P < 0.001), but comparable with that of the DSA-positive patients in the stable allograft group (P = 0.074). The AUC-ROC of cfDNA was 0.90 . When a cfDNA threshold of 1% was chosen, it had a sensitivity of 88.9% and a specificity of 73.7%. The PPV was 76.2% and the NPV was 87.5%.Totally 37 consecutive patients received kidney allografts, including 18 recipients in the ABMR group and 19 recipients in the stable allograft group (7 DSA-positive and 12 DSA-negative). All patients in the ABMR group were DSA positive and 7 patients in the stable group were DSA positive but had no pathologically proven ABMR. The median donor-derived plasma cfDNA fraction was 2.4% (Q1 1.52% -Q3 3.70%) in the ABMR group, and was significantly higher than that of the stable group (0.65%, Q1 0.57% -Q3 0.97%; Donor-derived plasma cfDNA fraction increased in kidney allograft recipients with ABMR. Detection of donor-derived plasma cfDNA fraction may contribute to the discrimination between ABMR and stable renal allograft function and may aid early recognition of earlier stage antibody-mediated injury. Antibody-mediated rejection (ABMR) is an important determinant of long-term outcome of kidney allografts . CurrentPrevious studies have demonstrated the presence of plasma donor-derived cell-free DNA (cfDNA) in allograft recipients , which cOnly scant literature is available on donor-derived cfDNA in kidney allografts. Studies have shown that donor-derived cfDNA levels are similar in renal function stable kidney allograft recipients and heart transplant recipients , 15. A sWe developed a single nucleotide polymorphism (SNP)-based method for detecting donor cfDNA, and have confirmed its use in monitoring acute rejection and infection in lung transplant patients . In the 2 and fluctuated within ±20% of the mean eGFR within the preceding year or from discharge from the hospital to enrollment in the study, whichever was shorter, and (3) who were HLA antibody negative, or who were DSA positive but had normal histology or no apparent pathological changes on percutaneous kidney biopsy. The ABMR group consisted of kidney allograft recipients (1) who were DSA positive, (2) who were indicated for percutaneous kidney biopsy because of elevated creatinine, proteinuria, positive DSA or follow-up examination after treatment, and (3) who had pathologic changes consistent with ABMR. The eGFR was determined using MDRD (≥16 years) or Schwartz (<16 years).In this prospective single center observational study, we enrolled consecutive kidney allograft recipients who received care between November, 2016 and September, 2017 at the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. The stable allograft group consisted of kidney allograft recipients (1) who had no proteinuria, within the preceding year or from discharge from the hospital to enrollment in the study, whichever was shorter, (2) whose eGFR was greater than 40 mL/min⋅1.73 mThe study protocol was approved by the local ethics committee at the authors’ affiliated hospital [No. (2017)171]. All study participants provided written informed consent. Kidney allografts from living or deceased organ donors who met the ethical guidelines for kidney donation were used.g 4°C within 4 hours of collection. The plasma supernatant was further clarified by centrifugation for 10 min at 16000 g to remove any remaining cells. The cells and the clarified plasma were stored at −80°C until use.Whole peripheral venous blood (5 mL) was collected at admission, 6, 12, and 24 months post kidney allograft surgery or at the time of each kidney allograft biopsy. Whole blood was centrifuged for 10 min at 1600 Plasma cfDNA was isolated using the QIAmp Circulating Nucleic Acid Kit  according to the manufacturer’s protocol. We measured cfDNA using a targeted next-generation sequencing assay  that empTM DNA software  to determine HLA genotype.Cellular DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen) as instructed by the manufacturer. HLA alleles  were detected using the Luminex platform and sequence-specific oligonucleotide (SSO) technique using the LIFECODES HLA-SSO kit  as instructed by the manufacturer. Specific sequences were analyzed using MATCHIT!TM ANTIBODY software. A mean fluorescence intensity <1000 was considered negative, between 1000 and 4000 weakly positive, between 4000 and 10000 intermediately positive, and >10000 strongly positive.Anti-HLA antibodies including antibodies against class IHLA-A, -B, and -C, and class II HLA-DRB1, -DQA1, -DQB1, -DPA1, and -DPB1 antigens were detected using the Luminex platform (Immucor Transplant Diagnostics) as instructed by the manufacturer. The mean fluorescence intensity of HLA antibodies was then calculated by normalization against the negative control. Data were analyzed using the LIFECODES MATCHIT!Pathological diagnosis of rejection was made according to the 2015 Banff Kidney Rejection Classification  by two eClinicians who were blinded to the cfDNA results chose treatment protocols for ABMR based on clinical conditions. Treatments included one or more of the following: plasma exchange, intravenous immunoglobulin, rituximab, and bortezomib. Glucocorticoid pulse therapy or anti-thymocyte globulin (ATG) was given if concurrent TCMR was present. The kidney allograft function-stable group and the DSA group were closely monitored and did not receive specific treatment. If ABMR developed, they were treated as described above.In additional to renal biopsy, routine blood tests and urinary test, biochemistries and hepatic and renal function tests were done regularly. Measurement of plasma concentrations of immunosuppressive drugs and anti-HLA antibodies were undertaken regularly. Complications, loss of graft function and death were recorded.t test was used for comparing differences between two groups for normally distributed data; Mann-Whitney U test was used for non-normally distributed data. One-way ANOVA or Kruskal-Wallis H test was used for comparison of continuous variables among more than two groups. A mixed linear model was used for repeated measurements of eGFR. P < 0.05 was considered statistically significant. Correlation between cfDNA and eGFR, and DSA MFI was evaluated by Spearman correlation analysis. The diagnostic performance of the cfDNA fraction for ABMR was evaluated using the receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC-ROC) was determined using the bootstrapping method to estimate median and 95% confidence interval (95% CI). The sensitivity, specificity and Youden index, positive predictive value (PPV) and negative predictive value (NPV) were calculated for specific cfDNA fractions . The optimal threshold was the cfDNA fraction corresponding to the maximal Youden index:All statistical analysis was done using R for Windows 3.6.1. Continuous variables were expressed as mean ± SD if normally distributed or median (the first quartile Q1 – the third quartile Q3) if non-normally distributed. Categorical data were expressed as frequency and percentage. Student’s P = 0.045). The ABMR group had significantly higher mean fluorescence intensity of DSA than the stable allograft group (P < 0.001). Furthermore, the ABMR group had a significantly lower mean eGFR than the stable allograft group . The two groups were comparable in other demographic and baseline variables.The study flowchart is shown in In addition, the Banff pathologic lesions of the ABMR groups are shown in P < 0.001, P = 0.016) (P = 0.074).The median donor-derived plasma cfDNA fraction was 2.4% (1.52–3.70%) in the ABMR group, and was significantly higher than that of the stable allograft group  . HoweverP > 0.05) . Furthermore, there was no statistical difference in cfDNA fractions among patients in the ABMR group with different Banff lesion grades (p = 0.791).The median time from renal transplant surgery to study enrollment was 3 years (range 1.0 – 4.4 years). The patients were followed up for median duration of 1.6 years (range 1.5 – 1.8 years). No patients in the cohort were lost to follow up. Except at the baseline, the eGFR was comparable among the ABMR group, DSA (+) subgroup and DSA (−) subgroup in the stable allograft group ( > 0.05) . Four (4n grades . There wP > 0.05) (We further used the median cfDNA fraction (2.4%) to categorize ABMR recipients into the low and high cfDNA fraction subgroup. Though the eGFR was numerically higher in the high cfDNA fraction subgroup than the low cfDNA fraction subgroup at each follow up measurement, no statistical difference was observed ( > 0.05) . FurtherTo define the AUC-ROC performance of cfDNA, we included all cfDNA results of the 37 allograft recipients. The AUC-ROC of cfDNA was 0.90  . When a Our prospective observational study revealed that donor-derived plasma cfDNA fraction well discriminated ABMR from stable renal allograft function in post kidney allograft transplant recipients. The ABMR group had a significantly higher median cfDNA fraction than the stable allograft group. The DSA negative patients in the stable allograft group had the lowest median cfDNA fraction. Our ROC analysis further showed an AUC of 0.90 , suggesting that donor-derived plasma cfDNA fraction is of good diagnostic performance.At the cfDNA fraction optimal threshold of 1% for ABMR, the test had a sensitivity of 88.9% and a specificity of 73.7%, a PPV of 76.2%,and a NPV of 87.5%, which is similar to the findings by Bloom et al. who identified an AUC-ROC value of 0.74  for donor-derived cfDNA fraction in discriminating active rejection including TCMR and ABMR and no active rejection and a AUC-ROC value of 0.87  in discriminating between ABMR and no ABMR and the optimal threshold for discriminating ABMR is 1.0%, with a PPV of 44% and a NPV of 96% . OelleriOur study and three other studies all showed that cfDNA fraction has a high NPV for ABMR diagnosis, which means most of patients with negative cfDNA fraction (below the threshold) do not have ABMR, suggesting that donor-derived cfDNA fraction measurement may avoid unnecessary kidney transplant needle biopsy when the other types of allograft injury can be excluded, especially in patients on anticoagulation medication or patients for whom it is inconvenient to perform kidney needle biopsy. For example, for patients with positive DSA but no clinical manifestations, if cfDNA fraction is negative, biopsy may be avoided temporarily and close follow-up should be performed.It remains controversial whether ABMR and TCMR could be discriminated by cfDNA fraction. Bloom et al.  found thUp to now, the diagnostic performance of cfDNA as a complement to DSA detection and biopsy in ABMR is not clear. Jordan et al. carried out a secondary analysis of the DART study  that incFew studies have investigated the relationship between donor-derived cfDNA and severity of pathologic injury and prognosis of ABMR. Our study did not show any correlation between cfDNA fraction and the mean fluorescence intensity of DSA in ABMR recipients, Banff pathologic categories and prognosis of kidney transplant, indicating that cfDNA fraction may not be used for assessing severity of acute and chronic lesions in ABMR as well as prognosis of ABMR. There were 3 patients in the low cfDNA fraction subgroup and 1 patient in the high cfDNA fraction subgroup who had graft loss. Among the three patients with low cfDNA (< median 2.4%), the pathological damage was serious , and thuUnder the current evidence, dd-cfDNA cannot be used alone as a diagnostic tool for a certain disease, and the diagnosis needs to be made in combination with medical history, clinical manifestations and other test results. To better apply the published evidence to the clinical practice, there are several aspects requiring clarification. First, the study population will affect the results interpretation. The risk of ABMR is variable in different study population, which will have an impact on the performing characteristics of cfDNA in diagnosis of ABMR. Second, the accuracy of the pathological diagnosis may have an effect on the results. The biopsy, which remains the gold standard for diagnosis of ABMR, reflects just a small part of the lesions and the pathological diagnosis is closely related to the location of the puncture, the quality of the specimen, and the experience of the pathologist. Third, up to now, no studies have investigated the association of cfDNA and the type of DSA . It has been realized that there is a difference in the pathogenicity of DSA, and 60–70% of patients with DSA do not develop ABMR. What causes the difference is still controversial. This is one of the directions for future research. However, it may be restricted by the high variability of DSA which requires large sample size and high cost to identify the effects. Fourth, detection timing will also affect the results. Evidence has confirmed that cfDNA will increase early in the postoperative period, and about 5 days after the will operation, cfDNA decrease to the normal value . If the Our study has several limitations. First, the sample size in this single-center study was not large enough. Nevertheless, the number of patients in the experimental groups  was comparable with that of the previously published studies , 28. SecIn conclusion, donor-derived plasma cfDNA fraction increased in kidney allograft recipients with ABMR and the detection of donor-derived plasma cfDNA fraction may contribute to the discrimination between the recipients with ABMR and those with stable renal allograft function. The optimal threshold of cfDNA fraction for diagnosing ABMR is 1%, with a PPV of 76.2% and an NPV of 87.5%. Donor-derived cfDNA fraction may aid early recognition of earlier stage antibody mediated injury.https://db.cngb.org/cnsa/) of CNGBdb with accession code CNP0000807.The data that support the findings of this study have been deposited in the CNSA (The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University. Written informed consent to participate in this study was provided by the participants and/or their legal guardian/next of kin.HZ, MY, and LL designed the study. HZ, CZ, and XL analyzed data and co-wrote the manuscript. CZ, XL, QS, and HH performed the experiments. JL and QF enrolled patients and collected data. LZ and ZL performed bioinformatic analyses. JW and BX collected and cleaned data. MY, LL, and CW supervised the research and critically reviewed the manuscript. All authors revised and approved the final manuscript. MY and LL was responsible for the decision to submit for publication.CZ, LZ, ZL, and MY were employed by the company BGI-Shenzhen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intraoperatively the hemoglobin level dropped further and reached 11.8 g/dL at 60 min in the absence of blood loss. Conclusions: Hypotensive epidural anesthesia and the resulting fluid substitution resulted in an average hemoglobin drop of 2.1 g/dL within the first 60 min. This needs to be taken into account when evaluating the need for blood transfusions after primary joint replacement surgery under HEA.Introduction: Hypotensive epidural anesthesia (HEA) is used in total joint arthroplasty as a safe and effective blood-saving modality. In order to maintain the blood pressure and heart rate patients, receive 1000 to 1500 mL of lactated Ringer’s solution during surgery. While HEA reduces the intraoperative blood loss, the effect of intravenous fluid loading on hemoglobin levels is not fully understood. The current study investigates the effect of HEA on perioperative hemoglobin levels. Materials and Methods: The study included 35 patients operated on by a single surgeon undergoing primary total knee arthroplasty under HEA. Intraoperatively, at least 300 mL of intravenous fluid were given every 15 min over the first 60 min after HEA. Blood samples were drawn before entering the operating room, after HEA, as well as after inflation of the tourniquet, every 15 min thereafter, as well as in the recovery room and on postoperative days one and two. In addition, fluid in- and outtake was recorded. Results: Patients received a mean 1275 mL during the 60 min of tourniquet time. The mean arterial pressure (MAP) 5 min after HEA dropped to 60 mmHg and reached a constant level of around 58 mmHg 15 min after HEA. The average hemoglobin level dropped from 13.9 g/dL prior to HEA, to 12.5 g/dL immediately after HEA ( Total knee arthroplasty (TKA) is a highly successful treatment option for end-stage arthritis of the knee, however it can result in significant blood loss ,2. The aHypotensive epidural anesthesia (HEA) is a readily available and effective method to reduce intraoperative blood loss . The tecDuring HEA, patients undergoing primary total hip replacement require an average of 1000–1500 mL of lactated Ringer’s solution to control hypotension . Fluid sThe current study investigates the effect of intraoperative fluid substitution during HEA on hemoglobin levels, during and after primary TKA.Between January and August 2014, thirty-five patients who underwent primary total knee arthroplasty were included in this prospective study. Out of these patients, 3 patients had to be excluded because of inadequate hypotensive anesthesia, resulting in too high blood pressure levels during surgery, leaving 32 patients, 22 female and 10 male patients for inclusion in the study. The senior author performed all surgical procedures. The institutional review board approved the study and all patients gave written consent prior to enrolling on the study. Demographic data , side of surgery, American Society of Anesthesiologists (ASA) score, diagnosis, procedure time, intraoperative blood loss in mL, blood pressure during surgery, administered intravenous fluid in mL every 15 min during the first 60 min of the procedure, hemoglobin and hematocrit levels and the amount of urine output and drain output were recorded.The first blood sample was drawn from an arterial line in the holding area prior to anesthesia. The patient was then transferred to the operating room and hypotensive epidural anesthesia (HEA) was performed aiming for an upper thoracic level such as T4 sensory block. 5 mL of blood were drawn from the arterial line to measure hemoglobin and hematocrit levels after HEA prior to inflation of the tourniquet and 15 min, 30 min, 45 min and 60 min after tourniquet inflation. In addition, blood was drawn immediately upon arrival in the postoperative care unit as well as postoperative day (POD) 1. Patients were required to receive at least 300 mL of lactate ringer every 15 min of tourniquet time.Blood pressure and heart rate were recorded preoperatively, prior to HEA, and every 5 min until the end of the procedure. In addition, the intravenous fluids were documented at 15 min intervals during the first 60 min, and every hour thereafter until POD 3. Furthermore the intraoperative blood loss and urine and drain output were recorded until discharge.Patients with non-inflammatory degenerative joint disease, between the ages of 40 and 80 years and undergoing primary total knee arthroplasty were included. Patients underwent neuraxial anesthesia and had to be rated grade 1 to 3, as per the American Society of Anesthesiologists (ASA) Physical Classification System. Exclusion criteria were patient refusing the participation in the study, contraindication to combined spinal epidural anesthesia , patients with congestive heart failure (at least one medication to treat congestive heart failure), coronary artery disease , kidney insufficiency (creatinine > 1.5 mg/dL), aortic or mitral valve disease and pulmonary hypertension. In addition, patients were excluded if they received less than 1200 mL of intravenous fluid during 60 min tourniquet time and if HEA resulted in inadequate levels of hypotension.The tourniquet was used during the first 60 min of the procedure. Therefore, there was no blood loss during the first 60 min of the surgery. In conclusion, the drop in hemoglobin level should be primarily related to the decrease in blood pressure and hemodilution secondary to fluid loading during HEA. The tourniquet always remained in place for 60 min.s) was used for nonparametric correlations. p-values < 0.05 were considered significant. Statistical tests were carried out using SPSS 22 software for windows .The distributions of variables were tested in exploratory data analysis. The Kolmogorov–Smirnov test was performed to test for the normal distribution of variables. Absolute mean values and differences of hemoglobin were expressed in grams per deciliter with standard deviation (SD). Hematocrit levels were described in volume percentage (vol.%) with SD. As not all variables met the criteria for normal distribution, the Wilcoxon signed ranks were used to compare differences of variables in time. Spearman rank correlation (r2 (range 20.7 to 42.4 kg/m2). Sixty-nine percent of the patients were female (n = 22), thirty-one percent male (n = 10). Twelve patients (37%) underwent left and 20 patients (63%) right TKA. In all patients, the ASA (American Society of Anesthesiologists) score was assessed, two patients were rated ASA 1, 22 patients ASA 2 and 8 patients ASA 3. All three patients, whom had to be excluded from the study because of insufficient drop of the blood pressure, belonged to ASA 3 group. Mean hemoglobin and hematocrit levels are displayed in The average age at the time of surgery was 66 years (range: 44 to 79 years) and the average BMI was 30.6 kg/mPatients received a mean of 1275 mL  lactated Ringer’s solution during surgery, of which 440 mL were given preoperatively. Altogether, a mean of 2947 mL  lactated Ringer’s solution were substituted at the day of surgery .The mean arterial pressure (MAP) prior to HEA was 87 mmHg. Five minutes after HEA, the mean MAP had dropped to an average of 60 mmHg and 15 min after HEA, the mean MAP dropped to an average of 58 mmHg . p < 0.001). After initiation of hypotensive epidural anesthesia, hemoglobin levels dropped by a mean of 1.4 g/dL (SD 0.5) and hematocrit levels dropped by a mean of 4.3% (SD 2.2). The differences of hemoglobin and hematocrit levels prior and after initiation of HEA were highly significant (p < 0.001). The release of the tourniquet resulted in a slight increase of Hb  and hematocrit  compared to levels 60 min after tourniquet inflation. In addition, the current study reports no difference at any timepoint regarding the intraoperative drop in hemoglobin and hematocrit in patients with a BMI < 30 and >30 kg/m2.The mean hemoglobin levels continued to drop during the 60 min of tourniquet time by an average of 0.7 g/dL (SD 0.4), the mean hematocrit dropped by an addition 2.1% (SD 1.3), respectively . The mean difference of hematocrit levels between PACU and POD 2 also correlated well with the total drain volume . Excellent correlations of preoperative hemoglobin levels and hemoglobin levels prior to tourniquet inflation with Hb levels on postoperative day 1 and postoperative day 2 were observed (The average drain output until POD 1 was 334 mL (range: 0–1100 mL). The mean difference in hemoglobin levels comparing POD 1 and POD 2 was 0.7 g/dL (SD 0.9 g/dL), the mean Hematocrit difference was 2.5% (SD 2.5%), respectively. The mean total drain volume was 334 mL (range: 0–1100). The mean difference in hemoglobin levels between time in PACU and POD 2 significantly correlated with the total drain volume  using student’s t-test for independent samples with parametric data, no significant differences were shown. Furthermore, the length of surgery showed very little variance with a mean of 61 (range 60–70) and the 95th percentile was 67 min with no significant differences.We compared hematocrit at the different intraoperative timepoints for the respective subgroups  since the blood volume is not measured it is not clear whether hemodilution is the underlying mechanism for the hemoglobin drop, (2) hemoglobin levels are determined from an arterial line and this could result in dilution of the blood samples. However, all samples were retrieved using the same technique, (3) the application of the tourniquet could result in changes of the overall blood volume, however, exsanguination of the leg should not have an impact on hemoglobin or hematocrit levels. The slight increase in hemoglobin and hematocrit after the release of the tourniquet suggests mixture of non-diluted blood from the operated extremity with diluted blood in the remaining body. (4) After the release of the tourniquet changes in blood loss are likely the result of bleeding from the surgical site and decreased hemodilution after discontinuation of the HEA.p < 0.001), however, continued changes in hemoglobin levels despite continued fluid substitution remained rather small (0.7 g/dL). The data suggest that initially after the HEA a considerable amount of fluid is retained or mobilized from the extravascular space to balance out the hypotensive effect of a high epidural anesthesia and its blockage of the sympathetic nerve system. The continued infusion of epinephrine probably minimizes the hemodilution associated with continued intravenous fluid infusion and might be the main reason for the reduced effect of HEA on hemoglobin levels later in surgery. After the release of the tourniquet, the hemoglobin level slightly increased by 0.2 g/dL, which might be explained by the mixture of undiluted blood from the operated extremity following the release of the tourniquet. In order to maintain blood pressure and heart rate, the patients received a mean of 1275 mL lactated ringer solution during surgery. This acute volume loading had an effect on the hemoglobin level during the use of the tourniquet. The immediate hemoglobin drop after initiation of the HEA in the absence of blood loss was statistically significant  to 50 mmHg has been found to decrease intraoperative blood loss by up to 40%  and reduSharrock et al. suggest that HEA does not increase the risk for acute renal dysfunction . HEA hasA randomized study comparing HEA and spinal anesthesia in patients scheduled for TKA reported no differences in renal function and no cardiac or neurologic complications. In addition, no increase in complications has been reported for the elderly .The most recent study concerning HEA in orthopedic surgery reported less blood loss, fewer transfusions and no increase in complications in sacral and pelvic bone tumor resections .Reports on negative side effects of HEA are rare, and include temporary decline in postoperative cognitive, hepatic or renal function . In addiWhile the perioperative anemia secondary to HEA has to be taken into consideration when judging the need for blood transfusion, its overall extend does not seem to outweigh its reported benefits on blood loss and morbidity. While hypotensive epidural anesthesia (HEA) has a significant immediate effect on patients’ hemoglobin levels; the continued drop in hemoglobin, although statistically significant, is rather low. Approximately 2.1 g/dL drop in hemoglobin in the first 60 min after surgery appear to be the result of the hemodilution from the intravenous fluid substitution during HEA. This needs to be taken into consideration when assessing the need for perioperative blood transfusions in the first 24 h after surgery performed under HEA.


Coronary heart disease (CHD) is one of the highest mortality diseases in the world, which seriously threatens human health and quality of life (QOL). The purpose of this study is to systematically analyze the effects of mind-body exercise on cardiopulmonary function, blood pressure and QOL in CHD patients, and to provide scientific evidence-based exercise prescription for patients with coronary heart disease.This research review will include the following electronic databases from its establishment to December 2020: PubMed, EMBASE, Web of Science, Cochrane Library, the Chinese National Knowledge Infrastructure, the Chinese Science and Technology Periodical Database, and Wanfang. Objective to search randomized controlled trials (RCTs) about the effects of mind-body exercise on cardiopulmonary function, blood pressure and QOL in patients with coronary heart disease.This systematic review and meta-analysis will provide strong evidence for the efficacy and safety of mind-body exercise in patients with coronary heart disease.INPLASY202120016.Ethical approval will not be necessary since this systematic review and meta-analysis will not contain any private information of participants or violate their human rights. Nearly every 25 seconds, an American will have a coronary event related to CHD, and every minute someone will die from one. It is well documented that exercise prevents and even reverses CHD. The U.S. Department of Health and Human Services (2008) formulated a recommendation on health promotion and disease prevention, in which reducing cardiovascular deaths and encouraging exercise are the 2 main goals. Therefore, health care providers are encouraged to reduce the risk of CHD by recommending appropriate aerobic exercises to patients to promote a healthy lifestyle. Some studies have shown that exercise training can effectively strengthen the aerobic metabolism of patients with coronary heart disease, which is conducive to the regulation of blood pressure, blood glucose, and blood lipid of patients. In addition, it can also improve the cardiopulmonary function of patients with myocardial infarction, reduce the incidence of acute coronary artery events and readmission rate of patients, and then improve the quality of life (QOL) of patients.–9 Ornish et al showed that the risk of sudden death in acute myocardial infarction patients receiving cardiac rehabilitation decreased by 45% within 1 year. As a way to promote physical and mental health, mind-body exercise has attracted the attention of many scholars in recent years. Mind-body exercise focuses on mind, body, psychology, and behavior, which is an effective combination of breathing and physical activities,–12 emphasizing the advantages of mind-body exercise at the same time.In recent years, the treatment of coronary heart disease in addition to conventional treatment also includes some nonpharmacological alternative therapy. Regular physical activity in the form of exercise has been used as an alternative therapy for many cardiovascular diseases, particularly CHD.2Objective to systematically evaluate the effects of mind-body exercise on cardiopulmonary function, blood pressure, and QOL in patients with coronary heart disease (CHD). We expect to provide reference for mind-body exercise treatment of coronary heart disease.33.1 statement guidelines.This review protocol is registered in the International Platform of Registered Systematic Review and Meta-analysis Protocols, registration number INPLASY202120016. This systematic review protocol will be conducted and reported strictly according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)3.23.2.1The inclusion criteria were randomized controlled trials (RCTs), with mind-body exercise as the main form of intervention. The language of literature only includes Chinese or English.3.2.21.Repeated publication;2.Animal experiment;3.Incomplete literature;4.Nonrandomized controlled trials;5.Systematic review and meta-analysis.3.33.3.11.New York Heart Association (NYHA) classification I–III;2.40 years old ≤ age < 70 years old;3.no hemodynamic disorder;4.good compliance.3.3.21.Patients with severe cardiopulmonary insufficiency and severe diseases;2.patients with severe limb and joint diseases affecting sports rehabilitation training;3.patients with severe infectious diseases and malignant tumors;4.patients with uncontrolled severe complications.3.4The intervention methods of the experimental group were mind-body exercise, including Tai Chi, yoga, Qigong, meditative movement, etc. The control group included usual care, no physical activity, and no-intervention control group and other different types.3.53.5.1The indexes of cardiopulmonary function were maximal oxygen uptake, maximal heart rate, and left ventricular ejection fraction.3.5.2The following were defined as secondary outcomes:1.Atherosclerosis;2.Systolic and diastolic blood pressure;3.QOL .3.6This research review will include the following electronic databases from its establishment to December 2020: PubMed, EMBASE, Web of Science, Cochrane Library, the Chinese National Knowledge Infrastructure, the Chinese Science and Technology Periodical Database, and Wanfang. The search terms will include “mind-body exercise,” and “yoga” or “taichi” or “tai ji quan” or “Taiji boxing” or “tai chi chuan” “qigong” or “health qigong” or “meditation,” with CHD terms including “coronary heart disease” or “CHD.”3.73.7.1  will independently check the title and abstract of the search results and preliminarily screen the articles. After reading the full text of the preliminary selected articles, 2 independent reviewers selected eligible studies according to our predetermined inclusion criteria. Finally, the article selected by 2 independent commentators is integrated after putting forward repetitive parts. When 2 independent reviewers disagree, the third reviewer(Yang) will make the decision. The details of selection process will be displayed in the PRISMA flow chart Fig. .3.7.2We will generate a funnel chart to evaluate the reporting bias. For continuous variables, Egger test will be used to test the asymmetry of funnel plot. It is generally believed that asymmetric funnel plot will present publication bias, which is one of the reporting bias, but it also means that there may be other reasons, such as methodological quality differences or real heterogeneity of intervention effect. We will analyze the possible reasons and give a reasonable explanation for the asymmetric funnel diagram.3.7.3The risk ratio (CIS) of 95% confidence interval was used. Odds ratio (OR) and relative risk (RR) are commonly used for dichotomy outcome data. For continuous variables, the mean or standard deviation of 95% CI is calculated.3.7.4The accuracy of the data may affect the results of the study, and even lead to different conclusions. Therefore, we will contact the corresponding authors to supplement the research results with insufficient or missing data. If the corresponding author cannot be contacted, the data will be processed separately, and the potential impact of incomplete data on the results will be analyzed and reported.3.7.5 to evaluate the methodological quality of each eligible study. The assessment includes the following 11 items: Item1 = explicit inclusion criteria; Item2 = randomization; Item3 = allocation concealment; Item4 = Similar at baseline; Item5 = Subjects blinded; Item6 = Therapists blinded; Item7 = Assessors blinded; Item8 = more than 85% retention; Item9 = Intention-totreat analysis; Item10 = Between-group comparisons; Item11 = Point measures, and variability data. The score of each item is 1 (clearly described in the article), or 0 (absent or unclear). According to different total scores, each study will be divided into 3 quality levels: high (9–11), moderate (5–8), and low (0–4). Any disagreement will be resolved through discussion and negotiation with a third experienced reviewer (Yang).Two independent reviewers (Duan and Wang) will use the Modified Physical Therapy Evidence Database (PEDro) scale3.7.6I2 value were used to evaluate the heterogeneity. I2 values of 25%, 50%, and 75% are considered as low, moderate, and high heterogeneity, respectively.The heterogeneity assessment will be conducted by the Review Manager (v.5.3.5). According to the Cochrane Handbook, Chi-Squared test and 3.7.7If there are enough studies available, we will evaluate the funnel plot to report bias.3.7.8I2 value is less than 50%, it means that the relative heterogeneity is small, and the fixed effect model should be used. Otherwise, the random effect model will be used.The Review Manager (v.5.3.5) software will be used for quantitative data analysis, including mapping overall forest plot, heterogeneity analysis, regression analysis, and subgroup analysis. If the 3.7.9Considering the possibility of high heterogeneity, we will conduct subgroup analysis to explain the potential causes of heterogeneity when necessary. First of all, the analysis will be based on the age, gender, course of disease of patients with coronary heart disease. In addition, according to the different intensity, time and duration in the process of mind-body exercise intervention.3.7.10I2 value will be used to quantify statistical heterogeneity.Sensitivity analysis will be used to assess the robustness of the results. After data synthesis, we will exclude the combined studies one by one for sensitivity analysis to observe whether the comprehensive results have significant changes. If there are significant changes indicating that the exclusion study has a significant impact on the results, it should be reevaluated. On the contrary, the comprehensive data results are reliable. Chi-Squared test and 3.7.11An internationally recognized scoring system will be used to assess the quality of our evidence. The evidence evaluation of all results will be summarized by the suggested assessment, development and assessment (GRADE) method. The level of evidence will be divided into high, moderate, low, and very low quality.4 and then reduce acute cardiovascular event mortality and all-cause mortality. Some studies have also shown that cardiovascular disease can lead to autonomic nervous regulation function damage, mainly manifested as chronotropic insufficiency of the heart. Heart rate and blood pressure may increase during the attack. Heart rate and blood pressure are effective indicators reflecting the autonomic nervous regulation function of the heart. Long term mind-body exercise can also enhance the ability of cardiac autonomic nerve regulation, reduce the sympathetic nerve activity of patients with coronary heart disease, improve the tension of vagus nerve, and keep the blood pressure and heart rate in the normal range.Patients with CHD often due to coronary artery disease and lead to the corresponding myocardial blood supply, oxygen shortage, myocardial asynchronous contraction, cardiac stroke volume and circulating blood volume decreased, and then affect the cardiopulmonary function of patients. Previous studies have shown that mind-body exercise can increase coronary blood flow, improve myocardial ischemia and hypoxia, reduce cardiovascular risk factors,In recent years, various mind-body exercise intervention methods have been increasingly used in the treatment of chronic diseases. A large number of studies have explored the effects of mind-body exercise on the cardiopulmonary function, blood pressure, heart rate, and QOL in patients with coronary heart disease. The specific results may be different due to differences in individual conditions such as the age, gender, and course of the intervention samples. At the same time, the time, intensity, frequency, specific methods, and duration of the intervention will also affect the results. Therefore, the purpose of this meta-analysis is to evaluate the effects of mind-body exercise on the cardiopulmonary function, blood pressure, heart rate, and QOL in patients with coronary heart disease, and to provide specific exercise prescriptions about the time, intensity and frequency of mind-body exercise for patients with coronary heart disease.Conceptualization: Fei-xing Duan, Hui-xin Yang, Fei Dai, Lin Wang.Data curation: Qi Wu, Yi-fan Zuo.Formal analysis: Qi Wu, Yi-fan Zuo.Funding acquisition: Qi Wu, Yi-fan Zuo, Lin Wang.Methodology: Fei-xing Duan, Hui-xin Yang, Fei Dai.Resources: Lin Wang.Software: Fei-xing Duan, Fei Dai.Supervision: Hui-xin Yang, Lin Wang.Validation: Fei-xing Duan.Writing – original draft: Fei-xing Duan.Writing – review & editing: Hui-xin Yang, Lin Wang.


In th0 ,4. We wo0 aim to correct for. Notably, different individuals with different blood pressures may show a different PWV due to (1) pressure dependency of PWV, and/or (2) intrinsic  differences in arterial stiffness [0 only correct—and should only correct—for the first effect. Differences in intrinsic arterial stiffness between individuals, e.g., due to hypertensive remodeling, age, obesity, etc., should be reflected in CAVI/CAVI0, thus allowing for the use of CAVI and CAVI0 to study the effect of such phenomena on arterial stiffness. After all, if a stiffness index did not vary with any of those phenomena, there would be no use in measuring it, and it would not predict any outcome.Due to the nonlinear elastic behavior of the artery wall, a higher blood pressure results in a higher pulse wave velocity (PWV) at the time of measurement ,6,7. It tiffness . CAVI an0 in a large population of normotensive and hypertensive individuals [0 was measured only once. Therefore, relations with blood pressure observed in such a study will reflect both intrinsic  blood pressure dependency and between-subject differences in intrinsic arterial stiffness. As elaborated in the previous paragraph, CAVI and CAVI0 are only meant to correct the former and not the latter effect. It is clear, therefore, that cross-sectional studies, while very useful to study population patterns and to derive reference values, cannot be used to assess the performance of CAVI and CAVI0 in correcting for the intrinsic blood pressure dependency of PWV [In a recent study, Shirai et al. compared CAVI and CAVIividuals . While ty of PWV . Such st0 and diastolic blood pressure (DBP) in healthy individuals, which Shirai et al. then used as a reason to label CAVI0 as “inappropriate” [0 on average have a lower DBP. This could be explained as follows. Subjects with  stiffer arteries typically have a larger pulse pressure than subjects with less stiff arteries. As these subjects were classified as healthy , their systolic blood pressure was probably approximately normotensive. The DBP of the individuals with stiffer arteries, however, could well have been lower than that of those with less stiff arteries, which potentially explains the negative cross-sectional correlation between CAVI0 and DBP as observed by Shirai et al.Alizargar et al. point out that Shirai et al. observed a negative, “inexplicable” correlation between CAVIopriate” ,9. Again0 were never aimed to be BMI-independent. In our initial study, we simulated subjects with differing values of intrinsic stiffness. Such differences could be interpreted as being due to BMI, age, calcification, arteriosclerosis, etc. However, the aim of our study was not to investigate the effects of these particular phenomena; it was merely to illustrate how, in a typical study cohort with (patho)physiological differences in intrinsic stiffness among subjects, blood pressure fluctuations could  influence CAVI and CAVI0 measurements.Alizargar et al. also mention that in our initial study, we did not take into account physiological properties of individuals, such as body mass index (BMI) ,4. Againa and b) that were recently disclosed to the public [0 [a/b parameters are used for different CAVI ranges [0 does not use such scale parameters. Considering these criticisms, as well as the CAVI-CAVI0 discussion, Alizargar et al. suggest that normal PWV and stiffness index β may be more reliable indices than the (less established) CAVI and CAVI0 [0 are all much less blood pressure dependent than PWV [Calculation of CAVI involves the use of two scale parameters (e public . This die public ) conversublic [0 . DiffereI ranges , the effI ranges . Notablynd CAVI0 . We agrethan PWV ,10,14, a0 [To conclude, we would like to re-emphasize that we consider the development and practical application of CAVI, as extensively described by Dr. Shirai and colleagues ,10, to b0 ,16,17,18


Hypertension is a major source of cardiovascular morbidity and mortality. Recent evidence from mouse models, genetic, and cross-sectional human studies suggest increased proportions of selected immune cell subsets may be associated with levels of systolic blood pressure (SBP).We assayed immune cells from cryopreserved samples collected at the baseline examination (2000–2002) from 1195 participants from the multi-ethnic study of atherosclerosis (MESA). We used linear mixed models, with adjustment for age, sex, race/ethnicity, smoking, exercise, body mass index, education, diabetes, and cytomegalovirus titers, to estimate the associations between 30 immune cell subsets (4 of which were a priori hypotheses) and repeated measures of SBP (baseline and up to four follow-up measures) over 10 years. The analysis provides estimates of the association with blood pressure level.++CD16−) was associated with 2.01 mmHG (95% CI 0.79–3.24) lower average systolic blood pressure. There were no associations of CD4+ T helper cell subsets with average systolic blood pressure.The mean age of the MESA participants at baseline was 64 ± 10 years and 53% were male. A one standard deviation (1-SD) increment in the proportion of γδ T cells was associated with 2.40 mmHg [95% confidence interval (CI) 1.34–3.42] higher average systolic blood pressure; and for natural killer cells, a 1-SD increment was associated with 1.88 mmHg (95% CI 0.82–2.94) higher average level of systolic blood pressure. A 1-SD increment in classical monocytes (CD14These findings suggest that the innate immune system plays a role in levels of SBP whereas there were no associations with adaptive immune cells. Hypertension is a major source of cardiovascular morbidity and mortality in the United States , 2. Majo+ T cells, and T helper type 1 (Th1) cells, analyzed as proportions of CD4+ cells, were associated with subclinical atherosclerosis [Human and animal studies suggest that both the innate and adaptive immune system may be related to hypertension –7 althouclerosis , 9. ThesTo investigate the links between immune cell subsets and cardiovascular disease (CVD), we used cryopreserved peripheral blood mononuclear cells (PBMC) to phenotype a range of immune cells by flow cytometry in the MESA cohort . We leveMESA is a community-based sample of 6814 men and women aged 45–84 years and recruited from 6 field centers . Participants self-identified as White, Black, Hispanic, or Chinese. Exclusion criteria for the MESA cohort included a self-reported medical history of myocardial infarction, angina, cardiovascular procedures, heart failure, cerebrovascular disease, active treatment for cancer, pregnancy or amputation. Details regarding design, recruitment, and objectives of MESA are described elsewhere . We desiThe original case cohort study of incident MI/angina was designed from the main MESA baseline cohort using cryopreserved cells stored at baseline 2000–2002) . The ori000–2002 Therefore, using weights derived from the case-cohort sampling to account for over-sampling of events, we included all participants with available data from this case-cohort study to investigate the associations of immune cell subsets with measures of systolic blood pressure over the course of the study. To be specific, we used MESA data from Exams 1–5 over 10 years of study follow-up, all of which included systolic blood pressure measures.University of Washington IRB Committee J reviewed the MESA cohort study and approved it as FWA #00006878.At the baseline (2000–2002) and follow-up clinical visits  standardized questionnaires and calibrated devices were utilized to obtain data on demographics, tobacco use, medical conditions, prescription medication usage, weight, and height. PBMCs, used in this study, were collected at baseline. At each MESA exam, resting, seated blood pressure was measured 3 times using a Dinamap automated oscillometric sphygmomanometer ; the last 2 measurements were averaged for analysis. To adjust for hypertension treatment (33% of MESA participants were treated for hypertension at baseline), we added 10 mmHg to systolic blood pressure for treated participants, based on an approach that is widely used in genome-wide association studies (GWAS) , 16, alt+  and CD8+ , cells were activated for 3 h at 37 °C with phorbal myristic acetate & ionomycin, and stained with live/dead stain. Cells were then surface labeled for CD4/CD8, fixed with paraformaldehyde and intracellularly stained for interferon gamma (IFN-γ), interleukin 4 (IL-4), and interleukin-17A (IL-17A) [Detailed methods for immune cell phenotyping and flow cytometry gating strategies have been published  and spec(IL-17A) . All oth+ Th1, Th, Th17 an(IL-17A) . Briefly(IL-17A) .Table We used linear mixed models to evaluate the associations between immune cell subsets measured at MESA baseline and systolic blood pressure levels over 10 years, across five MESA exams. Models were adjusted for covariates measured at baseline that were thought to be possible confounders, including age, sex, race/ethnicity, smoking, exercise, body mass index (BMI), college education, diabetes, and log transformed cytomegalovirus antibody titer , 9. Catep value threshold was 0.05/4 = 0.0125 for our primary hypotheses, and for our secondary hypotheses was 0.05/30 = 0.0017. Additionally, a false-discovery rate (FDR) approach [These mixed models handled within-person correlation due to each subject having up to five systolic blood pressure measurements with subject-specific random intercepts. Inverse probability of sampling weights were used to account for the case-cohort sampling scheme. Confidence intervals used robust (sandwich) standard error estimates to ensure validity in the presence of the sampling weights. We examined the residuals of the linear regression model to determine whether the residuals were normally distributed and thus estimation using linear mixed models was appropriate. We used Bonferroni correction as our primary approach for controlling for multiple testing , despiteapproach  was implPrimary analyses used all available data for each immune cell subset, under the assumption that missing data were missing completely at random. We included the SBP value at each of the five MESA exams as a repeated measure of blood pressure and thus estimates of association are with the average level of blood pressure, not with changes in blood pressure over time. Mixed models were used to handle irregular data sampling, differences in timing between measures, and the same participant contributing between one and five measures. Figure We performed exploratory analysis for immune cell interactions by age, sex, race, and BMI for the primary hypotheses and any secondary hypothesis that was deemed significant after multiple testing correction. We tested for effect measure modification by antihypertensive medication use, using medication exposure as a time-varying covariate.The case-cohort study sample included 1195 MESA participants, with a mean (SD) age of 64 (± 10) years, and 53% of whom were male. The mean (SD) baseline systolic blood pressure was 130.0 mmHG (21.7 mmHg) at the Exam 1 baseline visit (Table + cells measured. The numbers of participants with data on any particular immune cell phenotype ranged from 770 to 1113.The proportions of immune cell subsets and the number of participants contributing data to each are presented in Table +IFN-γ+), Th17 (CD4+IL-17A+), and Tregs (CD4+CD25+CD127−)), only γδ T cells were significantly associated with systolic blood pressure  was associated with a 2.01 mmHG (95% CI 0.79–3.24) lower level of systolic blood pressure. While barely missing the Bonferroni threshold, a 1-SD increment in non-classical monocytes (characterized as CD14dimCD16++) was associated with a 1.82 mmHG (95% CI 0.64–3.00) higher level of systolic blood pressure. In sensitivity analyses, using a false discovery rate approach [After adjusting for multiple comparisons, three other immune cell subsets, included in our exploratory secondary analyses, were also associated with systolic blood pressure; two were significant using a Bonferroni criteria , while using FDR added one additional immune cell subset . A 1-SD increment in the proportion of natural killer cells was associated with a 1.88 mmHG (95% CI 0.82–2.94) higher level of systolic blood pressure during follow-up. The two other immune cell subsets associated with systolic blood pressure were both monocytes. A 1-SD increase in classical monocytes (characterized as CD14approach  instead p = 0.0203)  Table . For allThe main finding of this study is the association of γδ T cells, natural killer cells, and two monocyte populations with average systolic blood pressure during 10 years of measurement in a large multi-ethnic population. Three of these immune-cell subsets-γδ T cells, natural killer cells, and non-classical monocytes-are pro-inflammatory cells, which may suggest an association between innate immune cell-mediated inflammation and systolic blood pressure in a multi-ethnic cohort with no baseline cardiovascular disease.One of our a priori hypotheses, the association between higher proportions of γδ T cells and higher systolic blood pressure replicates the association seen in Caillon et al.  and furtIn our exploratory analysis, we discovered an association between increased proportions of natural killer cells and levels of systolic blood pressure. Like γδ T cells, NK cells play a critical role in viral infection . NaturalMonocytes are an important component of the innate immune system and previous studies have implicated monocytes in atherosclerosis . MonocytThese data support other recent studies showing important links between the immune system and diseases, including both cardiovascular and kidney disease –33. ThisThe strengths of this study include the large sample size, the large panel of cell subsets evaluated and the long term, longitudinal measures of systolic blood pressure. The limitations include the observational nature of the data and technical issues with complex samples which resulted in some missing data. The use of cryopreserved PBMCs may result in different absolute levels of some of the subsets as compared with whole blood , althougIn conclusion, this study provides evidence that γδ T cells, natural killer cells and classical and non-classical monocytes are associated with systolic blood pressure levels in a large multi-ethnic cohort. These data support previous animal studies that show a role for innate immune cells in affecting systolic blood pressure levels, and the consequent possibility of hypertension.Additional file 1. Sample SAS code used to build the dataset used in this analysis and the exact statistical models used.Additional file 2. Supplmentary tables.Additional file 3. Sample MESA informed consent form.